Prokaryotic Abundance and Community Composition in a Freshwater Iron-Rich Microbial Mat at Circumneutral pH

The abundance, diversity and composition of bacterial and archaeal communities in a freshwater iron-rich microbial mat were investigated using culture-dependent and culture-independent methods. The sampling site is a mixing zone where ferrous-iron-rich fluids encounter oxygen-rich environments. Quantitative PCR analysis shows that Bacteria dominated the mat community (>99% of the total cell numbers). Phylotypes related to iron-oxidizers in Gallionellaceae, methano/methylotrophs in Methylophilaceae and Methylococcaceae, sulfide-oxidizers in Sulfuricurvum and an uncultured clone group, called Terrestrial group I or the 1068 group, in the Epsilonproteobacteria were detected in the clone library from the original sample and/or the enrichment cultures. This result suggests that these members may play a role in Fe, S and C cycling in the mixing zone. Although Archaea were minor constituents numerically, phylogenetic analysis indicates that unique and diverse yet-uncultivated Archaea are present in the iron-rich mat. The phylotypes of these yet-uncultivated Archaea belong to environmental clone groups that have been recovered from other mixing zones in terrestrial and marine environments, and some of our phylotypes have significantly low similarity (80% or lower) with the archaeal clones reported previously. Our results provide further insights into the bacterial and archaeal communities in a microaerobic iron-rich freshwater environment in mixing zones.     

Modulation of lipid metabolism with the overexpression of NPC1L1 in mouse liver

Niemann-Pick C1-like 1 protein (NPC1L1), a transporter crucial in intestinal cholesterol absorption, is expressed in human liver but not in murine liver. To elucidate the role of hepatic NPC1L1 on lipid metabolism, we overexpressed NPC1L1 in murine liver utilizing adenovirus-mediated gene transfer. C57BL/6 mice, fed on normal chow with or without ezetimibe, were injected with NPC1L1 adenovirus (L1-mice) or control virus (Null-mice), and lipid analyses were performed five days after the injection. The plasma cholesterol levels increased in L1-mice, and FPLC analyses revealed increased cholesterol contents in large HDL lipoprotein fractions. These fractions, which showed α-mobility on agarose electrophoresis, were rich in apoE and free cholesterol. These lipoprotein changes were partially inhibited by ezetimibe treatment and were not observed in apoE-deficient mice. In addition, plasma and VLDL triglyceride (TG) levels decreased in L1-mice. The expression of microsomal triglyceride transfer protein (MTP) was markedly decreased in L1-mice, accompanied by the reduced protein levels of forkhead box protein O1 (FoxO1). These changes were not observed in mice with increased hepatic de novo cholesterol synthesis. These data demonstrate that cholesterol absorbed through NPC1L1 plays a distinct role in cellular and plasma lipid metabolism, such as the appearance of apoE-rich lipoproteins and the diminished VLDL-TG secretion.     

Enzymatic Production of l-Tryptophan from l- and dl-5-Indolyl-methylhydantoin by Newly Isolated Bacterium

Bacteria which can hydrolyze dl-5-indolylmethylhydantoin to l-tryptophan were isolated from various soils. The dl-5-indolylmethylhydantoin-hydrolyzing enzymes were found to be inducible and intracellular. With intact cells, 50 mg/ml as wet base, of newly isolated bacterial strain T-523, 10 mg/ml of dl-5-indolylmethylhydantoin dissapeared and 7.4 mg/ml of l-tryptophan in a molar yield of 82% was produced after 35 hr incubation. Tryptophan produced was confirmed to be l-form regardless of the stereoisomer of the substrates used. A mechanism of asymmetric hydrolysis of dl -5-indolyImethylhydantoin was discussed.     

A Further Separation and Some Properties of β-Lactamase I

A carbohydrate binding protein was found in mid-lactating rat mammary gland. This rat mammary gland lectin agglutinated trypsinized rabbit erythrocytes and the hemagglutination was inhibited by the addition of β-d-galactosides such as lactose, melibiose, UDP-galactose and thio-d-galactoside. The lectin was partially purified by affinity chromatography on a column of Sepharose 4B to which asialo-fetuin had been covalently linked. Rat mammary gland lectin is a glycoprotein with a molecular weight of 14,800, estimated from SDS-PAGE, or 16,800 from gel filtration.

The occurrence of two glycoproteins, C4-casein and α-lactalbumin, is known in rat milk. Bovine κ-casein is a well-characterized glycoprotein. These glycoproteins were found to be bound by the rat mammary gland lectin, when they were desialylated by the action of neuraminidase. Neuraminidase-untreated α-lactalbumin also bound to the lectin but to a lesser extent. The level of the lectin in rat mammary gland was greatly reduced during regression of the gland after weaning.     

