Synthesis of human endothelin-1 precursors in Escherichia coli

Endothelin, the most potent vasoconstrictor found in nature, is thought to be important in the regulation of blood pressure and/or local blood distribution. Human placenta cDNA fragment encoding preproendothelin-1 (preproET-1) and its carboxyl terminal mature precursor (C-matured precursor) was expressed in E. coli. These products were characterized by both enzyme immunoassay and Western blot analysis.     

Occurrence of a novel cardiac natriuretic peptide in rats

We established a specific radioimmunoassay for the ring structure of "iso-ANP" and detected iso-ANP[23-46]-like immunoreactivity (-LI) in the rat atrium (2.76 +/- 0.5 micrograms/g) and ventricle (13.9 +/- 5.7 ng/g). High performance-gel permeation chromatography revealed that iso-ANP[23-46]-LI in the rat heart was composed of two components with molecular weights of 10K and 5K. In reverse phase-high performance liquid chromatography, the retention times of these components were clearly different from that of synthetic iso-ANP. The 5K peptide was demonstrated to be present in the perfusate from isolated rat hearts and possessed binding ability to ANP receptors. This natriuretic peptide was, however, not detectable in other tissues including the brain. We conclude that the novel cardiac natriuretic peptide distinct from iso-ANP and ANP occurs in the rat heart and is secreted from the heart.     

Isolation and sequence determination of rat cardiac natriuretic peptide

We have isolated a cardiac natriuretic peptide of 5K daltons from the rat atrium and determined its amino acid sequence. The 5K cardiac natriuretic peptide was elucidated to be a 45-amino acid peptide with the sequence of S-Q-D-S-A-F-R-I-Q-E-R-L-R-N-S-K-M-A-H-S-S-S-C-F-G-Q-K-I-D-R-I-G-A-V-S-R- L-G-C-D - G-L-R-L-F by sequencing the native peptide and its lysyl endopeptidase digests. The sequence of this peptide was identical to the amino acid sequence [51-95] of the rat brain natriuretic peptide (BNP) precursor deduced from the cDNA sequence. The 5K cardiac natriuretic peptide, or BNP[51-95], was identified as the major storage and secretory form derived from the BNP precursor in the rat heart.     

Endothelin in human plasma and culture medium of aortic endothelial cells--detection and characterization with radioimmunoassay using monoclonal antibody

We have developed monoclonal (KY-ET-1-I) and polyclonal (ET-F5) antibodies against endothelin-1 (ET-1) and established sensitive radioimmunoassays (RIAs) with different specificities. The RIA with KY-ET-1-I detected ET-1, ET-2 and ET-3, while the RIA with ET-F5 recognized ET-3 very weakly. Using these RIAs, we have investigated the concentration and molecular forms of ET-1-like immunoreactivity (-LI) in culture medium of bovine aortic endothelial cells and human plasma. Culture medium of endothelial cells contained two major components compatible with big ET and ET-1. ET-1-LI was also detected in human plasma. ET-1-LI in human plasma consisted of apparent two components, the small molecular form emerging at the position of ET-1 and the large form with the peak eluting at the preceding fraction of the elution position of big ET. The concentration of the small form of ET in human plasma was about 5 pg/ml.     

Detection and characterization of endothelin-1-like immunoreactivity in rat plasma

Using two radioimmunoassays (RIAs) for endothelin-1 (ET-1) with and without a substantial cross-reactivity with ET-3, we have measured the plasma ET-1-like immunoreactivity (-LI) level in rat plasma. ET-1-LI was detected in plasma from male Wistar rats. ET-1-LI in rat plasma consisted of three components with molecular weights of 6K, 4K and 2.5K daltons by gel permeation chromatography. Two of the components were eluted at positions of big ET (4K) and synthetic ET-1 (2.5K). The remaining component was eluted at the preceding fraction (6K). No difference was observed in ET-1-LI of the small molecular form of ET (2.5K) between the two RIAs. Thus, there is little or no ET-3 in rat plasma, which has the sequence found originally in the rat genome. The concentration of the small molecular form of ET, presumably ET-1, in rat plasma was about 4 pg/ml.     

