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The role of major cellular serine/threonine-specific protein phosphatases, protein phosphatase 1 and 2A, was investigated during chicken cartilage differentiation under in vitro conditions. Activity of protein phosphatase 2A decreased parallel to differentiation of chondrogenic cells, whereas activity of protein phosphatase 1 remained unchanged as assayed in the supernatants of the homogenised chicken limb bud micromass cell cultures. When okadaic acid, a potent inhibitor of protein phosphatase 1 and 2A was applied in 20 nM concentration for 4 h during the second and third culturing days, it significantly increased the size of metachromatic cartilage areas measured in 6-day-old colonies. Following okadaic acid treatments, a significant inhibition in the activity of protein phosphatase 2A was found, while the activity of protein phosphatase 1 was unaffected as measured an days 2 and 3. TRITC-phalloidin labelling demonstrated that okadaic acid disorganised actin filaments and induced rounding of chondrogenic cells. This deterioration of actin filaments was reversible. Electron microscopy and biochemical analysis of colonies revealed that the ultrastructure and major components of cartilage matrix remained unchanged under the effect of okadaic acid. Okadaic acid-treatment applied to cultures containing predominantly differentiated chondrocytes (after day 4) did not influence the cartilage formation. 3H-thymidine and bromodeoxyuridine incorporation-assays demonstrated enhanced cell proliferation in the okadaic acid-treated colonies compared to that of the untreated ones. Our results indicate, for the first time, that protein phosphatase 2A is involved in the regulation of chondrogenesis. Inhibition of protein phosphatase 2A with okadaic acid may result in increased chondrogenesis via modulation of proliferation and cytoskeletal organisation, as well as via alteration of protein kinase A-signaling pathway of the chondrogenic cells.

Previously, several studies attempting to analyze olfactory functions using dissociated culture systems of the olfactory bulb (OB) have been reported. Reciprocal dendrodendritic synapses between secondary neurons (mitral/tufted cells) and interneurons (periglomerular/granule cells) are considered to play the most important role in signal processing in the OB. However, it is unclear whether these reciprocal synapses are formed in vitro in the same way as they are in the intact OB. Thus, we synaptologically investigated the nature of cultured OB neurons. These neurons from embryonic rats were classified into four groups based on the size of their somata and their glutamic acid decarboxylase (GAD) immunoreactivity. At 14 days in vitro, most of the neurons synchronously showed spontaneous intracellular Ca2+ oscillations that were reversibly inhibited by application of D-APV and CNQX. Moreover, the frequency of the oscillations decreased and their amplitude became larger following application of bicuculline. These results suggest functional glutamatergic synaptic coupling and inhibitory GABAergic synaptic modulation. Immunocytochemical staining revealed many dot-like products (puncta) that were immunoreactive to GAD as well as to synaptophysin surrounding the cultured neurons. These results strongly indicate the presence of GABAergic synapses. The existence of synaptic contacts in OB neuron cultures was also confirmed by electron microscopy. Two types of synapses, symmetrical and asymmetrical, were morphologically recognizable. Moreover, we could also identify peculiar synapses resembling the in vivo reciprocal dendrodendritic synapses. The use of these primary culture systems will facilitate the elucidation of mechanisms underlying olfactory functions.

An 11-year-old boy, severely affected with polyostotic fibrous dysplasia, showed radiographically rapid expansion of a cystic lesion in his right humerus. At biopsy, there was an extraordinarily thin shell of bone and a cavity encapsulated by a hypertrophic fibrous membrane and filled with yellow serous fluid. Histologically, in addition to typical features of fibrous dysplasia, the fibrous capsule membrane was composed of proliferated mesenchymal cells characteristic of the affected bone. Ultrastructurally, many secretory granules were observed in numerous cytoplasmic vacuoles in the capsular cells as well as in the cultured cells isolated from the evacuated fluid.

A fully differentiated epidermis generated in vitro by culturing normal human keratinocytes at the air-liquid interface shares many similarities with native tissue. However, in contrast to native epidermis, its stratum corneum consists of a larger number of compactly packed corneocyte layers, indicating that the processes involved in corneocyte desquamation are disturbed. Although the tri-lamellar appearance of desmosomes in viable cell layers of both types of epidermis is similar, abnormalities in the transformation of desmosomes into the corneosomal plug in the stratum corneum of reconstructed epidermis have been observed. In the native epidermis, desmosomes are transformed into corneosomes at the stratum granulosum/stratum corneum interface. This process is retarded in vitro, since desmosomal structures with preserved lamellar appearance are present in the lower parts of the stratum corneum. Moreover, the corneosome frequency in reconstructed epidermis is significantly higher than in native human skin. A comparison of reconstructed epidermis with hyperproliferative epidermis, such as UV-irradiated epidermis, psoriatic epidermis and recently healed burn-wounds treated with cultured epidermal autografts, has revealed that only the structure of the stratum corneum derived from psoriatic skin is similar to that of reconstructed epidermis. The stratum corneum organization in the UV-treated epidermis and in recently healed burn-wound is, however, close to that seen in native skin.

