Harold Hillman's 'Unanswered Questions in Biology'

47 Unanswered Questions in Biology... First posted by Rae West in 1996 (to cellbiol and other Usenet groups) as an online debate on the foundations of biology, these questions, designed to highlight errors in post-1945 biology, remain unanswered. Some of the evasions are quite amusing; see for example Azpiroz's and Brookes's emails. May the truth prevail!
	... e-mail exchanges with biologists. (Hillman highlit in green). Ricardo Azpiroz, University of Arizona \| Chris Barry, Lawrence Livermore \| Beverley Barton, Schering-Plough Research Institute \| Paul S Brookes, Cambridge University \| Tom Chappell, University College, London \| Alex Dain?, UW Australia \| Richard Delorme , Lyon University \| Greg Fraley, Washington \| Richard van Frank, [not known] \| Peter French, Sydney. \| Warren Gallin, Alberta \| Brad Harris, Utah \| Richard Kondo, UCLA \| Cornelius Krasel, Würzburg (LONG!) \| John Joseph Ladasky, Berkeley \| Kevin McKenna, Northwestern University, Il. \| Ian Musgrave, Monash, Australia \| Paul Page, UWLAX? \| Anthony J Pelletier, Scripps Research Institute
Notes: These emails are all unedited and complete apart from a few very trivial points, e.g. where my name or email caused confusion. The numbering references may be slightly wrong, as the original list of 48 questions was found to have one duplicate. Apologies for the time taken to put these on the web. I hope the arrangement is as clear as possible; to avoid endless repetitions I've posted every other email, highlighting Harold's newest additions in green. © presumably Harold Hillman and others. Rae West 18 April 2000. \| Big Lies Home Page \| How much Modern Biology is a Fraud?

47 UNANSWERED QUESTIONS IN BIOLOGY. 27 May 1996. June/July 1996

Dr Harold Hillman writes:–
The only real guarantee of the progress of knowledge is for academics to be ready to enter into unlimited dialogue about their research and theories, especially about those which they have published. An academic who is not prepared to discuss or correspond with other interested parties is behaving improperly, and such conduct should not be tolerated by the academic community. Some colleagues seem to think that if they ignore the awkward questions about their disciplines, or are hostile to those who ask them, the contradictions or anomalies in their work will somehow or other resolve themselves, and their research can progress. On the contrary, such an attitude inhibits the examination of the fundamental aspects of their disciplines, and thus delays substantial progress.
The following questions have never been answered satisfactorily, several of them never at all:—
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Question 1: Can one obtain an enriched fraction of a subcellular organelle or cell type?
Question 2: How does one know that the disruptive procedure does not change the biochemistry of the fraction significantly?
Question 3: Why does one assume that homogenisation and centrifugation do not change the entropy, and therefore the free energy and the equilibria of reactions in subcellular particles? Why are not controls always carried out for subcellular fractionation, except for total recoveries relative to the crude homogenates?
Question 4: Why is it believed that each biochemical pathway or cycle has its own structural compartment when prokaryotes can carry out virtually all the same reactions in only one compartment?
Question 5: Does the finding that a chemical substance or activity is located in the same subcellular fraction and a structure identified by electron microscopy mean that the same chemical activity was located in that particular organelle in the living cell of the intact animal or plant.
Question 6: How is intracellular movement possible, and why is the viscosity of cytoplasm so low in the intact cell, if there is a cytoskeleton present?
Question 7: Where do protein synthesis and acid hydrolysis occur in cells in which ribosomes and lysosomes cannot be seen?
Question 8: What is the evidence that the microsomal fraction consists of cell membranes and endoplasmic reticulum?
Question 9: Why is it assumed that homogenisation and centrifugation do not affect the chemistry of receptors, or their affinities for transmitters, hormones, drugs, ligands, toxins?
Question 10: Can a particle and a vacuole both be lysosomes?
Question 11: Can one calibrate substances originating from tissues using pure solutions in simple salines of approximately the same concentrations?
Question 12: How can one study membranes by electron microscopy, when they are believed to contain lipids which the procedure extracts?
Question 13: What is the real evidence that rapid deep freezing for electron microscopy causes less shrinkage and distortion of tissues, cells and organelles, than classical transmission electron microscopy?
Question 14: Why do those who calculate dimensions from electron micrographs not take into account the shrinkage during preparation and examination of their sections, cells and organelles?
Question 15: Do membranes in cells appear to be normal to the plane of section more often than solid geometry would permit?
Question 16: Can one know the thickness in life of any biological membrane?
Question 17: Why should it be necessary to tilt the stage of the electron microscope to see randomly orientated membranes in all orientations, when this is not necessary with the light microscope?
Question 18: How can carriers assist the passage of ions, aminoacids, etc. across membrane, when the combination must be bigger than the substance carried?
Question 19: Why have few or no carriers been isolated?
Question 20: What is transport?
Question 21: Why are receptors and channels, which have been characterised, sequenced and their sizes measured or calculated, not seen on membranes by transmission electron microscopy?
Question 22: Can an electron microscopist looking at a metal deposit on a biological structure derive any information about its chemistry?
Question 23: Why do the lamellae of the myelin sheath appear to be equal distances apart irrespective of the thickness or depth of the longitudinal section cut?
Question 24: Is the repeating distance of the lamellae in the myelin sheath sufficient to regard it as a good model for the cell membrane?
Question 25: Since the myelin sheath is believed to consist of a scroll of membranes, and membranes appear darker by light microscopy than cytoplasm, why does not the myelin sheath appear darker than the axoplasm?
Question 26: Why is it assumed that the receptors for transmitters, hormones, messengers, antibodies, drugs and toxins are on the surface of the cell membrane?
Question 27: How valid is the use of agonists, antagonists and ligands to detect receptors, instead of the transmitters, hormones, antigens, drugs and toxins themselves?
Question 28: Why are the dimensions and numbers of synapses different by light and electron microscopy?
Question 29: Why are there no light micrographs in the literature showing the connection of one cell body by a dendritic pre-synaptic fibre to a synapse on another cell body?
Question 30: Does the chemical theory of synaptic transmission contain unprovable and unproved hypotheses?
Question 31: Why is it assumed that evidence derived from experiments on neuromuscular junctions is relevant to transmission in the central nervous system?
Question 32: If nuclear pores allow RNA to pass through, how do they prevent smaller molecules and ions going through at the same time, and why is there a potential difference across the nuclear membrane?
Question 33: What is the evidence that each cell of a particular plant or animal contains the same quantity of DNA?
Question 34: If the cell membrane is fluid mechanically, how can cells maintain their integrity?
Question 35: In immunocytochemistry, is it assumed that the fixatives, dehydrating reagents, washings, and primary and secondary antibodies, do not change the reaction of the antibody to the antigen believed to be in a particular cell or part of a cell?
Question 36: Is it reasonable to believe that processes or dendrites contain different antigens from the cell bodies from which they arise?
Question 37: Under what conditions can tissue cultures be used in the study of the tissues from which they originated?
Question 38: Is it warrantable to assume that growth of tissues in culture does not change their morphology, biochemistry, or immuno-reactivity?
Question 39: Does not the use of the term neuroglia imply that the authors can not distinguish between astrocytes, oligodendrocytes, and microglia?
Question 40: Why are the individual types of neuroglial cells so rarely seen by light microscopy of healthy central nervous systems?
Question 41: Since the latter three alleged cell types were described by classical histological techniques during the first half of the twentieth century, does this not imply that anyone using antibodies to mark them specifically must first identify them by these criteria?
Question 42: Why is there no common agreement about the staining procedures, which are supposed to identify astrocytes, oligodendrocytes and microglia histologically?
Question 43: Why is it necessary to use tissue cultures of the alleged cell types to identify them and their markers?
Question 44: If each cell in an organism contains the same DNA, but some produce different proteins, is the existence of suppressor genes the only possible explanation for the difference of the proteins?
Question 45: In diseases believed to be auto-immune, either organ-specific or tissue-specific, why does the body not reject the specific organ or tissue, as it rejects incompatible transplanted hearts, or blood of the wrong group, often making the patients ill, or even killing them?
Question 46: Why are pure proteins used for calibration, when different tissues contain different mixtures of proteins, which have different calibration curves?
Question 47: Why do synapses seen by electron microscopy appear so much smaller than those seen by light microscopy?
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These questions have been raised in previous publications, and there have been few serious responses to them. I feel it my duty, therefore, to put them on Internet, to stimulate colleagues, especially young ones, to address them seriously, or to explain why they are unwilling to do so. If, as I suspect, there will be few or no responses to these proper questions, they will remain for future generations to demonstrate their integrity by addressing them, and perhaps as a consequence, to change their views. Any of these questions may be quoted, and/or used in examination questions, preferably with acknowledgement of their source. I will answer all correspondence while I am physically capable of doing so.
Unity Laboratory of Applied Neurobiology,
76 Epsom Road,
GUILDFORD
Surrey
GU1 2BX
U.K.
Fax: UK 1483 31110
Telephone: UK 1483 568332
Hillman, H. *Certainty and Uncertainty in Biochemical Techniques* (1972), Surrey University Press, Henley-on-Thames, U.K.
Hillman, H. & Sartory, P. *The Living Cell* (1980), Packard Publishing, Chichester.
Hillman, H. *The Cellular Structure of the Mammalian Nervous System* (1986), MTP Press, Lancaster.
Hillman, H. *The Case for New Paradigms in Cell Biology and Neurobiology* (1991), Mellen Press, Lampeter.
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Harold's RESPONSE1 sent to Usenet groups cellbiol & neuroscience
1. Discourtesy, assumptions of ignorance, and emotive remarks are no substitute for measured argument and evidence. Each of these questions highlights a contradiction *within* current views; for example, (a) everyone agrees that intracellular movements can be seen by low power light microscopy in living cells, yet most people also believe that there is a cytoskeleton, which would not permit such movements; (b) most people believe in the Second Law of Thermodynamics, yet in subcellular fractionation they change the entropy of their systems (homogenise and centrifuge), and assume that this does not change the free energy, which drives all the biochemical reactions they are studying, and at the same time, they have refused for fifty years to do the necessary control experiments to find out by how much; (c) most people would agree that the laws of solid geometry must be obeyed, while in their electron micrographs - as opposed to their diagrams - they do not see a random selection of orientations, including oblique views of cell membranes, nuclear membranes, myelin lamellae, synapses, nuclear pores, etc.
2. In my publications cited, and in about 120 other full-length papers, I have shown, in detail, with evidence: (a) That one can not yet derive conclusions from subcellular fractionation about the chemistry of organelles, which are relevant to their original states in the intact, living organisms; (b) That the following structures do not exist in the living cells: endoplasmic reticula, Golgi bodies, lysosomes, nuclear pores, mitochondrial cristae, the cytoskeleton, actin filaments and synaptic knobs, either because they would not permit the evident intracellular movements, or because they disobey the laws of solid geometry. Transmembrane molecules and receptors can not be seen on the cell membranes by transmission electron microscopy, although sequencing shows them to be 2-3 times the diameter of the cell membrane, which *can* be seen by electron microscopy;
(c) That in the central nervous system, the only cells are neurons and microglia; astrocytes and oligodendrocytes do not exist in the whole intact mammalian nervous system;
(d) The chemical transmission hypothesis contains many untested and untestable hypotheses, and was worked out for neuromuscular junctions; it has been assumed to be relevant to synapses - especially since the latter term has been extended from its original meaning (nerve-nerve connections) to include neuromuscular junctions (nerve-muscle connections).
3. I have always suggested alternative and testable hypotheses, not open to the criticisms of current views, for example, how to localise biochemical activities without disruption of tissue, the structure of the living cell, the cellular structure of the central nervous system, the passage of excitability from one neuron to another, etc, etc.
4. The fundamental questions I must raise with the Internet cytologists are:
'By what criteria are questions improper?'
'Do all academics have a duty to address the difficulties and apparent contradictions of their own views?'
'Do they believe that progress can be made without examining their own views?'
'Would they disagree that a good academic should answer all these questions in the affirmative?'
5. It seems to me to be absolutely essential that any experimental project to list as many as possible of the assumptions inherent in:-
(a) the use of the experimental procedures;
(b) the processing of raw data into the results to be published;
(c) the interpretation of the results in the light of previous theories and new ones being generated.
It is simply not good enough to support one's case with other peoples' findings without examining the validity of the findings cited. One is responsible not only for the interpretation of one's own results but also for the validity of experiments or interpretations of other authors whose results one uses to interpret one's own findings. The validity of an experiment depends upon the warrantability of *every* statistically potentially significant assumption, both those recognised and those not recognised or ignored. Like a chain, its overall strength depends upon its weakest link. The validity and value of an experiment in pursuit of truth depends upon the warrantability of the weakest assumption. Indeed, *any* wrong assumption, whose error would make a significant difference to the result of an experiment, renders the whole experiment invalid. Of course, it becomes worse if the assumptions are testable but have never been tested, or are untestable. An assumption does not disappear just because research workers singly or collectively do not recognise it, or do not wish to do so.
6. I wish to repeat that I am prepared to enter into personal dialogue with anyone about any of these questions, and I have dealt with each of them in *detail* in my publications. I have answered the Internet responses received so far.
7. We are talking here about intellectual integrity, and not about promotion, grant applications, casuistry, verbal acrobatics, scoring points, theology or dogma - at least, I hope we are!

