Free and polymerized tubulin in cultured bone cells and Chinese hamster ovary cells: the influence of cold and hormones

A low pH method of liposome-membrane fusion (Schneider et al., 1980, Proc. Natl. Acad. Sci. U. S. A. 77:442) was used to enrich the mitochondrial inner membrane lipid bilayer 30-700% with exogenous phospholipid and cholesterol. By varying the phospholipid-to- cholesterol ratio of the liposomes it was possible to incorporate specific amounts of cholesterol (up to 44 mol %) into the inner membrane bilayer in a controlled fashion. The membrane surface area increased proportionally to the increase in total membrane bilayer lipid. Inner membrane enriched with phospholipid only, or with phospholipid plus cholesterol up to 20 mol %, showed randomly distributed intramembrane particles (integral proteins) in the membrane plane, and the average distance between intramembrane particles increased proportionally to the amount of newly incorporated lipid. Membranes containing between 20 and 27 mol % cholesterol exhibited small clusters of intramembrane particles while cholesterol contents above 27 mol % resulted in larger aggregations of intramembrane particles. In phospholipid-enriched membranes with randomly dispersed intramembrane particles, electron transfer activities from NADH- and succinate-dehydrogenase to cytochrome c decreased proportionally to the increase in distance between the particles. In contrast, these electron- transfer activities increased with decreasing distances between intramembrane particles brought about by cholesterol incorporation. These results indicate that (a) catalytically interacting redox components in the mitochondrial inner membrane such as the dehydrogenase complexes, ubiquinone, and heme proteins are independent, laterally diffusible components; (b) the average distance between these redox components is effected by the available surface area of the membrane lipid bilayer; and (c) the distance over which redox components diffuse before collision and electron transfer mediates the rate of such transfer.     

Glomerular basement membrane proteoglycans are derived from a large precursor

The basement membrane heparan sulfate proteoglycan produced by the Englebreth-Holm-Swarm (EHS) tumor and by glomeruli were compared by immunological methods. Antibodies to the EHS proteoglycan immunoprecipitated a single precursor protein (Mr = 400,000) from [35S]methionine-pulsed glomeruli, the same size produced by EHS cells. These antibodies detected both heparan sulfate proteoglycans and glycoproteins in extracts of unlabeled glomeruli and glomerular basement membrane. The proteoglycans contained core proteins of varying size (Mr = 150,000 to 400,000) with a Mr = 250,000 species being predominant. The glycoproteins are fragments of the core protein which lack heparan sulfate side chains. Antibodies to glomerular basement membrane proteoglycan immunoprecipitated the precursor protein (Mr = 400,000) synthesized by EHS cells and also reacted with most of the proteolytic fragments of the EHS proteoglycan. This antibody did not, however, react with the P44 fragment, a peptide situated at one end of the EHS proteoglycan core protein. These data suggest that the glomerular basement membrane proteoglycan is synthesized from a large precursor protein which undergoes specific proteolytic processing.     

Evidence for the Involvement of Serine Transhydroxymethylase in Serine and Glycine Interconversions in SALMONELLA TYPHIMURIUM

Salmonella typhimurium can normally use glycine as a serine source to support the growth of serine auxotrophs. This reaction was presumed to occur by the reversible activity of the enzyme, serine transhydroxymethylase (E. C. 2. 1. 2. 1; L-serine: tetrahydrofolic-5, 10 transhydroxymethylase), which is responsible for glycine biosynthesis. However, this enzyme had not been demonstrated to be solely capable of synthesizing serine from glycine in vivo. The isolation and characterization of a mutant able to convert serine to glycine but unable to convert glycine to serine supports the conclusion that a single enzyme is involved in this reversible interconversion of serine and glycine. The mutation conferring this phenotype was mapped with other mutations affecting serine transhydroxymethylase (glyA) and assays demonstrated reduced activities of this enzyme in the mutant.     

Influence of methionine biosynthesis on serine transhydroxymethylase regulation in Salmonella typhimurium LT2.

The enzyme serine transhydroxymethylase (EC 2.1.2.1; L-serine:tetrahydrofolate-5,10-hydroxymethyltransferase) is responsible both for the synthesis of glycine from serine and production of the 5,10-methylenetetrahydrofolate necessary as a methyl donor for methionine synthesis. Two mutants selected for alteration in serine transhydroxymethylase regulation also have phenotypes characteristic of metK (methionine regulatory) mutants, including ethionine, norleucine, and alpha-methylmethionine resistance and reduced levels of S-adenosylmethionine synthetase (EC 2.5.1.6; adenosine 5'-triphosphate:L-methionine S-adenosyltransferase) activity. Because this suggested the existence of a common regulatory component, the regulation of serine transhydroxymethylase was examined in other methionine regulatory mutants (metK and metJ mutants). Normally, serine transhydroxymethylase levels are repressed three- to sixfold in cells grown in the presence of serine, glycine, methionine, adenine, guanine, and thymine. This does not occur in metK and metJ mutants; thus, these mutations do affect the regulation of both serine transhydroxymethylase and the methionine biosynthetic enzymes. Lesions in the metK gene have been reported to reduce S-adenosylmethionine synthetase levels. To determine whether the metK gene actually encodes for S-adenosylmethionine synthetase, a mutant was characterized in which this enzyme has a 26-fold increased apparent Km for methionine. This mutation causes a phenotype associated with metK mutants and is cotransducible with the serA locus at the same frequency as metK lesions. Thus, the affect of metK mutations on the regulation of glycine and methionine synthesis in Salmonella typhimurium appears to be due to either an altered S-adenosylmethionine synthetase or altered S-adenosylmethionine pools.     

