Involvement of CCAAT/enhancer-binding protein and nuclear factor-kappa B binding sites in interleukin-6 promoter inhibition by estrogens.

Bone loss observed in postmenopausal women is clearly associated with a decrease in estrogen levels. Interleukin 6 (IL-6), a multifunctional cytokine involved in osteoclast differentiation, is secreted by osteoblasts and appears to be a key molecule in the osteoporotic process. As previous reports have shown that the human IL-6 promoter is inhibited by estradiol, we investigated the mechanism of estradiol (E2)-mediated IL-6 inhibition in human cells. Analysis of the IL-6 secretion as a function of time in osteoblastoma Saos-2 cells, using an IL-6 ELISA test, showed that a maximal E2 inhibition of tumor necrosis factor-alpha (TNF alpha) induction could be monitored between 2 and 24 h of treatment. IL-6 inhibition was clearly estrogen agonist-specific in Saos-2 and MCF7 cells. Transient transfections of HeLa cells with a pIL-6/CAT plasmid and an estrogen receptor (human ER) expression vector, confirmed the role of the human ER in inhibition of the IL-6 promoter. Deletion and mutational analysis of the promoter highlighted the role of the -185/-60 region and showed that in both MCF7 and HeLa cells, the nuclear factor-IL 6 (NF-IL6) site cooperates with the nuclear factor-kappa B (NF-kappa B) motif to produce maximal induction by TNF alpha, whereas the CCAAT/enhancer-binding protein (C/EBP) site displayed different cooperative effects toward NF-kappa B depending on the cell line used. In HeLa cells, but not in MCF7 cells, we defined an essential role for the C/EBP site by showing that the E2 sensitivity was clearly dependent on its integrity. In these cell lines, the NF-kappa B site mutation abrogated both the TNF alpha-and E2- sensitivity of the construct.

Characterization of the 5'-flanking region of the human preprogalanin gene.

Expression of the human galanin gene was analysed using a 3.5-kb DNA fragment comprising the 5'-flanking sequence of the gene. This sequence contains a TATA box (ATATATA) preceded by numerous potential binding sites for transcription factors such as SP1, AP2, and NF kappa B. Three half-palindromic estrogen response elements (EREs, GGTCA) are also found at positions -1,162, -361, and -122 bp relative to the transcription start site. To localize functionally important portions of the promoter region, several shorter fragments of the galanin 5'-flanking region were placed upstream from the chloramphenicol acetyltransferase (CAT) reporter gene. In transient transfection assays, all constructs demonstrated substantial transcriptional activity in both rat glioma/mouse neuroblastoma hybrid cells (NG108-15) and Chinese hamster ovary (CHO-K1) cells. Comparison of the basal expression levels of the different constructs suggests the presence of a negative modulator between positions -1,891 and -207. When cotransfected into NG108-15 cells with the human estrogen receptor cDNA, estrogen did not induce transcription of the human galanin gene at physiological levels of estrogen receptor, although transcription was induced up to 30-fold in the presence of high levels of receptor.

Localisation of mRNA encoding the protein precursor of galanin in the monkey hypothalamus and basal forebrain.

The hypothalamic and basal forebrain sites of synthesis of preprogalanin mRNA were identified in three adult monkeys (Macaca fascicularis) by in situ hybridisation performed with a radiolabelled cRNA probe transcribed from human preprogalanin cDNA. With stringent hybridisation conditions, the cRNA probe was hybridised to free-floating sections containing structures contiguous with the rostral hypothalamus through to the caudal limit of the hypothalamus as defined by the mammillary bodies. Specific hybridisation of the preprogalanin cRNA probe occurred throughout the hypothalamus but was particularly intense in the arcuate, paraventricular (parvicellular and magnocellular portions), and dorsomedial nuclei. Moderate hybridisation was found in the periventricular nucleus and scattered hybridisation in the medial preoptic nucleus. The medial preoptic area and the anterior and lateral hypothalamic areas showed moderate to intense hybridisation in scattered cells. A few cells in the tuberal portion and dorsal cap of the anterior portion of the supraoptic nucleus were labelled. Isolated cells were also labelled in the zona incerta. There was little labelling in the dorsal hypothalamic area but moderate labelling in the posterior hypothalamic area. Structures contiguous with the rostral hypothalamus including the diagonal band of Broca, bed nucleus of stria terminalis, substantia innominata, and basal nucleus of Meynert showed intense hybridisation. These data indicate a widespread distribution of preprogalanin mRNA in the monkey hypothalamus. A comparison with the previously reported distribution of preprogalanin mRNA in the rat, as well as with the distribution of galanin-like immunoreactivity in the rat and human, suggests some important species differences. Of particular interest were differences in the supraoptic, suprachiasmatic, and dorsomedial nuclei. The intense hybridisation throughout the paraventricular nucleus and in the rostral arcuate nucleus suggests that galanin may play a role in the regulation of both posterior and anterior pituitary function.

Localization of preprogalanin mRNA in the monkey hippocampal formation.

The existence of neurons expressing preprogalanin mRNA in the monkey hippocampal formation was demonstrated using in situ hybridization of a radio-labelled cRNA probe transcribed from human preprogalanin cDNA. Specific hybridization occurred in neurons of the hilus of the dentate gyrus, fields CA1-3 in Ammon's horn, subiculum, presubiculum, parasubiculum and occasionally in neurons of the entorhinal cortex. These findings suggest that galanin is synthesized by neurons intrinsic to the monkey hippocampal formation.

