Isolation and structural characterization of pituitary adenylate cyclase activating polypeptide (PACAP)-like peptide from the brain of a teleost, stargazer, Uranoscopus japonicus

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a novel neuropeptide consisting of 38-residue (PACAP 1-38) and a truncated form with 27 residues (PACAP 1-27) that plays several roles in tetrapods. We isolated a highly purified PACAP-like peptide from the brain of a teleost, the stargazer, by extracting of acetone-dried powder with acetic acid followed by high-performance liquid chromatography (HPLC) on gel-filtration, cation-exchange, and reverse-phase columns. Purification was monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting analysis using an anti-PACAP 1-27 antiserum. The PACAP-like peptide thus obtained had a molecular mass of 4,623, determined by mass spectrometry, and its amino acid sequence showed 89 and 87% identity with those of ovine and frog PACAPs, respectively. These results indicate that a PACAP-like peptide, which is a highly homologous with tetrapod PACAP, is present in the teleost brain.     

Botulinolysin, a thiol-activated hemolysin produced by Clostridium botulinum, inhibits endothelium-dependent relaxation of rat aortic ring

The effects of botulinolysin (Blyn), a thiol-activated hemolysin produced by Clostridium botulinum, on contractility of rat aortic ring were studied in order to clarify an underlying mechanism of vasoconstriction by the toxin observed previously as an increase in perfusion pressure in isolated rat organs. Blyn (30 hemolytic units/ml; HU/ml) itself did not elicit any apparent change in resting tension of the ring. Contractile tension elicited by a high concentration of phenylephrine in endothelium-intact rings increased significantly after treatment with Blyn (30 HU/ml), while phenylephrine-induced contraction of endothelium-denuded rings was not influenced by toxin treatment. In rings with intact endothelium, acetylcholine (ACh)-induced relaxation was significantly inhibited after treatment with Blyn (30, 10, 1 HU/ml). In contrast, relaxation of denuded rings by sodium nitroprusside was not affected by toxin treatment (30 HU/ml). Arginine (10(-4) M) partly reversed the inhibition of ACh-induced relaxation by the toxin (1 HU/ml). Endothelium-dependent relaxation by histamine or adenosine triphosphate was also inhibited by Blyn (1 HU/ml), but the relaxation elicited by calcium ionophore A23187 was not influenced by the toxin. The results indicate that Blyn acts on endothelium and inhibits agonist-induced endothelium-dependent relaxation of blood vessels.     

Enhancement of guanine-nucleotide exchange activity of C3G for Rap1 by the expression of Crk, CrkL, and Grb2

Crk is an adaptor protein that consists almost entirely of SH2 and SH3 domains. We have previously demonstrated, by using in vivo and in vitro systems, that C3G, which was identified as a Crk SH3 domain-binding guanine nucleotide exchange factor, specifically activates Rap1. C3G also binds to other adaptor proteins, including CrkL and Grb2. In the present study, we analyzed the effect of Crk, CrkL, and Grb2 on the C3G-Rap1 pathway. Expression of Crk, CrkL, and Grb2 with C3G in Cos1 cells significantly increased the ratio of GTP/GDP bound to Rap1. Both the SH2 and SH3 domains of Crk were required for this activity. However, Crk did not stimulate the guanine nucleotide exchange activity of C3G for Rap1 in vitro, suggesting that Crk does not activate C3G by an allosteric mechanism. The requirement of the SH2 domain of Crk for the enhancement of guanine nucleotide exchange activity for Rap1 could be compensated for by the addition of a farnesylation signal to Crk, indicating that Crk enhanced the guanine nucleotide exchange activity of C3G by membrane recruitment of C3G. These results demonstrate that Crk, CrkL, and Grb2 positively modulate the C3G-Rap1 pathway primarily by recruiting C3G to the cell membrane.     

