Expression of the Raf-1 protein in rat brain during development and its hormonal regulation in hypothalamus

To study mechanisms involved in the sexual differentiation of the rat brain, the expression of the protein product of the proto-oncogene c-raf-1 (Raf-1) was examined. Biochemical and immunocytochemical analyses localized Raf-1 in embryonic rat brain regions and demonstrated hormonally induced changes in Raf-1 expression. For this study an affinity-purified anti-peptide antiserum specific for Raf-1 (NH-44) was used. Western blots revealed an approximately 77 kD polypeptide isolated in the cytosol of developing rat brains. Raf-1 levels were highest in the embryonic (E) day 22 female hypothalamus (HYP), and approximately twofold higher than levels detected in male HYP at E22 as determined by quantitative protein dot blot and semiquantitative Western blot analyses. Raf-1 levels in HYP were greater than those in either brain stem (BS) or cortex. Immunocytochemical analysis revealed high levels of Raf-1 in selective brain regions (e.g., the ventromedial nucleus in the HYP, the mitral cell layers in the main and accessory olfactory bulbs (OB), and the locus coeruleus) at E22 and postnatal (P) day I. Lower levels of immunoreactivity were observed in many areas of the perinatal neuraxis. To test hormonal regulation of Raf-1, testosterone propionate (TP) was administered to pregnant rats on E17; male and female fetuses were examined on E22. This treatment significantly decreased Raf-1 levels in female HYP, but not in male HYP, as determined by Western blot analysis. No significant sex difference or response to prenatal hormone treatments were observed in either brain stem or cortex. No significant sex difference was noted postnatally, and administration of TP 3 h after birth did not change Raf-1 levels examined 24 h later. In summary, Raf-1 was localized within selective regions of the rat brain, and its expression was altered by exogenous prenatal hormonal stimulation. One role for Raf-1 in signal transduction may be to delimit hormonal critical periods in sexual differentiation of the brain.     

Impaired hormonal regulation of enzyme activity during aging.

A general feature of all aging populations is the progressively impaired ability to adapt to changes in the surrounding environment. Biochemical expressions of adaptive response include modifications in the rates of enzyme synthesis and degradation, as well as alterations in physiological activity. Therefore, the effects of aging on enzyme adaptation were surveyed in an attempt to explore fundamental biochemical mechanisms in the deterioration of responsiveness. The ability to stimulate adaptive increases in the activity of a large number of enzymes is impared during aging in a variety of tissues from several different species. The impaired capability for liver enzyme adaptation in a rigorously controlled colony of aging male Sprague-Dawley rats probably reflects alterations in hormonal control mechanisms. The present article reviews and evaluates our interest in understanding the effects of aging on regulation of liver enzyme activity by the hormones, insulin and corticosterone. Specific areas currently under investigation include: (1) the regulation of their concentrations in blood; (2) the integrity of their receptor systems in liver; and (3) effectiveness of the endogenous hormone pools fromthe viewpoints fothe availability of physiological antagonists and the potential for alterations in molecular structure.     

Expression of the Raf-1 protein in rat brain during development and its hormonal regulation in hypothalamus.

To study mechanisms involved in the sexual differentiation of the rat brain, the expression of the protein product of the proto-oncogene c-raf-1 (Raf-1) was examined. Biochemical and immunocytochemical analyses localized Raf-1 in embryonic rat brain regions and demonstrated hormonally induced changes in Raf-1 expression. For this study an affinity-purified anti-peptide antiserum specific for Raf-1 (NH-44) was used. Western blots revealed an approximately 77 kD polypeptide isolated in the cytosol of developing rat brains. Raf-1 levels were highest in the embryonic (E) day 22 female hypothalamus (HYP), and approximately twofold higher than levels detected in male HYP at E22 as determined by quantitative protein dot blot and semiquantitative Western blot analyses. Raf-1 levels in HYP were greater than those in either brain stem (BS) or cortex. Immunocytochemical analysis revealed high levels of Raf-1 in selective brain regions (e.g., the ventromedial nucleus in the HYP, the mitral cell layers in the main and accessory olfactory bulbs (OB), and the locus coeruleus) at E22 and postnatal (P) day 1. Lower levels of immunoreactivity were observed in many areas of the perinatal neuraxis. To test hormonal regulation of Raf-1, testosterone propionate (TP) was administered to pregnant rats on E17; male and female fetuses were examined on E22. This treatment significantly decreased Raf-1 levels in female HYP, but not in male HYP, as determined by Western blot analysis. No significant sex difference or response to prenatal hormone treatments were observed in either brain stem or cortex. No significant sex difference was noted postnatally, and administration of TP 3 h after birth did not change Raf-1 levels examined 24 h later. In summary, Raf-1 was localized within selective regions of the rat brain, and its expression was altered by exogenous prenatal hormonal stimulation. One role for Raf-1 in signal transduction may be to delimit hormonal critical periods in sexual differentiation of the brain.     

