Effects of Tween 80 and sodium fluoride on extracellular glucosyltransferase production and membrane lipids of Streptococcus mutans

1. Both Tween 80 and sodium fluoride significantly enhanced total extracellular glucosyltransferase activities of Streptococcus mutans. 2. Water-insoluble and water-soluble glucan formation were uniformly increased by Tween 80, whereas fluoride stimulated only water-soluble glucan formation. 3. Elevated glucan formation was due to an increase in enzymes secreted from bacterial cells. 4. Fatty acid composition and phospholipid content in bacterial membrane were changed by Tween 80, although sodium fluoride scarcely showed these changes. 5. Comparative results suggest that modulation of membrane lipids participates in mutansucrase production but not in dextransucrase production of S. mutans.     

Possibility of cytoplasmic pre-tRNA splicing: the yeast tRNA splicing endonuclease mainly localizes on the mitochondria

Pre-tRNA splicing has been believed to occur in the nucleus. In yeast, the tRNA splicing endonuclease that cleaves the exon-intron junctions of pre-tRNAs consists of Sen54p, Sen2p, Sen34p, and Sen15p and was thought to be an integral membrane protein of the inner nuclear envelope. Here we show that the majority of Sen2p, Sen54p, and the endonuclease activity are not localized in the nucleus, but on the mitochondrial surface. The endonuclease is peripherally associated with the cytosolic surface of the outer mitochondrial membrane. A Sen54p derivative artificially fixed on the mitochondria as an integral membrane protein can functionally replace the authentic Sen54p, whereas mutant proteins defective in mitochondrial localization are not fully active. sen2 mutant cells accumulate unspliced pre-tRNAs in the cytosol under the restrictive conditions, and this export of the pre-tRNAs partly depends on Los1p, yeast exportin-t. It is difficult to explain these results from the view of tRNA splicing in the nucleus. We rather propose a new possibility that tRNA splicing occurs on the mitochondrial surface in yeast.     

Role of telomere in endothelial dysfunction in atherosclerosis

PURPOSE OF REVIEW: Telomeres consist of repeats of G-rich sequence at the end of chromosomes. These DNA repeats are synthesized by enzymatic activity associated with an RNA protein complex called telomerase. In most somatic cells, telomerase activity is insufficient, and telomere length decreases with increasing cell division, resulting in an irreversible cell growth arrest, termed cellular senescence. Cellular senescence is associated with an array of phenotypic changes suggestive of aging. Until recently, cellular senescence has largely been studied as an in-vitro phenomenon; however, there is accumulating evidence that indicates a critical role of telomere function in the pathogenesis of human atherosclerosis. This review attempts to summarize recent work in vascular biology that supports the "telomere hypothesis". We discuss the possible relevance of telomere function to vascular aging and the therapeutic potential of telomere manipulation. RECENT FINDINGS: It has been reported that many of the changes in senescent vascular cell behavior are consistent with known changes seen in age-related vascular diseases. Introduction of telomere malfunction has been shown to lead to endothelial dysfunction that promotes atherogenesis, whereas telomere lengthening extends cell lifespan and protects against endothelial dysfunction associated with senescence. Indeed, recent studies have demonstrated that telomere attrition and cellular senescence occur in the blood vessels and are associated with human atherosclerosis. SUMMARY: Recent findings suggest that vascular cell senescence induced by telomere shortening may contribute to atherogenesis and may provide insights into a novel treatment of antisenescence to prevent atherosclerosis.     

A synthesis of 3′,4-́dideoxykanamycin B

3′,4′-Dideoxykanamycin B, the kanamycin B derivative that is active against resistant bacteria, was prepared from kanamycin B viaN-tosylation, 3′,4′-O-sulphonylation, 3′,4′-unsaturation, and hydrogenation. The unsaturated intermediate was obtained from the 3′,4′-di-O-sulphonyl derivatives by the action of sodium iodide in N,N-dimethylformamide; if zinc dust was added in this reaction, aziridine derivatives were formed, Removal of the tosyl group was successfully performed by using sodium in ammonia-ethylamine.     

Reaction mechanism of the Co2+-activated multifunctional bromoperoxidase-esterase from Pseudomonas putida IF-3.

The reaction mechanism of the Co2+-activated bromoperoxidase-esterase of Pseudomonas putida IF-3 was studied. Site-directed mutagenesis suggested that the serine residue of the catalytic triad conserved in serine hydrolases participates in the bromination and ester hydrolysis reactions. The enzyme released a trace amount of free peracetic acid depending on the concentration of H2O2, which had been considered the intermediate in the reaction of nonmetal haloperoxidases to oxidize halide ions to hypohalous acid. However, the formation of free peracetic acid could not explain the enzyme activation effect by Co2+ ions which completely depleted the free peracetic acid. In addition, the kcat value of the enzymatic bromination was 900-fold higher than the rate constant of free peracetic acid-mediated bromination. Those results strongly suggested that the peracetic acid-like intermediate formed at the catalytic site is the true intermediate and that the formation of free peracetic acid is only a minor reaction involving the enzyme. We propose the possible reaction mechanism of this multifunctional enzyme based on these findings.     

