Reactivity of asparagine residue at the active site of the D105N mutant of fluoroacetate dehalogenase from Moraxella sp. B

Fluoroacetate dehalogenase from Moraxella sp. B (FAc-DEX) catalyzes cleavage of the carbon–fluorine bond of fluoroacetate, whose dissociation energy is among the highest found in natural products. Asp105 functions as the catalytic nucleophile that attacks the α-carbon atom of the substrate to displace the fluorine atom. In spite of the essential role of Asp105, we found that site-directed mutagenesis to replace Asp105 by Asn does not result in total inactivation of the enzyme. The activity of the mutant enzyme increased in a time- and temperature-dependent manner. We analyzed the enzyme by ion-spray mass spectrometry and found that the reactivation was caused by the hydrolytic deamidation of Asn105 to generate the wild-type enzyme. Unlike Asn10 of the l-2-haloacid dehalogenase (L-DEX YL) D10N mutant, Asn105 of the fluoroacetate dehalogenase D105N mutant did not function as a nucleophile to catalyze the dehalogenation.     

Multilocus Sequence Typing and Phylogenetic Analyses of Pseudomonas aeruginosa Isolates from the Ocean

Recent isolation of Pseudomonas aeruginosa strains from the open ocean and subsequent pulsed-field gel electrophoresis analyses indicate that these strains have a unique genotype (N. H. Khan, Y. Ishii, N. Kimata-Kino, H. Esaki, T. Nishino, M. Nishimura, and K. Kogure, Microb. Ecol. 53:173-186, 2007). We hypothesized that ocean P. aeruginosa strains have a unique phylogenetic position relative to other strains. The objective of this study was to clarify the intraspecies phylogenetic relationship between marine strains and other strains from various geographical locations. Considering the advantages of using databases, multilocus sequence typing (MLST) was chosen for the typing and discrimination of ocean P. aeruginosa strains. Seven housekeeping genes (acsA, aroE, guaA, mutL, nuoD, ppsA, and trpE) were analyzed, and the results were compared with data on the MLST website. These genes were also used for phylogenetic analysis of P. aeruginosa. Rooted and unrooted phylogenetic trees were generated for each gene locus and the concatenated gene fragments. MLST data showed that all the ocean strains were new. Trees constructed for individual and concatenated genes revealed that ocean P. aeruginosa strains have clusters distinct from those of other P. aeruginosa strains. These clusters roughly reflected the geographical locations of the isolates. These data support our previous findings that P. aeruginosa strains are present in the ocean. It can be concluded that the ocean P. aeruginosa strains have diverged from other isolates and form a distinct cluster based on MLST and phylogenetic analyses of seven housekeeping genes.     

Preparation of 3-Methyl-3-(substituted phenyl)-pyruvic Acid

The relationships between dietary levels of the essential amino acids and hepatic polysome profiles of rats were investigated with special attention to the amino acid requirement pattern for the maximum rat growth as determined by other investigators. The basal diet contained a 7% essential amino acid mixture and a 3% non-essential amino acid mixture, with appropriate amounts of other nutrients. Rats were fed test diet for 5 hours and then the polysome profile was determined. The amounts of essential amino acids needed for maximum aggregation of polysome were low for methionine-cystine, leucine and tryptophan as compared with requirements for maximum growth. But in other essential amino acids, the amounts were in almost the same range as those reported for maximum growth by others. The differences between the amino acid requirement patterns for maximum aggregation of hepatic ribosomes and for maximum growth of rats might be due to a difference in amino acid requirements of the liver and whole body. Therefore, the hepatic polysome profile might be used to measure the effect of amino acid supplementation on dietary proteins. The requirement pattern of essential amino acids in other organs may be studied by polysome profile determination.     

