Activity manifestations of Hassall's corpuscles in vitro as revealed by cinematography

Summary Thymic explants from newborn rats were cultivated in Rose chambers under dialysis membranes. With the use of phase contrast, time-lapse cinematography, the following activity manifestations were observed: 1. Several corpuscles showed rotatory movements; 2. nuclear rotation was observed in some cells of the corpuscles; 3. some individual cells in the 15-day culture showed pulsatile activity which might be involved in the mechanism of the rotatory movements of the cellular aggregates; 4. the corpuscles increased in size by the addition of cells and/or by mitosis; 5. the elements at the periphery of the more mature corpuscles (7- and 9-day cultures) were viable, while the central area appeared to be composed of necrotic tissue or hyalinized material.Grateful acknowledgement is made to Dr. C. M. Pomerat for encouragement in the course of this study. Mr. Charles Raiborn aided in the preparation of cultures and Messrs. C. G. Lefeber and Robert Olson rendered indispensable assistance with the photographic work.Aided by an American Cancer Society Student Fellowship (USC-IDC) and in part by Grant No. G-14091 from the National Science Foundation administered by C. M. Pomerat.     

Observations on neurons and neuroglia from the area of the mesencephalic fifth nucleus of the cat in vitro

Summary Neurons from the mesencephalic root of the fifth nucleus from the kitten have been maintained in vitro for periods up to 70 days. During this time they retained their characteristic form and structure as seen in sectioned material. Axonic processes as long as 8 mm have been measured. The occurrence of recurrent axons intertwining with dendritic arborizations of the same neuron is discussed. Modulations are described for astrocytes in relation to their possible dependence on the scarcity of connective tissue elements and a lack of an outer mesenchymal tissue zone in cultures of mesencephalic explants.Grateful acknowledgment is made to Mrs. Walther Hild and Mrs. C. C. Morris for their generous contribution in the management of the tissue cultures and in executing staining procedures. Messrs G. Lefeber and E. Pitsinger offered indispensable aid with photographic work.This investigation was supported by a grant [PHS B-364 (C3)] from the National Institute of Neurological Diseases and Blindness, of the National Institutes of Health, Public Health Service, administered by C. M. Pomerat.     

Ependymal cells in tissue culture

Summary Ciliated ependymal cells from various locations of the ventricular system of mammals showed outgrowth either in the form of epithelial sheets or in the form of pools and elongated double rows, dependant on the site from which the ependyma was taken and on the original orientation of the cells in respect to the cover slip.The ependymal cilia in such cultures were shown to be moving at a rate of 51/2 to 6 times per second. The movement of the cilia of adjacent cells was apparently well coordinated causing the surrounding fluid to flow in a particular direction.The effect of physiological saline, morphine sulfate, ethyl and methyl alcohol on the ciliary motility has been studied in living cells with phase contrast cinematography.Grateful acknowledgement is made to Messers G. Lefeber, E. Pitsinger and J. Carnes for indispensible aid with photographic work. Mrs. J. Finerty and Mrs. W. Hild contributed generously in the management of the tissue cultures and in executing staining procedures.This investigation was supported by a research grant [PHS B-364 (C3)] from the National Institute of Neurological Diseases and Blindness, of the National Institutes of Health, Public Health Service, administered by C. M. Pomerat.     

Harvesting and culture of epithelial cells from hollow organs of the female reproductive system

Summary The technique of trypsinization of cells lining the hollow organs applied to the female reproductive tract has proven successful in harvesting living cells which produced luxuriant cultures in vitro.Elements from the oviduct, endometrial cavity and vagina were obtained from 6 rabbits, 2 dogs and 6 surgical specimens by means of bathing the lumen of the organs with the use of a 0.5% solution of trypsin in calcium and magnesium free Gey's balanced salt solution for 30 to 40 min, employing continued gentle agitation provided by a syringe.Epithelial cells so harvested were grown in Rose chambers and in T-30 flasks for as long as 30 days, when they were fixed for study with special stains.No fibrocytic contamination of the cultures was observed.Further applications of the method are under investigation and are briefly outlined in the discussion.This work was performed in the Department of Cellular Biology, Pasadena Foundation for Medical Research, Pasadena, California, and aided in part by a grant from the U. S. Public Health Service, No. 2 G 279 and from the U. S. Army Medical Research and Development Command, Department of the Army, under Research Grant No. DA-MD-49-193-63-G80 administered by C. M. Pomerat.     

