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.     

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.     

Heme A of cytochrome c oxicase. Structure and properties: comparisons with hemes B, C, and S and derivatives.

Heme A, isolated from bovine heart muscle by procedures which include extractions into pyridine/chloroform and two-phase, liquid-liquid chromatography on Celite, has been converted to several derivatives. Examination of the proton nuclear magnetic resonance (PMR) spectra and other properties of these derivatives reveals heme A to be the iron complex of 8-formyl-6,-m-bis(2'-hydroxycarbonylethyl)-2-(1'-hydroxy-5',9',13'-trimethyl-4',8',12'-trans,trans-tetradecatrienyl)-1,3,5-trimethyl-4-vinylporphin. Substituents at the 2,4, and 8 positions are replaced by hydrogen in a resorcinol melt to give cytodeuteroporphin (8-demethyldeuteroporphyrin IX)...     

An N-terminal GFP tag does not alter the functional expression to the plasma membrane of red cell and kidney anion exchanger (AE1) in mammalian cells

Two isoforms of the band 3 anion exchanger are expressed in mammalian cells, a 911 residue protein (B3) in red cells, and a truncated protein (KB3) in the &#102 -intercalated cells of the kidney. Mutants of both isoforms are known to be associated with human disease, and mistargeting of the mutated proteins has been suggested as the mechanism of pathogenesis in several cases but has been difficult to prove. The present study demonstrates the feasibility of using confocal microscopy for investigating the targeting of homozygous and heterozygous B3 and KB3 mutants. K562 erythroleukemia cells offer several advantages for the stable expression of B3, but have not previously been used for its visualization. A wide range of cell attachment factors, growth conditions, fixation reagents and primary antibodies were investigated to enable imaging of B3 and endogenous GPA by immunofluorescence confocal microscopy in stable K562/B3 clones. B3 co-localized with GPA at the cell surface and also in an intracellular compartment. Functional cell surface expression of KB3 in stable K562 clones was also obtained. Importantly, both B3 and KB3 could be expressed as stable fusion proteins tagged with green fluorescent protein (GFP) in K562 cells, and it was demonstrated that N-terminal GFP-tagging does not affect the targeting or chloride transport properties of B3 or KB3. The use of GFP as well as double-labelling methods developed for immunostaining will be invaluable for investigating the interactions of band 3 with itself and other proteins during its trafficking in erythroid and kidney cells. This will help elucidate how band 3 mutations can cause human diseases such as hereditary spherocytosis and distal renal tubular acidosis.     

Identification of Leptospira biflexa by real-time homogeneous detection of rapid cycle PCR product

Sequence analysis of 16S rRNA genes extracted from nucleic acids databases enabled the identification of a Leptospira biflexa (L. biflexa) signature sequence, against which a reverse primer designated L613, was designed. This primer, when used in conjunction with a universal bacterial specific forward primer designated Fd1, enabled the development of a LightCycler-based PCR protocol in which fluorescence emission due to binding of SYBR Green I dye to amplified products could be detected and monitored. A melting temperature (Tm), determined from the melting curve of the amplified product immediately following the termination of thermal cycling, confirmed that the product was that of L. biflexa. Agarose gel electrophoresis therefore was not necessary for identification of PCR products. The PCR protocol was very rapid, and consisted of 30 cycles with a duration of 20 s for each cycle with the monitoring of the melting curve requiring an additional 3 min. The whole protocol was completed in less than 20 min. The PCR protocol was also specific and enabled the identification of 18 strains of L. biflexa, whilst excluding 14 strains of L. interrogans and Leptonema illini. Two examples of its utility in improving work flow of a Leptospira reference laboratory are presented in this article. The use of a simple boiling method for extraction of DNA from all the members of the Leptospiraceae family DNA further simplifies the procedure and makes its use conducive to diagnostic laboratories.     

Phosphorylation of threonine 156 of the mu2 subunit of the AP2 complex is essential for endocytosis in vitro and in vivo

The clathrin-coated pit is the major port of entry for many receptors and pathogens and is the paradigm for membrane-based sorting events in higher cells [1]. Recently, it has been possible to reconstitute in vitro the events leading to assembly, invagination, and budding off of clathrin-coated vesicles, allowing dissection of the machinery required for sequestration of receptors into these structures [2-6]. The AP2 adaptor complex is a key element of this machinery linking receptors to the coat lattice, and it has previously been reported that AP2 can be phosphorylated both in vitro and in vivo [7-10]. However, the physiological significance of this has never been established. Here, we show that phosphorylation of a single threonine residue (Thr156) of the mu2 subunit of the AP2 complex is essential for efficient endocytosis of transferrin both in an in vitro coated-pit budding assay and in living cells.