Moshe Wolman

Polarized light microscopy in the study of the molecular structure of collagen and reticulin

SummaryAlthough collagen structure has been studied by polarized light microscopy since the early 19th century and continued since, modern studies and reviews failed to correlate the conclusions based on data obtained by the techniques with those of polarized light microscopy. Collagen I is intensely positively birefringent in respect to length of the fibres; the positive intrinsic birefringence indicates a quasi-crystalline alignment parallel to the fibre and molecule axis of the amino acid residues of the polypeptide chains. This would not have been compatible with a helical structure but has been achieved by similar tilt angles and opposite directions of the coiling and supercoiling.Birefringence characteristics of collagen are also affected by chemical treatments, extractions and staining procedures. Attachment of chemical groups to the anionic charges present on the surface of collagen molecules results in increased positive birefringence in the case of bipolar molecules attached to two or more anionic residues. Unipolar attachment to the same groups, or to the cationic groups of the associated proteoglycans, as well as sulfation or acetylation of hydroxyls of the protein and/or the carbohydrate, reduced or reversed the sign of birefringence. Increased birefringence caused by stretching cannot be due to intramolecular events and is caused by intermolecular changes. The same applies to changes in collagen during aging.Reticulin is a group of different substances which mostly contain collagen III. The pliability and deformability of this collagen is related to its weakly negative birefringence due to large side chains and presence of different and greater amounts of interstitial proteoglycans and other molecules. The so-called reticulin of healing wounds differs in its constitution from other reticulins but is also rich in intermolecular carbohydrate components.

Correlative histochemical and morphological study on the maturation of sensory ganglion cells in the rat

SummaryIn neonatal rats the sensory ganglion cells are uniform in size and in their stainability with hematoxylin and eosin. At this stage the cells differ, however, in the intensity of staining for RNA and for various enzyme activities. With maturation the ganglion cells differentiate into “light” (mostly large) cells, and “dark” (mostly small) cells. The differentiation is accompanied by changes in intensity of various enzyme activities. In sections stained for acid phosphatases and acetylcholine esterase, maturation was associated with a higher activity in the small than in the large cells, whereas with thiamine pyrophosphatase it was associated with a higher activity in the large than in the small neurones. With non-specific cholinesterase, maturation of all cells was accompanied by loss of activity in perikarya and increased activity in axons and satellite cells. With monoamine oxidase, the changes during maturation differed in the trigeminal from the spinal ganglion cells.The findings indicate that the difference between small and large cells might have a functional significance, the nature of which is discussed.

Wolman Disease and Its Treatment

and hepatosplenomegaly. Radiographic examination reveals calcification of both adrenal glands. The calcification appears to delineate the outline of both glands, and this sign appears to be pathognomonic for the disease, and for its less severe counterpart, cholesterol ester storage disease (CESD). The only test needed to corroborate the diagnosis is a biochemical or histochemical demonstration

Structure of the myelin sheath as a function of concentration of ions

Abstract Homogenization of cerebral white matter or of peripheral nerve with 1 M NaCl caused the formation of micellar complexes bounded on the outside by a hydrophilic layer (“oil-in-water”). Homogenization of such tissues in 0.5 M CaCl 2 caused the formation of lipophilic complexes. Histochemical experiments yielded comparable results. The implications of these observations for the study of the structure of myelin are discussed. Formation of oil-in-water and water-in-oil systems were associated respectively with the formation of aqueous channels or of oily pillars across the lamellae of myelin or across cell membrane layers. The possible formation of such channels in vivo as a result of localized changes in lipophilic-hydrophilic ratio may be used to explain a number of biological phenomena.

A Fluorescent Histochemical Procedure for Gamma-Aminobutyric Acid*

SummaryGamma aminobutyric acid (GABA) was selectively demonstrated in slide tests by the production of a fluorescent compound with ninhydrin in a non-aqueous medium (octanol). Thirty other related pure compounds either failed to yield a fluorescent product or produced distinguishable fluorescent products. The reaction was tested on sections of several organs of normal mice, mice with experimentally increased GABA levels and mice injected topically with GABA.In normal mice and in animals injected with AOAA intense GABA fluorescence was found in the brain in groups of cells in the cerebellum, in the hippocampus, and in some mesencephalic and hypothalamic nuclei. In most places flourescent lines surrounded perikarya. Fluorescence of lesser intensity was found around some cortical cells. Intense fluorescence, more marked after AOAA injection, but partly “masked” by autofluorescence, was found in erythrocytes, leptomeninges, choroid plexus and retina.Outside the brain GABA-fluorescence was found in some cells of the adrenal medulla and in isolated cells of sensory ganglia. After intraperitoneal GABA injection GABA fluorescence was noted in Kupffer cells, in renal tubular cells and in the adrenal.Topical injections of GABA resulted in non-specific uptake by glial and also neuronal cells of the brain and presumably in the retina.