The fine structure of the olfactory mucosa and nerve in the teleost Carassius carassius L.

Summary Morphological studies on teleost olfactory mucosa confirm the findings of previous authors regarding the general arrangement of conventional cell types, viz. receptor, sustentacular, mucous and basal, but teleosts show certain distinct differences. The receptor cells have the general mammalian bipolar shape but their peripheral dendrite does not project beyond the epithelial surface. In addition to numerous typical cilia, an exceptional ciliary formation was observed in which the filaments, instead of forming individual cilia, are grouped together in clusters and are enveloped in a single limiting membrane.At the junction between the finger-like process and the mucosal fold myelinated nerve fibres are observed within the subepithelial stroma.Within the postero-medial zone of the mucosa is a conspicuous well-differentiated new cell type. A thick rim of electron-dense cytoplasm, bounded by an outer trilaminar membrane, encloses prominent foliate (leaf-like) organelles, a basal nucleus, numerous mitochondria and vacuolar spaces. These foliaceous cells communicate with the external environment through a small stoma, their close association with epithelial components suggesting a possible secretory or absorptive function. Their intricate morphology, however, suggests that they may be receptors, but their role and neural connections still require definition.Supported by Grant 5 RO 5 TWOO 154-02 from the National Institutes of Health, United States Public Health Service.The authors are indebted to Dr. A. S. Wilson for his helpful criticism and gratefully acknowledge the photographic technical assistance of Mr. J. Simmons and Mr. S. Frank.     

Outgrowth of dentinal fibers observed in tissue culture

Summary In hanging-drop cultures of dental pulp of the permanent canines and molars of newborn kittens, outgrowth of dentinal fibers from the outer surface of young dentin was observed when the enamel organ was removed.The outgrowth started soon after explantation and the fibers reached a considerable length in a short period of time.Outgrowing fibers were not ramified, but usually showed a knot near their distal end.The potency of growth at the peripheral end of the dentinal fibers was positively established.This investigation was supported by the Medical Research and Development Board, Office of the Surgeon General, Department of the Army, under Contract Nos. DA-49-007-MD-447 and DA-49-007-MD-32, administered by Doctors T. G. Blocker, jr. and C. M. Pomerat respectively.     

Effects of protein kinase C modulation on NMDA receptor mediated regulation of neurotransmitter enzyme and c-fos protein in cultured neurons

The role of protein kinase C (PKC) in N-methyl-d-aspartate (NMDA) receptor-mediated biochemical differentiation and c-fos protein expression was investigated in cultured cerebellar granule neurons. The biochemical differentiation of glutamatergic granule cells was studied in terms of the specific activity of phosphate-activated glutaminase, an enzyme important in the synthesis of the putative neurotransmitter pool of glutamate. When the partially depolarized cells were treated with NMDA for the last 1 to 3 days (between 2 and 5 days in vitro), it elevated the specific activity of glutaminase. In contrast, NMDA had little effect on the activity of aspartate aminotransferase or of lactate dehydrogenase. Treatment of 10-day old granule neurons with NMDA also resulted in a marked increase in the immunocytochemically measured expression of c-fos protein. The increases in both the activity of glutaminase and the steady state level of c-fos protein were specific to the activation of NMDA receptors, as they were completely blocked byd,l-2-amino-5-phosphonovaleric acid. The specific stimulation of NMDA receptors in PKC-depleted granule neurons or in the presence of reasonably specific PKC inhibitors also produced significant elevation in the activity of glutaminase and the expression of c-fos protein. These increases were similar in magnitude to those observed in the granule neurons of the respective control groups. Our findings demonstrate that PKC is not directly involved in the NMDA receptor-mediated signal transduction processes associated with biochemical differentiation and c-fos induction in cerebellar granule neurons.     