Enzymological characterization of EpoA,a laccase-like phenol oxidase produced by Streptomyces griseus

Laccase is an enzyme that catalyzes the oxidation of phenolic compounds by coupling the reduction of oxygen to water. While many laccases have been identified in plant and fungal species, enzymes of prokaryotic origin are poorly known. Here we report the enzymological characterization of EpoA, a laccase-like extracytoplasmic phenol oxidase produced by Streptomyces griseus. EpoA was expressed and purified with an Escherichia coli host-vector system as a recombinant protein fused with a C-terminal histidine-tag (rEpoA). Physicochemical analyses showed that rEpoA comprises a stable homotrimer containing all three types of copper (types 1-3). Various known laccase substrates were oxidized by rEpoA, while neither syringaldazine nor guaiacol served as substrates. Among the substrates examined, rEpoA most effectively oxidized N,N-dimethyl-p-phenylenediamine sulphate with a Km value of 0.42 mM. Several metal chelators caused marked inhibition of rEpoA activity, implying the presence of a metal center essential for the oxidase activity. The pH and temperature optima of rEpoA were 6.5 and 40 degrees C, respectively. The enzyme retained 40% activity after preincubation at 70 degrees C for 60 min. EpoA-like activities were detected in cell extracts of 8/40 environmental actinomycetes strains, which suggests that similar oxidases are widely distributed among this group of bacteria.     

Birth of offspring after transfer of Mongolian gerbil (Meriones unguiculatus) embryos cryopreserved by vitrification

The Mongolian gerbil (Meriones unguiculatus) has been used as a laboratory species in many fields of research, including neurology, oncology, and parasitology. Although the cryopreservation of embryos has become a useful means to protect valuable genetic resources, its application to the Mongolian gerbil has not yet been reported. In this study, we investigated the in vitro and in vivo developmental competence of Mongolian gerbil embryos cryopreserved by vitrification. In vivo-fertilized embryos were vitrified on the day of collection using the ethylene glycol (EG)-based solutions EFS20 and EFS40, which contained 20% and 40% EG, respectively, in PB1 containing 30% (w/v) Ficoll 70 and 0.5 M sucrose. First, we compared one-step and two-step vitrification protocols. In the one-step method, the embryos were directly transferred into the vitrification solution (EFS40), whereas in the two-step method, the embryos were exposed serially to EFS20 and EFS40 and then vitrified. After liquefying (thawing), late two-cell embryos (collected on day 3) vitrified by the two-step method showed significantly better rates of in vitro development to the morula stage compared to those vitrified by the one-step method (65% vs. 5%, P < 0.0001). We then examined whether the same two-step method could be applied to early two-cell embryos (collected on day 2), four-cell embryos (day 4), morulae (day 5), and blastocysts (day 6). After liquefying, 87%-100% of the embryos were morphologically normal in all groups, and 23% and 96% developed to the compacted morula stage from early two- and four-cell embryos, respectively. After transfer into recipient females, 3% (4/123), 1% (1/102), 5% (4/73), and 10% (15/155) developed to full-term offspring from vitrified and liquefied early two-cell embryos, late two-cell embryos, morulae, and blastocysts, respectively. This demonstrates that Mongolian gerbil embryos can be safely cryopreserved using EG-based vitrification solutions.     

Lipase-catalyzed enantioselective hydrolysis of N-protected racemic non-protein amino acid esters

Porcine pancreatic lipase (PPL)-catalyzed enantioselective hydrolysis of N-benzyloxycarbonyl-dl-amino acid esters (Z-dl-AA-ORs) was studied for the optical resolution of a variety of non-protein amino acids. The ester moiety (R) of the substrate affected the rate of hydrolysis significantly. The glyceryl (Gl) and carbamoylmethyl (Cam) esters were found to be highly reactive substrates. The hydrolysis of the Gl esters (Z-dl-AA-OGls) of both aliphatic and aromatic amino acids was examined in acetonitrile containing 70% (v/v) of 0.02 M phosphate buffer (pH 7.0) at 30°C. With all amino acids tested, the corresponding l-enantiomers were hydrolyzed preferentially. PPL favored aromatic amino acids, such as phenylalanine and p-chlorophenylalanine, leading to completion of the hydrolysis within 20 min with excellent enantioselectivities (E>100). The PPL-catalyzed hydrolysis of the corresponding Cam esters (Z-dl-AA-OCams) was also examined under the same reaction conditions. Although the hydrolysis of the Cam esters was rapid, the l-enantioselectivities were rather poor with aromatic amino acids, such as 2-phenylglycine and homophenylalanine.