Harold's RESPONSE2 sent to Usenet Group Cellbiol posted Wed 26 June 1996
General Response to Comments of Greg Fraley and others on 'Unanswered Questions in Biology', posted by H Hillman.
[1] There have been extraordinarily few responses to the 47 questions which I have posed.
[2] Instead of answering the questions, most respondents have accused me of ignorance, not knowing the literature, not being critical of my own views, etc.; none have indicated that they have read any of my publications.
I would like to make it abundantly clear that in my books and papers I have answered at length all the points at issue. I hope they do not teach their students the unacceptable practice of indulging in polemics, when one has not read the evidence of those with whom one disagrees. So far, no one has asked me for any publications.
[3] Even if all my assertions and conclusions are wrong, cytologists should address the 47 questions, because they are central to their beliefs in cell structure. These questions may be included in examinations, and I would suggest that both undergraduate and postgraduate students should press their teachers and supervisors to answer them. Intellectual dishonesty, casuistry or avoidance of awkward questions will not advance science.
[4] I have never denied the view that tissues consist of cells, which have membranes around them. However, it has not been realised that a line is a geometrical abstraction with position but no thickness. Any real membrane has two sides, and any metal deposit must appear as *two* lines for every real membrane, if it is magnified enough. This applies to cell, nuclear and mitochondrial membranes, and cristae.
[5] I have made VHS tapes in PAL format of my views on the structure of the cells, and the cellular anatomy of the mammalian nervous system, which I am prepared to lend to anyone who will pay the postage and return them. I have lectured on these questions at over 120 institutions and universities in Britain, continental Europe, United States, Australia and Canada, and have answered in detail and in publication every objection I have heard to my views.
[6] Everywhere I have lectured, cytologists have alleged that they have electron micrographs of 'unit' membranes, endoplasmic reticulum, Golgi bodies, nuclear pores, microtubules, cristae of mitochondria, myelin lamellae and thylakoid membranes in a random selection of orientations in a single cell.
(One could not select a cell in which a section went normally thorough the membranes around the cell, nucleus, mitochondria, and endoplasmic reticulum, because the section does not know from which direction a microtome knife will come.)
In 24 years, no one has sent me a micrograph or any reference to any micrograph in the literature, showing any of the structures in a random series of orientations. Oblique views should be more frequent than transverse sections. Occasionally, people have drawn my attention to a minute piece of membrane *not* apparently cut in transverse section, but that is not good enough. I did not invent the laws of solid geometry or thermodynamics, and I do not believe that one can defy them without risk.
[7] Colleagues and students, I earnestly request you to address these proper questions. Their importance is quite independent of your views of me or my work. However, I stress that I have published full and critical answers to all the questions.
I will be pleased to send reprints to interested parties, who are prepared to enter into dialogue.
Harold Hillman
Unity Laboratory of Applied Neurobiology
76 Epsom Road
Guildford
Surrey
GU1 2BX
U.K.
Tel: (441) 483 568332
Fax: (441) 483 31110

Ricardo Azpiroz
Department of Biochemistry University of Arizona Tucson AZ
Date: Fri, 7 Jun 1996
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#I will confess that I didn't read your entire post addressing the rather
#rude remarks you have received, but I do agree with you that emotive
#responses don't help the respondents' case. They are also uncalled for.
#Now, it seems to me that you are not a biologist by training. Not because
#you don't believe the current cell theory but because it is clear that
#you don't know as much as you could in order to address these questions.
#For example: In electron microscopy, one DOES see various orientations of
#membranes (oblique and tangential sections). One simply doesn't publish
#them. Not to hide what one doesn't like, but because true cross-sections
#tend to be more informative, and are technically more demanding, which
#means that it's harder to slip a poor image in. Also, the presence of a
#cytoskeleton in no one's mind precludes intracellular movement. In fact,
#intracelluar movement REQUIRES the cytoskeleton. My guess is that your
#view of what the cytoskeleton is or should be is at odds with the current
#models.
#A point that is hard to explain is that in science arguments based on
#language carry absolutely no weight. If a particular biologist concludes
#that a rigid cytoskeleton would make movement impossible, her/his
#colleagues would assume that the person in question has not considered
#all the possible ways one could have both things. And most importantly,
#experimental evidence always has the final word. So in the absence of
#actual observations all arguments are just that. Therefore, what
#biologists do when they find evidence of BOTH a cytoskeleton AND
#intracellular movement is scratch their heads and try to imagine how this
#could be. Then they walk into their labs and design experiments to test
#their ideas. The standard behavior in science is that nothing is given
#much importance until it is proven correct or incorrect by experimentation.
#I hope I don't appear condescending; I don't know who you are and I
#haven't read your publications. I just hope to give you a scientist's
#perspective.
#----------------------
#Ricardo Azpiroz

I have excellent qualifications in biology and have carried out research for forty years.

I challenge you to send me a micrograph of any unit membrane appearing in a random selection of orientations in a single cell.

There is no explanation in the literature of how intracellular movement could occur by means of the cytoskeleton. You seem to think that you can be a scientist, and *not* read publications making out a case against your own beliefs, e.g. Hillman and Sartory (1980) The Structure of the Living Cell, Packard Publishers, Chichester, U.K.
Harold Hillman

#Re: Unanswered Questions
#Date: Tue, 18 Jun 1996
#From: Ricardo Azpiroz
#Very well, if you don't wish to friendly about this, consider the following:
#1) The reason I don't read Hillman is that I have never heard of
#him/her/you and the work cited. I have read dialectical critiques of
#modern science, and agree with much of them. So, I do have an open mind.

1) You should be prepared and willing to answer my questions without reference to my publications. I was only pointing out that I have published answers.

#2) If I sent you a micrograph with what you call random orientation of a
#bilayer, you would, rightly, claim it to be uninterpretable; such
#micrographs are only interpretable in the context of serial sections.

2) *I challenge you* as I did anyone to send me an electron micrograph with the cell, nuclear and mitochondrial membranes, the cristae, the endoplasmic reticulum, the synapses in a random selection of orientations. *You* can put arrows showing me and all the other Usenet users every orientation in *one* micrograph. I repeat this challenge to any electron microscopist in the world.

#3) Read about axonal transport of synaptic vesicles. There are VIDEOS
#showing vesicles moving down microfilaments. Movement and cytoskeleton,
#right before your eyes.

3) Synaptic vesicles can only be seen by electron microscopy of *dead tissues* in which intracellular movements do not occur. Therefore the movement of *vesicles* can not be seen. Sheetz' beautiful videos do not show mitochondria being pulled along. His microtubules can be seen by light microscopy (with a resolution of 200-250 nm under *best* conditions), but microtubules (see Dustin Amos and others) are <25 nm and are therefore not the same structures.
Harold Hillman.

Date: Fri, 28 Jun 1996
From: Ricardo Azpiroz <azpiroz@U.Arizona.EDU#>
cc: cellbiol@net.bio.net
To all: I, personally, am a bit tired of this discussion; this is going nowhere. Furthermore, I think at this point it is time to get off the cellbiol group and continue, if so desired, at the individual level. All of us who have participated are quite convinced we are right. I'm sorry it's not working out. Just a final note to Hillman: If I remember correctly, this thread started when you posed a number of questions and asked for some answers. That is well and good. However, I fail to see how it seems to have become OUR responsibility to address your issues and meet your "challenges". We, sir/madam, have no problem with our views and are not especially interested in having you agree with us. It is YOU who initiated this, which is why all our responses are in the tone of "you should read this or that". Do not expect us to read your publications. You can ask, you can offer reprints, but accusing us of not reading your stuff is out of place, and will not put us in a good frame of mind.
I am out of this thread, and I ask all others to follow suit.
Ricardo Azpiroz

*I CHALLENGE YOU* AS I DID ANYONE TO SEND ME AN ELECTRON MICROGRAPH WITH THE CELL, NUCLEAR AND MITOCHONDRIAL MEMBRANES, THE CRISTAE, THE ENDOPLASMIC RETICULUM, THE SYNAPSES IN A RANDOM SELECTION OF ORIENTATIONS. *YOU* CAN PUT ARROWS SHOWING ME AND ALL THE OTHER USENET USERS EVERY ORIENTATION IN *ONE* MICROGRAPH.
I REPEAT THIS CHALLENGE TO ANY ELECTRON MICROSCOPIST IN THE WORLD.

Harold Hillman
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From: Chris Barry <chbarry@llnl.gov>
Organization: Lawrence Livermore National Lab
Newsgroups: bionet.cellbiol, bionet.neuroscience
Subject: Re: *unanswered questions H Hillman
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Date: Wed, 03 Jul 1996
Hello Harold,
I think that you have several good points. May I make one suggestion though. Could you please post each question in a separate post on the newsgroup. When you post a large diatribe on a regular basis, It doesn't give people the opportunity to address the questions themselves and you come across as a bionet pariah.
Chris

Dear Chris,
My good friend Rae West put the questions on the network for me. I thought it would be more economical to put them all on together. They are a summary of questions that I have raised over 40 years. Each one still deserves an answer, even if cytologists want to ignore mine. I would be interested to hear any answers from you, your colleagues, your students.
Kind regards,
Harold.
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From: beverly barton
Date: Fri, 28 Jun 96
Organization: Schering-Plough Research Institute
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#Re your question #4--Archaebacteria are prokaryotes in which there is subcellular localization of biochemical processes. Eubacteria do have some subcellular localization, in that they have ribosomes (albeit 30
#and 50S, rather than 40 and 60S subunits); their energy-producing cycles
#localize to part of the membrane, and their genetic material is found in
#DNA and episomal material, such as plasmids.

All cells have the ability to synthesise proteins. Many have no visible 'ribosomes' or 'rough endoplasmic reticulum'. Therefore, the synthesis can occur *without* visible ribosomes.
(i) How do you know when you see them that they are the structures which contain the necessary enzymes;
(ii) when you don't see them, then the enzymes are there, *clearly* the structure (the ribosome) is not necessary for protein synthesis.

# Additionally, their #membranes form discrete organelle-like structure called mesosomes.
# Also, they have cilia-like structures in some species (flagellae, fimbriae, etc.) which serve both in locomotion and propagation (in pathogenic species).

So what?

Subject: questions?Date: 7/5/96
>
In answer to your question #35, it is known that fixation may change the>
avidity of an antibody for an antigen-----but that doesn't change the>
idiotypic specificity of the antibody for its antigen!!!!!!!!!!!!!!!
>
An autoimmune disease by definition is a "rejection" response to>
antigens in organs or tissues. An organism which could reject its>
entire antigenic repertoire of organs & tissues would not live to>
reproduce. Conversely, even in autoimmune individuals, at least some>
self-tolerance mechanisms are maintained.

Dear Dr Barton,
Most experiments in immunocytochemistry assume that the antigen-antibody response is *not* affected by the agents used in fixation, dehydration or mounting. As you say "An auto-immune disease ... is a rejection response to antigen in organs and tissues," so my question remains, "Why does not a person with rheumatoid arthritis reject his/her own joints?" Your note that "even in auto-immune individuals, some self-tolerance mechanisms remain," ignores the facts that (i) some apparently compatible transplanted tissues - which must have few incompatible antigens - subsequently are rejected, (ii) unmatched blood need have only one incompatible agglutinogen to be rejected. Thus the idea that many incompatible antigens are needed for rejection seems not to be the case. Thus the original question remains unanswered.

[... I wonder if I could ask your view on this: .. "partial immunity" >
or be "partially immune" to what extent is this expression just a way of >
saying that the mechanism of immunity isn't known?]-RW
>
I interpret "partial immunity" to mean that an organism mounts part of an >immune response, but not the full panoply. In humans this is exemplified by the >administration of gamma-globulin, in the days before vaccines to measles, >hepatitis, etc. were available. Giving gamma globulin was giving the partial >immune response, the antibody component. People became ill, but their disease >was attenuated. Contrast this with the result of vaccination--a complete immune response is mounted, and people do not get the disease. In the case of gamma >globulin, the antibodies are passively administered, but there is no T cell >response--in many viral diseases, measles and hepatitis among them, the T cell >response is the major protective response. So I do not agree that the term >means the protective mechanism is not known.>
You ask a lot of questions which lead me to believe you don't have a thorough >grounding in certain areas of physical chemistry and immunology. I could be >wrong, but most if not all of your questions are answered in standard texts, >that is how accepted the findings are. For example, it is not entropy which >drives a reaction to completion, but the decrease in Gibbs' free energy. >
Entropy may actually go either way. See for example the chapter in a textbook >such as Biochemistry by Albert Leninger, second edition. In my graduate-level >physical chemistry course at The Johns Hopkins University, I learned that the >entire universe may be considered the closed system in determining the entropy >of a reaction, not just the test tube. Many people make this mistake, and point out the same apparent paradox you do. It just shows they never took >thermodynamics in college.

It is true that the entire universe is a closed system, but in terms of subcellular fractionation, a test-tube is also. Furthermore, I ask you whether you believe that a subcellular fraction at the end of an experiment - for example a mitochondrial preparation - has the *same* activities which are distributed the same in an intact animal. Please cite me experiments showing this or examining the effect of the preparation procedure on these two parameters. No controls means unfinished experiments, not worthy of being interpreted.
Harold Hillman.

No, Dr. Hillman, the closed system referred to in the laws of thermodynamics is not the test tube but the universe. If you don't accept that, you will find "violations" of the laws of thermodynamics everywhere.Beverly Barton

Thanks for your e-mail of Monday 8th July. You make a point about thermodynamics, but don't seem to have answered Harold's point about "auto-immune diseases."
Best wishes, Rae West.

Does Harold have a point about autoimmune diseases? I already answered him in this respect. I don't think he's read the literature on autoimmune diseases.
The causes are multifactorial, the immune responses observed resemble rejection in some diseases but not others. It's really difficult to speak globally about autoimmune diseases; better to talk about a particular one.
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REPLY TO PAUL S. BROOKES psb@mole.bio.cam.ac.uk [Molecular biology dept, Cambridge University]
Re: UNANSWERED QUESTIONS
Date: Tue, 28 May 1996
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#A few answers to a few questions. I sincerely hope not all of these questions were your own, as some of them show a basic misunderstanding of simple biochemistry.
##Question 1: Can one obtain an enriched fraction of a subcellular organelle or cell type?
#Yes, by differential centrifugation on density gradients - try "methods in enzymology" for starters.

This is done to examine biochemistry, therefore assumes that procedure does NOT change biochemistry, see HH (1972).

##Question 2: How does one know that the disruptive procedure does not change the biochemistry of the fraction significantly?
#We don't, we just assume it doesn't affect the properties we're interested in, and usually test these properties in the isolates to make sure.

The Second Law of Thermodynamics tells us that this assumption *must be untrue*

##Question 4: Why is it believed that each biochemical pathway or cycle has its own structural compartment when prokaryotes can carry out virtually all the same reactions in only one compartment?
#Compartmentalisation is part of evolution. It makes processes more efficient, otherwise we'd all still be glooping around in pre-biotic soup.

There are hundreds of reactions and only 11 compartments but the fact that eukaryotes can do all biochemistry with only *one* compartment shows that the compartmentation is unnecessary.

##Question 5: Does the finding that a chemical substance or activity is located in the same subcellular fraction and a structure identified by electron microscopy mean that the same chemical activity was located in that particular organelle in the living cell of the intact animal or plant.
#Co-purification usually means this, yes, but there have been a few clangers in the past (someone one once said ox-phos was located in the outer membrane of mitochondria!) - we're getting better at it now.

Diffusion occurs during dying, homogenisation, and centrifugation, so movement of enzymes or cofactors will give false locations.

##Question 6: How is intracellular movement possible, and the cytoplasmic viscosity is low in life, if there is a cytoskeleton present?
#Ever heard of actin filaments, MTOC's e.t.c. Diffusion across a cell is quite fast too, even in the viscous environment.

This answer is offensive, and does not answer the question of how mitochondria and granules can be seen by light microscopy to be moving if there is a cytoskeleton.

##Question 9: Why is it assumed that homogenisation and centrifugation do not affect the chemistry of receptors, or their affinities for transmitters, hormones, drugs, ligands, toxins?
#It isn't - it usually gets tested in situ - see answer question 2

This has *never* been tested. Please cite publications.

##Question 11: Can one calibrate substances originating from tissues
## using pure solutions in simple salines of approximately the same concentrations?
#YES!

The extraction chemicals, other substances extracted at the same time and the vehicles affect the colour, absorption, permeability of membranes and fluorescence of substances studied.

##Question 12: How can one study membranes by electron microscopy, when they are believed to contain lipids which the procedure extracts?
#Depends which procedure you use. Not all of them do damage the lipids.

Do alcohols, acetone, xylene, extract lipids? Alcohols are used in *all* electron microscopic preparations.

##Question 13: What is the real evidence that rapid deep freezing for electron microscopy causes less shrinkage and distortion of tissues, cells and organelles, than classical transmission electron microscopy?
#It's faster, so causes less shrinkage, simple physics. The water freezes too quickly for the membranes e.t.c. to shrink appreciably.

Please cite publications showing this rather than just believing it.

##Question 15: Do membranes in cells appear to be normal to the plane of section more often than solid geometry would permit?
#The plane of section is usually along the cell membrane, as it is a line of weakness in the prep, so of course it will appear more often.

How can the plane of section cut through randomly orientated nuclei, mitochondria, cell membranes? Are you trying to explain why the laws of geometry do not apply?

