STUDIES ON LYMPHOCYTES

Some of the time parameters of the cell cycle in bovine thoracic duct lymphocytes have been estimated by analysing labeled mitoses curves, and by double labeling. The two methods gave similar estimates of T_s. Thus, T_s measured directly from labeled mitoses curves varied from 4 to 6 hr, while the estimates from double labeling were 4.8 and 4.5 hr. T _G measured directly from labeled mitoses curves was 5 hr, and estimates of T_G from the values of T_s ranged from 6.3 to 7.7 hr. The present data confirm the short generative cycle of large thoracic duct lymphocytes. Extracorporeal irradiation of the lymph (ECIL) had no detectable effect on the cell cycle or the fractional production rate of the lymphocytes. However, the calculated absolute production was reduced following ECIL, due to a decrease in the absolute number of cells present. The grain count over mitoses after ECIL was approximately one-half that found before ECIL.     

Active Ion Transport Across Canine Blood Vessel Walls

Experiments giving evidence of active Na and Cl ion fluxes across large canine blood vessel walls (aorta, vena cava) in vitro have been presented. The information has been obtained using ion tracer techniques after Ussing and with diffusion cells of the Hogben type. The available data suggest that the membranes are satisfactorily oxygenated by the bathing solutions saturated with oxygen at atmospheric pressure. Evidence is offered which indicates that active ion transport does occur across the aorta and vena cava in in vitro experiments. Under the conditions of the experiment net Na and Cl flux takes place from intima to adventitia across the aorta, and from adventitia to intima across the vena cava at low measured potential differences. The possible relationships of derangement of active ion transport mechanisms, produced by electric currents and tissue injury potential differences, to intravascular thrombosis are alluded to. It would appear that sodium and chloride fluxes across large blood vessel walls in vitro occur at least in part as the result of metabolic processes and cannot be explained simply on the basis of diffusion across a semipermeable membrane.     

Tissue-specific sorting of the human LDL receptor in polarized epithelia of transgenic mice

The distribution of human low density lipoprotein (LDL) receptors was studied by immunofluorescence and immunoelectron microscopy in epithelial cells of transgenic mice that express high levels of receptors under control of the metallothionein-I promoter. In hepatocytes and intestinal epithelial cells, the receptors were confined to the basal and basolateral surfaces, respectively. Very few LDL receptors were present in coated pits or intracellular vesicles. In striking contrast, in the epithelium of the renal tubule the receptors were present on the apical (lumenal) surface where they appeared to be concentrated at the base of microvilli and were abundant in vesicles of the endocytic recycling pathway. Intravenously administered LDL colloidal gold conjugates bound to the receptors on hepatocyte microvilli and were slowly internalized, apparently through slow migration into coated pits. We conclude that (a) sorting of LDL receptors to the surface of different epithelial cells varies with each tissue; and (b) in addition to a signal for clustering in coated pits, the LDL receptor may contain a signal for retention in noncoated membrane that is manifest in hepatocytes and intestinal epithelial cells, but not in renal epithelial cells or cultured human fibroblasts.     

Astrocytes induce neural microvascular endothelial cells to form capillary-like structures in vitro

Astrocytes maintain a unique association with the central nervous system microvasculature and are thought to play a role in neural microvessel formation and differentiation. We investigated the influence of astroglial cells on neural microvascular endothelial differentiation in vitro. Using an astroglial-endothelial coculture system, rat brain astrocytes and C6 cells of astroglial lineage are shown to induce bovine retinal microvascular endothelial (BRE) cells to form capillary-like structures. Light microscopic evidence for endothelial reorganization began within 48 hours and was complete 72-96 hours following the addition of BRE cells to 1-day-old astroglial cultures. The extent of BRE reorganization was quantitated by computer-assisted analysis and shown to be dependent upon the density of both the BRE and C6 cells within the cocultures. Coculture conditions in which BRE cells were separated from C6 cells by porous membranes failed to generate this endothelial cell change. Likewise, C6-conditioned media and C6-endothelial coculture conditioned media did not induce BRE cell reorganization. Extracellular laminin within the C6-endothelial cocultures, identified by indirect immunofluorescence, was concentrated at the endothelial-astroglial interface of capillary-like structures consistent with incipient basement membrane formation. Astroglial cells accumulated adjacent to capillary-like structures suggesting the presence of bidirectional influences between the reorganized endothelial cells and astroglia. This is the first demonstration of astroglial induction of angiogenesis in vitro and these findings support a functional role for perivascular astrocytes in the vascularization of neural tissue such as retina and brain.     

Hormonal control of neuron number in sexually dimorphic spinal nuclei of the rat: IV. Masculinization of the spinal nucleus of the bulbocavernosus with testosterone metabolites

The spinal nucleus of the bulbocavernosus (SNB) is a sexually dimorphic motor nucleus in the rat lumbar spinal cord. SNB motoneurons and their perineal target muscles are present in adult males but reduced or absent in females. This sexual dimorphism is due to the presence of androgen during development; females treated with testosterone (T) perinatally have a masculine SNB system. To assess whether masculinization of the SNB could involve the conversion of testosterone into its active metabolites, dihydrotestosterone (DHT) and estrogen, we examined the development of the SNB in females treated perinatally with estrogen alone or in combination with dihydrotestosterone. Counts of motoneurons in the developing SNB in all groups showed the typical prenatal increase followed by a differential postnatal decline; the incidence of degenerating cells reflected this decline. Motoneuron numbers and the frequency of degenerating cells in females treated with estrogen (E) alone did not differ from those of normal females, with both groups losing large numbers of motoneurons and having a high incidence of degenerating cells. In contrast, females treated with both estrogen and dihydrotestosterone did not show the female-typical decline in motoneuron number and had a low, masculine incidence of degenerating cells. By postnatal day 10, females treated with estrogen and dihydrotestosterone had a fully masculine SNB motoneuron number, suggesting that dihydrotestosterone alone or in conjunction with estrogen may be involved in the development of the sexually dimorphic SNB system.