The influence of dietary vitamin C and E supplementation on the physiological response of pirarucu, Arapaima gigas, in net culture

This study evaluated the efficacy of dietary vitamin C (ascorbic acid or AA), vitamin E (alpha-tocopherol or alpha-T), and C+E supplementation on the blood parameters of Arapaima gigas grown in net cages for 45 days. Four treatments were tested: control (commercial feed); C800; E500 and C+E (800+500) with supplementation of 800 mg AA kg(-1), 500 mg alpha-T kg(-1) and 800+500 mg AA+alpha-T kg(-1), respectively. Hematocrit (Ht), red blood cells (RBC), and hemoglobin concentration (Hb) (oxidative status indicators), thrombocytes and leukocytes (immunological indicators), plasma protein and glucose were evaluated. Fish fed vitamin C and C+E supplemented diets showed greater weight gain and survival. Dietary vitamin C and C+E diet supplementation resulted in increased Ht, Hb, RBC, MCHC, total leukocytes, total proteins, thrombocytes and eosinophils compared to the control and alpha-T. The alpha-tocopherol-supplemented diet reduced the number of total thrombocytes, lymphocytes and neutrophils and increased glucose and eosinophils relatively to the control. In general, leukocytes and thrombocytes were good indicators of the efficiency of vitamin on the defense mechanism of the A. gigas reared in cages. Results indicate that high alpha-T diet supplementation provides no benefit for the maintenance of the oxidative or the immunological status of A. gigas. However, it was demonstrated that high dietary AA improves A. gigas immunological status. Red blood cell indices and immune system indicators showed no synergistic effect between the vitamins after supplementing the A. gigas diet with alpha-T+AA.     

Analysis of various conditions in order to measure electromyography of isometric contractions in water and on air

The aim of this study was to verify if there are differences in the amplitude of signals from surface electromyography (EMG) during maximal and submaximal voluntary isometric contraction (MVC and 50% MVC, respectively) under different conditions, in our case, water and air, with and without extra protection (water-resistant tape) on the electrode. The isometric force and muscle activation of the MVC and 50% MVC of the biceps brachial muscle of nine healthy trained men were measured simultaneously, performed in water and on air, with and without protection of the EMG electrode. The multivariate analysis of variance with a post hoc Tukey test was applied to detect significant differences between the levels of muscular force. For the amplitude values of the EMG signal, the Wilcoxon signed rank test was applied to compare all experimental conditions in order to detect a significance of p < 0.05. The values of isometric force were not significantly different among conditions (MVC and 50% MVC). The results showed a significant difference among conditions in the water without extra protection compared to the conditions on air with and without extra protection and in water with extra protection. Reduced EMG amplitude was seen in water without extra protection from 37.04% to 55.81% regarding the other conditions. However, no significant difference was seen among conditions in water with extra protection in relation the conditions on air (with and without extra protection). This study suggest that it is necessary to use a water-resistant tape as an extra protection on the electrode when using EMG underwater, to avoid having a significant decrease in the EMG amplitude underwater and not to suffer interference from the water. There was no significant difference among the recordings of EMG with and without the use of protection on air; therefore, the protection does not influence the recording of EMG amplitude and isometric force on air.     

Leishmania tropica: intraspecific polymorphisms in lipophosphoglycan correlate with transmission by different Phlebotomus species

Lipophosphoglycan (LPG) is a dominant surface molecule of Leishmania promastigotes which has been shown to be critical for parasite-sand fly vector interactions. To provide additional evidence for its importance in transmission, the LPGs from three Leishmania tropica strains that differ in their capability to infect sand flies, were biochemically characterized. One of these strains, ISER/IL/98/LRC-L747, was isolated from a Phlebotomus sergenti female collected in the Judean Desert close to Jerusalem. The other strains originated from a different focus in the Galilee region of northern Israel. One was isolated from a patient (MHOM/IL/02/Ofri-LRC-L863) and the other from a naturally infected Phlebotomus arabicus female (IARA/IL/00/Amnunfly1-LRC-L810). The LPG structures of the isolates from the Galilee (L863 and L810) were similar to each other, but differed in the LPG repeat units from the Judean Desert isolate (L747). The terminal sugar in the side chains of the repeat units of LPG purified from L863 and L810 was beta-galactose and was not capped with glucose, unlike L747 and a previously characterized L. tropica strain from Iraq (L36). Since L810 was isolated from P. arabicus and L747 from P. sergenti, variations in the structure of their LPGs may explain their capacity to infect different sand fly species.     