Effects of human, rat and porcine galanins on cardiac vagal action and blood pressure in the anaesthetised cat.

Galanin (GAL) is distributed in sympathetic nerves in the cat, and exogenous GAL inhibits cardiac vagal action and lowers blood pressure in this species. This study on anaesthetised cats compares the effects on cardiac vagal action and blood pressure of human, rat and porcine GAL. Human GAL has only recently been sequenced. It is of particular interest as it is not C-terminally amidated, unlike porcine and rat GAL. Many regulatory peptides require a C-terminal amide group for their action. However, human GAL showed similar biological activity to the other (amidated) GALs here. Omission of a single amino acid (Ser6) from rat GAL significantly attenuated both cardiovascular actions studied here.

Human galanin: molecular cloning reveals a unique structure.

Galanin, a novel neuropeptide/hypothalamic hormone originally identified and isolated by virtue of its carboxy-terminal amide group, has recently been shown to have a diverse range of biological activities, including potent effects on the secretion of insulin and growth hormone. The physiological role of galanin remains unclear, with different effects being observed when porcine and rat galanin have been used in various animal model systems and in human studies. Molecular cloning of cDNA encoding human galanin and galanin mRNA associated peptide (GMAP) from both pituitary and neuroblastoma sources has revealed a unique and unexpected structure. In contrast to porcine, bovine and rodent galanin, human galanin lacks a carboxy-terminal amide. By analogy to other neurohormones, the absence of carboxy-terminal amidation would be expected to have significant effects on functional properties such as affinity for different receptor subtypes and physiological half life, and may be responsible for the species specificity observed in the action of galanin.

Dynamics of Baculovirus Growth and Dispersal in Mamestra brassicae L. (LepidopteraNoctuidae) Larval Populations Introduced into Small Cabbage Plots.

The nuclear polyhedrosis virus of Mamestra brassicae has been studied in larval populations of the moth introduced into small plots of cabbages. Primary dispersal of virus from single foci of infected larvae resulted from enhanced movement of the larvae, which colonized new plants logarithmically. Virus growth within the host population was quantified, and infection of young larvae in the following generation was related directly to the concentration of virus produced during the primary phase. Secondary cycling of virus resulted in dispersal of inoculum from multiple foci, and a large proportion of plants were ultimately colonized by infected larvae. The dynamics of virus growth during secondary dispersal were quantified and contrasted with results from the primary phase. The significance of these results is discussed in relation to possible control of insect pests through dispersal of virus by the host insect.

The influence of larval maturation on responses of Mamestra brassicae L. (Lepidoptera: Noctuidae) to nuclear polyhedrosis virus infection.

Responses of mature Mamestrae brassica larvae, in the fifth and sixth instars, to nuclear polyhedrosis virus (NPV) have been quantified. There was an 86 fold decrease in susceptibility to virus, related linearly to increasing body weight, for the larval weight range tested. Although the rate of decreasing response to weight was constant, three phases of susceptibility were identified by shifts in the positions of the regression line of log LD50 on log body weight. Larvae above 700 mg were proportionately less susceptible than those between 250 and 700 mg which were, in turn, proportionately less susceptible than those below 250 mg. LT50 responses were inversely related to virus dosage but increased with larval age. This was accompanied by an increase in the proportion of infected larvae that eventually died in the pupal stage. The ratio of LT50 to time remaining for larval development provided a good predictor of the time course of NPV infection for all larval stages. Extensive comparison with a previous study on responses of immature M. brassicae larvae to NPV infection (6) have been included.

Biological and biochemical investigations on five European isolates of Mamestra brassica nuclear polyhedrosis virus.

Five multiply enveloped European isolates of Mamestra brassicae nuclear polyhedrosis virus (Oxford, German, French, Dutch and Danish) were found to be very closely related serologically using the enzyme linked immunosorbent assay (ELISA) double antibody sandwich method and immunodiffusion. By SDS-polyacrylamide gel electrophoresis of viral proteins and restriction endonuclease analysis of DNA using seven enzymes there appeared to be two variants as the Oxford and German isolates were distinct from the other three. The German isolate was shown to be more susceptible to Nonidet P40 detergent treatment affecting some nucleocapsid structural polypeptides which also reduced antigenicity in gel immunodiffusion plates. In bioassays of polyhedra, the Dutch isolate showed a higher LD50 than the other viruses although this was not statistically significant.

Growth of nuclear polyhedrosis virus in larvae of the cabbage moth, Mamestra brassicae L.

Growth of nuclear polyhedrosis virus (NPV) in 5 larval instars of cabbage moth, Mamestra brassicae, has been quantified using 2 methods. Numbers of polyhedra were estimated by light microscope counts while concentrations of virus protein antigen were estimated using ELISA. Virus growth was rapid initially but slowed during its later stages, although ELISA protein concentrations decreased once a peak had been reached. There was a linear correlation between polyhedral counts and virus protein during the initial growth phase. Maximum polyhedral production ranged from 2 x 107 (first instar) to 3.4 x 109 (fifth instar) and could be correlated directly to increasing larval weight. Using ELisa, virus antigen was detectable at least 24 hours before polyhedra were observed under the light microscope. Productivity ratios ranged from 83,500 in the first instar to 1352 in the fifth instar.