Immunohistochemical localization of proteoglycans in interstitial elements of human pancreas and biliary system

Summary The immunohistochemical localization of large proteoglycan and small proteoglycan was observed, using antibodies 2B1 and 6B6 (Sobueet al., 1988, 1989a), in fetal and adult pancreas and biliary system as well as in tumour tissues, obtained from 11 autopsies and 74 biopsies. The distribution of chondroitin 4- and 6-sulphate side chains, type I and IV collagen and elastin were also studied. In adult pancreas and all the biliary tracts examined, periductal fibrous tissues consisted mainly of dermatan sulphate small proteoglycan with networks of fibrous elements, which were composed of large proteoglycan, elastin, type I collagen and type IV collagen. In the interstitial components of cystadenoma of pancreas and biliary duct carcinoma, similar small proteoglycan-rich components were relatively abundant, although large proteoglycan was present in much larger amounts than that in non-neoplastic adult tissues. In some cholangiomas, the extra-and intracellular hyaline globules formed by the carcinoma cells were found to contain chondroitin sulphate large proteoglycan, laminin and fibronectin.The distribution of proteoglycans was observed to be different in the arterial walls of the interlobular tissues of the adult and the fetal pancreas. The biological significance of large and small proteoglycans in the interstitial connective tissues was discussed.     

Hypoxia-induced fibre type transformation in rat hindlimb muscles Histochemical and electro-mechanical changes

Twelve male Sprague-Dawley rats (21 days old) were randomly assigned into two experimental groups: sea level control (CONT) and hypobaric hypoxia (HYPO). The HYPO rats were kept in an hypobaric chamber maintaining a simulated altitude of 4000 m (61.1 kPa). After 10 weeks of treatment, the rat hindlimb muscles [soleus (SOL) and extensor digitorum longus (EDL)] were subjected to histochemical and electro-mechanical analyses. Results indicated that compared to CONT the HYPO SOL muscle had a significantly greater relative distribution of fast-twitch-oxidative-glycolytic (FOG) fibres (28.9% SEM 2.0 vs 18.3% SEM 1.8, P less than 0.01) with a significant decrease in slow twitch oxidative fibre distribution (69.5% SEM 2.4 vs 82.9% SEM 3.1, P less than 0.01). Compared to CONT the HYPO EDL muscle also manifested a significant increase in FOG fibre distribution (51.6% SEM 0.8 vs 46.6% SEM 1.1, P less than 0.01), but this was accompanied by a significant decrease in fast twitch glucolytic fibres (44.3% SEM 0.9 vs 49.2% SEM 1.7, P less than 0.05). These histochemical fibre type transformations accompanied significant and expected changes in the electro-mechanical parameters tested in situ, e.g. maximal twitch force, maximal rate of force development, contraction time, half relaxation time, force: frequency curve, and fatigability. It was concluded that chronic hypobaric hypoxia could have a potent influence upon the phenotype expression of muscle fibres.     

Protection of mice against Sendai virus pneumonia by non-neutralizing anti-F monoclonal antibodies

Nine monoclonal antibodies (MAbs) directed to F protein of Sendai virus were obtained and characterized for their protective ability against Sendai virus infection in mice. None of the MAbs showed hemagglutination-inhibition (HI), hemolysis-inhibition (HLI), or neutralization (NT) activities in vitro when assayed by standard methods. Some of the MAbs, however, showed complement-requiring NT (C-NT) and complement-requiring hemolysis (C-HL) activities when assayed in the presence of complement. Passive immunization experiments revealed that the MAbs with higher C-NT and C-HL activities showed protective activity against Sendai virus pneumonia in mice, and that some MAbs with IgG1 isotype having neither C-NT nor C-HL activity also showed the protective activity. Digestion of the MAbs with pepsin which split immunoglobulin molecules into F(ab')2 and Fc fragments greatly suppressed the protective activity. These results suggest that not only complement-mediated immunological responses such as immune virolysis but also antibody-dependent cellular cytotoxicity (ADCC) and/or immune phagocytosis, in which complement system is not necessarily involved, play an important role in the protection of mice from Sendai virus infection.