##Question 16: Can one know the thickness in life of any biological membrane?
#Yes, by NMR, read some papers by John Nagle & others

Nearly all current measurements in the literature are made from electron micrographs of tissue which has been *dehydrated*.

##Question 19: Why have few or no carriers been isolated?
#Membrane proteins are very difficult to isolate as they need to be co-purified with native lipid, or lots of detergent. There has been some success though.

Is one sure that the detergent has no biochemical effects?

##Question 20: What is transport?
#Please, what sort of question is that for a newsgroup!

'Transport' means movement, and diffusion occurs in aqueous and other liquids.

##Question 21: Why are receptors and channels, which have been characterised, sequenced and their sizes measured or calculated, not seen on membranes by transmission electron microscopy?
#Ever seen an e/m of the mitochondrial inner membrane - ooh look, what are all those little stalky things... proton translocating ATP synthases. Most transporters are IN the membrane, hence you can't see tham as they're buried.

Sequencing of so-called receptors and channels show them to be 3x width of cell membrane which can be seen by electron microscopy, so why not the receptors and channels themselves?

##Question 26: Why is it assumed that the receptors for transmitters, hormones, messengers, antibodies, drugs and toxins are on the surface of the cell membrane?
#Its not! There are loads of receptors inside the cell (steroids are one example everyone is taught at undergraduate level biochemistry). Toxins act all over the place - mitochondria, DNA/RNA synthesis, protein synthesis, ion transport.

Is he assuming that diffusion will not occur during dying, homogenisation, centrifugation, fixation, dehydration etc.

##Question 27: How valid is the use of agonists, antagonists and ligands to detect receptors, instead of the transmitters, hormones, antigens, drugs and toxins themselves?
#As valid as using a copyed key to open a door - it works as well as the original in opning the lock, but it might interact differently with the lock at a minute level.

Why is not acetylcholine used to seek its receptors, or adrenalin, glutamate, gaba, etc? The ligand is chemically *different* from the transmitter.

##Question 33: If nuclear pores allow RNA to pass through, how do they prevent smaller molecules and ions going through at the same time,
## and why is there a potential difference across the nuclear membrane?
#Selective ion transport - a universal process. Line the pore with amino acids that will satisfy the bonding of the RNA and it can go through, but not other things, as their bonding isn't satisfied. A delta-psi is used for ion and other transports all over the cell, not just the nucleus!

You have obviously not compared the size of the pores (20-120 nm in the literature) to those of ions (<1 nm).

##Question 34: What is the evidence that each cell of a particular plant or animal contains the same quantity of DNA?
#It's been extracted and measured.

What animal or plant, and how many tissues?

##Question 35: If the cell membrane is fluid mechanically, how can cells maintain their integrity?
#Depends on the temperature, and the type/amount of protein present. I think the word "fluid" is being used very loosely here. Lipids can certainly migrate throughout the plane of the membrane, as well as "flip-flop" between the two leafelts of the bilayer. This doesn't seem to compromise cell integrity.

Singer and Nicholson use the word 'fluid' - I did not.

##Question 38: Under what conditions can tissue cultures be used in the study of the tissues from which they originated?
#Normal lab conditions, provided the cellular parameter you wish to study is no different in the cultured cells. Controls can be done to show this.

Cells when they are cultures change their environments, shapes, source of oxygen, staining, pressure, etc.

##Question 39: Is it warrantable to assume that growth of tissues in culture does not change their morphology, biochemistry, or immuno-reactivity?
#- repeat of Q 38!

##Question 45: If each cell in an organism contains the same DNA, but some produce different proteins, is the existence of suppressor genes the only possible explanation for the difference of the proteins?
#No... other mechanisms exist, such as post-translational modification of proteins, post-transcriptional modification of mRNA, and higher levels of control that an embryologist could probably explain better.

These are hypotheses.

##Question 46: In diseases believed to be auto-immune, either organ-specific or tissue-specific, why does the body not reject the specific organ or tissue, as it rejects incompatible transplanted hearts, or blood of the wrong group, often making the patients ill, or even killing them?
#Badly worded question. Auto immune disease is usually against structures inside the cell, but immune rejection is usually against the cell surface molecules, which are more strongly recognised as foreign, so rejected. Not sure on this one, but hey... who cares... so long as we can cure both of them where's the problem in them having different mechanisms?

1. You have not answered the question
2. Rheumatoid arthritis, multiple sclerosis, and many other diseases are meant to recognise and reject their own proteins.

##Question 47: Why are pure proteins used for calibration, when different tissues contain different mixtures of proteins, which have different calibration curves?
#It's the best we can do, given current extraction procedures. If you want to devote your entire life to isolating viable protein mixtures, please do, but don't expect great thanks from the scientific community when the mixture you just spent 20 years isolating is obsolete as its individual components work just as well!

It is not the *best*. You can do recovery calibrations with *all* the tissue constituents present.

## These questions have been raised in previous publications, and there have been few serious responses to them.
#All science is serious, given the materials/resources/knowledge of the time - what do you want, blood?
## I feel it my duty, therefore, to put them on Internet, InterNet (capital N)

Trivial.

##If, as I suspect, there will be few or no responses to these proper questions, they will remain for future generations.....
#These, if any, will be the reasons for few responses:-
#1. The length of the original mailing (11k) - instant wastebasket material
#2. The fundamental misunderstanding of basic biochemistry in some questions
#3. The "save the world" attitude of anyone who thinks putting such questions on the InterNet will change the way research is conducted world-wide.
#4. The patronising/condescending nature of the posting.
#I hope you think a bit more before placing another such posting!

Extremely rude and offensive and untrue - I have a PhD in biochemistry.
#Regards
#PSB
Dr Harold Hillman
Unity Laboratory of Applied Neurobiology

FROM Paul. S. Brookes.
Re: UNANSWERED QUESTIONS
Date: Fri, 7 Jun 1996
I sympathise entirely with some of the arguments you put in your mail.
However, the majority of biochemists recognise the simple fact that our whole field is based on models.
If I may get religious for a moment, most sensible people would agree that God does not exist, and is just a three letter euphemism for something we have absolutely no hope of ever explaining or rationalising. It is in our natural psyche as human beings to put things away in little boxes in order to understand them.
I think the same is true for science. For all we know cells may not exist; it's just there's just an overwhelming body of evidence that when we look down the microscope the pattern of light rays hitting our retina is conveniently boxed away as "a cell".
The whole of science is based upon such models, and ever will be. If earth today evolved from a speck of dust 46 billion years ago, then it's taken a long time to get here. Science as we know it is about 200 years old, so mankind cannot ever hope to understand everything in such a short time frame. In order to speed up this understanding, we must move via a series of approximations, of which the biochemical model is just one.
If scientists stopped at every stage to do complete controls, the pace of science would grind to a halt. We may miss out by saying "we know enough, let's move on", but that's a calculated risk. Unfortunately current science is driven by immediate needs such as the desire to end human suffering by understanding and fighting disease. I'm afraid the romantic notion of "blue skies" research for the sake of it just doesn't fit into the current global financial climate.
We'd all love to answer your unanswered questions but just can't afford to.
Regards
PSB

It is quite UNTRUE that we have ever doubted the existence of the cell.
I can only disagree with you that science can advance without doing complete controls.
Is it poverty in money or intellect which makes you unable to answer my questions? Nevertheless, you admit you are not answering them, and science will continue to suffer from its failure to address them.
Harold Hillman
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From: John Joseph Ladasky Jr. BA Biochemistry, U.C. Berkeley, 1989 (PhD perhaps 1998???)
Location: Stanford University, Dept. of Structural Biology, Fairchild D-105
Newsgroups: bionet.cellbiol
Subject: Re: UNANSWERED QUESTIONS
Date: 27 May 1996
[Back to top]
[48 questions deleted]

# These questions have been raised in previous publications, and there have been few serious responses to them. I feel it my duty, therefore, to put them on Internet, to stimulate colleagues, especially young ones, to address them seriously, or to explain why they are unwilling to do so.

O.K., I'm a young researcher. I'm unwilling to address these questions at the present time because they're tangential to my discipline of choice (immunology), and my PI would not appreciate such diversions. Not that I don't consider many of your questions to be interesting and relevant. However, the tone of your letter... e.g., #If, as I suspect, there will be few or no responses to these proper questions, they will remain for future generations to demonstrate their integrity by addressing them... implies that everyone who reads it should share your research priorities (neurobiology, apparently), or else be found lacking in integrity.
I could write a similar list of unanswered questions in immunology.
Would it be fair of me to regard you with disdain if you did not choose to drop what you are doing to answer my questions? Of course not.
Unique ID : Ladasky, John Joseph Jr.
======================
Subject: Re: UNANSWERED QUESTIONS
Date: 3 Jun 1996
Organization: Stanford University, CA 94305, USA
Tom Chappell [University College, London] wrote:
[Back to top]
##UNANSWERED QUESTIONS IN BIOLOGY. 27 May 1996
###Question 34: What is the evidence that each cell of a particular
### plant or animal contains the same quantity of DNA?
###
##Given the tone of the original question, I think I'll take the opportunity
##to rip into this one...
##
##There is no evidence because it's a false hypothesis.

It is neither false nor true - it is untested.

##Someone asking this question does not understanding (as a start):

This is not true

##1) differences between the G1, S and G2 phases of the cell cycle
##2) meiosis
##3) differentiation of either red blood cells or striated muscle
##4) polytene chromosomes
##5) keratinized epithelium
##6) mitochondria biogenesis
##7) endomitosis
##8) malignant and nonmalignant tumors
##9) apoptosis
##10) gene amplification
##11) retroviruses
##
##Buy a copy of Molecular Biology of the Cell and start at page 1.

This is rude

#On the other hand, if you confine yourself to the examination
#of healthy, diploid G0/G1 cells (which defines an awful lot of tissue),
#there is good evidence that the amount of DNA in each cell agrees to
#within the limits of measurement. The measurements are made using
#quantitative fluorescence techniques (e.g., flow cytometry) and a DNA
#fluorochrome.

Any references to your 'good evidence'?
Harold Hillman
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From: aledain@receptor.pharm.uwa.edu.au (Dr. Alex)
Newsgroups: bionet.cellbiol
Subject: Re: UNANSWERED QUESTIONS
Date: 28 May 1996
Organization: Pharmacology - UWA
[Back to top]
# ... questions snipped ...
Hmm. There will always be more questions than answers in any given universe. A fundamental law states that if ever all the questions and answers are known in a particular universe then it will disappear in a puff of logic. Somehing to do with the number 42 I suspect ...
On a more serious note ... is it possible that:
A. the questions haven't been satisfactorily answered in your opinion.
B. further reading in the correct sources will answer the questions.
C. the answers already at hand disagree with your own theories (see A).
D. you're not conversant enough with the fields to which your questions are aimed.
E. these questions are not relevant to scientists in other fields.
F. these questions are not relevant to scientists in your field.
G. these questions are not relevant.
Given infinite resources, infinite time, and more than one lifetime, all scientists would eventually answer all questions. At this point in time, to debate what is clarified or not clarified is irrelevant ... because given a reasonable pace to scientific discovery all(well many) things can be disproven or proven beyond doubt(well enough for most). That is why we hypothesise, make assumptions, debate and then our hyhpothesis can become a theory. Although, some will never attain the theory level ... poor Avagadro.
cheers, Alex.
P.S. And trolling will always get you a few bites :-)

[Harold's reply seemed not worth sending]
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Richard Delorme Laboratoire de Cytologie Analytique. univ-lyon.fr
Subject: Re: * UNANSWERED QUESTIONS: RESPONSE *
Date: Fri, 14 Jun 1996
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## (b) That the following structures do not exist in the living
##cells: endoplasmic reticula, Golgi bodies, lysosomes, nuclear pores,
##mitochondrial cristae, the cytoskeleton, actin filaments and synaptic knobs, either
##because they would not permit the evident intra-cellular movements, or because they disobey the laws of solid
##geometry. Transmembrane molecules and receptors can not be seen on
##the cell membranes by transmission electron microscopy, although
##sequencing shows them to be 2-3 times the diameter of the cell
##membrane, which *can* be seen by electron microscopy;
#It is possible to see some of this structure IN the living cells. For
#example, DIOC6 is a fluorescent dye that stains the endoplasmic reticula in
#the living cell, rhodamine 128 stains mitochondria, etc... If you are a web
#surfer, you can find some movies showing these structures moving inside the
#cells.

Nearly all fluorescence, if not all, is done in the fixed, dehydrated and mounted sections in which movements can not occur. Do you do immuno-fluorescence?

#Of course, if you are blind against any kind of evidences...

Rude. You have not read my publications, so how do you know?
[Back to top]

Greg S. Fraley
Date: Fri, 7 Jun 1996
Subject: Re: * UNANSWERED QUESTIONS: RESPONSE *
Washington State College of Veterinary Medicine
[Back to top]
## 1. Discourtesy, assumptions of ignorance, and emotive remarks are no
## substitute for measured argument and evidence. Each of these questions
##highlights a contradiction *within* current views; for example, ...
#Any discourtesy, assumptions of ignorance, and emotive remarks, real or
#implied were probably due to the antagonistic and arrogant wording of the
#original posts. And quite frankly, many of the questions make statments
#which are grossly inaccurate. This will be pointed out.
## (a) everyone agrees that intracellular movements can be
##seen by low power light microscopy in living cells, yet most people also
## believe that there is a cytoskeleton, which would not permit such
## movements;
#Very untrue. In fact, the cytoskeleton plays an intricate role in
#intracellualr movements. For example, look up ina ny text book or pier
#reviewed review article of the procees of axonal transport and the
#synergism between cytoskeleton and kinesins and dyniens.

No one has ever proposed a mechanism whereby all the elements of the cytoskeleton attached to mitochondria, for example, could move the latter.
The role of the various actin, tubulin, microtubules in movement is an unprovable hypothesis.
Do diffusion, Brownian movement, streaming, and convection seen in ground glass in water require cytoskeletons to move them? Please see Hillman & Sartory (1980) 'The Living Cell'

I have listed in 'certainty and Uncertainty..' (1972) 7 kinds of controls.
One has to show that all the chemical activities *and* their location have not been changed by the procedure.

## (c) most people would agree that the laws of solid geometry must
## be obeyed, while in their electron micrographs - as opposed to their
## diagrams - they do not see a random selection of orientations, including oblique views of cell membranes, nuclear membranes, myelin
## lamellae, synapses, nuclear pores, etc.
##
#Anyone who does electron (or light for that matter) always has examples
#of oblique sections through a cell/tissue. This is why, for example,
#many correction factors must be utilized when doing cell counts that may
#contain over/underestimation errors due to oblique sections.
#Photomicrographs in journal articles are best representatives--pictures
#are used that demonstrate the point being made in the text so the best
#possible orientation is shown. As far as digrams, if you are trying to
#learn the structure of a cell would you prefer a straigth forward
#diagram or something by MC Escher? Why would you ever propose a random
#distribution of cellular contents??? Is there random distributions of
#any organsimic structures??? Is your heart, lungs, BRAIN, in the same
#general place as any other homo sapien, or any other mammal for that
#matter?

In over 120 lectures in Europe, USA, and Canada, I have CHALLENGED anyone show me ANY micrograph of a whole cell with ANY 'unit' membrane appearing at right angle to its normal section. Everyone SAYS they have some - no one has. Have you?

