Purification and Properties of Penicillin Acylase from Kluyvera citrophila

Penicillin acylase (EC 3.5.1.11) of Kluyvera citrophila KY7844 was purified approximately 120-fold by DEAE-cellulose chromatography, hydroxyapatite chromatography and isoelectro-focusing fractionation. The purified enzyme, with an approximate molecular weight of 63,000, appeared to be homogeneous in disc electrophoretic analysis, and showed isoelectric point (Ip) 8.12 and 13.0 units/mg of specific activity for cephalexin hydrolysis. The Michaelis constant (Km) for cephalexin and for 7-[1-(1H)-tetrazolylacetamido]-desacetoxycephalosporanic acid ((1H) T-7ADCA) was 1.4 mM and 3.6 mM, respectively. This enzyme was capable of producing (1H) T-7ADCA in 80% yield from 1-(1H)-tetrazolylacetate methylester and 7-aminodesacetoxycephalosporanic acid.     

Comparison of Extracellular Polysaccharide Produced by Pullularia pullulans with Polysaccharides in the Cells and Cell Wall

The purified swep hydrolase appeared homogeneous on SDS disc electrophoresis, and the molecular weights of the enzyme and the subunit were 280,000 and 70,000, respectively. The isoelectric point of this enzyme was pH 4.8.     

Structural Uniformity of Pullulan Produced by Several Strains of Pullularia pullulans

Extracellular polysaccharides produced by 3 strains of Pullularia pullulans were fractionated by treating with cetyl trimethyl ammonium hydroxide into soluble and insoluble fractions, and the structure of the former fraction, i.e., pullulan, was studied. The yield and the ratio of 2 fractions varied widely according to the strains. But the structure of pullulan was found to be uniform irrespective of the strains used. All 3 samples of pullulan gave only glucose on complete acid hydrolysis, and 93~95% maltotriose and 5~7% maltotetraose after isoamylase (pullulanase) action. The ratio of α-1,4- to α-1,6-glucosidic linkages calculated from periodate oxidation data coincided very well with the value expected from the ratio of maltotriose to maltotetraose units. An evidence for the complete absence of branch structure in pullulan was presented from the results of hydrolysis by pullulan 4-glucanohydrolase.     

Nematocidal activity of 6-O-octanoyl- and 6-O-octyl-d-allose against larvae of Caenorhabditis elegans

ABSTRACT

The nematocidal activities of the fatty acid esters of d-allose were examined using the larvae of C. elegans. Among the fatty acid esters, 6-O-octanoyl-d-allose (3) showed significant activity. 6-O-octanoyl-d-glucose (5) showed no activity, indicating that the D-allose moiety is essential for the nematocidal activity of 3. A nonhydrolyzable alkoxy analog 6-O-octyl-d-allose (6) also showed activity equivalent to that of 3.     

Reduced cell number in the hindgut epithelium disrupts hindgut left–right asymmetry in a mutant of pebble,encoding a RhoGEF,in Drosophila embryos

Animals often show left–right (LR) asymmetry in their body structures. In some vertebrates, the mechanisms underlying LR symmetry breaking and the subsequent signals responsible for LR asymmetric development are well understood. However, in invertebrates, the molecular bases of these processes are largely unknown. Therefore, we have been studying the genetic pathway of LR asymmetric development in Drosophila. The embryonic gut is the first organ that shows directional LR asymmetry during Drosophila development. We performed a genetic screen to identify mutations affecting LR asymmetric development of the embryonic gut. From this screen, we isolated pebble (pbl), which encodes a homolog of a mammalian RhoGEF, Ect2. The laterality of the hindgut was randomized in embryos homozygous for a null mutant of pbl. Pbl is a multi-functional protein required for cytokinesis and the epithelial-to-mesenchymal transition in Drosophila. Consistent with Pbl’s role in cytokinesis, we found reduced numbers of cells in the hindgut epithelium in pbl homozygous embryos. The specific expression of pbl in the hindgut epithelium, but not in other tissues, rescued the LR defects and reduced cell number in embryonic pbl homozygotes. Embryos homozygous for string (stg), a mutant that reduces cell number through a different mechanism, also showed LR defects of the hindgut. However, the reduction in cell number in the pbl mutants was not accompanied by defects in the specification of hindgut epithelial tissues or their integrity. Based on these results, we speculate that the reduction in cell number may be one reason for the LR asymmetry defect of the pbl hindgut, although we cannot exclude contributions from other functions of Pbl, including regulation of the actin cytoskeleton through its RhoGEF activity.     