Involvement of Acidic Polysaccharide Ph-PS-2 and Protein in Initiation of Coccolith Mineralization,as Demonstrated by In Vitro Calcification on the Base Plate

Coccolithophorids, unicellular marine microalgae, have calcified scales with elaborate structures, called coccoliths, on the cell surface. Coccoliths generally comprise a base plate, CaCO₃, and a crystal coat consisting of acidic polysaccharides. In this study, the in vitro calcification conditions on the base plate of Pleurochrysis haptonemofera were examined to determine the functions of the base plate and acidic polysaccharides (Ph-PS-1, -2, and -3). When EDTA-treated coccoliths (acidic polysaccharide-free base plates) or low pH-treated coccoliths (whole acidic polysaccharide-containing base plates) were used, mineralization was not detected on the base plate. In contrast, in the case of coccoliths which were decalcified by lowering of the pH and then treated with urea (Ph-PS-2-containing base plates), distinct aggregates, probably containing CaCO₃, were observed only on the rim of the base plates. Energy dispersive X-ray spectroscopy (EDS) confirmed that the aggregates contained Ca and O, although X-ray diffraction analysis did not reveal any evidence of crystalline materials. Also, in vitro mineralization experiments performed on EDTA-treated coccoliths using isolated acidic polysaccharides demonstrated that the Ca-containing aggregates were markedly formed only in the presence of Ph-PS-2. Furthermore, in vitro mineralization experiments conducted on protein-extracted base plates suggested that the coccolith-associated protein(s) are involved in the Ca deposition. These findings suggest that Ph-PS-2 associated with the protein(s) on the base plate rim initiates Ca²⁺ binding at the beginning of coccolith formation, and some other factors are required for subsequent calcite formation.     

The efficacy of a herbal medicine (Mao-to) in combination with intravenous natural interferon-beta for patients with chronic hepatitis C, genotype 1b and high viral load: a pilot study.

Patients with chronic hepatitis C, with a high serum viral load (> or = 1 Meq/ml) and genotype 1b seem to be resistant to interferon (IFN) therapy. To evaluate the efficacy of a herbal medicine (Mao-to) in combination with natural IFN-beta for the treatment of these patients, eighteen Japanese patients were enrolled in this study. Every patient received 6 million units (MU) of IFN-beta intravenously daily for 8 weeks. Mao-to was given orally 3-4 times a day during the IFN-beta administration, Sixteen of the 18 patients (89%) became negative for serum HCV RNA at the end of treatment, but only 2 of them (11%) remained negative for the virus RNA at 6 months of follow-up. Serum ALT levels normalized in 17 patients (94%) at 2 weeks of follow-up after the cessation of therapy, and 11 patients (61%) retained normal ALT levels for more than 6 months of follow-up. This rate of biochemical response was high as compared with that of therapy with IFN-beta alone (19%) in the largest IFN-beta trial in Japan. Serum hyaluronic acid levels were decreased significantly from 147.0 +/- 110.5 ng/ml to 77.4 +/- 67.4 ng/ml in the sustained biochemical response group (P = 0.003). None of the patients needed to interrupt therapy because of side effects of IFN-beta. Thus, Mao-to administration together with IFN-beta treatment could increase the sustained biochemical response rate, and reduce liver fibrosis.     

Cross-linking of plasmalemmal cholesterol in lymphocytes induces capping, membrane shedding, and endocytosis through coated pits.

By use of a nicked and biotinylated perfringolysin O (BCtheta), which binds to cholesterol specifically, we studied consequences of cross-linking cholesterol in lymphocytes. When bound with BCtheta and then with labeled avidin or streptavidin, capping occurred in most cells within 30 min at 37 degrees C. It was inhibited by cytochalasin D or NaN3, but not by nocodazole. When BCtheta-cholesterol was capped, Thy-1 and transferrin receptor, a GPI-anchored protein and a transmembrane protein, respectively, remained evenly distributed. By fluorescence and electron microscopy, a cluster of small vesicles bound with BCtheta were observed in the cap. They were then shed in the medium or internalized through coated pits. The result indicates that cross-linking of cholesterol in lymphocytes induces capping, but does not affect distribution of membrane proteins, and that the capped cholesterol molecules are either shed as vesicles or endocytosed.