Genetic influences on plasma cytokine variation in a parasitized population

The soil-transmitted helminths are the most common helminthic infections, affecting about one-fourth of the world's population. There is a significant genetic component to susceptibility to infection with these organisms. Substantial changes in plasma cytokine levels are associated with helminthic infections, and there may be significant genetic components to this cytokine variation. Six plasma cytokine levels were assessed for 367 members of a single pedigree from the Jirel population of eastern Nepal. This population experiences moderate rates of infection with geohelminths. Sex, age, helminthic infection, infection with Giardia, and presence of a household latrine were considered as covariates in all analyses of the cytokine data. The analyses of the single Jirel pedigree revealed significant heritabilities for IFN-gamma (h2 = 0.654+/-0.096), TNF-alpha (h2 = 0.458+/-0.101), IL-2 (h2 = 0.583+/-0.101), IL-4 (h2 = 0.700+/-0.095), IL-5 (h2 = 0.676+/-0.087), and IL-10 (h2 = 0.597+/-0.093). The ratios of IL-4 to IFN-gamma and of IL-10 to IFN-gamma were used as indicators of the degree of type 2 bias in immunological response; analyses of these variables indicated that approximately 40-60% of the variation (h2 = 0.400-0.577) in these derived measures of relative type 2/type 1 response is due to genetic factors.     

Hemocyanin of the molluscan Concholepas concholepas exhibits an unusual heterodecameric array of subunits

We describe here the structure of the hemocyanin from the Chilean gastropod Concholepas concholepas (CCH), emphasizing some attributes that make it interesting among molluscan hemocyanins. CCH exhibits a predominant didecameric structure as revealed by electron microscopy and a size of 8 MDa by gel filtration, and, in contrast with other mollusc hemocyanins, its stabilization does not require additional Ca(2+) and/or Mg(2+) in the medium. Polyacrylamide gel electrophoresis studies, analyses by a MonoQ FPLC column, and Western blots with specific monoclonal antibodies showed that CCH is made by two subunits noncovalently linked, named CCH-A and CCH-B, with molecular masses of 405 and 350 kDa, respectively. Interestingly, one of the subunits undergoes changes within the macromolecule; we demonstrated that CCH-A has an autocleavage site that under reducing conditions is cleaved to yield two polypeptides, CCH-A1 (300 kDa) and CCH-A2 (108 kDa), whereas CCH-B remains unchanged. The CCH-A nick occurs at 4 degrees C, increases at 37 degrees C, and is not inhibited by the addition of protease inhibitors and/or divalent cations. Since the CCH structure is a heterodimer, we investigated whether subunits would be either intermingled, forming heterodecamers, or assembled as two homogeneous decamers. Light scattering and electron microscope studies of the in vitro reassociation of purified CCH subunits demonstrated that the sole addition of Mg(2+) is needed for its reassembly into the native decameric molecule; no homodecamer reorganization was found with either CCH-A or CCH-B subunits alone. Our evidence showed that C. concholepas hemocyanin is an unusual example of heterodecameric organization.     

Role of the histidine triad-like motif in nucleotide hydrolysis by the rotavirus RNA-packaging protein NSP2

Octamers formed by the nonstructural protein NSP2 of rotavirus are proposed to function as molecular motors in the packaging of the segmented double-stranded RNA genome. The octamers have RNA binding, helix unwinding, and Mg(2+)-dependent NTPase activities and play a crucial role in assembly of viral replication factories (viroplasms). Comparison of x-ray structures has revealed significant structural homology between NSP2 and the histidine triad (HIT) family of nucleotidyl hydrolases, which in turn has suggested the location of the active site for NTP hydrolysis in NSP2. Consistent with the structural predictions, we show here using site-specific mutagenesis and ATP docking simulations that the active site for NTP hydrolysis is localized to residues within a 25-A-deep cleft between the C- and N-terminal domains of the NSP2 monomer. Although lacking the precise signature HIT motif (H?H?H?? where ? is a hydrophobic residue), our analyses demonstrate that histidines (His(221) and His(225)) represent critical residues of the active site. Similar to events occurring during nucleotide hydrolysis by HIT proteins, NTP hydrolysis by NSP2 was found to produce a short lived phosphorylated intermediate. Evaluation of the biological importance of the NTPase activity of NSP2 by transient expression in mammalian cells showed that such activity has no impact on the ability of NSP2 to induce the hyperphosphorylation of NSP5 or to interact with NSP5 to form viroplasm-like structures. Hence the NTPase activity of NSP2 probably has a role subsequent to the formation of viroplasms, consistent with its suspected involvement in RNA packaging and/or replication.     