Community-wide plasmid gene mobilization and selection

Plasmids have long been recognized as an important driver of DNA exchange and genetic innovation in prokaryotes. The success of plasmids has been attributed to their independent replication from the host''s chromosome and their frequent self-transfer. It is thought that plasmids accumulate, rearrange and distribute nonessential genes, which may provide an advantage for host proliferation under selective conditions. In order to test this hypothesis independently of biases from culture selection, we study the plasmid metagenome from microbial communities in two activated sludge systems, one of which receives mostly household and the other chemical industry wastewater. We find that plasmids from activated sludge microbial communities carry among the largest proportion of unknown gene pools so far detected in metagenomic DNA, confirming their presumed role of DNA innovators. At a system level both plasmid metagenomes were dominated by functions associated with replication and transposition, and contained a wide variety of antibiotic and heavy metal resistances. Plasmid families were very different in the two metagenomes and grouped in deep-branching new families compared with known plasmid replicons. A number of abundant plasmid replicons could be completely assembled directly from the metagenome, providing insight in plasmid composition without culturing bias. Functionally, the two metagenomes strongly differed in several ways, including a greater abundance of genes for carbohydrate metabolism in the industrial and of general defense factors in the household activated sludge plasmid metagenome. This suggests that plasmids not only contribute to the adaptation of single individual prokaryotic species, but of the prokaryotic community as a whole under local selective conditions.     

Characterization of comparative genome‐derived simple sequence repeats for acanthopterygian fishes

Simple sequence repeats (SSRs) have become one of the most popular molecular markers for population genetic studies. The application of SSR markers has often been limited to source species because SSR loci are too labile to be maintained in even closely related species. However, a few extremely conserved SSR loci have been reported. Here, we tested for the presence of conserved SSR loci in acanthopterygian fishes, which include over 14 000 species, by comparing the genome sequences of four acanthopterygian fishes. We also examined the comparative genome‐derived SSRs (CG‐SSRs) for their transferability across acanthopterygian fishes and their applicability to population genetic analysis. Forty‐six SSR loci with conserved flanking regions were detected and examined for their transferability among seven nonacanthopterygian and 27 acanthopterygian fishes. The PCR amplification success rate in nonacanthopterygian fishes was low, ranging from 2.2% to 21.7%, except for Lophius litulon (Lophiiformes; 80.4%). Conversely, the rate in most acanthopterygian fishes exceeded 70.0%. Sequencing of these 46 loci revealed the presence of SSRs suitable for scoring while fragment analysis of 20 loci revealed polymorphisms in most of the acanthopterygian fishes. Population genetic analysis of Cottus pollux (Scorpaeniformes) and Sphaeramia orbicularis (Perciformes) using CG‐SSRs showed that these populations did not deviate from linkage equilibrium or Hardy–Weinberg equilibrium. Furthermore, almost no loci showed evidence of null alleles, suggesting that CG‐SSRs have strong resolving power for population genetic analysis. Our findings will facilitate the use of these markers in species in which markers remain to be identified.     

Injury due to extravasation of thiopental and propofol: Risks/effects of local cooling/warming in rats

Inadvertent leakage of medications with vesicant properties can cause severe necrosis in tissue, which can have devastating long-term consequences. The aim of this study was to evaluate the extent of extravasation injury induced by thiopental and propofol, and the effects of cooling or warming of local tissue on extravasation injury at macroscopic and histopathologic levels. Rats were administered intradermally thiopental (2.5 mg/100 µL) or propofol (1.0 mg/100 µL). Rats were assigned randomly to three groups: control (no treatment), cooling and warming. Local cooling (18–20 °C) or warming (40–42 °C) was applied for 3 h immediately after agent injection. Lesion sizes (erythema, induration, ulceration, necrosis) were monitored after agent injection. Histopathology was evaluated in skin biopsies taken 24 h after agent injection. Thiopental injection induced severe skin injury with necrosis. Peak lesions developed within 24 h and healed gradually 18–27 days after extravasation. Propofol induced inflammation but no ulceration, and lesions healed within 1–2 days. Local cooling reduced thiopental- and propofol-induced extravasation injuries but warming strongly exacerbated the skin lesions (e.g., degeneration, necrosis) induced by extravasation of thiopental and propofol. Thiopental can be classified as a “vesicant” that causes tissue necrosis and propofol can be classified as an “irritant”. Local cooling protects (at least in part) against skin disorders induced by thiopental and propofol, whereas warming is harmful.     

Proteomic analysis of bone proteins adsorbed onto the surface of titanium dioxide

Osseointegration is the structural and functional connection between bone tissues and implants such as titanium dioxide (TiO₂). The bone-TiO₂ interface is thought to contain proteoglycans. However, exhaustive analysis of the proteins in this layer has not been performed. In this study, we evaluated the bone protein adhered on the surface of TiO₂ comprehensively. Pig bone protein was extracted by sequential elutions with guanidine, 0.1 M EDTA, and again with guanidine. The proteins obtained from these extractions were allowed to adhere to an HPLC column packed with TiO₂ and were eluted with 0.2 M NaOH. The eluted proteins were identified by LC/MS/MS and included not only proteoglycans but also other proteins such as extracellular matrix proteins, enzymes, and growth factors. Calcium depositions were observed on TiO₂ particles incubated with bone proteins, guanidine-extracted proteins adhered to TiO₂ displayed significantly high amounts of calcium depositions.