A murine cDNA encodes a pan-epithelial glycoprotein that is also expressed on plasma cells.

Using a subtractive cDNA approach, we have identified a number of genes expressed in murine plasmacytomas, but not B or pre-B lymphomas. One of these genes, 289A, expresses a 1.8-kb microsomally localized mRNA that encodes a 314-amino-acid protein containing a signal sequence and a hydrophobic transmembrane domain. Sequence comparison suggests that the predicted protein is the murine homologue of a human cell surface pan-epithelial glycoprotein known variously as EGP, GA733-2, KSA, and KS1/4, recognized by mAb HEA125, GA733, KS1/4, CO17-1A, M74, and 323/A3. The 289A mRNA is highly expressed in normal murine tissues containing epithelial cells, and at a low level in plasma cells induced by LPS stimulation of spleen B lymphocytes. It is expressed in 15 of 16 plasmacytomas, but at a much lower level, if at all, in pre-B or B lymphomas. In human B cell lines, 289A detects a 1.5-kb mRNA in the myeloma cell line 8226, but not in Burkitt's lymphoma or lymphoblastoid cell lines. Subsequent FACS analysis of human cell lines with the mAb GA733 and KS1/4 demonstrated concordant expression of the mRNA and the protein. We conclude that 289A is the murine homologue of EGP, GA733-2, KSA, and KS1/4 Ag. Although its expression was previously thought to be restricted to epithelial cells, it is also expressed in plasma cells and is a B lymphocyte differentiation Ag. Because of the multiplicity of names, we propose calling the human gene hEGP314, and the murine gene mEGP314.     

Radiation chemical studies of Gly-Met-Gly in aqueous solution

Abstract

Important biological consequences are related to the reaction of HO^? radicals with methionine (Met). Several fundamental aspects remain to be defined when Met is an amino acid residue incorporated in the interior of peptides and proteins. The present study focuses on Gly-Met-Gly, the simplest peptide where Met is not a terminal residue. The reactions of HO^? with Gly-Met-Gly and its N-acetyl derivative were studied by pulse radiolysis technique. The transient absorption spectra were resolved into contributions from specific components of radical intermediates. Moreover, a detailed product analysis is provided for the first time for Met-containing peptides in radiolytic studies to support the mechanistic proposal. By parallel radiolytical and electrochemical reactions and consequent product identification, the formation of sulfoxide attributed to the direct HO^? radical attack on the sulfide functionality of the Met residue could be excluded, with the in situ generated hydrogen peroxide responsible for this oxidation. LC–MS and high resolution MS/MS were powerful analytical tools to envisage the structures of five products, thus allowing to complete the mechanistic picture of the overall Met-containing peptide reactivity.     

Experimental insights into the importance of aquatic bacterial community composition to the degradation of dissolved organic matter

Bacteria play a central role in the cycling of carbon, yet our understanding of the relationship between the taxonomic composition and the degradation of dissolved organic matter (DOM) is still poor. In this experimental study, we were able to demonstrate a direct link between community composition and ecosystem functioning in that differently structured aquatic bacterial communities differed in their degradation of terrestrially derived DOM. Although the same amount of carbon was processed, both the temporal pattern of degradation and the compounds degraded differed among communities. We, moreover, uncovered that low-molecular-weight carbon was available to all communities for utilisation, whereas the ability to degrade carbon of greater molecular weight was a trait less widely distributed. Finally, whereas the degradation of either low- or high-molecular-weight carbon was not restricted to a single phylogenetic clade, our results illustrate that bacterial taxa of similar phylogenetic classification differed substantially in their association with the degradation of DOM compounds. Applying techniques that capture the diversity and complexity of both bacterial communities and DOM, our study provides new insight into how the structure of bacterial communities may affect processes of biogeochemical significance.     

Isolation of skeletal muscle nuclei

A method for isolation of nuclei from rat skeletal muscle is described. The tissue is homogenized in the presence of Triton X-100, resulting in release of the nuclei from the muscle fibers. A crude nuclear pellet is prepared by differential centrifugation, and further purification is accomplished by centrifugation through dense sucrose. Bovine serum albumin is used to stabilize the nuclei during the isolation procedure. The isolated nuclei have been characterized microscopically and by chemical and enzymatic assay procedures.     

