* unanswered.. debate with C Krasel

Margaret Fowler 101722.35 at CompuServe.COM
Thu Jun 27 16:28:55 EST 1996

Re: Unanswered Questions
Cornelius Krasel,INTERNET:krasel at wpxx02.toxi.uni-wuerzburg.de
Newsgroups: bionet.cellbiol
Subject: Re: Unanswered Questions
To: 101722.35 at CompuServe.COM
Date: Wed, 19 Jun 1996 19:47:36 +0200 (MET DST)
Organization: CC University of Hohenheim (not responsible for contents)

Sorry for the late reply.

>>>Harold Hillman (101722.35 at CompuServe.COM) wrote:
>>>> 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?

>>> 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 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 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.)

Please let me have references to AFAIK, firstly what it is an abbreviation
for. 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 pout together.

>>>> 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?

>>> 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

>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) see by transmission electron

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, hor-
>>>>     mones, antigens, drugs and toxins themselves?

>>> You lost me here. What's the difference between certain ligands, agonists
>>> and transmitters?

>> Why not use ach, adrenaline, yaba 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 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?

>>> 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.

Cornelius Krasel.

>/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
>/* D-97078 Wuerzburg, Germany   email: phak004 at rzbox.uni-wuerzburg.de  SP3 */
>/* "Science is the game we play with God to find out what His rules are."  */

Dr Krasel, I will send you a copy of one of our books, 'The Living Cell'
as it is out of print.

Harold Hillman.

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