Comparative study of binding of ovine complement factor H with different <Emphasis Type="Italic">Borrelia</Emphasis> genospecies

This study presents the binding of ovine factor H (fH) by various serotypes of Borrelia and simultaneously correlates their complement resistance to sheep serum. Affinity ligand binding assay was employed to study the binding of borrelial proteins to ovine recombinant fH and its truncated forms (short consensus repeat, SCR 7 and SCRs 19–20). From a repertoire of 17 borrelial strains, only two strains showed affinity to sheep fH. A ~28-kDa protein of Borrelia burgdorferi sensu stricto (B. burgdorferi s.s., strain SKT-2) bound full-length fH as well as SCRs 19–20. This fH-binding protein was further identified as complement regulator-acquiring surface protein of B. burgdorferi (BbCRASP-1) by MALDI-TOF analysis. Surprisingly, a ~26-kDa protein of Borrelia bissettii (DN127) showed affinity to full-length fH but not to SCR 7 and SCRs19–20. In complement sensitivity assay, both strains—SKT-2 and DN127—were resistant to normal sheep serum. Significant complement resistance of two Borrelia garinii strains (G117 and T25) was also observed; however, none of those strains was able to bind sheep fH. Our study underscores the need of further exploration of fH-mediated evasion of complement system by Borrelia in domestic animals.     

Phylogenetic analysis, homology modelling, molecular dynamics and docking studies of caffeoyl–CoA-O- methyl transferase (CCoAOMT 1 and 2) isoforms isolated from subabul (Leucaena leucocephala)

Caffeoyl coenzyme A O-methyltransferase (CCoAOMT) is an important enzyme that participates in lignin biosynthesis especially in the formation of cell wall ferulic esters of plants. It plays a pivotal role in the methylation of the 3-hydroxyl group of caffeoyl CoA. Two cDNA clones that code CCoAOMT were isolated earlier from subabul and in the present study; 3D models of CCoAOMT1 and CCoAOMT2 enzymes were built using the MODELLER7v7 software to find out the substrate binding sites. These two proteins differed only in two amino acids and may have little or no functional redundancy. Refined models of the proteins were obtained after energy minimization and molecular dynamics in a solvated water layer. The models were further assessed by PROCHECK, WHATCHECK, Verify_3D and ERRAT programs and the results indicated that these models are reliable for further active site and docking analysis. The refined models showed that the two proteins have 9 and 10 α-helices, 6 and 7 β-sheets respectively. The models were used for docking the substrates CoA, SAM, SAH, caffeoyl CoA, feruloyl CoA, 5-hydroxy feruloyl CoA and sinapyl CoA which showed that CoA and caffeoyl CoA are binding with high affinity with the enzymes in the presence and absence of SAM. It appears therefore that caffeoyl CoA is the substrate for both the isoenzymes. The results also indicated that CoA and caffeoyl CoA are binding with higher affinity to CCoAOMT2 than CCoAOMT1. Therefore, CCoAOMT2 conformation is thought to be the active form that exists in subabul. Docking studies indicated that conserved active site residues Met58, Thr60, Val63, Glu82, Gly84, Ser90, Asp160, Asp162, Thr169, Asn191 and Arg203 in CCoAOMT1 and CCoAOMT2 enzymes create the positive charge to balance the negatively charged caffeoyl CoA and play an important role in maintaining a functional conformation and are directly involved in donor-substrate binding.     

Effect of Moringa oleifera leaves on hematological profile of fluorosis affected rats

Fluorosis is a metabolic disease that is endemic in nearly 25 countries with India being one of the most affected. It primarily affects the bone and the teeth. Moringa oleifera (MO) leaves are known to reduce the effect of fluorosis on various tissues. Therefore, it is of interest to document the effect of Moringa oleifera leaves on the hematological profile of fluorosis affected rats. Twenty four Sprague Dawley rats were housed two per cage in a room with 12 hours light and 12 hours dark cycle. The rats were allowed to adjust to the laboratory environment for about one to two weeks before the beginning of the study. This study reveals that MO leaves is effective in reducing the plasma fluoride content. It also helps in improving the Hb % and RBC count in fluorosis affected rats. Data shows that Moringa olifera leaves powder is effective in reducing the plasma fluoride content. It also helps in improving the Hemoglobin percentage & Red Blood Cell count in fluorosis affected rats.     