## 2. In my publications cited, and in about
#120 other full-length papers, I have shown, in detail, with evidence:
## (a) That one can not yet derive conclusions from subcellular
## fractionation about the chemistry of organelles, which are relevant
## to their original states in the intact, living organisms;
#Please reference pier reviewed journal articles which support this claim.

My books 'Certainty and Uncertainty in Biochemical techniques' (1972) and 'The Case for New Paradigms..' (1991)

## (b) That the following structures do not exist in the living
## cells: endoplasmic reticula, Golgi bodies, lysosomes, nuclear pores,
## mitochondrial cristae, the cytoskeleton, actin filaments and synaptic knobs,
## either because they would not permit the evident intracellular movements,
## or because they disobey the laws of solid
## geometry. Transmembrane molecules and receptors can not be seen on
## the cell membranes by transmission electron microscopy, although
## sequencing shows them to be 2-3 times the diameter of the cell
## membrane, which *can* be seen by electron microscopy;
#Again, please give some real data thast shows these structures do not
#indeed exist. I have seen ER and golgi and mitochondria, so what is it
#that I am seeing? When you are in an airplane you can se roads, but
#can you resolve cars or tractor trailer on this road? No you cannot,
#this is the same idea as membrane bound molecules. However, ligands
#can be bound to the transmembrane receptors that contain elements (such
#as biotin, or gold, etc) that can be resolved--an, indeed, are seen at
#the EM level.

You see Golgi and e.r. in fixed dehydrated cells upon which heavy metals have been deposited. Please send me and give me references to transmission ems of transmembrane molecules, in situ.

## (c) That in the central nervous system, the only cells are
## neurons and microglia; astrocytes and oligodendrocytes do not exist
## in the whole intact mammalian nervous system;
#Again, this is almost absurd. I do almost exclusively whole animals
#model systems. I routinely see both in my own work and the work of my
#colleagues all of these glia types within the CNS.

Perhaps you would be prepared to read my 'Cellular Structure of the Mammalian Nervous System' (1986) MTP Publications where I have brought the evidence.

## (d) The chemical transmission hypothesis contains many untested
## and untestable hypotheses, and was worked out for neuromuscular
## junctions; it has been assumed to be relevant to synapses - especially since the latter term has been extended from its original meaning
## (nerve-nerve connections) to include neuromuscular junctions (nerve-muscle connections).
#Many people have asked you and I repeat, what hypotheses are untestable
#and untested? There are many simiarities between a axono-dendritic,
#axo-axonic, dendro-axonic, dendro-dendritic, dendro-somatic,
#axono-somatic, and neuromuscular type synapses. Yes, there are also
#differences. That is the nature of biology.

In my paper 'A Re-examination of the Vesicle Hypothesis' (1991) Physiol Chem Phys & NMR 23, 177-198 I have listed 23 hypotheses. If you can not obtain this please let me know - then perhaps inform *me* which of these assumptions are *not* made, and which are *not* untestable

## 3. I have always suggested alternative and testable hypotheses, not
## open to the criticisms of current views, for example, how to localise
## biochemical activities without disruption of tissue, the structure
## of the living cell, the cellular structure of the central
## nervous system, the passage of excitability from one neuron to
## another, etc, etc.
##
#Never, in this news group have you posted alternative hypotheses. Please
#do so if you feel they can stand up to scrutiny.

I have not posted these on Usenet because they would take up too much space. Are you prepared to examine my evidence?

## 4. The fundamental questions I must raise with the Internet
## cytologists are:
## 'By what criteria are questions improper?'
## 'Do all academics have a duty to address the difficulties and
## apparent contradictions of their own views?'
## 'Do they believe that progress can be made without examining
## their own views?'
## 'Would they disagree that a good academic should answer all
## these questions in the affirmative?'
#As I have often been told and also in turn tell my own student, there are
#no stupid (improper) questions in science. Change is a necessary part of
#science. there has been several cases throughout history where the
#commonly accepted principle turned out to be dead wrong. However, this
#was done through meticulous testing. Any antagonism you may have
#received was solely due to the way in which these questions were worded.
#You began this whole thread in a very antagonistic and accusitory manner.
# Any normal person will become defensive when they are attacked. And
#yes, your questions were aggressive as worded.

Do you consider that asking questions is attacking - I do not

#5. It seems to me to be absolutely essential that any experimental
## project to list as many as possible of the assumptions inherent in:-
## (a) the use of the experimental procedures;
## (b) the processing of raw data into the results to be published;
## (c) the interpretation of the results in the light of previous
## theories and new ones being generated.
#I think I see this in most pier reviewed published scientific articles.

By *pier* review, I think you mean *peer* review. Why do you not answer the questions?

## It is simply not good enough to support one's case with other
## peoples' findings without examining the validity of the findings
## cited. One is responsible not only for the interpretation of one's
## own results but also for the validity of experiments or interpretations of other authors whose results one uses to interpret one's own
## findings. The validity of an experiment depends upon the warrantability of *every* statistically potentially significant assumption,
## both those recognised and those not recognised or ignored. Like a
## chain, its overall strength depends upon its weakest link. The
## validity and value of an experiment in pursuit of truth depends upon
## the warrantability of the weakest assumption. Indeed, *any* wrong
## assumption, whose error would make a significant difference to the
## result of an experiment, renders the whole experiment invalid. Of
## course, it becomes worse if the assumptions are testable but have
## never been tested, or are untestable. An assumption does not disappear just because research workers singly or collectively do not
## recognise it, or do not wish to do so.
#True, but if scientists never trust each others interpretation and all we
#did was repeat eachothers work in order to prove/disprove the findings,
#what progress would be made? That is not science.

You are confusing two different things. I *never* said that you should not 'trust' other authors. I said that when you used their evidence to support your findings, you implicitly take on intellectual responsibility for the validity of their findings.
Harold Hillman
#GS Fraley
#"They say there's no devil, Jim, but there is..."#

Date: Wed, 12 Jun 1996
From: "Greg S. Fraley"#How many times are you going to post the same questions. They are
#continuously being answered in the same way. Continuously posting the
#same questions will not result in your getting the answers you want.

I will continue to ask these important questions until you or someone else answers them, with *any* answers, not necessarily the ones you might give to the student, or perhaps yourself!
Harold Hillman

#Date: Wed, 26 Jun 1996
#From: "Greg S. Fraley"
## I will continue to ask these important questions until you or someone
## else answers them, with *any* answers, not necessarily the ones you
## might give to the student, or perhaps yourself!
##
#In other words, until you get the answers you want to hear?? Not very
#good science, and quite unethical.

No, I wrote "until I get *any* answers." Dr Cornelius Krasel of Wurzburg has been the only person who has answered many of the questions.
You seem to be unwilling to answer them to me, Usenet, yourself, or your students. Why not? I have, in my publications.
Harold Hillman

Re: Unanswered questions contd.
Date: Thu, 27 Jun 1996
From: "Greg S. Fraley"
# No, I wrote "until I get *any* answers." Dr Cornelius Krasel of
# Wurzburg has been the only person who has answered many of the questions.
# You seem to be unwilling to answer them to me, Usenet, yourself, or
# your students. Why not? I have, in my publications.
#
I DID ANSWER THEM, I EVEN SUPPLIED REFERENCES!!! I posted the answers as
well as emailed them to you. As did at least six others on the
bionet.neuroscience usenet--look them up in the archives.

*** Below are all the questions/answers we have; as you see, you don't answer Harold's replies, nor are there any references. If something's been lost, please send it.
[Followed by the three Fraley reply files to date. No reply]
[Back to top]

FROM Richard van Frank
Wed, 29 May 1996
Newsgroups: bionet.cellbiol
Subject: Re: UNANSWERED QUESTIONS
[Back to top]
## much deleted
##
## The following questions have never been answered satisfactorily,
##several of them never at all:-
##
##
##Question 1: Can one obtain an enriched fraction of a subcellular
## organelle or cell type?
#Yes! This has been done by various methods and with various degrees of sucess
#for at least 25 yrs.

Subcellular fractionation is done to examine biochemistry of organelles, it assumes that the procedure does not *change* biochemistry, which the 2nd law of thermodynamics dictates it must.

##Question 2: How does one know that the disruptive procedure does not
## change the biochemistry of the fraction significantly?
#You don't. However, if appropriate controls are run and assays are done of the
#fractions you can get some idea if this is occurring.

Controls have never been published (see H Hillman (1972)

##Question 3: Why are not
## controls always carried out for subcellular fractionation, except
## for total recoveries relative to the crude homogenates?
#I don't know. Good science requires that this be done. Sometimes it is eye
#opening.

Controls (they) have never been published. Therefore all s/c fractionation is uncontrolled.

##Question 5: Does the finding that a chemical substance or activity
## is located in the same subcellular fraction and a structure identified by electron microscopy mean that the same chemical activity
## was located in that particular organelle in the living cell of the
## intact animal or plant.
#Not necessarily--that is why both enzyme assays of the fractions and electron
#microscopy should be used.

Yes.

##Question 8: What is the evidence that the microsomal fraction consists of cell membranes and endoplasmic reticulum?
#Electron microscopy, cytochemistry and enzyme assays. Also er induction experiments.

'Microsomes' are spherical, cell membrane appears 'trilaminar' by electron microscopy; they do *not* look the same.

Hillman, H. *Certainty and Uncertainty in Biochemical Techniques* (1972),
Surrey University Press, Henley-on-Thames, U.K.
Hillman, H. & Sartory, P. *The Living Cell* (1980), Packard
Publishing, Chichester.
Hillman, H. *The Cellular Structure of the Mammalian Nervous System*
(1986), MTP Press, Lancaster.
Hillman, H. *The Case for New Paradigms in Cell Biology and Neurobiology*
(1991), Mellen Press, Lampeter.
Dr Harold Hillman
Unity Laboratory of Applied Neurobiology,
76 Epsom Road,
GUILDFORD
Surrey
GU1 2BX
U.K.
Fax: UK 1483 31110
Telephone: UK 1483 568332
[Back to top]

From: Peter French, Centre for Immunology, St Vincent's Hospital, Sydney.
Date: Fri, 7 Jun 1996
Subject: Re: * UNANSWERED QUESTIONS: RESPONSE *
[Back to top]
Anyway, although some of your points have merit, what is the overall point you are trying to make? Important discoveries have been made using the reductionist approaches which you despise, despite their well acknowledged limitations.
Of all your statements, the one that is clearly the most ridiculous is that the cytoskeleton and actin filaments do not exist. The cytoskeleton (and actin filaments which in my definition is a component of the cytoskeleton) has been visualised by electron microscopy, by light microscopy, by fluorescence microscopy and by atomic force microscopy. Furthermore, there are well known mechanisms by which the cytoskeleton regulates organelle movement within the cytoplasm, so why would you not believe in its existence on the basis of "because they would not permit the evident intra-cellular movements, or because they disobey the laws of solid geometry". On that basis, you wouldn't believe in heavier than air flight because it disobeys gravitational laws.
The cytoskeleton, in the form of tubulin, exists in the mitotic spindle (where it controls chromosome movement) and in neurons where it is involved in axonal transport; in the form of actin it is almost universally found at the cell membrane.
Sometimes, Hillman/Fowler, we have to modify our theories to fit the observed data. If it looks like a camel, feels like a camel, and smells like a camel, it might be a horse, but probably is a camel Similarly, if you argue that you can see a cell membrane by em, and accept that, why not accept actin filaments? I don't understand your dilemma.
Peter French, Centre for Immunology, St Vincent's Hospital, Sydney.
President, ANZSCBI

Actin filaments cannot be seen by light microscopy, and would not permit intracellular movement. How could fine networks move large particles like mitochondria? It is a hypothesis for which no one has suggested a mechanism.
It is difficult to discuss these matters, when you have not read my evidence in H Hillman and P Sartory (1980) 'The Living Cell', Packard, Chichester (it is in Sydney Univ Library).
H.H. (1991) 'Some microscopic considerations about cell structure, Microscopy 36, 557-577.
H.H. (1991) 'The Case for New Paradigms in Cell Biology and in Neurobiology' Mellen Press, Lewiston.
Please enter into correspondence about particular questions. I note that you have only read one out of 47.
Harold Hillman
[Back to top]

Warren Gallin Department of Biological Sciences
Re: * UNANSWERED QUESTIONS: RESPONSE *
University of Alberta Edmonton, Alberta T6G 2E9 Canada
Date: Fri, 7 Jun 96
[Back to top]
#I would suggest that demonstrations of ignorance are not conducive to
#getting serious or respectful answers.

Rude. Please indicate any 'demonstrations of ignorance'.

##1. Discourtesy, assumptions of ignorance, and emotive remarks are no
##substitute for measured argument and evidence. Each of these questions
##highlights a contradiction *within* current views; for example,
## (a) everyone agrees that intracellular movements can be seen by
##low power light microscopy in living cells, yet most people also
##believe that there is a cytoskeleton, which would not permit such
##movements;
#This is ridiculuous. The cytoskeleton demonstrably acts as a substrate for
#organelle movement. Read a basic cell biology textbook.

Besides the rude comment, I suggest you read my evidence in Hillman & Sartory (1980) and H.Hillman (1991) Some microscopic considerations about cell structure. Microscopy 36, 557-577.

In HH (1991). I have listed 25 assumptions *inherent* in the use of the technique H Hillman (1991) The Case for New Paradigms in Cell Biology and in Neurobiology, Mellen Press, Lewiston. No one has denied that any of the assumptions I have listed in *not* inherent in the use of the technique.

Show me a random distribution of orientations of 'unit' membranes or nuclear pores.

##2. In my publications cited, and in about 120 other full-length
##papers, I have shown, in detail, with evidence:
## (a) That one can not yet derive conclusions from subcellular
##fractionation about the chemistry of organelles, which are relevant
##to their original states in the intact, living organisms;
## (b) That the following structures do not exist in the living
##cells: endoplasmic reticula, Golgi bodies, lysosomes, nuclear pores,
##mitochondrial cristae, the cytoskeleton, actin filaments and synaptic
##knobs, either because they would not permit the evident
##intra-cellular movements, or because they disobey the laws of solid
##geometry. Transmembrane molecules and receptors can not be seen on
##the cell membranes by transmission electron microscopy, although
##sequencing shows them to be 2-3 times the diameter of the cell
##membrane, which *can* be seen by electron microscopy;
#Ah, you are a disciple of J.D. Robertson? How about gap junctions?

No - it is irrelevant.

## (c) That in the central nervous system, the only cells are
##neurons and microglia; astrocytes and oligodendrocytes do not exist
##in the whole intact mammalian nervous system;
## (d) The chemical transmission hypothesis contains many untested
##and untestable hypotheses, and was worked out for neuromuscular
##junctions; it has been assumed to be relevant to synapses - especially
##since the latter term has been extended from its original meaning
##(nerve-nerve connections) to include neuromuscular junctions (nerve-muscle connections).
#Don't you read the literature?

I have been in research for forty years and *do*. Have you read any of mine?

##3. I have always suggested alternative and testable hypotheses, not
##open to the criticisms of current views, for example, how to localise
##biochemical activities without disruption of tissue, the structure of
##the living cell, the cellular structure of the central
##nervous system, the passage of excitability from one neuron to
##another, etc, etc.
##
##4. The fundamental questions I must raise with the Internet
##cytologists are:
## 'By what criteria are questions improper?'
#Questions are not improper, but they can be pointless, or based on
#ignorance. Most of yours clearly are.