The culture of epithelial cells lining human efferent ducts, obtained from prostatic carcinoma patients, is described. Ciliated cells were observed to beat for at least one month on plastic. On previous filters low cuboidal cells characterized the monolayers. Cells comprising monolayers over the filter were 5 to 9 microns in height whereas taller cells were found over the original fragments (14 microns). Some non-ciliated cells contained dark and light vacuoles, others were found to lack them. Both non-ciliated and ciliated cells maintained tight junctional complexes restricting the paracellular movement of horseradish peroxidase. Both types of cultured cells exhibited fluid-phase and adsorptive endocytosis from both apical and basal surfaces. It is reported for the first time that the monolayers form high resistance barriers (150 omega.cm2) that prevent the apical medium from draining to the basal compartment over 24 h.

By incubating multilayered primary cultures of human epidermal keratinocytes in a low calcium medium, the suprabasal layers can be stripped off leaving a basal cell monolayer. When this monolayer is refed normal calcium medium a reproducible series of cell kinetic, morphological and biochemical changes take place resulting in the regeneration of a multilayered tissue. The stripping procedure seems to induce the selective proliferation of a cohort of basal cells that is committed to vertical migration and rapid terminal differentiation. In contrast, when the basal cells are allowed to regenerate in the presence of the strong mitogen, cholera toxin, lateral growth and continued proliferation are favoured at the expense of the capacity of the cells to differentiate. Repeated stripping of the same cultures disclosed a considerable heterogeneity in the capacity of the basal cells to regenerate the suprabasal layers. The number of times the basal cells could restore the suprabasal layers after repeated stripping varied from four to nine times. A negative correlation between donor age and regenerative capacity was observed. The experiments with repeated stripping of the same cultures also showed that the capacity to proliferate and to restore the multilayering was fully retained for at least four cycles of stripping-regeneration, whereas the capacity to terminally differentiate was rapidly lost. It is suggested that the present system of regenerating epidermal tissue cultures may serve as an experimental model for the study of epidermal tissue homeostasis and cellular aging.

Cells isolated from the guinea-pig vestibular sensory epithelia were studied using light- and electron-microscopic techniques. The cells maintained their characteristic shapes when they had been separated. Mammalian vestibular cells are traditionally divided into two classes, type-I and type-II hair cells. It was, however, found that the population of isolated cells consisted of hair cells with a striking variability in shape and size. This was most conspicuous for the type-I hair cells. Isolated hair cells processed for electron microscopy showed that the isolation process caused minor ultrastructural damage but that the separation often was incomplete in that the large calyx-like nerve endings were still attached to type-I cells. The results suggest that the distinction of only two classes might be insufficient to describe mammalian vestibular hair cells.

Primary cultured sensory neurons prepared from adult mice were maintained for 8 days in vitro. Such cultures were exposed to either a range of ethanol concentrations (50-300 mM) or acetaldehyde (0.5-2 mM) in serum-free medium for up to 24 h. Treated neuronal cultures, together with untreated controls in both the presence and absence of serum, were prepared for transmission electron microscopy. Nuclear morphology was not changed following treatment with either substance at the doses studied. A number of changes were observed, however, in the cytoplasm of neurons, and these were intensified by an increase in concentration and the length of exposure. Acetaldehyde induced effects at a much lower concentration than was required to induce a response with ethanol. Myelin lamellae loosely wound around dense granular core material appeared in multivesicular bodies at low doses. The prevalence of these increased with concentrations of 100 mM ethanol and 1 mM acetaldehyde; the numbers of lamellae in each myelin figure also increased but the core material was less prominent. Electron-dense bodies were also evident at higher dosages together with evidence of vacuolation of the endoplasmic reticulum and Golgi complexes. Mitochondrial profiles similar to those in untreated neurons persisted throughout the exposure periods. The generation of these inclusions may reflect a mechanism of membrane turnover, both of internal systems and cell membrane cycling, as a response to alcohol and aldehyde treatment.

The major form of autosomal dominant polycystic kidney disease (ADPKD) in humans is linked to the PKD1 gene on chromosome 16p. The identity of the gene and the underlying pathogenetic mechanisms are not yet defined. Cyst-lining epithelial cells derived from a polycystic kidney were successfully grown in culture and designated MZ-PKD-1 cells. By linkage analysis, the related pedigree of the nephrectomized patient could be linked to the PKD1 gene on chromosome 16p. Thus, these cells exhibit the genotype of a mutated PKD1 gene and represent an in vitro culture model for ADPKD involving chromosome 16p. The antigenic phenotype was characterized immunohistologically by epithelial differentiation antigens and markers of individual nephron segments. An essentially identical antigenic pattern of proximal tubular cells was observed both in vitro and in fresh frozen tissue. Electron microscopy showed the formation of a microvillous-like coating. During growth phases in vitro successive changes in the cell shape were observed. MZ-PKD-1 cells exhibited a limited lifespan ending in replicative senescence. Northern blot analysis of kidney-growth-related genes, c-myc, TGF-alpha, TGF-beta 1, and EGF receptor revealed abundant expression of all of these genes in MZ-PKD-1 cells.