Molecular aspects of stress-gene regulation during spaceflight

Spaceflight-associated stress has been the topic of investigation since the first terrestrial organisms were exposed to this unique environment. Organisms that evolved under the selection pressures of earth-normal environments can perceive spaceflight as a stress, either directly because gravity influences an intrinsic biological process, or indirectly because of secondary effects imparted by spaceflight upon environmental conditions. Different organisms and even different organs within an organism adapt to a spaceflight environment with a diversity of tactics. Plants are keenly sensitive to gravity for directed development, and are also sensitive to other stresses associated with closed-system spaceflight environments. Within the past decade, the tools of molecular biology have begun to provide a sophisticated evaluation of spaceflight-associated stress and the genetic responses that accompany metabolic adaptation to spaceflight.     

Enterochromaffin cells in the chicken duodenum during prenatal development

Summary The development of enterochromaffin cells (EC) was studied in the duodenum of the chicken during prenatal development.Basally granular cells, i.e. EC were first found at the 14th incubation day and thereafter their number increased greatly during further development, as did also the number of specific osmiophilic granules in the cells.First empty or pale granules were found in the cytoplasmic area showing various kinds of saccules and vacuoles, possibly representing the poorly developed Golgi area. The primitive granules seemed to turn rapidly in to granules with high affinity for osmium tetroxide.The differentiation of EC occurred in the epithelial lining of the intestinum and no morphologically similar cells were found in other intestinal layers.This work was supported by a grant from the Yrjö Jahnsson Foundation, Helsinki. The electron microscopic observations were made in the Electron Microscope Laboratory, University of Helsinki; the skillful assistance of Mr. M. Nyholm is acknowledged.     

Spatial, temporal, and hormonal regulation of epidermal keratin expression during development of the frog, Xenopus laevis.

To study the mechanism of hormone-induced keratin expression in the epidermis during Xenopus metamorphosis, a monospecific antibody was raised against a unique carboxy-terminal peptide of the 63-kDa keratin. Immunohistological analysis demonstrated that the onset of 63-kDa keratin expression showed distinct regional and temporal differences. The expression started at stage 54 in the hindlimb epidermis, at stage 57 in the head, and over 1 month later at stage 63 in the tail. The amount of 63-kDa keratin was further regulated during epidermal stratification and differentiation. The 63-kDa keratin was expressed first in basal epidermal cells before stratification began. The outer layer of the larval epidermis (periderm) did not express the 63-kDa keratin. As the cells moved out of basal layer, they stained more intensely with the anti-keratin antibody indicating that 63-kDa keratin synthesis is up-regulated during differentiation. Similar results were obtained with cultures of purified epidermal cells grown in high calcium conditions. Since we have shown that thyroid hormone (T3) induces 63-kDa keratin gene expression and hydrocortisone (HC) modulates T3 action we examined the effects of T3 and HC at the single cell level with the anti-keratin antibody. Immunostaining demonstrated that T3 alone and T3 plus HC increased the number of 63-kDa keratin-positive cells as well as the amount of 63-kDa keratin per cell. Unexpectedly these hormones had the same effects on head and tail epidermal cells even though the latter cells degenerate during metamorphosis. The major difference between tail and head cells was that the percentage 63-kDa keratin-producing cells was much greater in the head than in the tail.     

Basigin expression and hormonal regulation in mouse uterus during the peri-implantation period

Basigin, a transmembrane glycoprotein belonging to the immunoglobulin superfamily, has been shown to be essential for fertilization and implantation. The aim of this study was to determine the expression and hormonal regulation of basigin gene in mouse uterus during the peri-implantation period. Basigin immunostaining and mRNA were strongly localized in luminal and glandular epithelium on day 1 of pregnancy and gradually decreased to a basal level from day 2-4 of pregnancy. Basigin mRNA expression in the sub-luminal stroma was first detected on day 3 of pregnancy and increased on day 4 of pregnancy. On day 5 of pregnancy, the expression of basigin protein and mRNA was only detected in the implanting embryos, and the luminal epithelium and sub-luminal stroma surrounding the embryos. A similar expression pattern of basigin was also induced in the delayed-implantation uterus which was activated by estrogen injection. On day 6-8 of pregnancy, although a basal level of basigin protein was detected in the secondary decidual zone, basigin mRNA expression was strongly seen in this location. Basigin mRNA was also highly expressed in the decidualized cells under artificial decidualization. Estrogen significantly stimulated basigin expression in the ovariectomized mouse uterus. A high level of basigin immunostaining and mRNA was also seen in proestrus and estrus uteri. These results suggest that basigin expression is closely related to mouse implantation and up-regulated by estrogen.     

Hormonal regulation of the production of serum albumin by cultured hepatocytes of rats during prenatal and postnatal period of development

The effects of several hormones on the production of immunoreactive serum albumin (SA) were examined in primary cultures of liver cells obtained from rat fetuses on 21-22 days of gestation of from 3-week old rats. Cortisol, bovine insulin and human growth hormone stimulated SA production in both types of liver cell cultures during 20 h-incubation. L-Triiodothyronine (T3; 10(-9)-10(-7) M) weakly stimulated SA production by hepatocytes from the rats, but markedly inhibited it in cultures of fetal rat liver cells in a dose-dependent manner. In contrast, T3 action on total RNA and protein biosynthesis, estimated as the incorporation of labelled precursors into macromolecules, was stimulatory one in both types of cell cultures. It is concluded that hormonal regulation of SA production is similar in cultured liver cells from fetal and early postnatal rats except for the action of T3. The physiological importance of striking developmental change of T3 action on SA production remains to be determined.