Topographical and ontogenetic study of the neurons producing growth hormone-releasing factor in human hypothalamus

Neurons producing growth hormone-releasing factor have been characterized and analyzed by immunohistochemistry in the hypothalami of human fetuses, neonates, infants and adults, using two antibodies against human pancreatic GRF (hpGRF). One of the antibodies recognized both the hpGRF(1-40)OH and hpGRF(1-44)NH2 in the mid portion (between the 28th and 39th amino acid), the other one specifically recognized the C-terminal end of hpGRF(1-44)NH2. These two antibodies stain a single neuronal system with cell bodies mainly located in the infundibular (arcuate) nucleus, and in the ventromedial and lateralis tuber nuclei. These neurons project to the median eminence where they give numerous endings in contact with portal vessels. These neurons are distinct from those containing LH-RH, somatostatin, CRF or pro-opiocortin. In fetuses, neurons immunoreactive with hpGRF antibodies are first detected at the 29th week. They display a neuroblastic aspect which persists after birth. Immunoreactive fibers are detectable in the median eminence after the 31st week. These results demonstrate that the infundibular nucleus plays a major role in control of GH secretion in man and that secretion of GRF appears late during fetal life; this suggests that the first stages of differentiation and development of GH producing cells in the human fetus do not depend on hypothalamic GRF secretion.     

Physico-chemical properties and evidence for electrophoretic variants of rat transcortin

Isolation of rat plasma transcortin was carried out by affinity chromatography, as previously described for human. The protein was shown to be pure by PAGE and one single N-terminal amino acid was identified (Ser), which suggested that the protein molecule has a single polypeptide chain. This assumption is supported by SDS-PAGE. The amino acid composition was reported and compared with the one of human transcortin. The purified protein always migrated in PAGE (with or without SDS) as a double band; the faster component being more intense than the slower one. Whether transcortin was free or bound to corticosterone, the same aspect was observed. Molecular weight of these two variants were determined by SDS-PAGE as 65,900 and 75,800. Polymers only appeared after irreversible denaturation of the protein, as previously described for human transcortin. Various other physical parameters were determined: a sedimentation coefficient of 3.71 S +/- 0.18 was calculated by ultracentrifugation in sucrose gradient, association constants at 4 degrees C for corticosterone and cortisol (2.7 X 10(9) M-1 and 4.2 X 10(8) M-1, respectively).     

A new enkephalin analogue: trans-4-hydroxycinnamoyl-glycyl-glycyl-phenylalanyl-leucine. Synthesis and biological properties

A new analogue of the leucine-enkephalin in which the N-terminal tyrosine has been replaced by trans-4-hydroxycinnamic acid, has been synthetized by liquid-phase coupling methods. The central cardiovascular effects of this analogue were investigated and the results discussed.     

Uptake of hyaluronan in hepatic endothelial cells is not directly affected by endotoxin and associated cytokines

The uptake of hyaluronan (HYA) labeled with 3H in its acetyl group was measured in cultured liver endothelial cells from normal rats and from rats previously treated with sublethal doses of Escherichia coli endotoxin (ET). Replicate cultures were also exposed to recombinant human tumor necrosis factor-alpha (TNF-alpha), interleukin-1 (IL-1) or interferon-gamma for 1 to 3 h before the measurement of hyaluronan uptake. Under all conditions, HYA was absorbed by endothelial cells at rates consistent with receptor-mediated absorption. In cells exposed to HYA 20 h after isolation, rate of uptake was less than half the rate in cells exposed 6 or 7 h after isolation. Cellular uptake of HYA was neither reduced nor enhanced by any of the treatments with cytokines. Prior exposure of the cell donors to ET caused a three-fold increase in their plasma HYA but did not alter the subsequent rate of cellular HYA uptake in vitro, either with or without added treatment with TNF-alpha or IL-1. It was concluded that the elevation of plasma HYA caused by septicaemia or by the experimental administration of ET or TNF-alpha cannot be attributed to direct interference with HYA receptors on hepatic endothelial cells.     

Control of terminal differentiation of adipose precursor cells by glucocorticoids.

