Stimulation of the hypothalamic-pituitary-adrenal axis and inhibition of growth hormone release via increased central noradrenaline neuronal activity by urethane anaesthesia in the rat: blockade by clonidine

Computerized gas chromatography-mass spectrometry was used to measure precisely the hypothalamic levels of noradrenaline (NA), dopamine and serotonin together with those of their major neuronal metabolites 3,4-dihydroxyphenylethyleneglycol (DHPG), 3,4-dihydroxyphenylacetic acid and 5-hydroxyindoleacetic acid in normal male rats 45 min after stimulation of hypothalamic-pituitary-adrenal function by urethane (1.3 g/kg) administration. Urethane treatment resulted in a significant elevation of central noradrenergic neuronal activity (NNA) as assessed from marked rises in hypothalamic DHPG concentrations and the ratio (DHPG/NA). At the same time there was significant stimulation of ACTH and corticosterone release and inhibition of growth hormone release. These hormonal and central effects of urethane (but not anesthesia) were inhibited when the alpha 2-agonist clonidine (150 micrograms/kg) was co-administered. Urethane had no major effect on hypothalamic dopamine or serotonin status. We propose that the release of ACTH and the suppression of growth hormone release following urethane anaesthesia is a result of activation of central NNA and suggest that the hormonal responses are mediated via hypothalamic noradrenergic facilitation of corticotrophin releasing factor and somatostatin release to the anterior pituitary.     

Isolation and structural analysis of a peptide containing the novel tyrosyl-glucose linkage in glycogenin.

The glucosylation site on glycogenin, the protein primer required for de novo glycogen synthesis, has been identified. The glucose is attached at position C1 in a glycosidic linkage with a unique tyrosine, and the sequence surrounding this residue was found to be: His-Leu-Pro-Phe-Ile-Tyr-Asn-Leu-Ser-Ser-Ile-Ser-Ile-Tyr(Glc)-Ser-Tyr-Leu -Pro- Ala-Phe-Lys. The same tyrosine residue is glycosylated whether glycogenin is isolated as a complex with the catalytic subunit of glycogen synthase, or covalently attached to glycogen. The possibility that insulin and growth factors may enhance glycogen synthesis via stimulation of the priming reaction is discussed.     

Different acute effects of the tyrosine hydroxylase inhibitors alpha-methyl-p-tyrosine and 3-iodo-L-tyrosine on hypothalamic noradrenaline activity and adrenocorticotrophin release in the rat

Computerized gas chromatography-mass spectrometry techniques using selected ion monitoring and deuterated internal standards were used to assay simultaneously the medial basal hypothalamic concentrations of dopamine (DA) and noradrenaline (NA) and their major metabolites in individual rats 30 min after the administration of two different inhibitors of tyrosine hydroxylase, alpha-methyl-p-tyrosine (alpha-MT) and 3-iodo-L-tyrosine (MIT). Consistent with inhibition of DA synthesis, administration of both alpha-MT and MIT resulted in marked reductions (P less than 0.005) in the hypothalamic concentrations of DA and its metabolite homovanillic acid as well as in highly significant increases in prolactin secretion. alpha-MT administration, but not MIT, resulted in a highly significant decrease in NA concentration and a highly significant increase in the concentration of the NA metabolite 3,4-dihydroxyphenylethyleneglycol (DHPG). The hypothalamic ratio DHPG/NA was thus markedly increased (P less than 0.005) by alpha-MT indicating increased NA neuronal activity. alpha-MT administration also resulted in increased ACTH secretion (P less than 0.0005), an effect not observed following MIT. It is proposed that the effects on hypothalamic NA activity and ACTH secretion caused by alpha-MT are stress-mediated and unrelated to tyrosine hydroxylase inhibition. MIT is devoid of these effects but exhibits blockade activity, thus indicating it to be a preferable drug for the acute inhibition of tyrosine hydroxylase in neuroendocrine investigations.     

Isolation and characterization of a large,neurite-associated glycoconjugate from neuroblastoma cells

