Brucella melitensis 16M: characterisation of the galE gene and mouse immunisation studies with a galE deficient mutant

The galE gene of Streptomyces lividans was used to probe a cosmid library harbouring Brucella melitensis 16M DNA and the nucleotide sequence of a 2.5 kb ClaI fragment which hybridised was determined. An open reading frame encoding a predicted polypeptide with significant homology to UDP-galactose-4-epimerases of Brucella arbortus strain 2308 and other bacterial species was identified. DNA sequences flanking the B. melitensis galE gene shared no identity with other gal genes and, as for B. abortus, were located adjacent to a mazG homologue. A plasmid which encoded the B. melitensis galE open reading frame complemented a galE mutation in Salmonella typhimurium LB5010, as shown by the restoration of smooth lipopolysaccharide (LPS) biosynthesis, sensitivity to phage P22 infection and restoration of UDP-galactose-4-epimerase activity. The galE gene on the B. melitensis 16M chromosome was disrupted by insertional inactivation and these mutants lacked UDP-galactose-4-epimerase activity but no discernible differences in LPS structure between parent and the mutants were observed. One B. melitensis 16M galE mutant, Bm92, was assessed for virulence in CD-1 and BALB/c mice and displayed similar kinetics of invasion and persistence in tissues compared with the parent bacterial strain. CD-1 mice immunised with B. melitensis 16M galE were protected against B. melitensis 16M challenge. Crown Copyright (C) 1999 Published by Elsevier Science B.V.

A spirochete isolated from a case of severe virulent ovine foot disease is closely related to a Treponeme isolated from human periodontitis and bovine digital dermatitis

The isolation of spirochetes from severe ovine foot disease has been reported recently by our research group. In this study we describe the preliminary classification of this spirochete based on nucleotide sequence analysis of the PCR-amplified 16S rRNA gene. Phylogenetic analysis of this sequence in comparison with other previously reported 16S rRNA gene sequences showed that the spirochete belonged to the treponemal phylotype Treponema vincentii which has been associated with bovine digital dermatitis and human periodontal disease. Further work is required to define the common virulence determinants of these closely related treponemes in the aetiology of these tissue destructive diseases. (C) 2000 Elsevier Science B.V. All rights reserved.

Sequence analysis and distribution in Salmonella enterica serovars of IS3-like elements

The genome of Salmonella enterica serovar Enteritidis was shown to possess three IS3-like insertion elements, designated IS1230A, B and C, and each was cloned and their respective deoxynucleotide sequences determined. Mutations in elements IS1230A and B resulted in frameshifts in the open reading frames that encoded a putative transposase to be inactive. IS1230C was truncated at nucleotide 774 relative to IS1230B and therefore did not possess the 3' terminal inverted repeat. The three IS1230 derivatives were closely related to each other based on nucleotide sequence similarity. IS1230A was located adjacent to the sef operon encoding SEF14 fimbriae located at minute 97 of the genome of S. Enteritidis. IS1230B was located adjacent to the umuDC operon at minute 42.5 on the genome, itself located near to one terminus of an 815-kb genome inversion of S. Enteritidis relative to S. Typhimurium. IS1230C was located next to attB, the bacteriophage P22 attachment site, and proB, encoding gamma-glutamyl phosphate reductase. A truncated 3' remnant of IS1230, designated IS1230T, was identified in a clinical isolate of S. Typhimurium DT193 strain 2391. This element was located next to attB adjacent to which were bacteriophage P22-like sequences. Southern hybridisation of total genomic DNA from eighteen phage types of S. Enteritidis and eighteen definitive types of S. Typhimurium showed similar, if not identical, restriction fragment profiles in the respective serovars when probed with IS1230A.

The impact of obesity-related single nucleotide polymorphisms on appetite and energy intake: a pilot study