Rude. Which ones are 'pointless'?

## 'Do all academics have a duty to address the difficulties and
##apparent contradictions of their own views?'
#Yes. You do too. Your mechanical denial of huge chunks of current
#knowledge would perhaps be the first thing you should address.

I am afraid that I have done this; unfortunately you have not read my publications

## 'Do they believe that progress can be made without examining
##their own views?'
#Of course not. I suggest you apply this idea to your own views.

I suggest that you read my publications before commenting on them.

## 'Would they disagree that a good academic should answer all
##these questions in the affirmative?'
#No.
##5. It seems to me to be absolutely essential that any experimental
##project to list as many as possible of the assumptions inherent in:-
## (a) the use of the experimental procedures;
## (b) the processing of raw data into the results to be published;
## (c) the interpretation of the results in the light of previous
##theories and new ones being generated.
##
#True. Exactly how far does one need to go. When you use a 1 M sucrose
#solution do you need to outlines the uncertainties in structure of the
#sucrose, thermodynamics of the water interaction with the sucrose molecules,
#whether molecules actually exist? At some point one has to assume that the
#reader is informed of the current state of knowledge in the field. If you
#are not, then it is your fault, not that of the author of the paper.

When you use 1 M sucrose in an enzyme assay preparation, you need to know if it affects the enzyme itself. It does. Did you know that?

##It is simply not good enough to support one's case with other
##peoples' findings without examining the validity of the findings
##cited. One is responsible not only for the interpretation of one's
##own results but also for the validity of experiments or interpretations
##of other authors whose results one uses to interpret one's own
##findings. The validity of an experiment depends upon the warrantability
##of *every* statistically potentially significant assumption,
##both those recognised and those not recognised or ignored. Like a
##chain, its overall strength depends upon its weakest link. The
##validity and value of an experiment in pursuit of truth depends upon
##the warrantability of the weakest assumption. Indeed, *any* wrong
##assumption, whose error would make a significant difference to the
##result of an experiment, renders the whole experiment invalid. Of
##course, it becomes worse if the assumptions are testable but have
##never been tested, or are untestable. An assumption does not disappear
##just because research workers singly or collectively do not
##recognise it, or do not wish to do so.
##
##6. I wish to repeat that I am prepared to enter into personal
##dialogue with anyone about any of these questions, and I have dealt
##with each of them in *detail* in my publications. I have answered
##the Internet responses received so far.
##
##7. We are talking here about intellectual integrity, and not about
##promotion, grant applications, casuistry, verbal acrobatics, scoring
##points, theology or dogma - at least, I hope we are!
#We are talking about the practicality of intellectual discourse here. I
#have had too many runins with people who felt that by simply asking
#questions without making any serious effort to become informed about the
#issues they were contributing to intellectual discourse. I disagree with
#that view.

To say that I am ignorant, have not addressed questions, or put forward alternative hypotheses, can only be based on your reading and assessing my evidence.
Harold Hillman.
[Back to top]

From: brad.harris@u.cc.utah.edu
[22 July; this wasn't answered until October]
[Back to top]
#I appreciate your questioning of the establishment, only by doing so
#can science progress. When we fail to question our assumptions we
#fail to move forward. Often we must make bold assumptions in order
#to make any progress, however, it is important that these assumptions
#are recognized and addmitted, they qualify any answers that are
#reached and must be addresses individually at some point.

I am not questioning the establishment. I am asking scientific questions to anyone believing the currently accepted view. It is not only important that the assumptions are recognised and admitted, but more important, they must be tested.

# I do have a nagging question of 'why are you posing these questions.'
#My untrusting nature makes me wonder if you somehow have a hidden agenda?

What is the relevance of my motives for asking questions? Is it not proper to ask questions?

# However, in good faith and persuit of truth and understanding:
#Most of your questions I have too little knowledge about in order to give an inteligent answer but:
##Question 26: Why is it assumed that the receptors for transmitters, hormones, messengers, antibodies, drugs and toxins are on the surface of the cell membrane?
#When cono-toxins (neuro toxins from cono-snails) are radiactively labeled, not only are they too large and too ionically charged to pass through the membrane, specifically there is no sign of them beyond the junction itself. When florecently labeled, they have an additional relatively huge florecent addition, but never the less, fully block all transmission, and again there is no sign of them beyond the junction.

You are failing to distinguish between where they are in life, and where they may be found after preparation, that is, you are assuming they do not move.

##Question 27: How valid is the use of agonists, antagonists and ligands to detect receptors, instead of the transmitters, hormones, antigens, drugs and toxins themselves?
#I am most familiar with Ach, with this qualification, we most often use toxins to identify receptors, however, since other means of identification give results that are the same, they are generally accepted as valid.

Please quote me *any* papers using acetylcholine to detect acetylcholine receptors, or any comparing the site of acetylcholine receptors, to say nicotine or muscarine receptors, histochemically by markers, or by electron microscopy.

##Question 30: Does the chemical theory of synaptic transmission contain unprovable and unproved hypotheses?
#I assume that you believe this is so, as this is the first that I have heard of strong qualifications on this theory, would you mind enlightening me?

Please see Hillman (1991) Phys & Med N.M.R. for *22* assumptions, many of them unprovable and unfalsifiable.
[Back to top]

From: Richard Kondo Newsgroups: bionet.cellbiol,bionet.neuroscience
Subject: Re: * UNANSWERED QUESTIONS: RESPONSE *
Date: Mon, 10 Jun 1996
Organization: UCLA Cardiovascular Research Lab
[Back to top]

# 1. Discourtesy, assumptions of ignorance, and emotive remarks are no
# substitute for measured argument and evidence. Each of these questions
# highlights a contradiction *within* current views; for example,
# (a) everyone agrees that intracellular movements can be seen by
# low power light microscopy in living cells, yet most people also
# believe that there is a cytoskeleton, which would not permit such
# movements;

rest deleted.
On the contrary, the cytoskeleton is most likely essential for intracellular movement of organelles. The role of microtubules, associated structural proteins and the ATP driven protein motors, kinesin and dynein have been elucidated over the past 15 years.
Brady et al., (1982) Science 'Fast axonal transport in extruded axoplasm from giant squid axon' 218:1129-31.
Hayden et al., (1983) Cell Motility 'Cytoplasmic transport in keratocytes: direct visualization of particle translocation along microtubules' 3:1-19
Schnapp et al., (1985) Cell 'Single microtubules from single axoplasm support bidirectional movement of organelles' 40:455-62
Vale et al., (1985) Cell 'Identification of a novel force generating protein, kinesin, involved in microtubule-based motility' 42:39-50
Sheetz et al., (1987) Annals of New York Academy of Sciences, 'Movement of vesicles on microtubules' 493:409-16
Schnapp and Reese (1989) PNAS 'Dynein is the motor for retrograde axonal transport of organelles' 85:1548-52.

I am sending you my paper (Hillman, 1991) 'Some microscopic considerations about cell structure - light versus electron microscopy' Microscopy *36*, 557-576, dealing with this question in detail.
Meanwhile, a few remarks. I am sure that you have seen micrographs of networks of tubulin, vimentin, spectrin, endoplasmic reticulum, microtrabeculae and actin. In these you do *not* see lysosomes, Golgi bodies or mitochondria, and with *all* these elements together, they would not allow *enough space* for relatively large bodies to move round. The hypothesis that, for example, actin can pull mitochondria requires a mechanism and attachments all round the mitochondria *and to other structures* otherwise they could not pull. The maximum resolution of the light microscope—under which intracellular movements are seen in living cells—is 200-250 nm, yet the electron microscopists describe them as 25nm. Therefore the microtubules are not the same structures; this also applies to those believed to be the spindles pulling the chromosomes to the poles during cell division. The hypothesis that the cytoskeleton pulls, say mitochondria, ignores the possibility—a simpler one—that Brownian movement, diffusion, streaming, convection movements (none requiring biological mechanisms) occur in fine granules in fluid.
Harold Hillman.
[Back to top]

From: Cornelius Krasel. Department of Pharmacology
Newsgroups: bionet.cellbiol
Subject: Re: UNANSWERED QUESTIONS
Date: 28 May 1996
Organization: CC University of Hohenheim (not responsible for contents)
[Back to top]
#In My Humble Opinion: nice troll. I think you look for somebody to do your homework.
#Then, I could be wrong. I only picked a few questions which I thought were interesting or funny. Note that I'm not a neurobiologist, therefore I don't feel competent to answer those questions.
## Question 3: Why does one assume that homogenisation and centrifugation do not change the entropy, and therefore the free energy and the equilibria of reactions in subcellular particles? Why are not controls always carried out for subcellular fractionation, except for total recoveries relative to the crude homogenates?
#To my knowledge, it is very difficult to quantitate the thermodynamics of such complex systems as living cells. However, you imply that homogenisation does not change the entropy which is most certainly wrong. The scientific community is well aware of this problem.

No one has ever published controls for subcellular fractionation, as listed in Hillman (1972). On the contrary, homogenisation *does* change entropy.

## Question 5: Does the finding that a chemical substance or activity is located in the same subcellular fraction and a structure identified by electron microscopy mean that the same chemical activity was located in that particular organelle in the living cell of the intact animal or plant.
#It probably depends on the type of "structure". As Far As I Know, enzymes cannot be visualized by electron microscopy until you use their enzymatic activity for a stain. (I know that e.g. myosin molecules *can* be visualized, but not in a cellular context.)

You have not answered this question.

## Question 6: How is intracellular movement possible, and the cytoplasmic viscosity is low in life, if there is a cytoskeleton present?
#AFAIK, cytoplasmic viscosity is considered to be high.

You have not answered this question.

## Question 7: Where do protein synthesis and acid hydrolysis occur in cells in which ribosomes and lysosomes cannot be seen?
#Are there cells which synthesize proteins and don't have ribosomes? Examples please.

All cells synthesise proteins, in many one cannot see ribosomes, e.g. muscle.

## Question 16: Can one know the thickness in life of any biological membrane?
#Yes -- use AFM on living objects.

Why are all measurements in books measured from transmission electron micrographs or deposits on dead dehydrated tissue?

## Question 20: What is transport?
#Consult your Webster's :-)

What is wrong with diffusion?

## Question 21: Why are receptors and channels, which have been characterised, sequenced and their sizes measured or calculated, not seen on membranes by transmission electron microscopy?
#Too small.

Every week in Nature, Science, Molecular Biology etc one sees sequencing of molecules 3x the width of the cell membrane, seen by em.

## Question 22: Can an electron microscopist looking at a metal deposit on
## a biological structure derive any information about its chemistry?
#About the chemistry of the metal or the chemistry of the biological
#structure?

The biological structure, of course.

## Question 26: Why is it assumed that the receptors for transmitters,
## hormones, messengers, antibodies, drugs and toxins are on the
## surface of the cell membrane?
#For the beta2-AR:
#1) Evidence from use of hydrophilic ligands.
#2) Evidence from epitope mapping.
#3) Evidence from AP fusion studies.
#4) Evidence from protease accessibility.
#5) Evidence from immunoelectron microscopy.

Are you assuming that diffusion does not occur during homogenisation, centrifugation, fixation, dehydration, embedding, etc?

Why not use ach, adrenaline, gaba or glutamate to look for their *own* receptors - why use ligands, which are different substances?

## Question 32: How is intracellular movement possible, and why is
## the viscosity of cytoplasm so low in the intact cell, if there
## is a cytoskeleton?
#See above (wasn't it Question 6?).

Sorry

## Question 33: If nuclear pores allow RNA to pass through, how do they
## prevent smaller molecules and ions going through at the same time,
## and why is there a potential difference across the nuclear membrane?
#We don't know yet. If you can contribute to solving this problem,
#more power to you.

Nuclear pores are artefacts. See Hillman & Sartory (1980).

## Question 34: What is the evidence that each cell of a particular
## plant or animal contains the same quantity of DNA?
#Honestly, I don't know.

This is an *assumption*

## Question 35: If the cell membrane is fluid mechanically, how can cells
## maintain their integrity?
#Because a fluid bilayer is intrinsically stable.

Glass is a *solid* mechanically but a fluid physicochemically.

## Question 36: In immunocytochemistry, is it assumed that the fixatives,
## dehydrating reagents, washings, and primary and secondary antibodies, do not change the reaction of the antibody to the antigen
## believed to be in a particular cell or part of a cell?
#Yes. That's why many antibodies do not work in immunocytochemistry.

Nearly all immunocytochemistry is done on fixed, dehydrated, and mounted sections.

## Question 45: If each cell in an organism contains the same DNA,
## but some produce different proteins, is the existence of
## suppressor genes the only possible explanation for the
## difference of the proteins?
#Probably not. There are always thousand explanations for a given fact.

Agreed

If diabetes were autoimmune, how do Islets continue to exist in that condition?

## Question 47: Why are pure proteins used for calibration, when
## different tissues contain different mixtures of proteins, which
## have different calibration curves?
#What kind of calibration?

Whenever one measures proteins in tissue or refers measurements to proteins.

#--Cornelius.

From: krasel@wpxx02.toxi.uni-wuerzburg.de (Cornelius Krasel)
Subject: Re: * UNANSWERED QUESTIONS: RESPONSE *
Date: 7 Jun 1996
## 1. Discourtesy, assumptions of ignorance, and emotive remarks are no
## substitute for measured argument and evidence. Each of these questions
## highlights a contradiction *within* current views; for example,
## (a) everyone agrees that intracellular movements can be seen by
## low power light microscopy in living cells, yet most people also
## believe that there is a cytoskeleton, which would not permit such
## movements;
#I still fail to understand why a cytoskeleton would not permit intracellular
#movements. In fact, it has been shown that at least certain intracellular
#movements such as those of mitochondria are based on the existence of a
#cytoskeleton.

(i) Because the cytoplasm is too full of cytoskeleton.
(ii) Because small particles move without actin, e.g. by diffusion, Brownian movement, streaming and convection.
(iii) We have brought much evidence in Hillman and Sartory (1980)'The Living Cell'

## (b) most people believe in the Second Law of Thermodynamics, yet in subcellular fractionation they change the entropy of their systems (homogenise and centrifuge), and assume that this does not change the free energy, which drives all the biochemical reactions they are studying,
#Wrong. As stated before, most biochemists are aware of the fact that
#destruction of a living cell changes the entropy of the system.
#However, it's very difficult if not impossible to reconstitute a
#system with the same entropy (since it is difficult if not impossible
#to actually quantitate this entropy).

Why use destructive techniques?

## (c) most people would agree that the laws of solid geometry must be obeyed, while in their electron micrographs - as opposed to their diagrams - they do not see a random selection of orientations, including oblique views of cell membranes, nuclear membranes, myelin lamellae, synapses, nuclear pores, etc.
#I'm not knowledgeable in electron microscopy, so I leave this mostly to others. However, it seems fairly obvious to me that a serial section of an embedded cell would, for example, yield some sections where the cell membrane is hit in its plane (assuming that this is what you mean with "oblique view"); however, such a picture would not give very much information and is therefore not published.

Any section of a whole cell should show organelles in random orientations. The 'unit' membranes, the nuclear pores, and the myelin lamellae are not.