HISTOCHEMISTRY OF DEMYELINATION AND MYELINATION

The myelin sheaths are tubular structures which surround axons in the central and peripheral nervous system. It has been known for many years from polarized light studies (26, 36, 46) that myelin has a concentrical lamellar structure in which the lipid molecules are arranged radially and the protein components at right angle to the lipids and parallel to the surface. Electron microscopic studies (25, 44) have shown the reason and meaning of the lameilar structure of myelin. The sheaths were shown to consist of plasma membranes of satellite cells (Schwann cells in the peripheral and oligocytes in the central nervous system) enveloping and surrounding a number of times the axon in a spiral fashion like a scroll rolled around a rod. For the purpose of the following discussion it is important to note that the periodicity seen in electron micrographs and in x-ray diffraction studies (20) is given by two distinct bands or lines. The one, the major dense band, is due to coalescence of two faces of the plasma membrane facing the cytoplasm and results from growth in length of the cell process associated, so to say, with squeezing out of the cytoplasm and adhesion of the two opposing surfaces of the plasma membrane. The other, the intraperiod or intermediate band, is the result of coalescence of two faces of the plasma membrane facing the extracellular space, also here, as if the extracellular substance was squeezed out from between the two leaves. Histochemical, histophysical and biochemical studies have shown that the myelin sheath is a well ordered semicrystahline structure which contains various proteins, lipids and a hexosamine-containing mucopolysaccharide.

Aging of wound healing in an experimental model in mice.

Healing of incised wounds in the backs of mice was studied to establish criteria that would allow the timing of stages during cutaneous wound healing. Starting from day 4 after the incisions were made, three mice were killed every 3 days until day 40. Sections from wounds were stained with hematoxylin-eosin, a reticulin procedure, and picrosirius red. Unstained sections were used for blue autofluorescence. The significance of the findings from the ultraviolet study was limited, but the picrosirius red-polarized light procedure was found to be useful for estimating the age of the repair process as polarization colors of greater wavelengths progressively increased with the age of the lesion.

Histochemical study of the nature of the asymmetry of cell membranes.

SummaryThe effect of pretreatment with M/1 KCNS on the histochemical demonstration of 19 different enzymatic activities was studied in cryostat sections. Reversal by KCl of the KCNS inhibitory effect which had been obtained with most enzymes was investigated and the inhibition and restoration of enzyme activities were compared with the effects of M/2 CaCl2 and of the sequence CaCl2 followed by KCl.With most enzymes the CNS ion had a similar effect to the Ca ion and in some instances the effects were similarly reversible, whereas with some enzymes reversal of the inhibition differed quantitatively after KCNS and CaCl2 inhibition.The findings are considered to indicate that: 1. the cation-sensitive and the anion-sensitive components of membranes are situated on the opposing sides of membranes; 2. almost all the enzymes studied histochemically in the present work are situated on the cation-sensitive surface of membranes; 3. the various cell membranes differ in their reaction to reversal of inhibition by ions and presumably, therefore, in constitution, but the nature of the difference is not clear.

Myelin breakdown in vitro

Abstract Three patterns of myelin breakdown in vitro have been observed and studied. In the first type the sheath appeared to increase in length with the formation of invaginations and evaginations and subsequent separation of granules and droplets. This pattern, termed the “granular change”, was associated with changes in pH of the milieu, changes in the Ca2+:Na+ ratio in the medium and with other changes known to affect the hydrophilic-lipophilic ratio of lipoprotein membranes. The second pattern of breakdown in which the lamellar structure of myelin became apparent, was associated with hypotonicity of the medium. In the third type of demyelination in vitro the pattern was similar to that observed in wallerian degeneration. The pathogenesis of this change is not clear, but some data point to the possibility that an enzyme, possibly a cathepsin, might be responsible for it.

The relation of various enzymes to cellular membranes

SummaryThe effect of pretreatment by M/1 NaCl or M/2 CaCl2 on 11 enzymatic activities was studied in cryostat sections. The various enzymes were found to differ in their-reactions to these ions. In addition, different enzymes varied in their sensitivity to various noxious agents after these two pretreatments.The results are interpreted in terms of phase reversals of emulsions that can be expected to form as the result of an interaction of membrane anionic lipids and ions, with formation of oil in water (o/w) systems with monovalent cations and water in oil (w/o) systems with divalent cations. This assumption leads to the following interpretation of the data: in most instances the sensitivity of enzymes which are probably situated in the hydrophilic layer in a o/w system was greater to noxious agents presumed to act in the aqueous layer; the sensitivity of those enzymes which are probably situated in the hydrophobic layer was greater to noxious agents which are presumed to act in the less hydrophilic layer when in a w/o system. Some discrepancies were noted and suggestions for explaining them were proposed.