An immunohistochemical and electron microscopic study of the peri-ureteric ganglia of the guinea-pig

Summary The neuropeptide- and catecholamine-synthesizing enzyme content and ultrastructure of the peri-ureteric ganglia of guinea-pigs were investigated. Small numbers of neuronal perikarya were present at frequent intervals forming ganglia close to, and along the entire length of, the ureter. Each of these ganglia was surrounded by a connective tissue capsule, and was located in the peri-ureteric connective tissues. Within each ganglion were typical nerve terminals and varicosities containing small, clear synaptic vesicles or synaptic vesicles with an electron-dense core, or a mixture of the two. In the ganglia, immunoreactivity to tyrosine hydroxylase, dopamine hydroxylase, neuropeptide tyrosine, or vasoactive intestinal peptide was present in neuronal perikarya; immunoreactivity to substance P or leucine enkephalin was present in nerve terminals and varicosities. Electron-microscopic immunogold studies indicated that there was no coexistence of substance P and enkephalin in the nerve terminals, unlike related ganglia in the pelvis of guinea-pigs.     

An electron microscopic study of the photochemical cross-linking of DNA in guinea pig epidermis by psoralen derivatives.

Albino guinea pigs were treated with psoralen derivatives plus 320--400 nm ultraviolet radiation, and DNA was extracted from their epidermis. The DNA was assayed for the presence of interstrand cross-links by standard denaturation-renaturation assays and by a new technique, electron microscopy of the DNA under totally denaturing conditions. The latter method allows individual cross-links to be directly observed and counted. When either 4,5',8-trimethylpsoralen or 8-methoxypsoralen was applied topically to the skin (8--20 microgram/cm2) or administered orally (10--12 mg/kg body weight), followed by exposure to 320--400 nm ultraviolet radiation, most of the epidermal DNA was found to contain a high frequency of cross-links. For example, oral or topical trimethylpsoralen treatment gave an average of one cross-link per 250 nucleotide pairs or about 3 . 10(5) cross-links per guinea pig chromosome. When the dose of either drug was decreased 20-fold to the level used in the clinical treatment of psoriasis, however, no cross-links coulld be detected in the epidermal DNA. The electron microscopic assay is sensitive enough that we can put an upper limit of 1 cross-link per 10(6) nucleotide pairs (80 cross-links per chromosome) for the low dose studies. The significance of these findings to the understanding of the effectiveness of psoralens in psoriasis therapy is discussed.     

Selective migration of terminally differentiating cells from the basal layer of cultured human epidermis

How terminally differentiating cells are selectively expelled from the basal layer of epidermis has been a source of interest and speculation for many years. The problem can now be studied in culture, using involucrin synthesis as an early marker of terminal differentiation in human keratinocytes. When keratinocytes are forced to grow as a monolayer by reducing the calcium ion concentration of the culture medium, they still begin to synthesize involucrin. Raising the level of calcium ions induces stratification, and cells that are synthesizing involucrin are selectively expelled from the basal layer. I have found that during calcium-induced stratification no new proteins or glycoproteins are synthesized, and the rate of cell division does not change. Movement of involucrin-positive cells out of the basal layer was found to be unaffected by cycloheximide, tunicamycin, or cytosine arabinoside. These results suggest that keratinocytes growing as a monolayer already have the necessary properties to determine their position when stratification is induced. Addition of calcium simply allows formation of desmosomes and other intimate cell contacts required for stratification. The properties of involucrin-positive cells that determine their suprabasal position include a reduced affinity for the culture substrate and preferential adhesion to other cells at the same stage of terminal differentiation. The molecular basis of these adhesive changes is discussed.     

An electron microscopic and histochemical study of the secretory cells inHydra viridis

Summary The fresh water coelenterateHydra viridis possesses a unique distribution of mucous and serous secretory cells in the gastrodermis. The mucous cells are found only in the hypostome, a region devoid of the serous zymogen cells. On the other hand, the zymogen cells are found extending from the tentacles to the peduncle. Histochemical stains indicated that the two hypostomal mucous cells, spumous and granular, secreted an acid mucopolysaccharide, and incorporated radiosulfate. The radiosulfate label was not sensitive to hyaluronidase digestion, but was removed by acid methanolysis. In contrast, the secretory product of the zymogen cell was rich in proteins and a PAS-positive moiety (unsulfated).The ultrastructure of these cells was correlated with their histochemical staining properties. It was demonstrated that glutaraldehyde preserved the ultrastructure of the secretory granules better than osmium, and also preserved more components within the granules. The mucous cell granules contained an electrolucent and an electron dense component. The cells were both PAS-positive and alcianophilic. After osmium fixation the dense component was lost and the cells were primarily alcianophilic. Osmium also failed to preserve the electron dense component in the zymogen cells.Observations of corresponding thick and thin sections showed a cell containing granules similar to the granules seen in mouse Paneth cells. The dense core was osmiophilic and the lighter halo was alcianophilic.These results lead us to conclude that the electrolucent filamentous component is an alcianophilic acid mucopolysaccharide and the dense granular component is probably a PAS-positive material.