An increasing number of studies have reported a heritable component for the regulation of energy intake and eating behaviour, although the individual polymorphisms and their ‘effect size’ are not fully elucidated. The aim of the present study was to examine the relationship between specific SNP and appetite responses and energy intake in overweight men. In a randomised cross-over trial, forty overweight men (age 32 (sd 09) years; BMI 27 (sd 2) kg/m2) attended four sessions 1 week apart and received three isoenergetic and isovolumetric servings of dairy snacks or water (control) in random order. Appetite ratings were determined using visual analogue scales and energy intake at an ad libitum lunch was assessed 90 min after the dairy snacks. Individuals were genotyped for SNP in the fat mass and obesity-associated (FTO), leptin (LEP), leptin receptor (LEPR) genes and a variant near the melanocortin-4 receptor (MC4R) locus. The postprandial fullness rating over the full experiment following intake of the different snacks was 17·2 % (P= 0·026) lower in A carriers compared with TT homozygotes for rs9939609 (FTO, dominant) and 18·6 % (P= 0·020) lower in G carriers compared with AA homozygotes for rs7799039 (LEP, dominant). These observations indicate that FTO and LEP polymorphisms are related to the variation in the feeling of fullness and may play a role in the regulation of food intake. Further studies are required to confirm these initial observations and investigate the ‘penetrance’ of these genotypes in additional population subgroups.

Heterozygosity and diversity analysis using mapped single nucelotide polymorphisms in a faba bean inbreeding programme

The aim of this study was to convert existing faba bean (Vicia faba L.) single nucleotide polymorphism (SNP) markers from cleaved amplification polymorphic sequences and SNaPshot® formats, which are expensive and time-consuming, to the more convenient KBiosciences competitive allele‐specific PCR (KASP) assay format. Out of 80 assays designed, 75 were validated, though a core set of 67 of the most robust markers is recommended for further use. The 67 best KASP SNP assays were used across two generations of single seed descent to detect unintended outcrossing and to track and quantify loss of heterozygosity, a capability that will significantly increase the efficiency and performance of pure line production and maintenance. This same set of assays was also used to examine genetic relationships between the 67 members of the partly inbred panel, and should prove useful for line identification and diversity studies in the future.

Distribution of mosquitoes in the South East of Argentina and first report on the analysis based on 18S rDNA and COI sequences

Although Mar del Plata is the most important city on the Atlantic coast of Argentina, mosquitoes inhabiting such area are almost uncharacterized. To increase our knowledge in their distribution, we sampled specimens of natural populations. After the morphological identification based on taxonomic keys, sequences of DNA from small ribosomal subunit (18S rDNA) and cytochrome c oxidase I (COI) genes were obtained from native species and the phylogenetic analysis of these sequences were done. Fourteen species from the genera Uranotaenia, Culex, Ochlerotatus and Psorophora were found and identified. Our 18S rDNA and COI-based analysis indicates the relationships among groups at the supra-species level in concordance with mosquito taxonomy. The introduction and spread of vectors and diseases carried by them are not known in Mar del Plata, but some of the species found in this study were reported as pathogen vectors.

Analyses of single nucleotide polymorphisms in selected nutrient-sensitive genes in weight-regain prevention: the DIOGENES study

BACKGROUND: Differences in the interindividual response to dietary intervention could be modified by genetic variation in nutrient-sensitive genes. OBJECTIVE: This study examined single nucleotide polymorphisms (SNPs) in presumed nutrient-sensitive candidate genes for obesity and obesity-related diseases for main and dietary interaction effects on weight, waist circumference, and fat mass regain over 6 mo. DESIGN: In total, 742 participants who had lost ≥ 8% of their initial body weight were randomly assigned to follow 1 of 5 different ad libitum diets with different glycemic indexes and contents of dietary protein. The SNP main and SNP-diet interaction effects were analyzed by using linear regression models, corrected for multiple testing by using Bonferroni correction and evaluated by using quantile-quantile (Q-Q) plots. RESULTS: After correction for multiple testing, none of the SNPs were significantly associated with weight, waist circumference, or fat mass regain. Q-Q plots showed that ALOX5AP rs4769873 showed a higher observed than predicted P value for the association with less waist circumference regain over 6 mo (-3.1 cm/allele; 95% CI: -4.6, -1.6; P/Bonferroni-corrected P = 0.000039/0.076), independently of diet. Additional associations were identified by using Q-Q plots for SNPs in ALOX5AP, TNF, and KCNJ11 for main effects; in LPL and TUB for glycemic index interaction effects on waist circumference regain; in GHRL, CCK, MLXIPL, and LEPR on weight; in PPARC1A, PCK2, ALOX5AP, PYY, and ADRB3 on waist circumference; and in PPARD, FABP1, PLAUR, and LPIN1 on fat mass regain for dietary protein interaction. CONCLUSION: The observed effects of SNP-diet interactions on weight, waist, and fat mass regain suggest that genetic variation in nutrient-sensitive genes can modify the response to diet. This trial was registered at clinicaltrials.gov as NCT00390637.