## 2. In my publications cited, and in about 120 other full-length papers, I have shown, in detail, with evidence:
## (a) That one can not yet derive conclusions from subcellular fractionation about the chemistry of organelles, which are relevant to their original states in the intact, living organisms;
#Since I do not have the time to search for your publications, it would be maybe nice to summarize what lead you to arrive at those conclusions.

The Second Law of Thermodynamics

## (b) That the following structures do not exist in the living cells: endoplasmic reticula, Golgi bodies, lysosomes, nuclear pores, mitochondrial cristae, the cytoskeleton, actin filaments and synaptic knobs, either because they would not permit the evident intra-cellular movements, or because they disobey the laws of solid geometry.
#I think your "laws of solid geometry" need reevaluation. It is fairly obvious that the cytoskeleton not only permits intracellular movement, it is necessary for it.

Euclid invented the laws of geometry, not Hillman

## Transmembrane molecules and receptors can not be seen on the cell membranes by transmission electron microscopy, although sequencing shows them to be 2-3 times the diameter of the cell membrane, which *can* be seen by electron microscopy; The cell membrane can only be seen in transmission electron microscopy because the cells are fixed with electron-dense material with high affinity for lipids; cell-membrane *and* transmembrane molecules can be visualized by, for example, atomic force microscopy on living cells or freeze-fracture electron microscopy.
## Why can not they be seen as gaps if the electron dense materials do not stain them?
#[snipped neuronal stuff and philosophical questions]
#Unfortunately I cannot answer to the hypotheses which you have brought forward, since our library does not seem to carry any of the books you gave nor any of the journals where you have published since 1990 (I did a quick Medline search to check them out). I would be interested in older references which might have been published in more "mainstream" journals :-)

#I will comment on the questions raised in your email later.

I should be pleased to send you reprints on any particular question.
Harold Hillman.

answered Questions
Cornelius Krasel, krasel@wpxx02.toxi.uni-wuerzburg.de
Subject: Re: Unanswered Questions
Date: Wed, 19 Jun 1996
Sorry for the late reply.
#### Question 3: Why does one assume that homogenisation and centrifugation
#### do not change the entropy, and therefore the free energy and
#### the equilibria of reactions in subcellular particles? Why are not
#### controls always carried out for subcellular fractionation, except
#### for total recoveries relative to the crude homogenates?
###
### To my knowledge, it is very difficult to quantitate the thermodynamics
### of such complex systems as living cells. However, you imply that
### homogenisation does not change the entropy which is most certainly
### wrong. The scientific community is well aware of this problem.
##
## No one has ever published controls for subcellular fractionation, as
## listed in Hillman (1972). On the contrary, homogenisation *does* change
## entropy.
#That's what I said.

It is difficult to calculate the energy, therefore one has to control the experiments. In Hillman H (1972) 'Certainty and Uncertainty in Biochemical Techniques', Surrey University Press, Henley on Thames, I have listed 7 different kinds of controls. Homogenisation, centrifugation, purification all change entropy therefore free energy, which drives biochemical reactions.
Therefore it is an illegal procedure without controls!

#### Question 5: Does the finding that a chemical substance or activity
#### is located in the same subcellular fraction and a structure identified by electron microscopy mean that the same chemical activity
#### was located in that particular organelle in the living cell of the
#### intact animal or plant.
###
### It probably depends on the type of "structure". AFAIK, enzymes cannot be
### visualized by electron microscopy until you use their enzymatic
### activity for a stain. (I know that e.g. myosin molecules *can* be
### visualized, but not in a cellular context.)

Why can not myosin molecules be seen in cells, if they can be seen by electron microscopy and of they are there?

## You have not answered this question.
#Then, what's your question? If I find an enzymatic activity in a subcellular
#fraction which I have identified as being Golgi previously, and I can also
#locate the activity in the Golgi by let's say immunelectron microscopy,
#the probability is high that the enzyme is indeed located in the Golgi.
#If I understand the question wrong, please try to rephrase it.

Subcellular fractionation is used for locating enzyme *activities* on the assumption that the procedure does not change *activity* or location - the latter assuming that diffusion does not occur.

#### Question 6: How is intracellular movement possible, and the cytoplasmic
#### viscosity is low in life, if there is a cytoskeleton present?
###
### AFAIK, cytoplasmic viscosity is considered to be high.
##
## You have not answered this question.
#You claim the cytoplasmic viscosity is low in life. It isn't.

Cytoplasmic viscosity is low. See Hillman & Sartory (1980) The Living Cell, Packard, Chichester, pp 55-57; viscosity in cytoplasm is usually less than glycerol.

#### Question 7: Where do protein synthesis and acid hydrolysis occur in
#### cells in which ribosomes and lysosomes cannot be seen?
###
### Are there cells which synthesize proteins and don't have ribosomes?
### Examples please.
##
## All cells synthesise proteins, in many one cannot see ribosomes, e.g.
## muscle.
#Is it possible to localize ribosomal proteins in these cells by cell
#fractionation or immunoblotting? Or is it possible to isolate ribosomes
#by cell fractionation? If yes, there are ribosomes -- you just can't
#see them in the EM because of whatever reason (I'm not an electron
#microscopist, so I don't know whether it is indeed impossible to see ribosomes in muscle cells).

'Ribosomal' activity is believed to be protein synthesis in ribosomes. All cells synthesise proteins, including prokaryotes, where ribosomes can not be seen.

#### Question 16: Can one know the thickness in life of any biological
#### membrane?
###
### Yes -- use AFM on living objects.
Please spell out 'AFM'
## Why are all measurements in books measured from transmission electron
## micrographs or deposits on dead dehydrated tissue?
#Because AFM is a relatively new technique (about ten years old). However,
#AFM measurements correlate well with sizes given in books derivated from
#other techniques.

All the figures in the literature about the thickness of cell membrane are from low angle diffraction or transmission electron microscopy.

#### Question 20: What is transport?
###
### Consult your Webster's :-)
##
## What is wrong with diffusion?
#Membrane transport, as you probably know, can be classified into facilitated
#diffusion and active transport. Transport is movement against a concentration
#gradient and needs energy to be accomplished (ATP or ion gradients).

'Transport' is a vague term, only meaning movement - not necessarily across membranes. Occam's Razor encourages one to consider that movement is by diffusion, Brownian movement, convection, *before* considering any other process, which - if claimed to be unique - should be faster or slower than all the above put together.

#### Question 21: Why are receptors and channels, which have been
#### characterised, sequenced and their sizes measured or calculated, not seen
#### on membranes by transmission electron microscopy?
###
### Too small.
##
## Every week in Nature, Science, Molecular Biology etc one sees sequencing
## of molecules 3x the width of the cell membrane, seen by em.
#It's pretty easy to visualize concentrated amounts of macromolecules.
#Check out Unwin's paper about nicotinic acetylcholine receptors from
#Torpedo electric organs. However, common receptors, such as most
#G-protein-coupled ones, are just to rare to be distinguishable from noise. The
#signal-to-noise ratio is much higher in AFM.

Unwin's nicotinic ach receptor is the *only* one anyone has claimed to see. Where are the others?

#Furthermore, transmission EM does not visualize the membrane in statu nascendi
#(AFAIK) but electron-dense material (osmium tetroxide, is that correct?) which happens to stain lipids.

If large molecules are present, but not stained, there should be a gap around them of unstained material - *there is not*.

#### Question 22: Can an electron microscopist looking at a metal deposit on
#### a biological structure derive any information about its chemistry?
###
### About the chemistry of the metal or the chemistry of the biological
### structure?
##
## The biological structure, of course.

It can not, it looks at heavy metal.

#If you think in terms of chemical composition, it's difficult. I've been
#told that with STM it is possible to see the chemical composition of
#surfaces. However, an electron microscopist will not be able to tell
#much about atomic composition of his stained images for several reasons.
#So, the global answer would be no.
#### Question 26: Why is it assumed that the receptors for transmitters,
#### hormones, messengers, antibodies, drugs and toxins are on the
#### surface of the cell membrane?
###
### For the beta2-AR:
### 1) Evidence from use of hydrophilic ligands.
### 2) Evidence from epitope mapping.
### 3) Evidence from AP fusion studies.
### 4) Evidence from protease accessibility.
### 5) Evidence from immunoelectron microscopy.
##
## Are you assuming that diffusion does not occur during homogenisation,
## centrifugation, fixation, dehydration, embedding, etc?
#Some of these experiments, e.g. the ligand binding experiments, are
#done with whole cells. Same goes for protease cleavage and epitope
#mapping.
#As others have pointed out, there are also receptors which are not
#located in membranes (e.g. for steroid hormones). There are also
#receptors in internal membranes, or receptors that are cycling
#between different compartments (e.g. transferrin receptor).

There is still the question about why these large macro-molecules whose size is known are represented *in diagrams* as 2-3 x width of cell membrane are not (except for Unwin's) seen by transmission electron microscopy.
Localisations are usually done by microscopy of dehydrated tissues or subcellular fractionation in both of which diffusion *must* occur therefore one can not decide localisation.

#### Question 27: How valid is the use of agonists, antagonists and
#### ligands to detect receptors, instead of the transmitters, hormones, antigens, drugs and toxins themselves?
###
### You lost me here. What's the difference between certain ligands, agonists
### and transmitters?
##
## Why not use ach, adrenaline, gaba or glutamate to look for their *own*
## receptors - why use ligands, which are different substances?
#Of course you can use adrenaline to look for adrenergic receptors. However,
#it binds fairly unspecific to several receptors. Other ligands bind
#more specifically, and since people are usually interested in the
#properties of one receptor, they use ligands specific for it. (But
#adrenaline is also a ligand for these receptors.)

Of course, you can use adrenaline to look for adrenaline receptors. Then *why* do people *not*?

#### Question 33: If nuclear pores allow RNA to pass through, how do they
#### prevent smaller molecules and ions going through at the same time,
#### and why is there a potential difference across the nuclear membrane?
###
### We don't know yet. If you can contribute to solving this problem,
### more power to you.
##
## Nuclear pores are artefacts. See Hillman & Sartory (1980).
#Don't know. Other people seem to have other opinions.

It is a question of evidence, not just opinions.

#### Question 34: What is the evidence that each cell of a particular
#### plant or animal contains the same quantity of DNA?
###
### Honestly, I don't know.
##
## This is an *assumption*
#Would you assume that each cell contains the same amount of chromosomes?

I would not. It has not been proven. It is an assumption.

#I think this has been very well proven. On the other hand, polytene
#chromosomes certainly contain more DNA than normal chromosomes, so
#there are cells which contain more DNA than others. Also, cells where
#chromosomes are defective contain different amounts of DNA compared to
#"normal" cells.

I do not agree that one should continue to accept an unproven assumption.

#### Question 35: If the cell membrane is fluid mechanically, how can cells
#### maintain their integrity?
###
### Because a fluid bilayer is intrinsically stable.
##
## Glass is a *solid* mechanically but a fluid physicochemically.
#You lost me here.

What evidence is there for that other than the *belief* that the cell membrane is a fluid bilayer?

#### Question 36: In immunocytochemistry, is it assumed that the fixatives,
#### dehydrating reagents, washings, and primary and secondary antibodies, do not change the reaction of the antibody to the antigen
#### believed to be in a particular cell or part of a cell?
###
### Yes. That's why many antibodies do not work in immunocytochemistry.
##
## Nearly all immunocytochemistry is done on fixed, dehydrated, and
## mounted sections.
#I know. So?

Therefore, one assumes that the fixative dehydrating agent and mounting agent do not affect the antigen-antibody reaction - an untested and very unlikely assumption.

#### Question 46: In diseases believed to be auto-immune, either
#### organ-specific or tissue-specific, why does the body not reject
#### the specific organ or tissue, as it rejects incompatible
#### transplanted hearts, or blood of the wrong group, often
#### making the patients ill, or even killing them?
###
### It does. That's where diabetes type I can come from.
##
## If diabetes were autoimmune, how do Islets continue to exist
## in that condition?
#I don't know the exact mechanism of diabetes type I but there has been
#recently a review published about it as an autoimmune disease in Cell.
#(I haven't had time yet to read it.)

I can not understand why anyone alleges a disease to be autoimmune if the main organs, e.g. the joints, the Islets of Langerhans, the brain (schizophrenia) are not *rejected*, as would incompatible blood be.

## ## Question 47: Why are pure proteins used for calibration, when
## ## different tissues contain different mixtures of proteins, which
## ## have different calibration curves?
## #
## #What kind of calibration?
##
## Whenever one measures proteins in tissue or refers measurements to proteins.
#You mean protein quantitation? I use BSA in my Bradford assays just because
#it is convenient. Everybody knows that Ovalbumin gives a completely different
#standard curve. It's just to standardize the assay to *something*.

What one needs to do is a separate recovery curve with your bovine serum albumin for *each* fraction.
Dr Krasel, I will send you a copy of one of our books, 'The Living Cell' as it is out of print.
Harold Hillman.

From: Cornelius Krasel
Subject: Re: Unanswered Questions
Date: Thu, 27 Jun 1996
Again I apologize for answering so late, but I have been quite busy (probably that's what all scientists say :-)
# (iii) We have brought much evidence in Hillman and Sartory (1980)
## 'The Living Cell'

I recently sent you a series of answers, and a copy of 'The Living Cell'. In the latter, I quote figures showing the intracellular velocity is *low*.

I don't know this paper. I am aware of Brownian movement, but as you say, it is likely to be substantially reduced because of the high viscosity #of the cytoplasm. I also don't think that organelle transport by microtubules has been demonstrated in living cells, but the dynamics of microtubules has been observed in living cells by injecting them with fluorescently labeled tubulin (which is included in microtubuli) and watching the cells over time with a fluorescence microscope. Pictures of this can be found in Alberts et al, Molecular biology of the cell, who also give references. Therefore, unless you can *prove* the contrary, I don't think your argumentation is valid (I've been taught that common-sense argumentation is not much worth in science :-).
Albers shows tubulin, and others show vimentin, spectrin, actin, microfilaments, microtrabeculae.

As I have shown, (i) the structures moving are much larger than the distances between fibres (ii) mitochondria (Golgi bodies) (lysosomes) are *not* seen between fibres.

## Why use destructive techniques?
#Because it's difficult to explore cells without noninvasive techniques?
#(Frankly, there are not much noninvasive techniques I know of. There is the use of optical tweezers; there is AFM; there is immunfluorescence microscopy, but only under certain circumstances. Do you count patch-clamp techniques as noninvasive?)

There is a large number of experiments, microdissection, prokaryotes, tissue culture, in vivo experiments, windows etc (see Hillman 1991, the case for new paradigms in cell biology and neurobiology).

## Any section of a whole cell should show organelles in random
## orientations. The 'unit' membranes, the nuclear pores, and the myelin
## lamellae are not.
#As stated, I have never ever done electron microscopy. Therefore, I don't
#know how common "unorthodox" views of a cell and its organelles are.
#However, I don't think that it is possible to conclude from published
#figures that the average electron micrograph is similar. These figures
#have been selected for clarity, to emphasize a certain point.

Please tell me *one* publication showing a lamella of endoplasmic reticulum or cell membrane in the plane of the section.

