Differences in growth response to hydrocortisone and ascorbic acid by human diploid fibroblasts

Summary The effects of hydrocortisone and ascorbic acid on growth parameters were measured in human diploid skin fibroblasts from fetal and adult donors. In the presence of culture medium containing 10% fetal bovine serum, 0.3 μM hydrocortisone produced a 20% increase in the population growth rate and a 50 to 70% increase in the confluent density of fibroblasts from adult donors. Daily addition of 28 μM ascorbic acid also stimulated the population growth rate and cell density at confluency. The effects of hydrocortisone and ascorbic acid on the final cell density were additive. The action of hydrocortisone was restricted to cells in log-phase growth, whereas ascorbic acid affected cells in both the log and the postconfluent phases of the growth cycle. In fibroblasts from fetal donors, ascorbic acid was stimulative but hydrocortisone was not. The data suggest that whereas both compounds stimulate cell growth in an additive manner, they do so by different cellular mechanisms. This investigation was supported in part by USPHS Grants AM 02456, AM 05020 and AM 15312, and by the Kroc Foundation, No. UW 63-2986. Dr. Rowe is a fellow of the Helen Hay Whitney Foundation. Dr. Fujimoto is a recipient of a Research Career Development Award, AM 47142, from NIAMDD.     

Duplication of the segment q12.2→qter of chromosome 22 due to paternal inversion 22(p13q12.2)

Summary A 1730-g male infant, born at 37 weeks gestation, had multiple congenital anomalies, consisting of microcephaly, hypertelorism, bilateral cleft lip and palate, micrognathia, lowset ears, and cryptorchidism. Chromosome analysis showed a recombinant 22 derived from the paternal inversion (22) (p13q12.2). The proband's karyotype is 46,XY,rec(22),dup q,inv(22)(p13q12.2)pat, which has a duplication of q12.2qter. An identical recombinant has been reported in a female infant in Mexico whose mother was a carrier of the inversion. Similar congenital anomalies present in these two patients demonstrate the phenotype of duplication of the distal long arm 22. This report also documents the occurrence of an identical inversion in two apparently unrelated Mexican families.     

Translation of human interleukin 2 mRNA in Xenopus laevis oocytes

Poly(A)-positive mRNA extracted from tonsillar mononuclear cells stimulated with phytohemagglutinin-M and 12-o-tetradecanoyl phorbol 13-acetate was successfully translated into biologically active interleukin 2 (IL-2) in Xenopus laevis oocytes, and secreted into the incubation medium. In control experiments, the extract of oocytes injected with either poly(A)-negative RNA or buffer did not show any IL-2 activity. By sucrose density gradient centrifugation analysis, IL-2 mRNA was found as a single peak corresponding to a sedimentation coefficient of 10-11S.     

Detection of brain-specific gene expression in brain cells in primary culture: a novel promoter assay based on the use of a retrovirus vector.

Promoter activities of the brain-specific genes for glial fibrillary acidic protein (GFAP) and myelin basic protein (MBP) were investigated in brain cells in primary culture with the use of a novel retrovirus vector, pIP200. With this vector, promoter activity can be expressed in terms of beta-galactosidase activity. Differentiation of the primary brain cells to mature glial cells was not affected by treatment with the pIP200 virus vector. The 256-bp 5'-flanking region of the GFAP gene directed astrocyte-specific expression of lacZ. It was silent in fibroblasts, even in multiple copies. The 1.3-kb 5'-flanking region of the MBP gene exhibited strict tissue (oligodendrocyte) specificity under the present assay method but showed some leakiness when integrated into the chromosome in multiple copies. Promoter regions conferring cell type specificity in brain were effectively identified by the present method.     

Gray and Red Fragments of the Egg of the Ascidian Ciona savignyi: Preferential Development of Muscle Cells from Gray Fragments

The egg of the ascidian Ciona savignyi is pinkish red with brownish myoplasm that contains the putative determinants responsible for differentiation of muscle cells. When dechorionated unfertilized eggs were centrifuged at moderate speed, eggs were divided into centripetal, small gray fragments and centrifugal, large red fragments. The former contained the female pronucleus and clear cytoplasm, while most of the latter was filled with yolk granules. An antibody raised against the myoplasm of C. intestinalis eggs extensively stained the cortical region of gray fragments, while the antibody stained only small regions of the red fragments. After insemination, both fragments cleaved and gave rise to partial embryos. When development of muscle and epidermal cells in the partial embryos was examined with specific antibodies, muscle development was conspicuous in gray partial embryos, while epidermal differentiation was extensive in red partial embryos. Furthermore, when expression of markers of differentiation was examined in cleavage-arrested gray and red fragments, the number of arrested gray fragments exhibiting the muscle marker was about three-fold greater than in controls. These results suggest that putative muscle determinants are concentrated into gray fragments.     