The reticular substance of the medulla oblongata of the albino rat

Summary The region of the reticular giganto-cellular nucleus, perfused with formalin and postfixed in osmium tetroxide, was studied with histochemical and electron microscopic techniques. The perikarya of the neurons have two zones. The peripheral cytoplasm contains Nissl bodies, mitochondria, and free RNP particles. The juxtanuclear cytoplasm contains the Golgi complex, mitochondria, RNP particles and dense bodies. The nucleus is indented and has a prominent nucleolus and a paranucleolar body. Dense bodies are found along the axon and dendrites as well. Three different types of synapses are described and two types of synaptic vesicles (spherical and ellipsoidal) are shown.The capillary endothelium shows microvilli and marginal flaps. The endothelial cytoplasm contains vacuoles, micropinocytotic vesicles, and a few dense bodies. Processes of pericapillary cells, surrounded by a basement membrane, also contain dense bodies. The dense bodies found in the neurons and endothelial cells show acid phosphatase activity. On the basis of their morphology and their enzymatic activity these bodies are identified as lysosomes.Partially supported by a school grant No RF 62051 from the Rockefeller Foundation, New York, USA, and Consejo Nacional de Investigaciones Científicas y Técnicas, Argentina.Fellows of the Consejo National de Investigaciones Científicas y Técnicas, Argentina. The authors wish to thank Dr. Mario H. Burgos for his constant interest and criticism, and to Dr. Eduardo Rodriguez Echandia and Dr. Fabio L. Sacerdote for their valuable assistance.     

Prolonged prostaglandin E1 stimulation of cyclic AMP production in transformed and normal WI-38 fibroblasts

Summary Long-term (48-hr) incubations of either the fibroblast strain WI-48 or its SV40-transformed counterpart, WI-38-VA13-2RA, in growth medium containing 1 μm prostaglandin E₁ (PGE₁) resulted in a sustained production and release of cyclic AMP from the cells into the medium. Despite the steady production, intracellular levels of the nucleotide decreased, reaching steady-state values within 4 hr of the initial exposure to PGE₁. These values were maintained for the remainder of the 48-hr experimental period. The steadystate levels of intracellular cyclic AMP were higher than those observed in unstimulated cells, and cyclic AMP-dependent protein phosphokinase was in a highly activated state as compared to controls. Under these conditions little change in the growth or morphology of either the normal or transformed cells was observed. In contrast, inhibition of growth, apparent cell death, and unusual morphological changes were observed in both normal and transformed cells when high concentrations of either PGE₁ (10 μm) or the phosphodiesterase inhibitor 1-methyl, 3-isobutylxanthine (0.5mm to 2mm) were used, which was indicative of toxic effects of the drugs. It was concluded that cyclic AMP-mediated activation of protein phosphokinase does not completely inhibit growth in WI-38 cells or restore normal growth and morphology to the SV40-transformed cells. This work was supported by Grants AM 13904 and CA 21612 from the National Institutes of Health, Department of Health, Education and Welfare.     