Expression of the Rap1 guanine nucleotide exchange factor, MR-GEF, is altered in individuals with bipolar disorder

In the rodent forebrain GABAergic neurons are generated from progenitor cells that express the transcription factors Dlx1 and Dlx2. The Rap-1 guanine nucleotide exchange factor, MR-GEF, is turned on by many of these developing GABAergic neurons. Expression of both Dlx1/2 and MR-GEF is retained in both adult mouse and human forebrain where, in human, decreased Dlx1 expression has been associated with psychosis. Using in situ hybridization studies we show that MR-GEF expression is significantly down-regulated in the forebrain of Dlx1/2 double mutant mice suggesting that MR-GEF and Dlx1/2 form part of a common signalling pathway during GABAergic neuronal development. We therefore compared MR-GEF expression by in situ hybridization in individuals with major psychiatric disorders (schizophrenia, bipolar disorder, major depression) and control individuals. We observed a significant positive correlation between layers II and IV of the dorso-lateral prefrontal cortex (DLPFC) in the percentage of MR-GEF expressing neurons in individuals with bipolar disorder, but not in individuals with schizophrenia, major depressive disorder or in controls. Since MR-GEF encodes a Rap1 GEF able to activate G-protein signalling, we suggest that changes in MR-GEF expression could potentially influence neurotransmission.

Expression of the guanine nucleotide exchange factor, mr-gef, is regulated during the differentiation of specific subsets of telencephalic neurons

We have performed a screen combining subtractive hybridization with PCR to isolate genes that are regulated when neuroepithelial (NE) cells differentiate into neurons. From this screen, we have isolated a number of known genes that have not previously been associated with neurogenesis, together with several novel genes. Here we report that one of these genes, encoding a guanine nucleotide exchange factor (GEF), is regulated during the differentiation of distinct neuronal populations. We have cloned both rat and mouse GEF genes and shown that they are orthologs of the human gene, MR-GEF, which encodes a GEF that specifically activates the small GTPase, Rap1. We have therefore named the rat gene rat mr-gef (rmr-gef) and the mouse gene mouse mr-gef (mmr-gef). Here, we will collectively refer to these two rodent genes as mr-gef. Expression studies show that mr-gef is expressed by young neurons of the developing rodent CNS but not by progenitor cells in the ventricular zone (VZ). The expression pattern of mr-gef during early telencephalic neurogenesis is strikingly similar to that of GABA and the LIM homeobox gene Lhx6, a transcription factor expressed by GABAergic interneurons generated in the ventral telencephalon, some of which migrate into the cortex during development. These observations suggest that mr-gef encodes a protein that is part of a signaling pathway involved in telencephalic neurogenesis; particularly in the development of GABAergic interneurons.

Tangeretin regulates platelet function through inhibition of phosphoinositide 3-Kinase and cyclic nucleotide signaling

OBJECTIVE: Dietary flavonoids have long been appreciated in reducing cardiovascular disease risk factors, but their mechanisms of action are complex in nature. In this study, the effects of tangeretin, a dietary flavonoid, were explored on platelet function, signaling, and hemostasis. APPROACH AND RESULTS: Tangeretin inhibited agonist-induced human platelet activation in a concentration-dependent manner. It inhibited agonist-induced integrin αIIbβ3 inside-out and outside-in signaling, intracellular calcium mobilization, and granule secretion. Tangeretin also inhibited human platelet adhesion and subsequent thrombus formation on collagen-coated surfaces under arterial flow conditions in vitro and reduced hemostasis in mice. Further characterization to explore the mechanism by which tangeretin inhibits platelet function revealed distinctive effects of platelet signaling. Tangeretin was found to inhibit phosphoinositide 3-kinase-mediated signaling and increase cGMP levels in platelets, although phosphodiesterase activity was unaffected. Consistent with increased cGMP levels, tangeretin increased the phosphorylation of vasodilator-stimulated phosphoprotein at S239. CONCLUSIONS: This study provides support for the ability and mechanisms of action of dietary flavonoids to modulate platelet signaling and function, which may affect the risk of thrombotic disease.

