Expression of human growth hormone-releasing factor in transgenic mice results in increased somatic growth

The neurohumoral regulation of growth hormone secretion is mediated in part by two hypothalamic peptides that reach the anterior pituitary via the hypothalamo-hypophysial portal blood system. Somatostatin inhibits the release of growth hormone, whereas growth hormone-releasing factor (GRF) positively regulates both growth hormone synthesis and secretion. Two forms of human GRF, 40 and 44 amino acids long, have been characterized from extra-hypothalamic tumours as well as from the hypothalamus. Analysis of human GRF complementary DNA and genomic clones indicates that the GRF peptides are first synthesized as a 107- or 108-amino-acid precursor protein. To examine the physiological consequences of GRF expression, we have established strains of transgenic mice containing a fusion gene including the promoter/regulatory region of the mouse metallothionein-I (MT-I) gene and the coding region of the human GRF gene. We report that expression of the human GRF precursor protein in these animals results in measurable levels of human GRF and increased levels of mouse growth hormone in plasma and accelerated growth rates relative to control littermates. These results demonstrate a direct role for GRF in the positive regulation of somatic growth. Unexpectedly, female transgenic mice carrying the MT-GRF fusion gene are fertile, in contrast to female transgenic mice expressing human or rat growth hormone, which are generally infertile. These transgenic mouse strains should provide useful animal models for the study of several types of human growth disorders.     

Expression of a microinjected immunoglobulin gene in the spleen of transgenic mice

Transgenic mice were produced by microinjection of a rearranged, functional immunoglobulin kappa gene into fertilized mouse eggs and implantation of the microinjected embryos into foster mothers. Mice that integrated the injected gene were mated and the DNA, RNA and serum kappa chains of their offspring were analysed. The data from offspring of three different transgenic mice indicate that the microinjected gene is expressed in the spleen, but not the liver of mice which inherited the injected gene.     

Leptin reverses insulin resistance and diabetes mellitus in mice with congenital lipodystrophy.

Congenital generalized lipodystrophy (CGL) is a rare autosomal recessive disorder characterized by a paucity of adipose (fat) tissue which is evident at birth and is accompanied by a severe resistance to insulin, leading to hyperinsulinaemia, hyperglycaemia and enlarged fatty liver. We have developed a mouse model that mimics these features of CGL: the syndrome occurs in transgenic mice expressing a truncated version of a nuclear protein known as nSREBP-1c (for sterol-regulatory-element-binding protein-1c) under the control of the adipose-specific aP2 enhancer. Adipose tissue from these mice was markedly deficient in messenger RNAs encoding several fat-specific proteins, including leptin, a fat-derived hormone that regulates food intake and energy metabolism. Here we show that insulin resistance in our lipodystrophic mice can be overcome by a continuous systemic infusion of low doses of recombinant leptin, an effect that is not mimicked by chronic food restriction. Our results support the idea that leptin modulates insulin sensitivity and glucose disposal independently of its effect on food intake, and that leptin deficiency accounts for the insulin resistance found in CGL.     

A simple method for counting nuclei in the preimplantation mouse embryo

An easy and rapid method of counting the number of cells in the preimplantation mouse embryo is described. The procedure increases the speed with which large numbers of embryos can be processed using a simple squash technique. Cell numbers are determined by exposing the embryos to the fluorescent DNA-binding dye, Hoechst 33258, removing the zona pellucida and simply squashing the embryo and counting the number of fluorescent nuclei. An increase in fluorescent intensity and maintenance of nuclear conformation of the squashed preparations are greatly improved by the use of the non-ionic detergent Triton X-100. Viability of dye-treated fertilized one-cell and blastocyst stage embryos is maintained at least up to day 13 of pregnancy following transfer of the embryos to the uteri of pseudopregnant recipients. Additional uses for this staining technique are discussed.     

Production of transgenic rabbits, sheep and pigs by microinjection

Direct microinjection has been used to introduce foreign DNA into a number of terminally differentiated cell types as well as embryos of several species including sea urchin, Candida elegans, Xenopus, Drosophila and mice. Various genes have been successfully introduced into mice including constructs consisting of the mouse metallothionein-I (MT) promoter/regulator region fused to either the rat or human growth hormone (hGH) structural genes. Transgenic mice harbouring such genes commonly exhibit high, metal-inducible levels of the fusion messenger RNA in several organs, substantial quantities of the foreign growth hormone in serum and enhanced growth. In addition, the gene is stably incorporated into the germ line, making the phenotype heritable. Because of the scientific importance and potential economic value of transgenic livestock containing foreign genes, we initiated studies on large animals by microinjecting the fusion gene, MT-hGH, into the pronuclei or nuclei of eggs from superovulated rabbits, sheep and pigs. We report here integration of the gene in all three species and expression of the gene in transgenic rabbits and pigs.     