A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as I(0) based on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. I(0) is found in the substratum-attached material or cell fraction of neurite-producing neuroblastoma cells, depending upon culture conditions. It is found in the substratum-bound fraction of B104 rat neuroblastoma cells during serum starvation and in the EGTA-detached cell fraction of B104 cells grown in chemically defined N2 medium. It occurs only in the cell fraction of the human neuroblastoma line Platt. Examination of behavioral variants of the B104 rat line further strengthens the association of I(0) with neurite production; the constitutive neurite-producing E(R)B9 variant contains I(0) while the non-neurite-producing E(R)A11 variant does not. I(0) is large, eluting in the void volume of sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows I(0) to be a cell surface component. Metabolic radiolabeling studies show that it contains a high proportion of polysaccharide to protein, does not contain mannose, and is unsulfated. Alkaline borohydride reduction release two size classes of large polysaccharide chain. The alkaline reduction results, along with the mannose incorporation studies, show the presence of O-glycosidic linkages and few, if any, N-linkages. Resistance to nitrous acid deamination, insensitivity to glycosaminoglycan lyases, and the absence of sulfation, indicate that I(0) does not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparin- sulfates. Affinity column chromatography reveals high binding affinity of I(0) to polyornithine and no binding to gelatin (collagen) or the glycosaminoglycans hyaluronate and heparin. These studies describe a unique high molecular weight glycoconjugate on the surface of neurite-producing neuroblastoma cell lines from two species.     

Relative apparent synapomorphy analysis (RASA). I: The statistical measurement of phylogenetic signal

We have developed a new approach to the measurement of phylogenetic signal in character state matrices called relative apparent synapomorphy analysis (RASA). RASA provides a deterministic, statistical measure of natural cladistic hierarchy (phylogenetic signal) in character state matrices. The method works by determining whether a measure of the rate of increase of cladistic similarity among pairs of taxa as a function of phenetic similarity is greater than a null equiprobable rate of increase. Our investigation of the utility and limitations of RASA using simulated and bacteriophage T7 data sets indicates that the method has numerous advantages over existing measures of signal. A first advantage is computational efficiency. A second advantage is that RASA employs known methods of statistical inference, providing measurable sensitivity and power. The performance of RASA is examined under various conditions of branching evolution as the number of characters, character states per character, and mutations per branch length are varied. RASA appears to provide an unbiased and reliable measure of phylogenetic signal, and the general approach promises to be useful in the development of new techniques that should increase the rigor and reliability of phylogenetic estimates.      

Molecular evolution of voltage-sensitive ion channel genes: on the origins of electrical excitability

We have analyzed nucleic acid and amino acid sequence alignments of a variety of voltage-sensitive ion channels, using several methods for phylogenetic tree reconstruction. Ancient duplications within this family gave rise to three distantly related groups, one consisting of the Na+ and Ca++ channels, another the K+ channels, and a third including the cyclic nucleotide-binding channels. A series of gene duplications produced at least seven mammalian homologues of the Drosophila Shaker K+ channel; clones of only three of these genes are available from all three mammalian species examined (mouse, rat, and human), pointing to specific genes that have yet to be recovered in one or another of these species. The Shaw-related K+ channels and the Na+ channel family have also undergone considerable expansion in mammals, relative to flies. These expansions presumably reflect the needs of the high degree of physiological and neuronal complexity of mammals. Analysis of the separate domains of the four-domain channels (Ca++ and Na+) supports their having evolved by two sequential gene duplications and implies the historical existence of a functional two-domain channel.      

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.      

促甲状腺激素对甲状腺细胞胞内游离钙和钙调蛋白的影响

本文研究TSH和forskolin对原代培养的猪甲状腺细胞[Ca~(2+)]_i和钙调蛋白的影响。结果表明,TSH可引起甲状腺细胞[Ca~(2+)]_1急性升高。此反应是剂量依赖关系,而与细胞外钙的存在与否无关。其反应性在细胞单层高于细胞是液,近汇合细胞单层高于汇合细胞单层。TSH作用3天,可使甲状腺细胞的钙调蛋白含量增高,此作用与TSH对甲状腺细胞数的影响无关。Forskolin对甲状腺细胞的[Ca~(2+)]_i和钙调蛋白均无明显的影响。     

Cytosol- and clathrin-dependent stimulation of endocytosis in vitro by purified adaptors

Using stage-specific assays for receptor-mediated endocytosis of transferrin (Tfn) into perforated A431 cells we show that purified adaptors stimulate coated pit assembly and ligand sequestration into deeply invaginated coated pits. Late events in endocytosis involving membrane fission and coated vesicle budding which lead to the internalization of Tfn are unaffected. AP2, plasma membrane adaptors, are active at physiological concentrations, whereas AP1, Golgi adaptors, are inactive. Adaptor-dependent stimulation of Tfn sequestration requires cytosolic clathrin, but is unaffected by clathrin purified from coated vesicles suggesting that soluble and assembled clathrin pools are functionally distinct. In addition to adaptors and cytosolic clathrin other, as yet unidentified, cytosolic factors are also required for efficient coated pit invagination. These results provide new insight into the mechanisms and regulation of coated pit assembly and invagination.