A novel species of alkaliphilic Bacillus that produces an oxidatively stable alkaline serine protease

A novel gram-positive, strictly aerobic, motile, sporulating, and facultatively alkaliphilic bacterium designated KSM-KP43 was isolated from a sample of soil. The results of 16S rRNA sequence analysis placed this bacterium in a cluster with Bacillus halmapalus. However, the level of the DNA-DNA hybridization of KSM-KP43 with B. halmapalus was less than 25%. Moreover, the G + C contents of the genomic DNA were 41.6 mol% for KSM-KP43 and 38.6 mol% for B. halmapalus. Because there were also differences in physiological properties and cellular fatty acid composition between the two organisms, we propose KSM-KP43 as a novel species of alkaliphilic Bacillus. This novel strain produces a new class of protease, an oxidatively stable serine protease that is suitable for use in bleach-based detergents. The enzyme contained 640 amino acid residues, including a possible approximately 200-amino-acid prepropeptide in the N-terminal and a unique stretch of approximately 160 amino acids in the C-terminal regions (434-amino-acid mature enzyme with a calculated molecular mass of 45,301 Da). The C-terminal half after the putative catalytic Ser255 and the contiguous C-terminal extension shared local similarity to internal segments of a membrane-associated serine protease of a marine microbial assemblage and the serine protease/ABC transporter precursors of the slime mold Dictyostelium discoideum, and to the C-terminal half of a cold-active alkaline serine protease of a psychrotrophic Shewanella strain.     

Identification and characterization of the UL56 gene product of herpes simplex virus type 2

The UL56 gene product of herpes simplex virus (HSV) has been shown to play an important role in viral pathogenicity. However, the properties and functions of the UL56 protein are little understood. We raised rabbit polyclonal antisera specific for the UL56 protein of HSV type 2 (HSV-2) and examined its expression and properties. The gene product was identified as three polypeptides with apparent molecular masses ranging from 32 to 35 kDa in HSV-2-infected cells, and at least one species was phosphorylated. Studies of their origins showed that the UL56 protein of HSV-2 is also translated from the upstream in-frame methionine codon that is not present in the HSV-1 genome. Synthesis was first detected at 6 h postinfection and was not abolished by the viral DNA synthesis inhibitor phosphonoacetic acid. Indirect immunofluorescence studies revealed that the UL56 protein localized to both the Golgi apparatus and cytoplasmic vesicles in HSV-2-infected and single UL56-expressing cells. Deletion mutant analysis showed that the C-terminal hydrophobic region of the protein was required for association with the cytoplasmic membrane and that the N-terminal proline-rich region was important for its translocation to the Golgi apparatus and cytoplasmic vesicles. Moreover, the results of protease digestion assays and sucrose gradient fractionation strongly suggested that UL56 is a tail-anchored type II membrane protein associated with lipid rafts. We thus hypothesized that the UL56 protein, as a tail-anchored type II membrane protein, may be involved in vesicular trafficking in HSV-2-infected cells.     

Replication of Deoxyribonucleic Acid in Escherichia coli C Mutants Temperature Sensitive in the Initiation of Chromosome Replication

An Escherichia coli HF4704S mutant temperature sensitive in deoxyribonucleic acid (DNA) synthesis and different from any previously characterized mutant was isolated. The mutated gene in this strain was designated dnaH. The mutant could grow normally at 27 C but not at 43 C, and DNA synthesis continued for an hour at a decreasing rate and then ceased. After temperature shift-up, the increased amount of DNA was 40 to 50%. When the culture was incubated at 43 C for 70 min and then transferred to 27 C, DNA synthesis resumed after about 50 min, initiating synchronously at a fixed region on the bacterial chromosome. The initiation step in DNA replication sensitive to 30 mug of chloramphenicol per ml occurs synchronously before the resumption of DNA replication after the temperature shift-down, being completed about 30 min before the start of DNA replication. When the cells incubated at 27 C in the presence of 30 mug of chloramphenicol per ml after the temperature shift-down to 27 C were transferred to 43 C with simultaneous removal of the antibiotic, no resumption of DNA replication was observed. When the culture was returned to 43 C after being released from high-temperature inhibition at 30 min before the start of DNA replication, no recovery replication was observed; whereas at 20 min, the recovery of replication was observed. These results indicated that HF4704S was temperature sensitive in the initiation of DNA replication. Analysis of HF4704S, by an interrupted conjugation experiment, indicated that gene dnaH was located at about 64 min on the E. coli C linkage map. In E. coli S1814 (a K-12 derivative), which was a dnaH(ts) transductant from HF4704S (C strain) with phage P1, the mutated gene (dnaH) was demonstrated to be closely linked to the thyA marker by conjugation and P1 transduction experiments and to be distinct from genes dnaA through dnaG.