Urea cycle of Fasciola gigantica: purification and characterization of arginase

The ornithine-urea cycle has been investigated in Fasciola gigantica. Agrinase had very high activity compared to the other enzymes. Carbamoyl phosphate synthetase and ornithine carbamoyltransferase had very low activity. A moderate enzymatic activity was recorded for argininosuccinate synthetase and argininosuccinate lyase. The low levels of F. gigantica urea cycle enzymes except to the arginase suggest the urea cycle is operative but its role is of a minor important. The high level of arginase activity may benefit for the hydrolysis of the exogenous arginine to ornithine and urea. Two arginases Arg I and Arg II were separated by DEAE-Sepharose column. Further purification was restricted to Arg II with highest activity. The molecular weight of Arg II, as determined by gel filtration and SDS-PAGE, was 92,000. The enzyme was capable to hydrolyze l-arginine and to less extent l-canavanine at arginase:canavanase ratio (>10). The enzyme exhibited a maximal activity at pH 9.5 and Km of 6 mM. The optimum temperature of F. gigantica Arg II was 40 degrees C and the enzyme was stable up to 30 degrees C and retained 80% of its activity after incubation at 40 degrees C for 15 min and lost all of its activity at 50 degrees C. The order of effectiveness of amino acids as inhibitors of enzyme was found to be lysine>isoleucine>ornithine>valine>leucine>proline with 67%, 43%, 31%, 25%, 23% and 15% inhibition, respectively. The enzyme was activated with Mn2+, where the other metals Fe2+, Ca2+, Hg2+, Ni2+, Co2+ and Mg2+ had inhibitory effects.     

DNase expression allows the pathogen group A Streptococcus to escape killing in neutrophil extracellular traps

The innate immune response plays a crucial role in satisfactory host resolution of bacterial infection. In response to chemotactic signals, neutrophils are early responding cells that migrate in large numbers to sites of infection. The recent discovery of secreted neutrophil extracellular traps (NETs) composed of DNA and histones opened a novel dimension in our understanding of the microbial killing capacity of these specialized leukocytes. M1 serotype strains of the pathogen Group A Streptococcus (GAS) are associated with invasive infections including necrotizing fasciitis (NF) and express a potent DNase (Sda1). Here we apply a molecular genetic approach of allelic replacement mutagenesis, single gene complementation, and heterologous expression to demonstrate that DNase Sda1 is both necessary and sufficient to promote GAS neutrophil resistance and virulence in a murine model of NF. Live fluorescent microscopic cell imaging and histopathological analysis are used to establish for the first time a direct linkage between NET degradation and bacterial pathogenicity. Inhibition of GAS DNase activity with G-actin enhanced neutrophil clearance of the pathogen in vitro and reduced virulence in vivo. The results demonstrate a significant role for NETs in neutrophil-mediated innate immunity, and at the same time identify a novel therapeutic target against invasive GAS infection.     

Cadherins and synaptic plasticity

Given their trans-synaptic localization, their persistent expression at mature synapses and their distinct biochemical and adhesive properties, cadherins are uniquely poised at the synapse to mediate synaptic plasticity, the ability to change synaptic function thought to underlie learning and memory. For example recent work suggests that cadherins may recruit and stabilize AMPA receptors at the synapse via direct interactions or through complex formation, revealing cross talk between postsynaptic signaling and adhesion. Moreover, the use of small interfering RNA knockdown of cadherin, the availability of N-cadherin-deficient embryonic stem cells and the acute disruption of cadherin function with peptide application in vivo have allowed for more precise dissection of the molecular mechanisms by which cadherins function in both structural and functional plasticity.     

Purification and properties of the Escherichia coli nucleoside transporter NupG, a paradigm for a major facilitator transporter sub-family

NupG from Escherichia coli is the archetype of a family of nucleoside transporters found in several eubacterial groups and has distant homologues in eukaryotes, including man. To facilitate investigation of its molecular mechanism, we developed methods for expressing an oligohistidine-tagged form of NupG both at high levels (>20% of the inner membrane protein) in E. coli and in Xenopus laevis oocytes. In E. coli recombinant NupG transported purine (adenosine) and pyrimidine (uridine) nucleosides with apparent K(m) values of approximately 20-30 microM and transport was energized primarily by the membrane potential component of the proton motive force. Competition experiments in E. coli and measurements of uptake in oocytes confirmed that NupG was a broad-specificity transporter of purine and pyrimidine nucleosides. Importantly, using high-level expression in E. coli and magic-angle spinning cross-polarization solid-state nuclear magnetic resonance, we have for the first time been able directly to measure the binding of the permeant ([1'-(13)C]uridine) to the protein and to assess its relative mobility within the binding site, under non-energized conditions. Purification of over-expressed NupG to near homogeneity by metal chelate affinity chromatography, with retention of transport function in reconstitution assays, was also achieved. Fourier transform infrared and circular dichroism spectroscopy provided further evidence that the purified protein retained its 3D conformation and was predominantly alpha-helical in nature, consistent with a proposed structure containing 12 transmembrane helices. These findings open the way to elucidating the molecular mechanism of transport in this key family of membrane transporters.     

Morphological variation in the horse: defining complex traits of body size and shape

Horses, like many domesticated species, have been selected for broad variation in skeletal size. This variation is not only an interesting model of rapid evolutionary change during domestication, but is also directly applicable to the horse industry. Breeders select for complex traits like body size and skeletal conformation to improve marketability, function, soundness and performance in the show ring. Using a well-defined set of 35 measurements, we have identified and quantified skeletal variation in the horse species. We collected measurements from 1215 horses representing 65 breeds of diverse conformation such as the American Miniature, Shetland Pony, Arabian Horse, Thoroughbred, Shire and Clydesdale. Principal components analysis has identified two key dimensions of skeletal variation in the horse. Principal component 1 is positively correlated with every measurement and quantifies overall body size. Principal component 2 captures a pattern of bone widths vs. lengths and thus quantifies variation in overall bone thickness. By defining these complex skeletal traits, we have created a framework for whole genome association studies to identify quantitative trait loci that contribute to this variation.     

WNT5A signaling affects pituitary gland shape

Wnt signaling is important in organogenesis, and aberrant signaling in mature cells is associated with tumorigenesis. Several members of the Wnt family of signaling molecules are expressed in the developing pituitary gland. Wnt5a is expressed in the neuroectoderm that induces pituitary gland development and has been proposed to influence pituitary cell specification. We discovered that mice deficient in Wnt5a display abnormal morphology in the dorsal part of the developing pituitary. The expression of downstream effectors of the canonical Wnt pathway is not altered, and expression of genes in other signaling pathways such as Shh, Fgf8, Fgf10 and Fgfr2b is intact. Prop1 and Hesx1 are also important for normal shape of the pituitary primordium, but their expression is unaltered in the Wnt5a mutants. Specification of the hormone-producing cell types of the mature anterior pituitary gland occurs appropriately. This study suggests that the primary role of Wnt5a in the developing pituitary gland is in establishment of the shape of the gland.     

我国生物柴油产业化现状及前景

生物柴油是石化柴油的一种优良替代品，发展生物柴油有助于缓解能源危机，减少污染物的排放。本文从原料、技术、现有生产能力等方面概述了生物柴油在我国的产业化发展现状，并针对我国生物柴油产业发展提出了建议。[编者按]