#I recently came across a paper which purposely shows cell membranes (i.e.
#"unit" membranes) in plane:
#@article{montesano:82,
# author = {R. Montesano and J. Roth and A. Robert and L. Orci},
# title = {Non-coated membrane invaginations are involved in
# binding and internalization of cholera and tetanus toxin.},
# journal = {Nature},
# volume = 296,
# pages = {651--653},
# year = 1982}

I will look this up and comment later

#However, also from the laws of geometry it should be clear that a membrane
#will be much more often displayed as a section. I.e., let us assume
#membrane orientation is random (which is most certainly wrong). Then
#even a cut through a membrane which is at a degree of 450 to the plane
#of the cut would show a "fuzzy" unit membrane, fuzzy, because the two
#sheets of the membrane would be broader than usual.

The laws of geometry dictate that a membrane and any and *every* structure should be seen in all orientations, because the tissue does not know from which direction the microtome will cut. Nuclear pores are seen in sections as cracks in side view and circles in face view but *never* in intermediate elipses or ovals. In so-called fractions of pores - they are always circles, *never* any other orientation. e.g. circles of different sizes, ovals, elipses. That is impossible in geometry.

## ## 2. In my publications cited, and in about 120 other full-length
## ## papers, I have shown, in detail, with evidence:
## ## (a) That one can not yet derive conclusions from subcellular
## ## fractionation about the chemistry of organelles, which are relevant
## ## to their original states in the intact, living organisms;
## #
## #Since I do not have the time to search for your publications, it would
## #be maybe nice to summarize what lead you to arrive at those conclusions.
##
## The Second Law of Thermodynamics
#A very precise answer. Maybe you could elaborate a bit.

Since you agree that homogenisation, centrifugation and separation *all* change entropy. Therefore free energy, which drives biochemical reactions, you should not carry out these manoevres and *assume* that an enzyme activity not be changed or relocated. Therefore results of subcellular fractionation experiments are not valid until controls are done.

## ## (b) That the following structures do not exist in the living
## ## cells: endoplasmic reticula, Golgi bodies, lysosomes, nuclear pores,
## ## mitochondrial cristae, the cytoskeleton, actin filaments and synaptic knobs, either because they would not permit the evident intra-cellular movements, or because they disobey the laws of solid
## ## geometry.
## #
## #I think your "laws of solid geometry" need reevaluation. It is fairly
## #obvious that the cytoskeleton not only permits intracellular movement,
## #it is necessary for it.
##
## Euclid invented the laws of geometry, not Hillman.
#Euclid discovered laws applicable in a mathematical context, not in
#a living cell.

Dr Krasel. I am sorry that you think that the laws of geometry are not applicable to living cells. I do.

## #The cell membrane can only be seen in transmission electron microscopy
## #because the cells are fixed with electron-dense material with high
## #affinity for lipids; cell-membrane *and* transmembrane molecules
## #can be visualized by, for example, atomic force microscopy on living
## #cells or freeze-fracture electron microscopy.
##
## Why can not they be seen as gaps if the electron dense materials
## do not stain them?
#I don't think that staining a slide is a very precise procedure. Why
#can you see transmembrane receptors in freeze-fracture microscopy
#of cell membranes? Why can you see them in AFM? Why can you see them
#with immunelectron microscopy or immunofluorescence (even on living
#cells)?

I think that you are saying that transmembrane molecules are there, but can not be seen. The simplest hypothesis for not being able to see them is that they are *not* there.

#If you have published an overview or summary of your ideas I would
#indeed appreciate a reprint of that particular article. I would prefer
#this over work to a particular question to get a better grasp of your
#ideas, for example, why the 2nd law of thermodynamics is incompatible
#with a cytoskeleton in your opinion.

Second law does not permit fractionation. Cytoskeleton is another question. Please read these as well as please see reprints I sent and my last answers.
Best wishes
Harold Hillman
[Back to top]

Kevin McKenna
Subject: Re: a selection of unanswered questions
Date: Mon, 03 Jun 1996
Organization: Northwestern University, Evanston, IL, US
[Back to top]
#[snip]
## The following questions have never been answered satisfactorily,
## several of them never at all:-
#Actually, that's wrong. Most of these have been answered extensively.
## Question 1: Can one obtain an enriched fraction of a subcellular
## organelle or cell type?
#Yes.

One does subcellar fractionation to study *biochemistry* of organelle on the assumption that the procedure does not change it.

## Question 2: How does one know that the disruptive procedure does not
## change the biochemistry of the fraction significantly?

The second law of thermodynamics dictates that it must

#One doesn't know this a priori. It must be established by experiments to
#validate the procedures.

Such experiments have *never*, *never* been published

## Question 3: Why does one assume that homogenisation and centrifugation do not change the entropy, and therefore the free energy and
## the equilibria of reactions in subcellular particles? Why are not
## controls always carried out for subcellular fractionation, except
## for total recoveries relative to the crude homogenates?
#Who is using subcellular or cell-free systems which have not been validated?

Everyone who does subcellular fractionation (see Hillman, 1972)

## Question 4: Why is it believed that each biochemical pathway or cycle
## has its own structural compartment when prokaryotes can carry out
## virtually all the same reactions in only one compartment?
#Could you rephrase this question? Are you claiming that any compartment
#can mediate any function?

Prokaryotes have only *one* compartment, yet to all biochemistry

## Question 5: Does the finding that a chemical substance or activity
## is located in the same subcellular fraction and a structure identified by electron microscopy mean that the same chemical activity
## was located in that particular organelle in the living cell of the
## intact animal or plant.
#It's certainly a good place to start.

This is assuming that diffusion does not occur during the procedure.

## Question 6: How is intracellular movement possible, and the cytoplasmic
## viscosity is low in life, if there is a cytoskeleton present?
#Because most intracellular movement is driven by active processes using
#the cytoskeleton. Some processes are diffusion-driven, but not most.

Are diffusion, Brownian movement and streaming driven by active processes?

## Question 7: Where do protein synthesis and acid hydrolysis occur in
## cells in which ribosomes and lysosomes cannot be seen?
#Because these cells still contain the necessary enzymes.

That is, the biochemical activities can occur in the absence of ribosomes and/or/lysosomes

## Question 8: What is the evidence that the microsomal fraction consists of cell membranes and endoplasmic reticulum?
#Examination of the biochemical constituents of the fraction and
#electronmicroscopic examination.

The microsomal fraction appears as circles; the membranes are trilaminar

## Question 9: Why is it assumed that homogenisation and centrifugation
## do not affect the chemistry of receptors, or their affinities for
## transmitters, hormones, drugs, ligands, toxins?
#Who assumes this?

Everyone who examines receptors in homogenates

## Question 10: Can a particle and a vacuole both be lysosomes?
#A particle of what?

Any particle. I have shown that lysosomes are artifacts.

## Question 11: Can one calibrate substances originating from tissues
## using pure solutions in simple salines of approximately the same
## concentrations?
#Sometimes.

This assumes that no other substance in the tissue affects the measurement.

## Question 12: How can one study membranes by electron microscopy, when
## they are believed to contain lipids which the procedure extracts?
#Membranes have many components in addition to the lipids.

About half the membrane is supposed to be lipid, and water soluble

membrane components will also be extracted
## Question 13: What is the real evidence that rapid deep freezing for
## electron microscopy causes less shrinkage and distortion of tissues, cells and organelles, than classical transmission electron
## microscopy?

None; it is a belief

## Question 14: Why do those who calculate dimensions from electron
## micrographs not take into account the shrinkage during preparation
## and examination of their sections, cells and organelles?
##
## Question 15: Do membranes in cells appear to be normal to the plane
## of section more often than solid geometry would permit?
#No.

Every electron microscopist, including one answering on Usenet, has

admitted they do.
## Question 16: Can one know the thickness in life of any biological
## membrane?
#Yes.

Are you not aware that electron microscopists dehydrate cells before examining them?

## Question 17: Why should it be necessary to tilt the stage of the
## electron microscope to see randomly orientated membranes in all
## orientations, when this is not necessary with the light microscope?

No answer

## Question 18: How can carriers assist the passage of ions, aminoacids,
## etc. across membrane, when the combination must be bigger than the
## substance carried?
#What?

No answer

## Question 19: Why have few or no carriers been isolated?
#Lots of them have.

Please cite these and the *evidence* that they are carriers

## Question 20: What is transport?
#The movement of substances across membranes by passive, facilitated and
#active processes.

It also includes diffusion, Brownian movement, streaming, convection, not necessarily across membranes

## Question 21: Why are receptors and channels, which have been characterised, sequenced and their sizes measured or calculated, not seen
## on membranes by transmission electron microscopy?
#Because their sizes are small relative to the resolution of typical EM.

All that are frequently illustrated in Nature, Science and *all* diagrams of 'transmembrane' molecules are at least 2x the membrane's diameter

## Question 22: Can an electron microscopist looking at a metal deposit on
## a biological structure derive any information about its chemistry?

No answer

## Question 23: Why do the lamellae of the myelin sheath appear to be
## equal distances apart irrespective of the thickness or depth of
## the longitudinal section cut?
#Because the lamellae are equal distances apart.

If you cut an orange at the diameter, its skin is thin; away from the diameter, it appears thicker.

## Question 24: Is the repeating distance of the lamellae in the myelin
## sheath sufficient to regard it as a good model for the cell
## membrane?
#A model for what aspect of membrane function?

Structure in *all* textbooks

## Question 25: Since the myelin sheath is believed to consist of a
## scroll of membranes, and membranes appear darker by light microscopy than cytoplasm, why does not the myelin sheath appear darker
## than the axoplasm?

No answer

## Question 26: Why is it assumed that the receptors for transmitters,
## hormones, messengers, antibodies, drugs and toxins are on the
## surface of the cell membrane?
#It is not assumed at all. Some receptors are on the surface, some are not.
#The location of the receptors has been *determined* by experimentation,
#not assumed.

Nearly all are - except steroids. Why are they never seem on em? (Q 21)

Why not use ach, adrenalin, noradrenalin, gaba or glutamate to look for receptors instead of ligands, *which are different*?

## Question 28: Why are the dimensions and numbers of synapses
## different by light and electron microscopy?
#Most synapses can't be resolved by light microscopy.

Large ones seen by light (3-8 mu m) are *never* seen by em - please explain that.

## Question 29: Why are there no light micrographs in the literature
## showing the connection of one cell body by a dendritic pre-synaptic fibre to a synapse on another cell body?
#Most synapses can't be resolved by light microscopy.

They were seen by light microscopy for 50 years before em. I am talking about *presynaptic fibres*

## Question 30: Does the chemical theory of synaptic transmission
## contain unprovable and unproved hypotheses?
#No.

A large number (Hillman 1991) Phys Chem Phys Med NMR 23, 177-198

## Question 31: Why is it assumed that evidence derived from experiments on neuromuscular junctions is relevant to transmission
## in the central nervous system?
#Who is doing all this assuming? Some characteristics of neuromuscular
#junctions are similar to CNS synapses, some are not.

Katz's hypothesis was about neuromuscular junctions. Please tell me who does *not*, other than me.

## Question 32: How is intracellular movement possible, and why is
## the viscosity of cytoplasm so low in the intact cell, if there
## is a cytoskeleton?
#You already asked this one.

Sorry

1. How do you know?
2. Ions are <1 nm, pores 20-120 nm wide
3. You can only stop diffusion at 0 degrees K

## Question 34: What is the evidence that each cell of a particular
## plant or animal contains the same quantity of DNA?

No answer

## Question 35: If the cell membrane is fluid mechanically, how can cells
## maintain their integrity?
#Glass is a fluid, too. How do your windows maintain their integrity?

Glass is a solid mechanically

Everyone who does immunocytochemistry on histological sections makes these assumptions.

## Question 37: Is it reasonable to believe that processes or dendrites
## contain different antigens from the cell bodies from which they
## arise?
#Yes. There's lots of evidence for segregation.
It is not reasonable because processes arise from somas.
## Question 38: Under what conditions can tissue cultures be used in the
## study of the tissues from which they originated?
#When it has been established that the phenomenon under study in culture
#provides useful insights for the in vivo situation.

See next answer

## Question 39: Is it warrantable to assume that growth of tissues in
## culture does not change their morphology, biochemistry, or
## immuno-reactivity?
#No, it is not. Tissue culture changes many aspects of cell phenotype.

The latter 3 are the commonest uses of tissue culture in neurobiology.

## Question 40: Does not the use of the term neuroglia imply that the
## authors can not distinguish between astrocytes, oligodendrocytes,
## and microglia?
#No.

Of course it does.

## Question 41: Why are the individual types of neuroglial cells so
## rarely seen by light microscopy of healthy central nervous systems?
#It depends on the stain you use.

Every author has a different view of specificity. See tables in Hillman (1986)

## Question 42: Since the latter three alleged cell types were described
## by classical histological techniques during the first half of the
## twentieth century, does this not imply that anyone using antibodies to mark them specifically must first identify them by
## these criteria?
#No.

Of course it does, unless you change the meaning of the word 'neuroglia'.

## Question 43: Why is there no common agreement about the staining
## procedures, which are supposed to identify astrocytes, oligodendrocytes and microglia histologically?
#Is this true?

I have several tables of references showing this (Hillman, 1986).

## Question 44: Why is it necessary to use tissue cultures of the
## alleged cell types to identify them and their markers?
#Is it?

Most identification of the alleged cell types and their markers are doing in tissue culture.

I agree

## Question 46: In diseases believed to be auto-immune, either
## organ-specific or tissue-specific, why does the body not reject
## the specific organ or tissue, as it rejects incompatible
## transplanted hearts, or blood of the wrong group, often
## making the patients ill, or even killing them?
##
## Question 47: Why are pure proteins used for calibration, when
## different tissues contain different mixtures of proteins, which
## have different calibration curves?
##
## Question 48: Why do synapses seen by electron microscopy appear so
## much smaller than those seen by light microscopy?
##
## These questions have been raised in previous publications, and
## there have been few serious responses to them. I feel it my duty,
## therefore, to put them on Internet, to stimulate colleagues,
## especially young ones, to address them seriously, or to explain why
## they are unwilling to do so. If, as I suspect, there will be few or
## no responses to these proper questions, they will remain for future
## generations to demonstrate their integrity by addressing them, and
## perhaps as a consequence, to change their views. Any of these
## questions may be quoted, and/or used in examination questions,
## preferably with acknowledgement of their source. I will answer all
## correspondence while I am physically capable of doing so.
#So what's your point?

See the response posted in Usenet

Dr Harold Hillman
[Back to top]

FROM Ian Musgrave Ph.D, Prince Henry's Institute of Medical Research. Monash. Australia
Newsgroups: bionet.cellbiol,bionet.neuroscience
Subject: Re: a selection of unanswered questions
Date: Tue, 4 Jun 1996
[Back to top]
#G'Day All
##[NOTE: 1 June 1996: since posting last weekend, five replies were
##received, two (Ladasky of Stanford, Alex of ?Western Australia) not
##taking the questions seriously; one from Paul S Brookes of Cambridge,
##long but with elementary replies; one short reply from Van Frank of
##?MHAFC looking at a couple of questions; and a more considered reply
##from Cornelius Krasel in Wuerzburg.
#[snip]
#You may be having trouble getting replies as most of your list of 48 questions are already answered in the open literature. Few poeple will spend their time trying to answer a grab bag of questions whose answers can be found with a little effort on the part of the asker.
##UNANSWERED QUESTIONS IN BIOLOGY.
#As I said, most of these have been answered. If you have problems with the existing answers, you should address them. To avoid band width wastage. I will briefly address a very few of them as a general illustration.
*[homily snipped]
## The following questions have never been answered satisfactorily,
##several of them never at all:-
#Then indicate which have been never answered and which have not been
#satisfactorily annswered. To my knowledge, most have infact been answered
#quite nicely indeed.