Ultrastructure of Human labial salivary glands

Summary Human labial salivary glands, obtained by biopsy from 32 subjects, were studied by light and electron microscopy. Intranuclear inclusions, unrelated to nucleoli, were present in many of the acinar nuclei in glands from 16 of the 32 donors. More than one inclusion was sometimes observed within a single nucleus. They measured about 1 in diameter, and were stainable in a variety of ways. They were eosinophilic, some were stained by Nile blue sulphate, some were PAS-positive, and all were Feulgen-negative. They were bounded by a single membrane, which never exhibited continuity with the nuclear envelope, and they showed considerable morphological variation. The more complex inclusions consisted of alternating shells of light and dark material with tiny dense granules embedded in the latter. The intranuclear inclusions, which apparently were non-viral in origin, were in some way related to the secretory cycle of the mucous cells, since they were found only in immature cells, and never in cells in which secretory products were abundant.This work was supported in part by grants from the Henry Spenadel Trust and the Max C. Fleischmann Foundation of Nevada, by grant CA-08748 from the National Cancer Institute, by grant 5 SO1 FR 05335-07 from the National Institutes of Health, by a grant from the National Cystic Fibrosis Research Foundation, and by an Institutional Grant to the School of Dental and Oral Surgery, Columbia University, from the National Institutes of Health.The authors are indebted to Dr.Louis Mandel for performing the biopsies used in this study. The expert technical assistance of Mrs.Mona Seggio is acknowledged.     

The liver of the slender salamander Batrachoseps attenuatus

Summary The structure of the crystalline inclusions found in Batrachoseps liver cells is described and it is shown that the most symmetric unit cell upon which the crystal lattice is built is a face-centered cube. Taking into consideration the physical properties of a face-centered cubic structure, an attempt is made to determine the nature of the macromolecules that comprise the crystal. It is concluded on the basis of available evidence that the macromolecules probably represent serum lipoproteins. The intracellular synthesis of the crystals and the possible functions they may subserve in the animal are discussed. A comparison is made between the crystals and granules in rat hepatocytes discussed by Bruni and Porter (1965).Research supported by grant RG-6729 from the Institute of General Medical Sciences, National Institutes of Health, United States Public Health Service.     

Organ culture of adult rat colonic mucosa on fibrin foam

Summary A system for maintaining adult rat colonic mucosa in organ culture for up to 28 days is described. Distal colonic mucosa physically separated from the muscle layers was cultured at 37°C on a substrate of human fibrin foam in HEPES- and bicarbonate-buffered Waymouth's MB 752/1 medium supplemented with 10% fetal bovine serum,l-glutamine, bovine albumin, ascorbic acid, hydrocortisone, insulin, and ferrous sulfate; the optimal atmosphere for culture was 95% O₂ and 5% CO₂. Viability of explants was demonstrated by tissue morphology with light microscopy, incorporation of [³H]thymidine and [³H]leucine into DNA and protein, [¹⁴C]glucosamine and [³H]fucose incorporation, and glycoprotein synthesis. Two days after initiation of culture, degeneration of surface and crypt cells was observed. Secreted mucosubstances covered the explants. Explants maintained in 95% O₂ retained a variable number of glandular crypts with normal columnar epithelium for 14 to 21 days in culture. At 28 days, explants contained a single layer of cuboidal surface epithelium and a rare cryptlike gland. This work was supported by the National Cancer Institute Contract N01-CP-75953 and in part by the International Cancer Research Data Bank Program of the National Cancer Institute, National Institutes of Health, under Contract N01-CO-65341 with the International Union Against Cancer.     

Fluorescence microscopic observations of catecholamines in cultures of the sympathetic chains

Summary A histochemical technique for the demonstration of catecholamines developed by Falck et al. has been successfully applied to the sympathetic chains of rats and mice maintained in vitro. Catecholamines were localized in the nerve fibers, showing identical green fluorescence as in tissue sections of healthy rats. The cultures 8 days in vitro exhibited positive reaction in a few terminals, whereas sister cultures 1 month in vitro showed strong fluorescence reaction in thicker proximal axons and networks of nerve fibers as well. Reactivity of neuron somas became positive after 1 month of cultivation. Application of reserpine in amount of 0.00025 mg/ml for 2 hours resulted in complete disappearance of fluorescence. Furthermore, cultures of spinal ganglia from fetal rat produced no fluorescence reaction with this technique. Therefore, the reaction is specific for sympathetic nervous tissue and reliable for the differentiation of sympathetic neurons from other types of nerve cells.This work was supported by research grant NBO 3173 from the National Institute of Neurological Diseases and Blindness, U.S. Public Health Service, and research grant No. 355 from the National Multiple Sclerosis Society, New York.     