The role of glucocorticoids on adipose conversion has been studied using confluent Ob1771 mouse preadipose cells maintained in a serum-free culture medium able to support the emergence of early but not that of late markers of differentiation. Under these culture conditions, glucocorticoids play, at physiological concentrations, a permissive role for terminal differentiation, characterized by glycerol-3-phosphate dehydrogenase expression and triacylglycerol accumulation within 12 days, whereas progesterone, testosterone, and estradiol are inactive. Glucocorticoids behave as mitogenic-adipogenic stimuli able to trigger growth-arrested, early marker-expressing cells to enter the terminal phase of the differentiation program and thus appear to mimic the mitogenic-adipogenic activity already described for arachidonic acid and cyclic AMP-elevating agents, especially prostacyclin. When compared to corticosterone alone, exposure of Ob1771 cells to both corticosterone and arachidonic acid leads to an additional increase in the glycerol-3-phosphate dehydrogenase activity and number of differentiated cells; this potentiation is further enhanced when the culture medium is supplemented with the cyclic AMP phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine. This suggests indirectly the involvement of prostacyclin as a metabolite of arachidonic acid able to induce cyclic AMP accumulation. In agreement with this hypothesis, it is found that a promoting effect is exerted by corticosterone on the metabolism of arachidonic acid, leading in turn to an increase in the production of prostacyclin. These findings allow a better understanding of the role of glucocorticoids on adipose cell differentiation and explain a posteriori the effectiveness of the combination of dexamethasone-isobutyl-methylxanthine used in innumerable studies.     

Functional differentiation and primary metabolism of mouse mammary epithelial cells in extended-batch and hollow-fiber culture

Growth, expression of functional differentiation (as characterized by synthesis and secretion of milk proteins), and primary metabolism were studied for a mouse mammary epithelial cell line, COMMA-1D, in extended-batch and hollow-fiber reactor cultures. Batch cultures were performed on Costar polycarbonate membrane inserts, allowing basal and apical exposure to medium. Protein production was induced in both batch and hollow-fiber cultures in hormonesupplemented medium. In batch cultures, high levels of protein production and secretion were maintained for 18 days. Once differentiation was induced, the rate of deinduction was low, even in medium containing epidermal growth factor (EGF) and serum; cells continued to express and secrete proteins for at least 12 days after prolactin and hydrocortisone were removed. Cells in both batch and hollow-fiber cultures were highly glycolytic and exhibited low rates of glutaminolysis. In batch culture on membrane inserts, cells showed polarized metabolism between the apical and basal side, maintaining significant gradients of glucose and lactate. Medium hormonal composition and subsequent differentiation affected both glucose uptake and lactate yield for COMMA-1D in batch culture. (c) 1992 John Wiley & Sons, Inc.     

Localization and synthesis of hyaluronic acid in the cumulus cells and mural granulosa cells of the preovulatory follicle.

Mural and cumulus granulosa cells synthesize hyaluronic acid (HA) and expand in vitro in response to follicle-stimulating hormone and a soluble factor(s) produced by fully grown oocytes. In the present study we examined HA synthesis and extracellular matrix organization by the two cell populations in vivo during the preovulatory period. After injection of human chorionic gonadotropin into pregnant mares' serum gonadotropin-primed animals, a progressive increase in HA synthesis was observed by the cumulus cell-oocyte complex (COC), and by the mural granulosa cells adjacent to the antrum (antral granulosa cells). The outermost layers of mural granulosa cells (peripheral granulosa cells) did not synthesize HA. Net HA synthesis was approximately 4 pg/cell for COCs isolated after full expansion induced either in vivo or in vitro, whereas the total HA content and cell number in the ovulated COC (approximately 11 ng HA and approximately 3000 cells per COC) were about threefold higher than for COCs expanded in vitro (approximately 4 ng HA and approximately 1000 cells per COC). The increased cell content of ovulated COCs appears to be primarily the result of inclusion of proximal mural granulosa cells which synthesize HA in response to the oocyte factor(s) and become incorporated in the expanded COC extracellular matrix mass. Media conditioned by oocytes enclosed in the cumulus cell mass (intact COCs) contained only 10-20% of the HA-stimulatory activity of media conditioned by an equal number of isolated oocytes when tested on mural granulosa cell cultures. Further, HA-stimulatory activity of media conditioned by isolated oocytes was dramatically reduced (approximately 70%) by preincubation for 5 hr with cumulus cells compared to preincubation in the absence of cells. The results suggest that differences in HA synthesis between subregions of membrana granulosa depend on a diffusion gradient of the oocyte factor(s).     

Study by in situ hybridization of the prevalence of herpes virus as cofactors of the tumoral development of papillomatous lesions of the anogenital region in seropositive and seronegative men against HIV]

Infection with specific types of HPV has emerged as necessary but not sufficient factor in the neoplastic transformation of anogenital condylomas. Some viruses (HIV, Herpes viridae: HSV, CMV, EBV) might act as cofactors in the neoplastic changes and cancer. To study the prevalence of these viral pathogens in anogenital lesions, biopsies were obtained from HIV seropositive or seronegative men and tested using in situ hybridization technique. Infection by "high risk" HPV, HSV and CMV are facilitated in patients immunocompromised by HIV. Presence of CMV is more frequent in high risk HPV-induced lesions than in low risk HPV lesions.