Dynamic behavior of histone H1 microinjected into HeLa cells

Histone H1 was purified from bovine thymus and radiolabeled with tritium by reductive methylation or with 125I using chloramine-T. Red blood cell-mediated microinjection was then used to introduce the labeled H1 molecules into HeLa cells synchronized in S phase. The injected H1 molecules rapidly entered HeLa nuclei, and a number of tests indicate that their association with chromatin was equivalent to that endogenous histone H1. The injected molecules copurified with HeLa cell nucleosomes, exhibited a half-life of approximately 100 h, and were hyperphosphorylated at mitosis. When injected HeLa cells were fused with mouse 3T3 fibroblasts less than 10% of the labeled H1 molecules migrated to mouse nuclei during the next 48 h. Thus, the intracellular behavior of histone H1 differs markedly from that of high mobility group proteins 1 and 2 (HMG1 and HMG2), which rapidly equilibrate between human and mouse nuclei after heterokaryon formation (Rechsteiner, M., and L. Kuehl, 1979, Cell, 16:901-908; Wu, L., M. Rechsteiner, and L. Kuehl, 1981, J. Cell Biol, 91: 488-496). Despite their slow rate of migration between nuclei, the injected H1 molecules were evenly distributed on mouse and human genomes soon after mitosis of HeLa-3T3 heterokaryons. These results suggest that although most histone H1 molecules are stably associated with interphase chromatin, they undergo extensive redistribution after mitosis.     

Effects of prostaglandins and arachidonic acid on baboon cerebral and mesenteric arteries

Effects of prostaglandins (PGs) E1, E2, F2 alpha and I2 in a wide range of concentration were examined in mesenteric and cerebral arteries isolated from mature baboons. PGs E1, E2 and F2 alpha at low concentrations (10(-10) to 10(-7) M) elicited relaxation in helically cut strips of cerebral arteries precontracted with phenylephrine. In contrast, the PGs did not cause relaxation in the mesenteric artery. PGI2 (10(-9) to 10(-6) M) produced marked relaxation in both arteries. The EC25 for PGI2 in the mesenteric artery was significantly lower than that in the cerebral artery. During baseline conditions, cerebral arteries contracted in response to high concentrations (greater than 10(-7) M) of PGs E1, E2 and F2 alpha. In mesenteric arteries, a large contraction was induced by PGs F2 alpha and E2 but not by PGE1. Arachidonic acid (10(-6) M) produced an aspirin-inhibitable relaxation in both arteries to a similar extent, so that the vasodilator PG(s) formed in the two different arterial walls appear to exert a similar relaxant action. Thus, the baboon mesenteric artery was more sensitive to PGI2 for the relaxant effect than was the cerebral artery, while PGs F2 alpha, E1 and E2 caused only a contraction in the mesenteric artery but both relaxation and contraction in the cerebral artery.     

Modulation of membrane composition of swine vascular smooth muscle cells by homologous lipoproteins in culture

Swine vascular smooth muscle cells were exposed to homologous low-density or high-density lipoprotein fractions for 24 h. Total cell membranes were isolated from the post-nuclear supernatant of the cell homogenates, fractionated by sucrose density gradient centrifugation and characterized by enzyme assays. The membrane fraction with the lowest density was enriched in plasma membrane marker enzymes. Cholesterol analysis showed that cells exposed to low-density lipoprotein had higher cholesterol-to-protein ratios in total cells, total cell membranes and individual membrane fractions than had the cells exposed to high-density lipoproteins. Cholesterol-to-phospholipid ratios of the plasma membrane-enriched fraction from cells exposed to low-density lipoprotein were higher than the same membrane fraction of cells exposed to high-density lipoprotein. Studies with iodinated lipoproteins showed that these compositional changes could not be due to lipoprotein contamination. Membrane microviscosity was determined by fluorescence depolarization with diphenylhexatriene and the microviscosity of the plasma membrane-enriched fraction was different in the cells exposed to the two different lipoprotein fractions. This difference in membrane microviscosity was significant only when the medium cholesterol content was 40 μg per ml or greater; cells exposed to low-density lipoprotein gave membranes with higher microviscosity.These results demonstrate that the properties of vascular smooth muscle cell membranes are influenced by exposure of the cells to homologous lipoprotein fractions.