a selection of unanswered questions

Margaret Fowler 101722.35 at CompuServe.COM
Sun Jun 2 18:05:01 EST 1996

[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.
   I'm only an intermediary in this and am myself awaiting Dr Hillman's
own responses; my profuse apologies for this exasperating delay.
   This posting was first made to cellular biology in Usenet; since
the posting also refers to neurobiology, this site, neuroscience, seems
suitable for other people who'd like to answer/ comment/ or add their
own doubts about the fundamentals of this subject.]


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 acad-
emic who is not prepared to discuss or correspond with other interest-
ed 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 examin-
ation 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:-

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 centrifugat-
    ion 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 ident-
    ified 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 the cytoplasmic
    viscosity is low in life, 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 con-
    sists 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 101:  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

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 tis-
    sues, cells and organelles, than classical transmission electron

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

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 character-
    ised, 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

Question 25: Since the myelin sheath is believed to consist of a
    scroll of membranes, and membranes appear darker by light micro-
    scopy 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, hor-
    mones, 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 experi-
    ments on neuromuscular junctions is relevant to transmission
    in the central nervous system?

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?

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?

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

Question 35: If the cell membrane is fluid mechanically, how can cells
    maintain their integrity?

Question 36:  In immunocytochemistry, is it assumed that the fixatives,
    dehydrating reagents, washings, and primary and secondary anti-
    bodies, do not change the reaction of the antibody to the antigen
    believed to be in a particular cell or part of a cell?

Question 37:  Is it reasonable to believe that processes or dendrites
    contain different antigens from the cell bodies from which they

Question 38: Under what conditions can tissue cultures be used in the
    study of the tissues from which they originated?

Question 39:  Is it warrantable to assume that growth of tissues in
    culture does not change their morphology, biochemistry, or

Question 40:  Does not the use of the term neuroglia imply that the
    authors can not distinguish between astrocytes, oligodendrocytes,
    and microglia?

Question 41:  Why are the individual types of neuroglial cells so
    rarely seen by light microscopy of healthy central nervous systems?

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 anti-
    bodies to mark them specifically must first identify them by
    these criteria?

Question 43:  Why is there no common agreement about the staining
    procedures, which are supposed to identify astrocytes, oligo-
    dendrocytes and microglia histologically?

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

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?

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.
       Unity Laboratory of Applied Neurobiology,
       76 Epsom Road,
       GU1 2BX
       Fax:  UK 1483 31110
       Telephone:  UK 1483 568332

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