The influence of early protein-calorie malnutrition on neuronal and glial protein synthesis

The effect of pre- and postnatal undernutrition, produced according to the method of Chow and Lee (3), on the rate of protein synthesis in the brains of rats 11, 21, 34 and 90 days of age was studied by measuring the incorporation ofl-[¹⁴C]valine in vivo andl-[³H]lysine in vitro. Both in vivo and in vitro experiments were performed with high concentration of the precursor to decrease the effects of pool variations and protein degradation. Particular interest was given to the effects of this form of early protein-calorie malnutrition (PCM) on neuronal and glial cells which were isolated from the brains by gradient centrifugation. Brain protein synthesis measured in vivo which showed a peak at 21 days in both animal series, was depressed by PCM at 11 days but stimulated at 34 days of animal age. Small effect was observed in the 90-day-old animals. A similar response as in whole brain was seen for neuronal cells, while glial cells showed a different reaction. Studies of in vitro protein synthesis did not reveal appreciable effects of undernutrition in whole brain. Both neuronal and glial cells showed a moderate but not statistically significant elevation of protein synthesis in animals subjected to early PCM.     

NADPH-Diaphorase-containing neurons in the medullary structures involved in generation of the respiratory activity in neonatal rats

Using a histochemical technique, we examined distribution of the neurons containing a marker of nitric oxide synthase (NOS), NADPH-diaphorase (NADPH-d), on frontal slices of the medulla and upper cervical spinal segments of 4-day-old rats. It was demonstrated that NADPH-d-positive cells are present within the dorsal and ventral medullary respiratory groups. The highest density of the labeled middle-size multipolar neurons (27.9±2.6 cells per 0.1 mm² of the slice) was observed in the rostral part of the ventral respiratory group, within the reticular lateral paragigantocellular nucleus. Similar NADPH-d-positive neurons were also observed in other reticular formation structures: rostroventrolateral reticular, gigantocellular, and ventral medullary nuclei, and in the ventral part of the paramedial nucleus. There were no labeled neurons in the lateral reticular nucleus. Single small and medium-size labeled neurons were found at all rostro-caudal levels of thenucl. ambiguous (nuclei retrofacialis, ambiguous, andretroam-biguous). Groups of NADPH-d-positive neurons were also revealed within the dorsal respiratory group, along the whole length of thenucl. tractus solitarii (mostly in its ventrolateral parts). Single labeled neurons were also observed in thenucl. n. hypoglossi, and their groups were observed in the dorsal motor part of thenucl. n. vagus. Involvement of the structures containing NADPH-d-positive neurons in the processes related to generation of the respiratory activity is discussed. Our neuroanatomical experiments prove that in early postnatal mammals NO is actively involved in generation and regulation of the medullary respiratory rhythm. Neirofiziologiya/Neurophysiology, Vol. 32, No. 2, pp. 128–136, March–April, 2000.     

A Catecholamine storage in the pancreatic A2-cells of owl monkey (Aotes trivirgatus)

Summary The fluorescence method ofFalck andHillarp was used to study the occurrence of biogenic monoamines in the islets of Langerhans of monkeys. A storage of a catecholamine was demonstrated in the A₂-cells of owl monkey, whereas no histochemically demonstrable amount of monoamines could be seen in the islet cells of marmoset,Rhesus monkey, squirrel monkey, andCebus monkey.Supported by grants from the Swedish Medical Research Council (No. B69-14x-712-04C) and by the National Institutes of Health (No. 06701-02).     