Expression of activin A is increased in cirrhotic and fibrotic rat livers

BACKGROUND & AIMS: Activin A, a member of the transforming growth factor (TGF)-beta superfamily, recently has been reported to suppress DNA synthesis and to induce apoptosis of hepatocytes. These biological functions are similar to those of TGF-beta1, which is overexpressed in liver cirrhosis. The aim of this study was to examine whether activin A is involved in liver cirrhosis and fibrosis. METHODS: Liver cirrhosis or fibrosis was induced by intraperitoneal injections of dimethylnitrosamine or porcine serum into rats. The kinetics of activin A messenger RNA (mRNA) expression in cirrhotic and fibrotic livers and primary cultured rat hepatocytes were assessed by Northern blotting, and the localization of activin A was determined immunohistochemically. Modulation of type 1 collagen mRNA expression by activin A in rat cultured Ito/fat-storing cells and fibroblasts was also examined. RESULTS: Northern blotting showed that activin A mRNA expression was enhanced in fibrotic livers. The numbers of hepatocytes expressing immunoreactive activin A were significantly greater, especially around the fibrotic areas. Activin A mRNA expression in cultured hepatocytes was increased significantly by TGF-beta1 and by activin A itself. Furthermore, type 1 collagen mRNA expression in cultured cells was enhanced by activin A and TGF-beta1 in a synergistic manner. CONCLUSIONS: Activin A is overexpressed in rat cirrhotic and fibrotic livers and may contribute to hepatic fibrogenesis.     

Gene transfer to vein graft wall by HVJ-liposome method: time course and localization of gene expression

BACKGROUND: A novel gene transfer method using liposomes with a viral envelope of hemagglutinating virus of Japan (HVJ) has been reported to be very effective for gene transfection into somatic cells and might be applicable to improve the patency of vein grafts. The present study examined the time course and localization of gene expression to assess the feasibility of ex vivo gene transfer into the vein graft by the HVJ-liposome method. METHODS: The HVJ-liposome complex containing either beta-galactosidase plasmid DNA (deoxyribonucleic acid) or no genes (controls) (experiment 1) or fluorescein isothiocyanate-labeled oligonucleotides either with or without HVJ-liposomes (experiment 2) was infused into rabbit vein grafts and allowed to incubate before autologous transplantation to carotid arteries. RESULTS: In experiment 1, all grafts incubated with beta-galactosidase plasmid with HVJ-liposomes showed the blue staining of X-gal 7 days after operation, whereas the controls did not. The blue granules were present in the medial and adventitial tissue and were still present after 14 days. In experiment 2, many fluorescein isothiocyanate-labeled nuclei were observed in the graft wall 2 and 4 days after operation and remained present mainly in the media of HVJ-liposome-treated grafts after 7 and 14 days, when no fluorescein isothiocyanate activity was observed without HVJ-liposome treatment. CONCLUSIONS: These results demonstrated the feasibility of ex vivo transfection to the medial and adventitial tissue of the vein graft by the HVJ-liposome method and suggest the possibility of its clinical application to prevent vein graft failure.     

Variable supply-voltage scheme for low-power high-speed CMOSdigital design

This paper describes a variable supply-voltage (VS) scheme. From an external supply, the VS scheme automatically generates minimum internal supply voltages by feedback control of a buck converter, a speed detector, and a timing controller so that they meet the demand on its operation frequency. A 32-b RISC core processor is developed in a 0.4-μm CMOS technology which optimally controls the internal supple voltages with the VS scheme and the threshold voltages through substrate bias control. Performance in MIPS/W is improved by a factor of more than two compared with its conventional CMOS design     

Hepatoprotective and nitric oxide production inhibitory activities of coumarin and polyacetylene constituents from the roots of Angelica furcijuga

