Human endothelin converting enzyme-2 (ECE2): characterization of mRNA species and chromosomal localization.

Generation of the functionally pleiotropic members of the endothelin vasoactive peptide family is critically catalyzed by unique type II metalloproteases, termed endothelin converting enzymes (ECE). Isolation of human ECE-2 (EC 3.4.24.71) cDNAs revealed deduced open reading frames of 787 and 765 amino acids with approximately 60% identity with human ECE-1. Characterization of mRNA variants revealed mRNA structural diversity at the 5'-terminus. Two mRNA species exist containing distinct first and second exons. Furthermore, in one of these species, an in-frame deletion of the intracytoplasmic domain removed 29 amino acids. Because of the previously reported human genetic diseases ascribed to germline mutations of member genes of the endothelin family, ECE2 was localized in human chromosomes with fluorescence in situ hybridization and radiation hybrid mapping to 3q28-q29 and SHGC-20171/D3S1571, respectively.     

Nonlethal G0-ts mutant tsJT60 becomes lethal at the nonpermissive temperature after transformation: a hint for new cancer chemotherapeutics

tsJT60 is a nonlethal temperature-sensitive (ts) mutant of a Fischer rat cell line (3Y1) classified as a G0 mutant; i.e., the ts defect is not expressed within the cell growth cycle but is expressed only between the G0 and S phase. tsJT60 clones transformed with oncogenes such as adenovirus E1A, polyoma large T, polyoma middle T, v-Ki-ras, and LTR activated c-myc, or with a chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine, grew well at 34 degrees C. However, most of these clones grew slowly at 40 degrees C, producing many floating dead cells, and some clones were killed at 40 degrees C. When they were cultured under conditions inadequate for growth of untransformed cells, such as high cell density or serum restriction, they were killed at 40 degrees C. These and previous results from SV40- and adenovirus-transformed tsJT60 clones favour the idea that transformed tsJT60 cells occasionally enter the G0 phase and are metabolically imbalanced at 40 degrees C during self-stimulation from the G0 to S phase. We propose that a drug which exclusively block, G0-G1 transition would be cytocidal to transformed cells but cytostatic to normal cells.     

A tandem enzymatic hydrolysis of 3-acetylthio-2-methylpropionic methy ester

Summary Enzymatic hydrolysis of racemic 3-acetylthio-2-methylpropionic methyl ester catalyzed by bovine pancreatic protease and Mucor javanicus lipase showed opposite enantioselecivity. A tandem hydrolysis of the ester catalyzed by these two enzymes gives enantiomerically enriched (S)-3-acetylthio-2-methylpropionic acid, a building block of captopril.     

Pr-SNTX,a short-chain three-finger toxin from Papuan pigmy mulga snake,is an antagonist of muscle-type nicotinic acetylcholine receptor (α2βδε)

Three-finger toxins (3FTxs) are one of the major components in snake venoms. In this study, we isolated a cDNA encoding a short-chain 3FTx, Pr-SNTX, from Pseudechis rossignolii. The amino acid sequence of Pr-SNTX is nearly identical to that of its ortholog in Pseudechis australis. Pr-SNTX protein inhibited muscle-type (α₂βδε), but not neuronal α7 nicotinic acetylcholine receptor (nAChR) activity.     

Protein translocation into Escherichia coli membrane vesicles is inhibited by functional synthetic signal peptides

A synthetic peptide corresponding to the signal sequence of wild type Escherichia coli lambda-receptor protein (LamB) inhibits in vitro translocation of precursors of both alkaline phosphatase and outer membrane protein A into E. coli membrane vesicles (half-maximal inhibition at 1-2 microM). By contrast, the inhibitory effect was nearly absent in a synthetic peptide corresponding to the signal sequence from a mutant strain that harbors a deletion mutation in the LamB signal region and displays an export-defective phenotype for this protein in vivo. Two peptides derived from pseudorevertant strains that arose from the deletion mutant and exported LamB in vivo were found to inhibit in vitro translocation with effectiveness that correlated with their in vivo export ability. Controls indicated that these synthetic signal peptides did not disrupt the E. coli membrane vesicles. These results can be interpreted to indicate that the presequences of exported proteins interact specifically with a receptor either in the E. coli inner membrane or in the cytoplasmic fraction. However, biophysical data for the family of signal peptides studied here reveal that they will spontaneously insert into a lipid membrane at concentrations comparable to those that cause inhibition. Hence, an indirect effect mediated by the lipid bilayer of the membrane must be considered.     

Synergism between eosinophil cationic protein and oxygen metabolites in killing of schistosomula of Schistosoma mansoni

To study the cytotoxic reactions responsible for mediating eosinophil damage to schistosomula of Schistosoma mansoni, we have used cytoplasts (eosinophil or neutrophil vesicles devoid of granules and nuclei, with an intact oxidase in their plasma membrane) in combination with purified eosinophil cationic protein (ECP) or major basic protein (MBP) in a cytotoxicity test toward schistosomula. Suboptimal concentrations of ECP (10(-6) M) or MBP (10(-6) M) resulting in less than 10% killing were used in combination with cytoplasts. Cytoplasts alone in the presence of immune serum tested over a wide range of cytoplast:schistosomula ratios generated superoxide and hydrogen peroxide, but were unable to damage schistosomula. However, when a suboptimal ECP concentration (10(-6) M) was combined with neutroplasts or eosinoplasts, 43.9% +/- 8.5 (n = 7) and 24.7% +/- 9.8 (n = 3), respectively, of the schistosomula were killed. Oxygen metabolites were responsible for the synergism, because cytoplasts from a patient with chronic granulomatous disease were unable to act in synergy with ECP. In contrast to ECP, no synergism was found between cytoplasts and MBP (10(-6) to 2 X 10(-5)M). These results show that oxygen metabolites are important for the killing of schistosomula by lowering the concentration of ECP needed to inflict damage.     

Stimulations of arachidonate release and inositol-1,4,5-triphosphate formation are mediated by distinct G-proteins in human platelets

Addition of fluoroaluminate to human platelet suspension stimulated thromboxane synthesis and inositol-1,4,5-triphosphate formation in a time and dose dependent manner. Neomycin inhibited markedly fluoroaluminate induced inositol-1,4,5-triphosphate formation without significantly affecting thromboxane synthesis. Preincubation of platelets with PGE1, also inhibited significantly inositol-1,4,5-triphosphate formation with modest reduction of thromboxane synthesis. On the contrary, pretreatment of platelets with pertussis toxin inhibited fluoroaluminate stimulated thromboxane synthesis without affecting inositol-1,4,5-triphosphate formation. Similarly, preincubation of platelets with phorbol ester, PMA, inhibited markedly thromboxane synthesis with modest reduction of inositol-1,4,5-triphosphate formation. These results indicate that inositol-1,4,5-triphosphate formation and arachidonate release and thromboxane synthesis are controlled separately and are mediated by different G-proteins which are coupled to phospholipase C and phospholipase A2 respectively in platelets.