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Zusammenfassung Der FrischwassercölenteratHydra viridis weist eine einzigartige Verteilung von mukösen und serösen sekretorischen Zellen in der Gasterodermis auf. Die mukösen Zellen finden sich nur im Hypostom, in welchem seröse Zymogenzellen fehlen. Die Zymogenzellen andereseits finden sich von den Tentaklen bis zum Pedunkulus. Histochemische Methoden zeigten, daß die zwei Typen hypostomaler muköser Zellen, d.h. spumöse und granuläre, ein saures Mukopolysaccharid ausscheiden und radioaktives Sulfat einbauen. Der Radiosulfat-Markierer war nicht sensitiv gegenüber Hyaluronidase, konnte aber entfernt werden mit saurem Methanol. Im Gegensatz dazu war das Produkt der Zymogenzellen reich an Proteinen und enthielt PAS-positives Material.Die Feinstruktur dieser Zellen war korreliert mit diesen histochemischen Befunden. Glutaraldehyd erhielt die Feinstruktur der Sekretgranula besser und fixierte mehr Komponenten als Osmium. Die Granula der mukösen Zellen enthielten elektronendichte und -durchsichtige Komponenten; diese Zellen färbten sich mit PAS und Alcyan. Nach Osmium-Fixierung war die elektronendichte Komponente abwesend und die Zellen waren hauptsächlich alcyanophil. Auch in den Zymogenzellen vermochte Osmium nicht, die elektronendichte Komponente zu erhalten. Beobachtungen an alternierenden dicken und dünnen Schnitten zeigten eine Zelle mit Körnern ähnlich den Granulen von Maus Paneth-Zellen. Das dichte Zentrum dieser Granula war osmiophil, der hellere Halo alcyanophil.Diese Resultate lassen uns schließen, daß die elektronen-durchsichtige filamentöse Komponente ein alcyanophiles Mukopolysaccharid ist; das dichte, zentrale Material ist wahrscheinlich PAS-positiv.

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This paper was prepared from a thesis submitted in partial fulfillment for the degree of Master of Arts.

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This work was supported by the National Institutes of Health grant no. GM-11218.

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I wish to thank Dr.Marcus Singer for permission to use the E. M. facilities in the Dept. of Anatomy, Case Western Reserve University, and Dr.Joseph A.Grasso for instruction in the techniques of electron microscopy and the use of his facilities.     

An electron microscopic study of the human infundibulum

Summary For a limited period during the oogenesis of Protopterus, blebs of the perinuclear cistern contain, in addition to other inclusions, a special kind of microtubular elements. Most of these blebs face parts of multiple nucleolar bodies that extend toward and make contact with the inner nuclear membrane. The microtubular lumen contains a finely dispersed material of moderate electron density which seems to be in contact with this nucleolar material.Aside from these intracisternal structures there are, within both the perinuclear cytoplasm and the nucleoplasm, similar microtubular arrays without apparent connection with the nuclear envelope. These are either enclosed by membranes derived from those of the envelope or unconfined, having escaped through breaks in their respective bounding membranes. Extracisternal tubules are presumed to have passed their period of putative functional activity and to be undergoing a process of regression and subsequent disintegration.Among possible roles attributable to the intracisternal microtubular apparatus are the following: (1) It may serve for the transport of special nucleolar components to the cytoplasm, possibly to be incorporated in the matrix of developing perinuclear mitochondria; (2) it may provide openings in the nuclear membranes for the direct passage of particulate elements between nucleus and cytoplasm; (3) it may be instrumental in the breakdown of parts of the nuclear envelope prior to its restitution during the subsequent phase of oogenesis.Supported by grants NB-00840 and NB-05219 from the U.S.P.H.S.     