Lipid Bilayer Composition Can Influence the Orientation of Proteorhodopsin in Artificial Membranes

Artificial membrane systems allow researchers to study the structure and function of membrane proteins in a matrix that approximates their natural environment and to integrate these proteins in ex vivo devices such as electronic biosensors, thin-film protein arrays, or biofuel cells. Given that most membrane proteins have vectorial functions, both functional studies and applications require effective control over protein orientation within a lipid bilayer. In this work, we explored the role of the bilayer surface charge in determining transmembrane protein orientation and functionality during formation of proteoliposomes. We reconstituted a model vectorial ion pump, proteorhodopsin, in liposomes of opposite charges and varying charge densities and determined the resultant protein orientation. Antibody-binding assay and proteolysis of proteoliposomes showed physical evidence of preferential orientation, and functional assays verified the vectorial nature of ion transport in this system. Our results indicate that the manipulation of lipid composition can indeed control orientation of an asymmetrically charged membrane protein, proteorhodopsin, in liposomes.     

The Growing Season,but Not the Farming System,Is a Food Safety Risk Determinant for Leafy Greens in the Mid-Atlantic Region of the United States

Small- and medium-size farms in the mid-Atlantic region of the United States use varied agricultural practices to produce leafy greens during spring and fall, but the impact of preharvest practices on food safety risk remains unclear. To assess farm-level risk factors, bacterial indicators, Salmonella enterica, and Shiga toxin-producing Escherichia coli (STEC) from 32 organic and conventional farms were analyzed. A total of 577 leafy greens, irrigation water, compost, field soil, and pond sediment samples were collected. Salmonella was recovered from 2.2% of leafy greens (n = 369) and 7.7% of sediment (n = 13) samples. There was an association between Salmonella recovery and growing season (fall versus spring) (P = 0.006) but not farming system (organic or conventional) (P = 0.920) or region (P = 0.991). No STEC was isolated. In all, 10% of samples were positive for E. coli: 6% of leafy greens, 18% of irrigation water, 10% of soil, 38% of sediment, and 27% of compost samples. Farming system was not a significant factor for levels of E. coli or aerobic mesophiles on leafy greens but was a significant factor for total coliforms (TC) (P < 0.001), with higher counts from organic farm samples. Growing season was a factor for aerobic mesophiles on leafy greens (P = 0.004), with higher levels in fall than in spring. Water source was a factor for all indicator bacteria (P < 0.001), and end-of-line groundwater had marginally higher TC counts than source samples (P = 0.059). Overall, the data suggest that seasonal events, weather conditions, and proximity of compost piles might be important factors contributing to microbial contamination on farms growing leafy greens.     

Methyl eugenol effects on Bactrocera dorsalis male total body protein,reproductive organs and ejaculate

Methyl eugenol (ME) and inclusion of protein into the adult diet increase the mating competitiveness of the Oriental fruit fly, Bactrocera dorsalis (Hendel). Exposing males to ME or protein is a promising post‐teneral treatment for males being released in the sterile insect technique (SIT). However, the effect of this post‐teneral treatment on male reproductive organs or the male ejaculate is unknown. During mating, males transfer sperm and accessory gland products (AGPs) to females and these compounds are reported to modulate female sexual inhibition. We studied the impact of male exposure to ME and a yeast hydrolysate (YH) diet on the protein reserves of males, male reproductive organ size, and the male ejaculate through sperm and AGPs. We show that males exposed to ME regardless of access to YH accumulated a greater amount of whole body protein. Males fed on YH also had increased total body protein and had bigger reproductive organs than YH‐deprived males, but no apparent effect of ME exposure was observed on reproductive organ size. Females stored less sperm when mated with males fed on YH and ME compared to males not fed on ME. YH and ME had no effect on male AGPs. Females injected with AGPs of males fed on YH and exposed to ME were just as likely to mate as females injected with AGPs of non‐treated males. However, females injected with AGPs of males exposed to ME mated faster than females injected with AGPs of non‐exposed males. We conclude that while exposure to ME increases male copulatory success and protein reserves in the male body, there seem to be some potential trade‐offs such as lower sperm stored by females. We discuss our results in terms of pre‐release protocols that may be used for B. dorsalis in SIT application.     