In order to elucidate the association between hyperglycemia and the vascular complications of diabetes, the effects of high glucose concentrations on the migration, proliferation and tube formation of bovine carotid artery endothelial cells were investigated. Cells treated with 16.7 and 33.3 mM glucose for 6 days showed 1.69- and 1.75-fold increase in serum-induced migration compared with cells treated with 5.6 mM glucose (p less than 0.05). The effect of glucose on cell proliferation was affected by serum concentration. When this was below 0.5%, a high glucose concentration stimulated cell growth to a maximum of 1.73 times that at a serum concentration of 0.05% (p less than 0.01) whereas at a serum concentration of 10%, growth was inhibited (p less than 0.05). Tube formation was studied by culturing the cells between two layers of collagen gel. Ultrastructurally, tubular structures were composed of one to several endothelial cells containing pinocytotic vesicles and cytoplasmic projections, and linked by junctional complexes. A basal lamina-like structure surrounded the abluminal surface. Treatment of the cells with 16.7 and 27.8 mM glucose for 4 days stimulated tubular elongation 1.85 and 1.71 times, respectively (p less than 0.01). Other osmogenic molecules such as mannitol and sucrose did not affect tube formation. These data imply that high glucose concentrations mimicking diabetic hyperglycemia may not inhibit the repair of endothelial injury and could act as a stimulator of neovascularization.

Actin-containing cytoplasmic fibers were visualized in the mesenteric mesothelial cells of the large intestine of bullfrog tadpoles by rhodamine-phalloidin staining of en face preparations of mesothelial cells. These fibers were concurrently stained by immunofluorescence using antibodies to myosin or alpha-actinin. Electron microscopy showed the presence of bundles of microfilaments in the basal cytoplasm of the cells. Such fibers in the mesothelial cells may be comparable to the stress fibers present in cultured cells. The mesothelial cells initially formed axially oriented stress fibers when they changed from a rhombic to a slender spindle-like shape. On the other hand, stress fibers disappeared as cells transformed from elongated to polygonal shapes during the period of metamorphic climax. Expression of stress fibers in these cells appears to be related to the degree of tension loaded on the mesentery, which may be generated by mesenteric winding. These stress fibers in the mesothelial cells may serve to regulate cellular transformation. They may also help to maintain cellular integrity by strengthening the cellular attachment to subepithelial tissue against tensile stress exerted on the mesentery.

Cultures of epithelial cells can be initiated with the sediment of voided urine of normal adults. Tightly or loosely packed colonies were formed by cells of diverse morphologic configuration. Ultrastructural studies revealed that the proliferating cells formed abundant desmosomes, imperfectly formed tight junctions and lamina densa, all typical of epithelial cells. Some cells were lined by the characteristic asymmetric unit membrane, thus confirming the urothelial derivation of the cultures. Peculiar, apparently hitherto not described multivesicular bodies, seemingly of cytoplasmic origin, were observed near the surfaces of some cells. The urinary cell culture system is a potentially useful tool for diagnostic and research purposes.

The fine structure of filamentous structures and lattice-like structures found in herpes simplex virus type 2-infected cells was studied. By specimen tilting experiments, it was demonstrated that the lattice-like structures were the cross-sectional views of the tubular structures and the filamentous structures were the longitudinal sections. The observation in negatively stained preparations of isolated tubular structures revealed that the structure was constructed by regular array of ring shaped elements. A hypothetical model is presented for tubular structures constructed on the basis of the present findings. The tubular structures appeared as early as 6 hours after infection and reached a maximum at 12 hours.

In vitro indole metabolism and ultrastructural morphology of the pineal gland of male rats were examined. A comparison of the effect of norepinephrine stimulation on indole synthesis in whole cultured glands and preparations of dispersed pineal cells is discussed. Our studies on the performance of dispersed cells during the first 24 h after preparation indicate a strong dependence of pineal cells upon physical attachment to the culture dish and probably also on cell-to-cell contact.

Specific anti-actin and anti-myosin antibodies were shown to react in single and double immunofluorescence sandwich tests with identical sites in non-muscle cells in frozen sections of tissues and in cultured cells. In tissues, both antibodies reacted with liver cell membranes, parts of renal glomeruli, brush borders and peritubular fibrils of renal tubules, brain synaptic junctions, and membranes of lymphoid cells in thymic medulla, lymph nodes and spleen. Both antibodies reacted strongly with long parallel cytoplasmic fibrils in cultured fibroblasts, and with disrupted fibrils in cytochalasin-B treated cells. In neuroblastoma cells both antibodies gave prominent staining of growth cones and microspikes. The observation that the distribution of myosin parallels that of actin in non-muscle cells argues strongly in favour of a functional interaction between the two molecules in the generation of contractile activity in non-muscle cells.

Acid phosphatase has been demonstrated ultrastructurally in 3T3 and SV40-3T3 mouse cells using sodium beta-glycerophosphate and p-nitrophenyl phosphate as substrate. The former substrate only demonstrates the enzyme in lysosomes and elements of the Golgi apparatus while the latter demonstrates it in the cisternae of the endoplasmic reticulum and in the cell surface as well as at lysosomal sites. The significance of surface acid phosphatase activity is discussed in terms of sublethal autolysis.