Harold Hillman's Response to replies to 47 'Unanswered Questions
in Biology'. 2 July 1996
I have received lengthy replies to several of the questions, to
which I have responded individually. The replies have usually been
polite and proper - but, alas, not always. Although some respondents
have implied that my questions have been pointless, improper or
ignorant, most have accepted them in good faith, and replied to
several of them, if only briefly. Lengthy replies have come from
Paul Brookes (Cambridge UK), Greg Fraley (Washington State Coll),
Richard Van Frank (MHAFC), Warren Gallin (Alberta), Cornelius Krasel
(Hohenheim), Kevin McKenna (North West University), Ian Musgrave
(Monash) and Anthony Pelletier (Scripps).
Most of those who have responded have reacted in the following ways
to the questions.
1. They believe that all those who carry out subcellular fraction-
ation or accept findings by this technique can either continue to
ignore the facts that purification, separation, homogenisation,
centrifugation and extraction change entropy, therefore free energy,
therefore the enzyme activity of any subcellular fraction. The only
way one can assess the degree of change is to do the *control*
experiments, which have never been done. (Hillman, H. Experientia 51,
no 7, p 757, 1995). The use of large volumes of unnatural reagents,
including ethanol, acetone, trichloracetic acid, phenol etc, and
energetic procedures such as homogenisation, centrifugation, freezing
etc will extract any substances soluble in the reagents, and generate
heat, respectively. The latter will increase diffusion of any soluble
material in the cells. Therefore, the mitochondria or nuclei at the end
of a procedure *will not* have the same properties quantitatively as
they had in the original intact organism. Therefore, it is absolutely
essential to do control experiments and to avoid disruptive techniques.
There are *many* non-disruptive techniques, mostly developed and used
before the 1940s. Paying lip-service to thermodynamics *is no substitute*
for measuring or calculating what the effects of one's analytical
procedures are on the system one is studying.
2. The laws of geometry must be applicable in biology, and at any
time the cell membrane, nuclear membrane, cristae, thylakoids, synaptic
clefts, endoplasmic reticulum, Golgi body, synaptic vesicles, should be
seen in all possible orientations in a whole cell, since the tissue does
not know from which direction it is going to be cut. Any explanation as
to why cell membranes, synaptic clefts, nuclear pores are not seen in
various orientations, is an admission that they are not, and an unadmitted
attempt to defy solid geometry.
3. No explanation has been given why one should seek receptors for
acetylcholine, adrenaline, gaba, etc by using ligands instead of the
transmitters themselves. *Obviously*, the ligands are chemically different
from the transmitters.
4. The vesicle hypothesis for chemical transmission contains so many
unproved and unprovable assumptions which I have listed elsewhere (H
Hillman, 1991, Physiol Chem Phy & Med NMR 23, 177-198) that I think I
have brought evidence that it is no longer tenable. If anyone would like
to take issue with this, please address the arguments I have put in the
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.
6. Although it is widely believed that intracellular movements *can*
occur in the presence of cytoskeletal elements, for example, by actin,
tubulin pulling the moving structures, this is impossible because
(i) the viscosity of cytoplasm in life is too low;
(ii) if one looks at light or electron micrographs of cells -
which have been fixed and dehydrated - one sees what are believed to be
extensive networks of microtubules, microfilaments, microtrabeculae,
actin, spectrin, etc, it is quite obvious that *there would be no room*
for mitochondria to move around, nor for the nuclei to rotate.
(iii) no one has shown how fibres attached to mitochondria could
move anything, since if they were attached at one edge, they would have
to detach to move the mitochondrion, and could only attach again if the
mitochondria went back to its previous position. The beautiful demon-
strations by Sheetz and others show vesicles visible by light microscopy
in living tissue cultures. Micromotors move vesicles along the fibres -
*they do not pull* mitochondria;
(iv) all the elements of the 'cytoskeleton' appear in the plane of
section virtually always, but, of course, they should appear normal to
that plane equally often;
(v) Brownian movement, diffusion, convectional movements appear in
all small particles in fluids of low viscosity, e.g. iron filings, pollen,
ground glass in water, *without* any network to pull them;
(vi) those who believe that movement of particles is compatible with
fine networks have not compared the minimum width of, say, mitochondria
(0.5 mu m) with the distance of the microtubules or microtrabeculae from
each other, (less than one tenth of this). People have suggested that
the mitochondria could swim through the cytoskeleton, because they have
not realised the relative dimensions of the moving particles and the
elements of cytoskeleton, or have not realised that moving particles
would have to secrete enzymes to dissolve the networks *extremely*
rapidly, and also 'know' in which direction they were going to break
down the cytoskeletal elements in front. Of course, glass powder indulg-
ing in Brownian movement in water, does not have such mechanisms.
In brief, these are the answers to some of the questions I have
raised, but complete evidence appears in my references as summaries,
and in full-length papers, which I will supply (if I have any left) or
will send you references (if I have not). I shall, of course, be pleased
to enter into correspondence with anyone who wishes to discuss these
questions seriously. For the benefit of those who have *not* seen them,
the questions are reposted below.
Cytology will not advance unless it attempts to resolve contradictions
in current belief.
Dr Harold Hillman.
[47 questions reposted]
UNANSWERED QUESTIONS IN BIOLOGY.
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
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 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 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 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
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
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: 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 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 36: Is it reasonable to believe that processes or dendrites
contain different antigens from the cell bodies from which they
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
Question 39: Does not the use of the term neuroglia imply that the
authors can not distinguish between astrocytes, oligodendrocytes,
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 anti-
bodies to mark them specifically must first identify them by
Question 42: Why is there no common agreement about the staining
procedures, which are supposed to identify astrocytes, oligo-
dendrocytes 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?
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,
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
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.