The methanolic extract from the roots of Angelica furcijuga KITAGAWA was found to exhibit protective effects on liver injury induced by D-galactosamine (D-GalN) and lipopolysaccharide (LPS). From the methanolic extract, seventeen coumarins, two phenylpropanoids, and two polyacetylenes were isolated and examined their in vitro and in vivo hepatoprotective effects and inhibitory activity of NO production in macrophages. A acylated khellactone, isoepoxypteryxin, showed protective activity against D-GalN-induced cytotoxicity in primary cultured rat hepatocytes. On the other hand, six acylated khellactones (hyuganins A, B, C, and D, anomalin, isopteryxin) and two polyacetylenes [(-)-falcarinol and falcarindiol] strongly inhibited NO production induced by LPS in cultured mouse peritoneal macrophages, and also other acylated khellactones (isoepoxypteryxin, pteryxin, and suksdorfin) and a coumarin glycosides (praeroside II) were found to show the activity. By comparison of the inhibitory activities for acylated khellactones with those for other coumarins, acyl groups were found to be essential to exerting potent activity.     

Increase of chemokine levels in sputum precedes exacerbation of acute asthma attacks

Basophils and eosinophils can be activated in vitro by several chemokines such as RANTES, monocyte chemotactic and activating factor (MCAF/MCP-1), macrophage inflammatory peptide-1 alpha (MIP-1 alpha), and interleukin-8 (IL-8). To explore the clinical relevance of the in vitro observations, we measured here the concentrations of these chemokines in sputa from asthmatic patients during acute attacks. Before the onset of a late-phase exacerbation, sputum MCAF/MCP-1, MIP-1 alpha, and IL-8 levels transiently but markedly increased from the basal levels in all of the patients with exacerbation, whereas the sputum levels of these chemokines remained unchanged during the course in the patients without a late-phase exacerbation. These results suggest the involvement of these chemokines in the late-phase exacerbation of asthma.     

Growth hormone inhibits apoptosis and up-regulates reactive oxygen intermediates production by human polymorphonuclear neutrophils

In a pilot study the safety and therapeutic effects of an immunostimulatory intralymphatic treatment with natural human interleukin-2 (IL-2) in combination with zidovudine were evaluated in nine patients with AIDS. Therapy with IL-2 consisted of one subcutaneous injection of 0.1 microgram/kg IL-2, followed by four intralymphatic IL-2 infusions of 0.1 microgram/kg each within a period of up to 15 days. Enlargement of lymph nodes was seen in six and a transient increase of CD4 cells in five out of nine persons in association with the IL-2 therapy. An increase of HIV p24-antigenemia was observed only in the two patients in whom zidovudine dosage had to be reduced because of side effects. Moderate clinical side effects occurred in eight of the nine patients. Four patients developed zidovudine associated anemia. Six participants showed a favourable course of disease with survival of 25 to 54 months (median 30 months) despite a previous diagnosis of manifest AIDS before IL-2 therapy. This pilot study demonstrates that a combination therapy with intralymphatic IL-2 and zidovudine can induce positive immunomodulatory effects, even in the presence of manifest AIDS. Further studies should explore the tolerability and effects of a prolonged therapy with IL-2 in combination with a more potent antiviral drug combination therapy.     

Toughness investigation on simulated weld HAZs of SQV-2A pressure vessel steel

In order to get detailed information about weld HAZs toughness of SQV-2A steel and determine the optimum welding and heat treatment parameters, the toughness of simulated CGHAZs (coarse grained heat affected zone) and CGHAZs (intercritically reheated CGHAZ) were systematically investigated. The influence of tempering thermal cycles on weld ICCGHAZs toughness was clarified. The effect of post weld heat treatments (PWHT) on weld CGHAZs toughness was also determined. The results showed that high toughness (absorbed energy >200 J) of weld HAZs could be achieved by selecting the optimum welding and PWHT parameters (cooling time Δt_8/5: 6–40 s, PWHT: 893 K, 3.6–7.2 ks). Tempering thermal cycles with peak temperature of above 573 K could remarkably improve the toughness of deteriorated ICCGHAZs and reduce the hardness, when cooling time Δt_8/5(2) of the reheating thermal cycle was 6 s, which implies that welding of SQV-2A without PWHT is possible, provided that low heat input welding is adopted and welding procedure is correctly arranged. Metallography and fractography revealed that M–A constituents in weld HAZs played an important role in controlling weld HAZ toughness.