Chaos of Wolbachia Sequences Inside the Compact Fig Syconia of Ficus benjamina (Ficus: Moraceae)

Figs and fig wasps form a peculiar closed community in which the Ficus tree provides a compact syconium (inflorescence) habitat for the lives of a complex assemblage of Chalcidoid insects. These diverse fig wasp species have intimate ecological relationships within the closed world of the fig syconia. Previous surveys of Wolbachia, maternally inherited endosymbiotic bacteria that infect vast numbers of arthropod hosts, showed that fig wasps have some of the highest known incidences of Wolbachia amongst all insects. We ask whether the evolutionary patterns of Wolbachia sequences in this closed syconium community are different from those in the outside world. In the present study, we sampled all 17 fig wasp species living on Ficus benjamina, covering 4 families, 6 subfamilies, and 8 genera of wasps. We made a thorough survey of Wolbachia infection patterns and studied evolutionary patterns in wsp (Wolbachia Surface Protein) sequences. We find evidence for high infection incidences, frequent recombination between Wolbachia strains, and considerable horizontal transfer, suggesting rapid evolution of Wolbachia sequences within the syconium community. Though the fig wasps have relatively limited contact with outside world, Wolbachia may be introduced to the syconium community via horizontal transmission by fig wasps species that have winged males and visit the syconia earlier.

Top single nucleotide polymorphisms affecting carbohydrate metabolism in Metabolic Syndrome: from the LIPGENE study

Rationale:Metabolic Syndrome (MetS) is a high prevalence condition characterized by altered energy metabolism, insulin resistance and elevated cardiovascular risk.Objectives:Although many individual single nucleotide polymorphisms (SNPs) have been linked to certain MetS features, there are few studies analyzing the influence of SNPs on carbohydrate metabolism in MetS.Methods:904 SNPs (tag SNPs and functional SNPs) were tested for influence in eight fasting and dynamic markers of carbohydrate metabolism, performing an intravenous glucose tolerance test in 450 participants of the LIPGENE study.Findings:From 382 initial gene-phenotype associations between SNPs and any phenotypic variables, 61 (a 16 % of the pre-selected) remained significant after Bootstrapping. Top SNPs affecting glucose metabolism variables were as follows: fasting glucose: rs26125 (PPARGC1B); fasting insulin: rs4759277 (LRP1); C peptide: rs4759277 (LRP1); HOMA-IR: rs4759277 (LRP1); QUICKI: rs184003 (AGER); SI: rs7301876 (ABCC9), AIRg: rs290481 (TCF7L2) and DI: rs12691 (CEBPA).Conclusions:We describe here the top SNPs linked to phenotypic features in carbohydrate metabolism among aproximately 1000 candidate gene variations in fasting and postprandial samples of 450 patients with MetS from the LIPGENE study.

Single nucleotide polymorphism rs6716901 in SLC25A12 gene is associated with Asperger syndrome

BACKGROUND: Autism Spectrum Conditions (ASC) are a group of developmental conditions which affect communication, social interactions and behaviour. Mitochondrial oxidative dysfunction has been suggested as a mechanism of autism based on the results of multiple genetic association and expression studies. SLC25A12 is a gene encoding a calcium-binding carrier protein that localizes to the mitochondria and is involved in the exchange of aspartate for glutamate in the inner membrane of the mitochondria regulating the cytosolic redox state. rs2056202 SNP in this gene has previously been associated with ASC. SNPs rs6716901 and rs3765166 analysed in this study have not been previously explored in association with AS. METHODS: We genotyped three SNPs (rs2056202, rs3765166, and rs6716901) in SLC25A12 in n?=?117 individuals with Asperger syndrome (AS) and n?=?426 controls, all of Caucasian ancestry. RESULTS: rs6716901 showed significant association with AS (P?=?0.008) after correcting for multiple testing. We did not replicate the previously identified association between rs2056202 and AS in our sample. Similarly, rs3765166 (P?=?0.11) showed no significant association with AS. CONCLUSION: The present study, in combination with previous studies, provides evidence for SLC25A12 as involved in the etiology of AS. Further cellular and molecular studies are required to elucidate the role of this gene in ASC.

Oligonucleotide compound and method for treating nidovirus infections

A method and oligonucleotide compound for inhibiting replication of a nidovirus in virus-infected animal cells are disclosed. The compound (i) has a nuclease-resistant backbone, (ii) is capable of uptake by the infected cells, (iii) contains between 8-25 nucleotide bases, and (iv) has a sequence capable of disrupting base pairing between the transcriptional regulatory sequences in the 5′ leader region of the positive-strand viral genome and negative-strand 3′ subgenomic region. In practicing the method, infected cells are exposed to the compound in an amount effective to inhibit viral replication.