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.     

Selective increase in cytokeratin synthesis in cultured rat hepatocytes in response to hormonal stimulation

Addition of a combination of insulin, dexamethasone and EGF at seeding time to cultured rat hepatocytes in serum-free medium caused a selective increase in the biosynthesis of particular cytokeratin components. This increase was prominent during the first day in culture. No significant increases were detected in the absence of hormones or in the presence of either hormones added alone or in pairs, except in the case of insulin plus dexamethasone, which yielded an effect close to that obtained with the three factors. Interestingly, the latter condition also maintained a high level of albumin production over a 6-day period in culture.     

Chromosomal assignment of amplified genes in hydroxyurea-resistant hamster cells

We have shown previously that cDNAs for the M1 and M2 subunits of ribonucleotide reductase, ornithine decarboxylase (ODC), and p5-8, a 55,000-Dalton protein, hybridize to amplified genomic sequences in a highly hydroxyurea-resistant hamster cell line. We have extended these observations to include two additional, independently isolated, hydroxyurea-resistant cell lines: SC8, a single-step hamster ovary cell line, and KH450, a multistep human myeloid leukemic cell line, have also undergone genomic amplification for sequences homologous to ODC and p5-8 cDNAs. However, neither SC8 nor KH450 contains amplified genomic sequences homologous to an M1 cDNA probe. A panel of mouse-hamster somatic cell hybrids was used to map sequences homologous to M1, M2, ODC, and 5-8 cDNAs in the hamster genome. The M2, ODC, and p5-8 cDNAs hybridized to DNA fragments that segregated with hamster chromosome 7. In contrast, M1 cDNA hybridized to DNA fragments that segregated with hamster chromosome 3. These data suggest that the genes RRM2, (M2), ODC, and p5-8, but not RRMI (M1), are linked and may have been co-amplified in the selection of the hydroxyurea-resistant hamster and human cell lines.     

The enzymes of phospholipid synthesis in Clostridium butyricum

We have examined extracts of Clostridium butyricum for several enzymes of phospholipid synthesis. Membrane particles were shown to catalyze the formation of CDP-diglyceride from [3H]CTP and phosphatidic acid. The reaction was dependent on Mg2+ and stimulated by monovalent cations. CDP-diglyceride formed in vitro was found to be a substrate for both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase. The formation of phosphatidylglycerophosphate from added CDP-diglyceride and [U-14C]sn-glycerol-3-phosphate was dependent on Mg2+ and Triton X-100. The dephosphorylation of endogenously-generated phosphatidylglycerophosphate to yield phosphatidylglycerol was observed to be pH-dependent. The formation of phosphatidylserine from CDP-diglyceride and L-[3-14C]serine was stimulated by Mg2+ and Triton X-100. dCDP-diglyceride was a suitable substrate for both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase. Phosphatidylserine decarboxylase activity was barely detectable in membrane particles from C. butyricum. The addition of E. coli membrane particles provided efficient phosphatidylserine decarboxylase activity in this system. Although plasmalogens are the principal lipids of C. butyricum, none of the products of phospholipid synthesis formed in vitro contained measurable amounts of plasmalogens. The subcellular distribution of both phosphatidylglycerophosphate synthetase and phosphatidylserine synthetase in C. butyricum was also studied. Both were found to be membrane-associated.     

Two-dimensional gel analysis of swine histocompatibility antigens

Miniature swine MHC antigens from three inbred herds were examined by two-dimensional gel electrophoresis. These antigens were found to constitute a series of complex glycoproteins displaying haplotype-specific patterns that allowed the distinction of both class I and class II molecules among the three haplotypes. Selected outbred pig antisera reacted with a subset of class I antigens, suggesting the presence of at least two distinct molecular species among these antigens. Similarly, alloantisera reacting with mouse Ia antigens and a monoclonal anti-human DR were shown to immunoprecipitate a subset of class II molecules. Examination of the cells from two recombinant haplotypes demonstrated that both independent recombinational events took place between the class I and class II genes.     

Identification of the 190 kD microtubule-associated protein in cultured fibroblasts and its association with interphase and mitotic microtubules

We previously investigated the biochemical characteristics of microtubule-associated proteins (MAPs) of the adrenal medulla and adrenal cortex and found that they contain a new kind of MAP with a molecular weight of 190,000 (190 kD MAP) as a major species (Kotani, S., H. Murofushi, S. Maekawa, C. Sato, and H. Sakai. Eur. J. Biochem. 156, 23-29, 1986). We now have used an affinity purified anti-(190 kD MAP) antibody and show by indirect immunofluorescent microscopy the association of this MAP with microtubules in situ in TIG-3 cells (human embryonic lung fibroblasts). The 190 kD MAP was present along the interphase and mitotic microtubules, and there was no marked difference between the staining pattern with anti-tubulin and that with anti-(190 kD MAP) antibodies, evidence that the localization of 190 kD MAP is not restricted to the subset of microtubules. We also isolated MAPs from TIG-3 cells and identified their 190 kD MAP as a major heat-stable component. Several other unidentified polypeptides were recovered in the MAP fraction specifically.     

Correlation between bilayer destabilization and activity enhancement by diacylglycerols in reconstituted Ca-ATPase vesicles

Using the reconstituted Ca-ATPase vesicles as a model system, we demonstrated that the presence of 1,2-dioleoyl-sn-glycerol (diolein) in the membrane introduces a pronounced enhancement in the Ca-transport function of Ca-ATPase, while the 1,2-dipalmitoyl-sn-glycerol (dipalmitin) does not. We also found by both 31P NMR and freeze-fraction electron microscopy that diolein destabilized lipid bilayers to a greater extent than did dipalmitin. We conclude that the tendency of diacylglycerols to destabilize the phospholipid bilayer is related to their capacity to enhance the activity of the membrane calcium pump.