AnAnolis skin melanophore assay suitable for photoaffinity labeling studies with α-MSH

Summary Visual determination of MSH-induced pigment migration in melanophores of small pieces ofAnolis carolinensis skin is standardized by first measuring photoelectrometrically the change in reflection/transmission of the whole dorsal skin in response to different hormone concentrations. This method allows the rapid and precise recording of time-response curves after photoaffinity labeling of MSH receptors or of dose-response curves of large series of synthetic compounds.Acknowledgments. We wish to thank Ms V. Jäggin, Ms C. Schulthess and Ms G. van Hees for excellent technical assistance, Dr. R. Andreatta, Ciba-Geigy AG, Basel, for his generous gift of -MSH and Prof. A Pletscher for his continuous interest. This work was supported by the Swiss National Science Foundation.     

HIV F/3' orf encodes a phosphorylated GTP-binding protein resembling an oncogene product

Apart from the retroviral gag, pol and env the HIV genome contains the F (3' orf) gene which encodes a polypeptide of 206 amino acids which is myristylated at the N-terminal and whose function is unknown. We have expressed the F gene in Escherichia coli and from a recombinant vaccinia virus, VVTGfHIV. The F-protein produced in VVTGfHIV-infected mammalian cells is myristilated, and is phosphorylated by protein kinase C at a residue close to the N-terminus like pp60-src (ref. 5). Purified bacterial F-protein also shows the GTPase, autophosphorylation and GTP-binding activities reported for the ras gene product. Furthermore, we show that expression of F in a CD4+ cell line down-regulates the CD4(T4) antigen. These results suggest that F is important in the pathophysiology of AIDS (acquired immune deficiency syndrome).     

Subcellular patterns of calcium release determined by G protein-specific residues of muscarinic receptors.

Calcium release from intracellular stores is a point of convergence for a variety of receptors involved in cell signaling. Consequently, the mechanism(s) by which cells differentiate between individual receptor signals is central to transmembrane communication. There are significant differences in timing and magnitude of Ca2+ release stimulated by the m2 and m3 muscarinic acetylcholine receptors. The m2 receptors couple to a pertussis toxin-sensitive G protein to activate phosphatidyl inositol hydrolysis weakly and to stimulate small, delayed and oscillatory chloride currents. In contrast, m3 receptors potently activate phosphatidyl inositol hydrolysis and stimulate large, rapid and transient chloride currents by a pertussis toxin-insensitive G protein pathway. Using confocal microscopy, we now show that the m2- and m3-coupled Ca2+ release pathways can also be spatially distinguished. At submaximal acetylcholine concentrations, both receptors stimulated pulses of Ca2+ release from discrete foci in random, periodic and frequently bursting patterns of activity. But maximal stimulation of m2 receptors increased the number of focal release sites, whereas m3 receptors invariably evoked a Ca2+ wave propagating rapidly just beneath the plasma membrane surface. Analysis of pertussis toxin sensitivity and hybrid m2-m3 muscarinic acetylcholine receptors confirmed that these Ca2+ release patterns represent distinct cell signalling pathways.     

Human growth hormone in urine: development of an ultrasensitive radiometric assay

An immunoradiometric assay for human growth hormone (HGH) has been developed which has a detection limit of 1 ng/l and can measure HGH in unextracted urine from normal children and adults. The assay is based on a two-step procedure, using a solid-phase goat-anti-HGH immunosorbent for immunoextraction and [125I]-labeled monoclonal HGH-antibody for detection and quantification. The assay is not affected by urea, NaCl or changes of pH from 5-8. The mean urine HGH concentration in normal children is 6.78 +/- 7.6 (SD) pg/ml, in patients with HGH-deficiency 1.3 +/- 0.9 pg/ml which increases to 11.7 +/- 13.4 pg/ml on the day of growth hormone injection.     

Interaction of cortisol and epinephrine in the regulation of leucine kinetics in man

To assess the interaction of the two major stress hormones epinephrine and cortisol in the regulation of leucine kinetics in man, epinephrine (50 ng/kg/min) was infused either alone or in combination with cortisol (2 micrograms/kg/min) into two groups of 6 postabsorptive normal male subjects during 180 min. Plasma leucine concentrations decreased by 28% (p less than 0.05) from baseline during epinephrine treatment (plasma levels 515 pg/ml); this was due to a decrease of leucine appearance (determined by 1-13C-leucine infusions) by 23% (p less than 0.025); leucine oxidation decreased by 29% (p less than 0.05). However, when plasma cortisol concentrations were elevated to supraphysiological levels (16.3 mumol/l) during epinephrine administration, the decreases of leucine plasma concentrations, appearance and oxidation were abolished. Plasma glucose and FFA concentrations were similarly elevated during both kinds of treatment. Since leucine appearance represents a measurement of total body protein breakdown and leucine disappearance into non-oxidative pathways reflects protein synthesis, the data indicate that plasma epinephrine concentrations during severe stress exert a protein anabolic effect in man which may counteract catabolic properties of elevated plasma cortisol.     

On the use of a peptidase inhibitor (trasylol) for storage of I125-labelled peptide hormones (HGH,TSH, ACTH,insulin, glucagon,PTH)

Zusammenfassung Von den untersuchten J¹²⁵-markierten Peptidhormonen sind HGH und TSH auch ohne Trasylol gut haltbar. Die mässige Degradation von Insulin und die rasch zunehmende Degradation von PTH und Glukagon kann durch Trasylol (500 E/ml) verhindert werden. Für ACTH ist eine Kombination von Trasylol mit 0.5% Mercaptoethanol erforderlich.