An electron microscopic study of the ultrastructure of microbial cells in extreme biotopes in situ

The ultrastructure of microbial cells was studied in situ in natural biotopes by high-resolution transmission electron microscopy using the known methods of cryofractography, thin sectioning, and the negative staining of total cell specimens, as well as new methods of the low-temperature fractionation of microbial cells (providing for the recovery of cells from natural sources and their concentration), the preparation of micromonoliths, and aimed electron microscopy. Among the natural biotopes studied were permafrost ground and oil sludge. Most of the microorganisms found in the 1- to 3-million-year-old permafrost ground represented resting forms (spores, cysts, and cyst-like cells with specific organo-mineral envelopes). Oil sludge older than 35 years contained bacteria of atypical morphology and ultrastructure, including various resting forms and ultramicrobacteria. The data obtained is indicative of considerable promise of high-resolution electron microscopy in studying microbial communities in situ.     

An electron microscopic study of the ultrastructure of microbial cells in extreme biotopes in situ

The ultrastructure of microbial cells was studied in situ in natural biotopes by high-resolution transmission electron microscopy using the known methods of cryofractography, thin sectioning, and the negative staining of total cell specimens, as well as the new methods of the low-temperature fractionation of microbial cells (providing for the recovery of cells from natural sources and their concentration), the preparation of micromonoliths, and aimed electron microscopy. Among the natural biotopes studied were permafrost ground and oil sludge. Most of the microorganisms found in the 1- to 3-million-year-old permafrost ground were represented by resting forms (spores, cysts, and cystlike cells with specific organomineral envelopes). Oil sludge older than 35 years contained bacteria of atypical morphology and ultrastructure, including various resting forms and ultramicrobacteria. The data obtained is indicative of considerable promise of high-resolution electron microscopy for studying microbial communities in situ.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 832–840.Original Russian Text Copyright © 2004 by Dmitriev, Suzina, Barinova, Duda, Boronin.     

A light and electron microscopic study of GAT-1 in the monkey basal ganglia

The distribution of the GABA transporter GAT-1 was studied by immunocytochemistry and electron microscopy in the monkey basal ganglia. Dense staining was observed in the globus pallidus externa and interna, intermediate in the subthalamic nucleus, and substantia nigra, and light staining in the caudate nucleus and putamen. Staining was observed in axon terminals, but not cell bodies. Electron microscopy showed that the GAT-1 positive axon terminals formed symmetrical synapses, suggesting that they were the terminals of GABAergic neurons. Comparison of areas high in GAT-1 protein with that of GABA showed a good correlation between the density in neuropil staining for GAT-1, and that of GABA.     

An electron microscopic study of the human epidermal keratinocyte

Summary 1. The epidermis of the flexor surface of the upper arm of human subjects was studied with the electron microscope. 2. The cytoplasm of the keratinocytes in the basal layer contained many tonofilaments, ribosomes and other cell organelles. The tonofilaments were arranged singly or in loose bundles and many were attached to the inner membrane of the desmosomes. Along the basal border of the cells pinocytotic vesicles could be seen at different stages of development. 3. The keratinocytes in the stratum spinosum differed from those in the basal layer in two main ways: (a) The tonofilaments were grouped together into large compact bundles known as tonofibrils and it was possible to determine a definite beading or cross banding along the length of some of the filaments. (b) The cells were assuming a flattened shape. 4. The keratinocytes in the stratum granulosum possessed large numbers of irregularly shaped keratohyaline granules. The granules were strongly osmiophilic and were always situated on a meshwork of tonofibrils. The keratohyaline granules had no internal structure. The nuclei and mitochondria showed evidence of degeneration. 5. The keratinocytes in the stratum corneum were long and flattened. The cell walls showed increased electron density and were considerably thickened. The cytoplasm was filled with closely packed fibres separated by a small amount of lucent matrix. The fibres were grouped together in bundles running in different directions within the flattened squames. The fibres had along their entire length alternating areas of high and low electron density. The keratohyalin granules had disappeared and nothing remained of the nuclei or the organelles. In the deepest cells of this region the fibres were sometimes loosely packed leaving large irregular open spaces. This area corresponded to the stratum lucidum. In the most superficial layers of the stratum corneum the fibres appeared to be breaking down so that little remained within the keratinocyte except large lucent spaces. The desmosomes showed distinct structural changes. 6. An attempt was made to correlate the structural changes in the different epidermal layers with the process of keratinization. The possible part that keratohyalin may play in the process of thickening of the cell walls was discussed. The relationship between the desmosome and its dynamic environment was considered.I wish to express my sincere thanks to Dr. David Hilding of the Department of Otolaryngology for the use of an R.C.A. electron microscope and other facilities in his laboratory. This research was supported by the United States Public Health Service and American Cancer Society grants. USPHS CA 04679-07, NB 03995.