Regulation by thyrotropin-releasing hormone (TRH) of TRH receptor mRNA degradation in rat pituitary GH3 cells.

In rat pituitary GH3 cells, thyrotropin-releasing hormone (TRH) down-regulates TRH receptor (TRH-R) mRNA (Fujimoto, J., Straub, R.E., and Gershengorn, M.C. (1991) Mol. Endocrinol. 5, 1527-1532), at least in part, by stimulating its degradation (Fujimoto, J., Narayanan, C.S., Benjamin, J.E., Heinflink, M., and Gershengorn, M.C. (1992) Endocrinology 130, 1879-1884). Here we show that TRH regulates RNase activity in GH3 cells and that specific mRNA sequences are needed for in vivo regulation of TRH-R mRNA by TRH. TRH affected RNase activity in a biphasic manner with rapid stimulation (by 10 min) followed by a decrease to a rate slower than in control lysates within 6 h. This time course paralleled the effects of TRH on degradation of TRH-R mRNA in vivo. The regulated RNase activity was in a polysome-free fraction of the lysates and was not specific for TRH-R RNA. A truncated form of TRH-R RNA that was missing the entire 3'-untranslated region (TRHR-R5) was more stable than full-length TRH-R RNA (TRHR-WT). In contrast to TRHR-WT mRNA, TRHR-R5 mRNA and TRHR-D9 mRNA, which was missing the 143 nucleotides 5' of the poly(A) tail, were not down-regulated by TRH in stably transfected GH3 cells as their rates of degradation were not increased. These data show that TRH regulates RNase activity in GH3 cells, that the 3'-untranslated region bestows decreased stability on TRH-R mRNA and that the 3' end of the mRNA is necessary for regulation by TRH of TRH-R mRNA degradation. We present an hypothesis that explains specific regulation of TRH-R mRNA degradation by TRH in GH3 pituitary cells.     

Localization of inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae

Activation of various receptors by extracellular ligands induces an influx of Ca2+ through the plasma membrane, but its molecular mechanism remains elusive and seems variable in different cell types. In the present study, we utilized mAbs generated against the cerebellar type I inositol 1,4,5-trisphosphate (InsP3) receptor and performed immunocytochemical and immunochemical experiments to examine its localization in several non-neuronal cells. By immunogold electron microscopy of ultrathin frozen sections as well as permeabilized tissue specimens, we found that a mAb to the type I InsP3 receptor (mAb 4C11) labels the plasma membrane of the endothelium, smooth muscle cell and keratinocyte in vivo. Interestingly, the labeling with the antibody was confined to caveolae, smooth vesicular inpocketings of the plasma membrane. The reactive protein, with an M(r) of 240,000 by SDS-PAGE, could be biotinylated with a membrane-impermeable reagent, sulfo-NHS-biotin, in intact cultured endothelial cells, and recovered by streptavidin-agarose beads, which result further confirmed its presence on the cell surface. The present findings indicate that a protein structurally homologous to the type I InsP3 receptor is localized in the caveolar structure of the plasma membrane and might be involved in the Ca2+ influx.     

Non-isopeptide-selective endothelin receptors in human Girardi heart cells.

We characterized the endothelin (ET) receptor in Girardi heart (GH) cells derived from human atrium. The ET isopeptides ET-1, ET-2 and ET-3 induced the monotonous and long-lasting rise in cytosolic free Ca2+ concentration [( Ca2+]i) with almost the same potency in GH cells. Scatchard analysis of [125I]ET-1 and [125I]ET-3 binding revealed that GH cells have almost the same number of binding sites for either labeled ligand. All ET isopeptides displaced either [125I]ET-1 or [125I]ET-3 binding in GH cells almost equipotently. These results reveal that the functional ET receptors in GH cells are of the ETB-type. GH cells are the first cell line to be found to express the functional ETB-receptor.