Which ones have been answered 'quite nicely'? I maintain *none*.

#[large snip]
##Question 18: How can carriers assist the passage of ions, aminoacids,
## etc. across membrane, when the combination must be bigger than the
## substance carried?
#Why on earth do you believe this to be a problem? There are several good
#reviews on structure and function of carriers (most relating to proton
#transporters). Look up a few issues of Trends in Biochemical Sciences, which
#should be easily available. If you have problems with these theories, what
#are they?

A carrier would make a molecule or ion bigger, and therefore would inhibit
passage across membrane.

##Question 19: Why have few or no carriers been isolated?
#Several proton transporters, five glucose transporters, two monoamine
#transporters (at least). These are the ones I know of, and I deliberately
#avoid the transporter literature. How many do you want?

These are hypotheses.

##Question 26: Why is it assumed that the receptors for transmitters,
## hormones, messengers, antibodies, drugs and toxins are on the
## surface of the cell membrane?
#It is not assumed, it has been experimentally demonstrated using several
#different techniques (differental centrifugation, autoradiography, antibody
#localization etc.) for some classes of receptor (heptahelicals, growth factor
#receptors etc.) and also experimentally demonstrated that others are
#intracellular (retinoid receptors, steroid receptors). Do you have specific
#reasons why these techniques, especially when used in combination, should be
#misleading us.

Are you assuming that diffusion and therefore possible relocation can not occur during centrifugation, homogenisation, fixation, embedding etc?

##Question 27: How valid is the use of agonists, antagonists and
## ligands to detect receptors, instead of the transmitters, hormones, antigens, drugs and toxins themselves?
#Very. There is extensive literature validating these techniques, and any
#number of basic "methods in.." books outlining the theories and results.
#Receptors are usually "detected" using a number of ligands, (these ligands
#usually include the appropriate native hormones/neurotransmitters as well), to
#be certain of their identity. Many synthetic agonists/antagonists have been
#specificallly synthesized to be specific for a particular receptor, so why do
#you see a problem using them to "detect" these receptors in new tissues,gene
#expression experiments etc.? Goodman and Gillman is a good place to start,
#followed by T. Kennakins publictions (see especially reviews of physiology and
#pharmacology)

Why not study ach receptors using ach, adrenaline, noradrenaline, gaba, glutamate, etc, when the ligands are *not* the transmitters themselves?

#[rest of article snipped]
#I could reply in detail to all of the above, but the information is
#already available in the open literature (and time is too short). There are
#many more examples in your list where the answers are already known, so it is
#hard to take the entire list seriously. If you have specific problems with the
#methods/techniques used to give the answers mentioned above, what are they?
#Questioning our assumptions at regular intervals is healthy for science. But
#posting a huge list of "no one has answered this" questions, when they have
#indeed been answered is not going to start a useful debate.

I am sorry, Dr Musgrave, that I have not put the four books I published on Internet, and more particularly, that you have not read them. For example, subcellular fractionation implies *22* inescapable assumptions (Hillman, 1971)

#Cheers! Ian
[Back to top]

Paul Page Received: from anx1p2.wi.centuryinter.net by students.uwlax.edu
Thu, 6 Jun 96
Subject: Re: * UNANSWERED QUESTIONS: RESPONSE *
[Back to top]
#Dr. Hillmanm,
# I would like to debate a bit on some of these proposed ideas you put forth.
##
## (a) everyone agrees that intracellular movements can be seen by
## low power light microscopy in living cells, yet most people also
## believe that there is a cytoskeleton, which would not permit such
## movements;
# Why would you say that the presence of a cyotskeleton would prevent
#intracellular movements? The presence of a cytoskeleton would more likely
#enhance the intracellular movement especially when considering that the
#cytoskeleton is most likely not a static entity, but rather a dynamic
#structure.
##
## (b) That the following structures do not exist in the living
## cells: endoplasmic reticula, Golgi bodies, lysosomes, nuclear pores,
## mitochondrial cristae, the cytoskeleton, actin filaments and synaptic knobs,
## either because they would not permit the evident intra-cellular movements,
## or because they disobey the laws of solid
## geometry. Transmembrane molecules and receptors can not be seen on
## the cell membranes by transmission electron microscopy, although
## sequencing shows them to be 2-3 times the diameter of the cell
## membrane, which *can* be seen by electron microscopy;
##
# How can you say that things simply by existing in the cell would prevent
#intracellular movement or the laws of solid geometry? Perhaps if you clarified
#the argument that you make for each example it would be easier to follow you
#logic. In addition, why do you consider the laws of solid geometry for these
#organelles which are not static, and are not solid? Wouldn't it be more
#appropriate to look at these items using a fluid dynamics approach?
#Paul.

Dear Paul Page,
The particles which move are 10-100 x the diameter of the space between the elements of the cytoskeleton. Please see Hillman and Sartory (1980) 'The Living Cell', Packard.
Can fish swim freely through a fishing net? Solid geometry applies because the cytoskeleton is considered to be a structure. Please address my questions, or write to me for references to any of my assertions.
Harold Hillman.
[Back to top]

Anthony J. Pelletier, Ph.D.
The Scripps Research Institute, La Jolla, CA
Date: Fri, 07 Jun 1996
Subject: Re: * UNANSWERED QUESTIONS: RESPONSE *
Newsgroups: bionet.cellbiol,bionet.neuroscience
[Back to top]
## 1. Discourtesy, assumptions of ignorance, and emotive remarks are no
## substitute for measured argument and evidence.
#Agreed.
## Each of these questions highlights a contradiction *within* current views; for example,
## (a) everyone agrees that intracellular movements can be seen by
## low power light microscopy in living cells, yet most people also
## believe that there is a cytoskeleton, which would not permit such
## movements;
#I don't understand your conlusion. Why do you conclude that the
#cytoskeleton would interfere with intracellular movement? On the one
#hand, you say below that the cytoskeleton does not exist. But, you seem
#pretty sure of its properties. Is it necessarily so that a network of
#fibers is refractory to intracellular movement? Personally, I can imagine
#many mechanisms wherein such a network, given the appropriate motor
#system, could facilitate intracellular movement. So it is in no way
#obvious that the presence of intracellular movement is inconsistent with a
#cytoskeleton. It is inconsistent if and only if the cytoskeleton is a
#rigid network whose effective pore size is smaller than the molecules that
#must diffuse through it. I've seen no evidence of this, and in fact much
#evidence to the contrary.
#As a somewhat strained analogy, particles the size of electrons have
#little problem passing through apparently solid material such as wood.
#In fact, a quick calculation of diffusion coefficients tells you that a
#molecule the size of a protein could not diffuse from one side of a cell
#to another in any time reasonable in comparison to the cells life.
#Moreover, movement must be directional. This would seem to make necessary
#the existence of some structure along which molecules can be transported
#actively.
#Then, there are the data to consider. Just to pick one, how do you
#explain the incorporation of fluorescently labeled tubulin into what
#appear to be fillements in the cell? what would you call these
#microtubules if not a cytoskeleton?
#It would take far too long to go into even a small fraction of the data.
#However, let's take my favorite molecules, Integrins, which we think
#violate two of your rules in that they are transmembrane proteins and that
#they associate with elements of the cytoskeleton. Biochemistry,
#immunofluorescence and genetic evidence all support the idea that these
#proteins have a portion outside the membrane and one inside, that the
#region inside assciates with proteins we consider part of the
#cytoskeleton, and that these associations are necessary to maintain cell
#shape. Are you saying all these data must be flawed because they don't
#fit with your first-principles argument?

Because according to cytologists the cytoplasm has a thick skein of microtubules, actin, spectrin, tubulin, vimantin, etc. These are found in dead, stained dehydrated tissue. Dried out salt solution contains similar strands as do snow flakes but liquid H2O does not have a network. The moving particles are 10-100 x the diameters of the space between the fibres. Please see Hillman and Sartory (1980) 'The Living Cell'. The cytoskeleton is a precipitate of the cytoplasm resulting from dehydration during preparation.

## (b) most people believe in the Second Law of Thermodynamics, yet
## in subcellular fractionation they change the entropy of their systems
## (homogenise and centrifuge), and assume that this does not change the
## free energy, which drives all the biochemical reactions they are
## studying, and at the same time, they have refused for fifty years to
## do the necessary control experiments to find out by how much;
#No biochemist worth a damn believes this. We all are accutely aware that
#removing the enzymes from their environment can grossly alter the
#reactions, for many reasons including but not limitted to the ones you
#cite. That's why we use all these hand-waving terms like "local high
#concentration" to account for an an entropy term cannot quantify.
#as for the "proper controls," we do try.

The use of s/c fractionation to find any enzyme activity *and its s/c distribution* implies 25 separate assumptions, several contrary to laws of thermodynamics. Unless these assumptions are shown to be warranted or their effects statistically insignificant, any results must be uncertain. Hillman (1991) 'The Case for New Paradigms', Mellen Press, Lewiston.

## 3. I have always suggested alternative and testable hypotheses, not
## open to the criticisms of current views, for example, how to localise biochemical activities without disruption of tissue, the structure of the living cell, the cellular structure of the central
## nervous system, the passage of excitability from one neuron to
## another, etc, etc.
#Many of us try to do this. What you have said above does not give me
#great confidence in your methods. However, I would love to read how, for
#example, we could better examine the biochemistry even something "simple"
#like glycolysis in the cell.

By the use of non-destructive techniques, which I have listed in H.H. (1991)

## 4. The fundamental questions I must raise with the Internet
## cytologists are:
## 'By what criteria are questions improper?'
#Your questions are not improper. They may assume facts not in evidence,
#or disregard many things for which there is evidence, but they are not
#improper.
#In science, the only time a question is improper (as a scientific
#question) is when the person asking it does not accept the possibility of
#being wrong.
#I hope you acknowledge at least the formal possibility that you may be wrong?

In all my publications I have examined my own assumptions.

## 'Do all academics have a duty to address the difficulties and
## apparent contradictions of their own views?'
#sure. That is a truism

I am pleased you agree.

## 'Do they believe that progress can be made without examining
## their own views?'
#no, of course not. not any good ones. Do you examine the possibility
#that your views are wrong?

Yes, always.

## 'Would they disagree that a good academic should answer all
## these questions in the affirmative?'
#well...your first question was not framed in a yes/no format, the answer
#to the second is "yes" and the answer to the third is "no."
#As for re-checking others findings, there is some limit,practically, in
#what I can do. I certainly do not take their conclusions without critical
#examination of the data. But I cannot repeat all their experiments.
#There are many published papers I have read whose conclusions I think are
#not supported by the data. I consider these as I design and interpret my
#experiments.
#One ground rule I will have, if we are to have any discussion:
#Disagreeing with you is not the same as failing to examine data critically.
#Ok, so, i can examine data, look at both side of the argument, and still
#think you are wrong, if that turns out to be the case, and my scientific
#integrity is still intact.
#--
#Anthony J. Pelletier, Ph.D.

Dear Dr Pelletier,
You are entitled to believe that I am wrong, if you have read my publications. I should be pleased to supply you with reprints on particular points. I would be more pleased if you would answer the 45 questions.

##Because according to cytologists the cytoplasm has a thick skein of
##microtubules, actin, spectrin, tubulin, vimantin, etc. These are found
##in dead, stained dehydrated tissue. Dried out salt solution contains
##similar strands as do snow flakes but liquid H2O does not have a
##network. The moving particles are 10-100 x the diameters of the space
##between the fibres. Please see Hillman and Sartory (1980) 'The Living
##Cell'. The cytoskeleton is a precipitate of the cytoplasm resulting
##from dehydration during preparation.
#I'll have to read your work to find out where you get the "10-100X" figure.
# But real-time measurment of "microdiffusion" of particles in living cells
#suggests that some of the large particles are in fact constrained for short
#periods within actin "cages" and move from place to place only when the
#actin skeleton rearranges locally to allow it. Treatment with actin
#de-polymerizing agents removes the "cage" effect.

Do you know of any electron micrographs showing mitochondria attached to any part of the cytoskeleton in such a way?

#Below, you ask me to answer your 45 questions. Why won't you answer one of
#mine? I asked you how you explain the visualization of microtubules in
#*living* cells.
# I understand your concern over fixation artifacts...we all are concerned
#with them. This is why alternate methods have been employed.
# So, one can micro-inject fluorescent tubulin, wait a while for the cell to
#recover, do all the correct controls to show that, following the
#microinjection, the cells continue to live, grow, divide etc, and
#visualize, directly, the microtubules in the living cell. Moreover, since
#the fluorescent dye photobleaches, you can bleach out the dye in a defined
#region and follow the incorporation of new tubulin into the network.
#Living cell...not dehydration artifact. Explain please?

I should be grateful for references to this. However, microtubules which can be resolved by light microscopy (200-250 nm) are 10x diameter of those seen by electrons (<25 nm) therefore could not be the same.

#Also, consider the mutations in the genes encoding the proteins of the
#cytoskeleton. The effects on cell morphology are as predicted from the
#model. How does your model account for the effects of mutations to
#cytoskeletal proteins on cell shape, motility etc. Indeed, how does your
#model account for cell shape and motility at all?
##Dear Dr Pelletier,
##You are entitled to believe that I am wrong, if you have read my
##publications. I should be pleased to supply you with reprints on
##particular points.
#Absolutely send me any reprints you feel are appropriate.
##I would be more pleased if you would answer the
##45 questions.
#Not to be rude, but you have not yet asked a question I find compelling.
#Perhaps your reprints will help with that. And, in the cases where I
#attempted to address you questions with data, you ignored them.

You do not have to find them compelling. They are reasonable questions to which you - as a teacher - should have answers.

Dr. Hillman,
I have recieved your reprints, thankyou.
I have a grant deadline I am facing at the moment, and likely will not read them for a week or two. But I wanted to tell you that I plan to do so at the first opportunity. Also, I hope this is a reasonable e-mail address for you. I notice that the account on which you are posting has changed.
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From: Jorg Kirberg
Subject: Re: *unanswered questions H Hillman
Date: Wed, 03 Jul 1996
Organization: nki. [A Dutch Institute?]
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# Harold Hillman's Response to replies to 47 'Unanswered Questions
# in Biology'. 2 July 1996# 5. No reason has been put forward why auto-immune diseases do not
# reject the joints, nerves, brains, etc as incompatible blood or trans-
# plants would be rejected if transfused or transplanted.Dear Harold,
well, maybe you could think about the possibility that an autoimmune reaction is not like a transplant rejection - and why should it ? (however, as stated before - and I don't know why you ignore this except when assuming you want to be ignorant - in young onset diabetes (IDDM) the immune system actually destroys those cells that secrete insulin. You don't see rejection here ?)
There are tremendous examples where the immune system's response ends up in some sort of chronic inflammation. And it might be so simple that (some forms of) autoimmunity are a reflection of such a form of immunity that inadequately is directed at self-constituents.
In any case, how can you address such a problem in such a simplyfied way when actually nobody kows why autoimmunity occurs ? The dilemma is one still has to find out what autoimmunity is - and it is quite clear that it doesn't reflect a normal infection that is cured (whereas transplants that are rejected apparently follow such normal rules).
jorg kirberg@nki.nl
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