The fine structure of the megakaryocyte of the bone marrow of the guinea pig

Summary The cytoplasm of megakaryocytes frequently exhibits bullate processes which protrude into the lumen of the sinusoids through small apertures in the reticular cells. These bullae differ morphologically from the platelet demarcation zones and are held to have a different function. It is concluded that the bullae detach from the megakaryocyte in a manner comparable to apocrine secretion and enter the blood stream.This investigation was supported by a PHS research grant (CA 05493) from the U.S. Public Health Service.Dedicated to Professor Friedrich Wassermann with admiration and affection on the occasion of his 80^th birthday.     

Effects of bulbar stimulation on smooth muscle tone in the feline airway

Changes induced in tracheal smooth muscle tone by bulbar electrical stimulation were investigated in 30 cats anesthetized with a chloralose-urethane mixture and paralyzed with succinyl choline bromide. Raised tonus was mainly observed during stimulation of the caudal section of the dorsal motor nucleus of the vagus nerve, the vicinity of the nucleus ambiguus, and the adjoining reticular formation structures. Attenuation, however, was produced by stimulating bulbar reticular formation nuclei at a level 1 mm caudal and 6 mm rostral to the obex. Raised tonus is thought to be connected with activation of efferent neurons belonging to the motor nucleus of the vagal nerve, as well as axons of nucleus ambiguus neurons in transit through the medial zone, whilst attenuation is connected with excitation of sympathotonic reticular neurons, inhibitory neurons activated by pulmonary stretch receptors, and possibly with vagal efferent neurons activating the non-adrenergic inhibitory nervous system of the bronchi.Medical Institute, Latvian Ministry of Health, Riga. Cardiology Research Institute. Latvian Ministry of Health, Riga. Translated from Neirofiziologiya, Vol. 21, No. 3, pp. 320–326, May–June, 1989.     

Peculiarities of ovine cells in culture

Summary The chromosome complement of two ovine (Ovis aries L.) kidney cell lines are described. Nuclei of MDOK, the older cell line, are generally larger than those of OK cells, they are hypo-tetraploid and show considerable chromosomal irregularities both in structure and behavior. The OK line observed since its initiation, is now hyper-diploid and exhibits a gradual accumulation of chromosomes. Both cell lines have in common the fact that they now maintain a larger proportion of chromosomes with interstitial centromeres than telocentric chromosomes. This observation parallels similar ones made earlier in bovine cells. However, the processes whereby this condition arose in the respective cultures are believed to be different and are discussed.Dedicated to Professor Dr. H. Bauer on the occasion of his 60th birthday.Supported in part by Contract No. PH 43-63-13 from the National Cancer Institute, National Institutes of Health, Public Health Service.     

Evidence of species differences in the ultrastructure of the Hepatic sinusoid

Summary Hepatic sinusoids in the calf have been studied by light and electron microscopy. The endothelial lining is shown to be continuous and to be associated with a prominent basement membrane. The perisinusoidal space (of Disse) contains collagen fibrils and is otherwise nearly filled with hepatic cell microvilli. Perisinusoidal cells resembling fibroblasts or reticular cells in morphology and distribution are present in moderate numbers. Hepatic sinusoids in the rat were examined for purposes of comparison. In the rat the sinusoidal lining is discontinuous and there is no identifiable basement membrane. The perisinusoidal space is larger than in the calf and contains more sparsely distributed collagen fibrils. Perisinusoidal cells have not been identified. It is suggested that the difference in structure is not artifactitious but represents a species variation. This species difference seems important to record since observations on the rat and other small laboratory animals provide the basis for most of the current generalizations about the organization of the mammalian liver sinusoid. The functional significance of the two types of sinusoidal arrangement is not clear.A preliminary report of this work was presented at the VII th International Congress of Anatomists (Wood 1960).Supported in part by Grant H-2698 from the National Institutes of Health, U. S. Public Health Service.