In article <4op5c1$190$1 at mhadf.production.compuserve.com>, Margaret Fowler
<101722.35 at CompuServe.COM> wrote:
[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.
> Question 2: How does one know that the disruptive procedure does not
> change the biochemistry of the fraction significantly?
One doesn't know this a priori. It must be established by experiments to
validate the procedures.
> 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?
Who is using subcellular or cell-free systems which have not been validated?
> 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?
> 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.
It's certainly a good place to start.
> 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.
> 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.
> Question 8: What is the evidence that the microsomal fraction con-
> sists of cell membranes and endoplasmic reticulum?
Examination of the biochemical constituents of the fraction and
electronmicroscopic examination.
> 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?
> Question 101: Can a particle and a vacuole both be lysosomes?
A particle of what?
> Question 11: Can one calibrate substances originating from tissues
> using pure solutions in simple salines of approximately the same
> concentrations?
Sometimes.
> 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.
> 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
> 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?
No.
> Question 16: Can one know the thickness in life of any biological
> membrane?
Yes.
> 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?
What?
> Question 19: Why have few or no carriers been isolated?
Lots of them have.
> Question 20: What is transport?
The movement of substances across membranes by passive, facilitated and
active processes.
> 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?
Because their sizes are small relative to the resolution of typical EM.
> 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?
Because the lamellae are equal distances apart.
> 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?
> 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?
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.
> 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?
In most cases, very valid.
> 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.
> 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.
> Question 30: Does the chemical theory of synaptic transmission
> contain unprovable and unproved hypotheses?
No.
> Question 31: Why is it assumed that evidence derived from experi-
> ments 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.
> 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.
> 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?
Because the passage is not by simple diffusion.
> 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?
Glass is a fluid, too. How do your windows 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?
No.
> 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.
> 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.
> 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.
> Question 40: Does not the use of the term neuroglia imply that the
> authors can not distinguish between astrocytes, oligodendrocytes,
> and microglia?
No.
> 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.
> 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?
No.
> Question 43: Why is there no common agreement about the staining
> procedures, which are supposed to identify astrocytes, oligo-
> dendrocytes and microglia histologically?
Is this true?
> Question 44: Why is it necessary to use tissue cultures of the
> alleged cell types to identify them and their markers?
Is it?
> 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.
> 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?