Specific expression of an elastase-human growth hormone fusion gene in pancreatic acinar cells of transgenic mice

Transfection of genes into tissue culture cell lines has demonstrated that relatively short DNA sequences can allow expression of immunoglobulin, insulin and chymotrypsin genes in their appropriate cell types. A definitive test of cell-specific gene expression, however, requires testing genes in every possible cell type, an experiment performed easily by introducing the gene in question into the germ line of an animal. Transfer of intact genes into mice has demonstrated that a mouse immunoglobulin kappa gene is expressed specifically in B lymphocytes, a rat elastase I gene is expressed specifically in pancreas and a chicken transferrin gene is expressed preferentially in liver. Mouse metallothionein-growth hormone fusion genes introduced into mice are preferentially expressed in the liver, consistent with the expression of endogenous metallothionein genes, but initial experiments with beta-globin genes have not revealed proper regulation. To identify the DNA elements required for pancreas-specific expression of the rat elastase I gene, we joined the 5'-flanking region of this gene to the human growth hormone (hGH) structural gene and introduced the fusion gene into mice. Here we demonstrate that a fusion gene containing only 213 base pairs (bp) of elastase I gene sequence directs expression of hGH in pancreatic acinar cells.     

Alpha-neurexins couple Ca2+ channels to synaptic vesicle exocytosis

Synapses are specialized intercellular junctions in which cell adhesion molecules connect the presynaptic machinery for neurotransmitter release to the postsynaptic machinery for receptor signalling. Neurotransmitter release requires the presynaptic co-assembly of Ca2+ channels with the secretory apparatus, but little is known about how synaptic components are organized. Alpha-neurexins, a family of >1,000 presynaptic cell-surface proteins encoded by three genes, link the pre- and postsynaptic compartments of synapses by binding extracellularly to postsynaptic cell adhesion molecules and intracellularly to presynaptic PDZ domain proteins. Using triple-knockout mice, we show that alpha-neurexins are not required for synapse formation, but are essential for Ca2+-triggered neurotransmitter release. Neurotransmitter release is impaired because synaptic Ca2+ channel function is markedly reduced, although the number of cell-surface Ca2+ channels appears normal. These data suggest that alpha-neurexins organize presynaptic terminals by functionally coupling Ca2+ channels to the presynaptic machinery.     

Influence of dihydroergotamine on the lipolytic system of isolated dog fat cells

Zusammenfassung Die Beeinflussung der Lipolyse isolierter Fettzellen des Hundes durch Dihydroergotamin (DHE) wurde untersucht. DHE stimuliert die Lipolyse ähnlich wie Noradrenalin. Insulin und Propranolol antagonisieren DHE. Die Adenyl-cyclase wird stimuliert, die Phosphodiesterase wird hingegen durch DHE nicht beeinflusst.     

Chiral recognition in dimerization of adsorbed cysteine observed by scanning tunnelling microscopy

Stereochemistry plays a central role in controlling molecular recognition and interaction: the chemical and biological properties of molecules depend not only on the nature of their constituent atoms but also on how these atoms are positioned in space. Chiral specificity is consequently fundamental in chemical biology and pharmacology and has accordingly been widely studied. Advances in scanning probe microscopies now make it possible to probe chiral phenomena at surfaces at the molecular level. These methods have been used to determine the chirality of adsorbed molecules, and to provide direct evidence for chiral discrimination in molecular interactions and the spontaneous resolution of adsorbates into extended enantiomerically pure overlayers. Here we report scanning tunnelling microscopy studies of cysteine adsorbed to a (110) gold surface, which show that molecular pairs formed from a racemic mixture of this naturally occurring amino acid are exclusively homochiral, and that their binding to the gold surface is associated with local surface restructuring. Density-functional theory calculations indicate that the chiral specificity of the dimer formation process is driven by the optimization of three bonds on each cysteine molecule. These findings thus provide a clear molecular-level illustration of the well known three-point contact model for chiral recognition in a simple bimolecular system.