Movements and associations of ribosomal subunits in a secretory cell during growth inhibition by starvation

In Chironomus tentans salivary gland cells, the cytoplasm can be dissected into concentric zones situated at increasing distances from the nuclear envelope. After RNA labeling, the newly made ribosomal subunits are found in the cytoplasm mainly in the neighborhood of the nucleus with a gradient of increasing abundance towards the periphery of the cell. The gradient for the small subunit lasts for a few hours and disappears entirely after treatment with puromycin. The large subunit also forms a gradient but one which is only partially abolished by puromycin. The residual gradient which which is resistant to the addition of the drug is probably due to the binding of some large ribosomal units to the membranes of the endoplasmic reticulum (J.-E. Edstrom and u. Lonn. 1976. J. Cell Biol. 70:562-572, and U. Lonn and J.-E. Edstrom. 1976. J. Cell. Biol. 70:573-580). If growth is inhibited by starvation, only the puromycin-sensitive type gradient is observed for the large subunit, suggesting that the attachment of these newly made subunits to the endoplasmic reticulum membranes will not occur. If, on the other hand, the drug-resistant gradient is allowed to form in feeding animals, it is conserved during a subsequent starvation for longer periods than in control feeding animals. This observation provides a further support for an effect of starvation on the normal turnover of the large subunits associated with the endoplasmic reticulum. These results also indicate a considerable structural stability in the cytoplasm of these cells worth little or no gross redistribution of cytoplasmic structures over a period of at least 6 days.     

Immune Complexes in Cystic Fibrosis

Circulating immune complexes were detected in serum and sputum of patients with cystic fibrosis (C.F.). There were extensive deposits of immunoglobulins and complement immune complexes in several of the C.F. organs, especially the respiratory and gastrointestinal tracts, but not in the kidneys. Significant concentrations of IgG and of complement complexes could be eluted from the lungs of the C.F. patients but not from those of controls. Studies involving immunoabsorption, autoradiography, and molecular sieving through Sephadex G-200 columns identified both bovine serum albumin and staphylococcal α-haemolysin as two of the antigens present in the immune complexes. The sedimentation constant of the immune complexes was about 8S to 11S. The clinical significance of these immune complexes and the wide variety of antibodies detected in C.F. patients are discussed.     

Production of N 2 O by Ammonia Oxidizing Bacteria at Subfreezing Temperatures as a Model for Assessing the N 2 O Anomalies in the Vostok Ice Core

It was suggested that the abnormally high N ₂ O values found in 130,000–160,000 year-old Vostok ice core samples, characterized by high δ ¹⁵ N and low δ ¹⁸ O values, resulted from in situ microbial N ₂ O production. To substantiate these observations we obtained new geochemical data from the last glacial period and showed the existence of additional small N ₂ O anomalies. To test the hypothesis that microbial metabolism could contribute to these anomalies, we developed protocols for examining the ability of Nitrosomonas cryotolerans cells to produce N ₂ O at subfreezing temperatures. Our results show that these model, frozen cultures produce N ₂ O at temperatures as low as ?32°C.     

A multiwell format assay for heparanase

This assay employs a biotinylated heparan sulfate glycosaminoglycan (HSGAG) substrate that is covalently linked to the surface of 96-well immunoassay plates. The ratio of biotin:HSGAG and the coating concentration of substrate bound to the wells have been optimized and allow removal of biotin HSGAG within 60 min of incubation at 37 degrees C in assay buffer with a standard dilution of bacterial heparitinase or platelet heparanase. Loss of biotin signal from the well surface is detected on incubation with peroxidase-streptavidin followed by color development using 3,3',5,5'-tetramethylbenzidine as the peroxidase substrate. The new assay allows specific detection of heparanase activity in multiple samples in a total time of 3 h including a 1-h substrate digestion step and is a significant improvement with regard to sensitivity, specificity, and ease of handling of multiple samples compared to other described assays. Heparanase specifically degrades the biotinylated HSGAG substrate, when used with an optimized assay buffer. A range of enzymes including collagenase, trypsin, plasmin, pepsin, chondroitinases, hyaluronidase, and neuraminidase show no effect on the substrate under optimized assay conditions. The covalent linkage of the substrate to the well prevents leaching of substrate and allows preparation and long-term storage of substrate-coated plates. The assay can be used to detect heparanase levels in clinical samples and cell culture supernatants and is ideal as a screening method for antagonists of enzyme activity.     

Phylogenetic analysis of anaerobic psychrophilic enrichment cultures obtained from a greenland glacier ice core

The examination of microorganisms in glacial ice cores allows the phylogenetic relationships of organisms frozen for thousands of years to be compared with those of current isolates. We developed a method for aseptically sampling a sediment-containing portion of a Greenland ice core that had remained at -9 degrees C for over 100,000 years. Epifluorescence microscopy and flow cytometry results showed that the ice sample contained over 6 x 10(7) cells/ml. Anaerobic enrichment cultures inoculated with melted ice were grown and maintained at -2 degrees C. Genomic DNA extracted from these enrichments was used for the PCR amplification of 16S rRNA genes with bacterial and archaeal primers and the preparation of clone libraries. Approximately 60 bacterial inserts were screened by restriction endonuclease analysis and grouped into 27 unique restriction fragment length polymorphism types, and 24 representative sequences were compared phylogenetically. Diverse sequences representing major phylogenetic groups including alpha, beta, and gamma Proteobacteria as well as relatives of the Thermus, Bacteroides, Eubacterium, and Clostridium groups were found. Sixteen clone sequences were closely related to those from known organisms, with four possibly representing new species. Seven sequences may reflect new genera and were most closely related to sequences obtained only by PCR amplification. One sequence was over 12% distant from its closest relative and may represent a novel order or family. These results show that phylogenetically diverse microorganisms have remained viable within the Greenland ice core for at least 100,000 years.