Role of Deacetylase Activity of N-Deacetylase/N-Sulfotransferase 1 in Forming N-Sulfated Domain in Heparan Sulfate

Heparan sulfate (HS) is a highly sulfated polysaccharide that plays important physiological roles. The biosynthesis of HS involves a series of enzymes, including glycosyltransferases (or HS polymerase), epimerase, and sulfotransferases. N-Deacetylase/N-Sulfotransferase isoform 1 (NDST-1) is a critical enzyme in this pathway. NDST-1, a bifunctional enzyme, displays N-deacetylase and N-sulfotransferase activities to convert an N-acetylated glucosamine residue to an N-sulfo glucosamine residue. Here, we report the cooperative effects between N-deacetylase and N-sulfotransferase activities. Using baculovirus expression in insect cells, we obtained three recombinant proteins: full-length NDST-1 and the individual N-deacetylase and N-sulfotransferase domains. Structurally defined oligosaccharide substrates were synthesized to test the substrate specificities of the enzymes. We discovered that N-deacetylation is the limiting step and that interplay between the N-sulfotransferase and N-deacetylase accelerates the reaction. Furthermore, combining the individually expressed N-deacetylase and N-sulfotransferase domains produced different sulfation patterns when compared with that made by the NDST-1 enzyme. Our data demonstrate the essential role of domain cooperation within NDST-1 in producing HS with specific domain structures.     

Development of simple and rapid elution methods for proteins from various affinity beads for their direct MALDI-TOF downstream application

Commercially available desalting techniques, necessary for downstream MALDI-TOF analysis of proteins, are often costly or time consuming for large-scale analysis. Here, we present techniques to elute proteins from various affinity resins, free from salt and ready for MALDI mass spectrometry. We showed that 0.1% TFA in 50% acetonitrile or 40% ethanol can be used as salt-free eluents for His-tagged proteins from variety of polyhistidine-affinity resins, while washing of resin beads twice with double-distilled water prior to the elution effectively desalted and recovered wide-range-molecular size proteins than commercially available desalting devices. Modified desalting and elution techniques were also applied for Flag- and Myc-tag affinity resins. The technique was further applied in co-precipitation assay, where the maximum recovery of wide-range molecular size proteins is crucial. Further, results showed that simple washing of the beads with double distilled water followed by elution with acetonitrile effectively desalted and recovered 150 kDa factor H protein of the sheep and its binding partner ~30 kDa BbCRASP-1 in co-precipitation assay. In summary, simple modifications in the desalting and elution strategy save time, labor and cost of the protein preparation for MALDI mass spectrometry; and large-scale protein purifications or co-precipitations can be performed with ease.     

Components of a fanconi-like pathway control pso2-independent DNA interstrand crosslink repair in yeast

Fanconi anemia (FA) is a devastating genetic disease, associated with genomic instability and defects in DNA interstrand cross-link (ICL) repair. The FA repair pathway is not thought to be conserved in budding yeast, and although the yeast Mph1 helicase is a putative homolog of human FANCM, yeast cells disrupted for MPH1 are not sensitive to ICLs. Here, we reveal a key role for Mph1 in ICL repair when the Pso2 exonuclease is inactivated. We find that the yeast FANCM ortholog Mph1 physically and functionally interacts with Mgm101, a protein previously implicated in mitochondrial DNA repair, and the MutSα mismatch repair factor (Msh2-Msh6). Co-disruption of MPH1, MGM101, MSH6, or MSH2 with PSO2 produces a lesion-specific increase in ICL sensitivity, the elevation of ICL-induced chromosomal rearrangements, and persistence of ICL-associated DNA double-strand breaks. We find that Mph1-Mgm101-MutSα directs the ICL-induced recruitment of Exo1 to chromatin, and we propose that Exo1 is an alternative 5'-3' exonuclease utilised for ICL repair in the absence of Pso2. Moreover, ICL-induced Rad51 chromatin loading is delayed when both Pso2 and components of the Mph1-Mgm101-MutSα and Exo1 pathway are inactivated, demonstrating that the homologous recombination stages of ICL repair are inhibited. Finally, the FANCJ- and FANCP-related factors Chl1 and Slx4, respectively, are also components of the genetic pathway controlled by Mph1-Mgm101-MutSα. Together this suggests that a prototypical FA-related ICL repair pathway operates in budding yeast, which acts redundantly with the pathway controlled by Pso2, and is required for the targeting of Exo1 to chromatin to execute ICL repair.