Interaction of peptides with binary phospholipid membranes: application of fluorescence methodologies

The application of fluorescence methodologies to obtain information about the extent, dynamics and topology of peptide interaction with binary phospholipid (mainly zwitterionic/anionic) mixtures is reviewed. First, general approaches based on peptide (tryptophan residues) fluorescence properties that give information about its partition, location and dynamics will be presented. Then, methodologies based on membrane probes fluorescence that report the influence of peptide binding and/or incorporation on the lateral organization (phase separation) of membrane phospholipids will be described. Specific examples taken from the literature that illustrate both situations are presented as well as formalisms for data analysis. It is shown that steady-state and time-resolved fluorescence data (particularly important in the case of fluorescence resonance energy transfer studies) give complementary information, allowing a molecular picture of peptide interaction with biphasic systems to be drawn.     

Two mammalian glucosamine-6-phosphate deaminases: a structural and genetic study

Glucosamine-6-phosphate deaminase (EC 3.5.99.6) is an allosteric enzyme that catalyzes the reversible conversion of D-glucosamine-6-phosphate into D-fructose-6-phosphate and ammonium. Here we describe the existence of two mammalian glucosamine-6-phosphate deaminase enzymes. We present the crystallographic structure of one of them, the long human glucosamine-6-phosphate deaminase, at 1.75 A resolution. Crystals belong to the space group P2(1)2(1)2(1) and present a whole hexamer in the asymmetric unit. The active-site lid (residues 162-182) presented significant structural differences among monomers. Interestingly the region with the largest differences, when compared with the Escherichia coli homologue, was found to be close to the active site. These structural differences can be related to the kinetic and allosteric properties of both mammalian enzymes.     

The InterPro Database, 2003 brings increased coverage and new features

InterPro, an integrated documentation resource of protein families, domains and functional sites, was created in 1999 as a means of amalgamating the major protein signature databases into one comprehensive resource. PROSITE, Pfam, PRINTS, ProDom, SMART and TIGRFAMs have been manually integrated and curated and are available in InterPro for text- and sequence-based searching. The results are provided in a single format that rationalises the results that would be obtained by searching the member databases individually. The latest release of InterPro contains 5629 entries describing 4280 families, 1239 domains, 95 repeats and 15 post-translational modifications. Currently, the combined signatures in InterPro cover more than 74% of all proteins in SWISS-PROT and TrEMBL, an increase of nearly 15% since the inception of InterPro. New features of the database include improved searching capabilities and enhanced graphical user interfaces for visualisation of the data. The database is available via a webserver (http://www.ebi.ac.uk/interpro) and anonymous FTP (ftp://ftp.ebi.ac.uk/pub/databases/interpro).     

Simple and reliable multiplex PCR assay for detection of blaTEM,bla(SHV) and blaOXA-1 genes in Enterobacteriaceae

Third-generation cephalosporin resistance is often mediated by TEM- and SHV-type beta-lactamases in Enterobacteriaceae. TEM-type and OXA-1 enzymes are the major plasmid-borne beta-lactamases implicated in amoxicillin-clavulanic acid resistance in Escherichia coli isolates. We have developed a rapid and simple multiplex polymerase chain reaction (PCR) which discriminates bla(TEM), bla(SHV) and bla(OXA-1) genes by generating fragments of 516, 392 and 619 bp respectively. Multiplex PCR analysis of 51 amoxicillin-clavulanate resistant E. coli isolates detected bla(TEM) and bla(SHV) genes in 45 and two strains, respectively, and only one strain harboured a bla(OXA-1) gene. Twenty-three of the 40 cefotaxime-resistant Enterobacteriaceae isolates produced amplicons with a size compatible with the presence of bla(TEM) (13 strains), bla(SHV) (six strains) genes or the association of both genes (four strains). These results were verified by colony hybridisation. Therefore, multiplex PCR is a suitable tool for initial rapid screening of bla genes in Enterobacteriaceae.