Restrictions to the adaptation of influenza A virus H5 hemagglutinin to the human host

The binding specificities of a panel of avian influenza virus subtype H5 hemagglutinin (RA) proteins bearing mutations at key residues in the receptor binding site were investigated. The results demonstrate that two simultaneous mutations in the receptor binding site resulted in H5 RA binding in a pattern similar to that shown by human viruses. Coexpression of the ion channel protein, M2, from most avian and human strains tested protected H5 RA conformation during trafficking, indicating that no genetic barrier to the reassortment of the H5 surface antigen gene with internal genes of human viruses existed at this level.

Rapid parallel expression in E-coli and insect cells: Analysis of five lef gene products of the Autographa californica multiple nuclear polyhedrosis virus (AcMNPV)

A number of strategies are emerging for the high throughput (HTP) expression of recombinant proteins to enable structural and functional study. Here we describe a workable HTP strategy based on parallel protein expression in E. coli and insect cells. Using this system we provide comparative expression data for five proteins derived from the Autographa californica polyhedrosis virus genome that vary in amino acid composition and in molecular weight. Although the proteins are part of a set of factors known to be required for viral late gene expression, the precise function of three of the five, late expression factors (lefs) 6, 7 and 10, is unknown. Rapid expression and characterisation has allowed the determination of their ability to bind DNA and shown a cellular location consistent with their properties. Our data point to the utility of a parallel expression strategy to rapidly obtain workable protein expression levels from many open reading frames (ORFs).

Distribution, gene sequence and expression in vivo of the plasmid encoded fimbrial antigen of Salmonella serotype Enteritidis

The pefA gene which encoded the serotype associated plasmid (SAP) mediated fimbrial major subunit antigen of Salmonella enterica serotype Typhimurium shared genetic identity with 128 of 706 salmonella isolates as demonstrated by dot (colony) hybridization. Seventy-seven of 113 isolates of Typhimurium and individual isolates of serotypes Bovis-morbificans, Cholerae-suis and Enteritidis phage type 9b hybridized pefA strongly, whereas 48 isolates of Enteritidis hybridized pefA weakly and one Enteritidis isolate of phage type 14b failed to hybridize. Individual isolates of 294 serotypes and 247 individual isolates of serotype Dublin did not hybridize pefA. Southern hybridization of plasmids extracted from Enteritidis demonstrated that the pefA gene probe hybridized strongly an atypical SAP of 80 kb in size harboured by one Enteritidis isolate of phage-type 9b, whereas the typical SAP of 58 kb in size harboured by 48 Enteritidis isolates hybridized weakly. One Enteritidis isolate of phage type 14b which failed to hybridize pefA in dot (colony) hybridization experiments was demonstrated to be plasmid free. A cosmid library of Enteritidis phage type 4 expressed in Escherichia coli K12 was screened by hybridization for the presence of pef sequences. Recombinant clones which were deduced to harbour the entire pef operon elaborated a PEF-like fimbrial structure at the cell surface. The PEF-like fimbrial antigen was purified from one cosmid clone and used in western blot experiments with sera from chickens infected with Enteritidis phage-type 4. Seroconversion to the fimbrial antigen was observed which indicated that the Enteritidis PEF-like fimbrial structure was expressed at some stage during infection. Nucleotide sequence analysis demonstrated that the pefA alleles of Typhimurium and Enteritidis phage-type 4 shared 76% DNA nucleotide and 82% deduced amino acid sequence identity.

Expression of the efflux pump genes cmeB, cmeF and the porin gene porA in multiple-antibiotic-resistant Campylobacter jejuni

Aims: In Escherichia coli, increased expression of efflux pumps and/or decreased expression of porins can confer multiple antibiotic resistance (MAR), causing resistance to at least three unrelated classes of antibiotics, detergents and dyes. It was hypothesized that in Campylobacter jejuni, the efflux systems CmeABC, CmeDEF and the major outer membrane porin protein, MOMP (encoded by porA) could confer MAR. Methods: The expression of cmeB, cmeF and porA in 32 MAR C. jejuni isolated from humans or poultry was determined by comparative (C)-reverse transcriptase (RT)-PCR and denaturing DHPLC. A further 13 ethidium bromide-resistant isolates and three control strains were also investigated. Accumulation of ciprofloxacin carbonyl cyanide-m-chlorophenyl hydrazone (CCCP) was also determined for all strains. Results: Although resistance to ethidium bromide has been associated with MAR, expression of all three genes was similar in the ethidium bromide-resistant isolates. These data indicate that CmeB, CmeF and MOMP play no role in resistance to this agent in C. jejuni. Six MAR isolates over-expressed cmeB, 3/32 over-expressed cmeB and cmeF. No isolates over-expressed cmeF alone. Expression of porA was similar in all isolates. All nine isolates that over-expressed cmeB contained a mutation in cmeR, substituting glycine 86 with alanine. All cmeB over-expressing isolates also accumulated low concentrations of ciprofloxacin, which were restored to wild-type levels in the presence of CCCP. Conclusions: These data indicate that over-expression of cmeB is associated with MAR in isolates of C. jejuni. However, as cmeB was over-expressed by only one-third (9/32) of MAR isolates, these data also indicate other mechanisms of MAR in C. jejuni.

Jupiter's and Saturn's convectively driven banded jets in the laboratory

The banded patterns of cloud and wind are among the most striking features of the atmospheres of Jupiter and Saturn, but their dynamical origin remains poorly understood. Most approaches towards understanding zonation so far (also in the terrestrial oceans) have used highly idealized models to show that it might originate from dynamical anisotropy in a shallow turbulent fluid layer due to the planetary β-effect. Here we report the results of laboratory experiments, conducted on a 14-m diameter turntable, which quantitatively confirm that multiple zonal jets may indeed be generated and maintained by this mechanism in the presence of deep convection and a topographic β-effect. At the very small values of Ekman number (≤2 × 10−5) and large local Reynolds numbers (≥2000, based on jet scales) achieved, the kinetic energy spectra suggest the presence of both energy-cascading and enstrophy-cascading inertial ranges in addition to the zonation near twice the Rhines wave number.

Effect of prolonged intravenous glucose and essential amino acid infusion on nitrogen balance, muscle protein degradation and ubiquitin-conjugating enzyme gene expression in calves

Background: Intravenous infusions of glucose and amino acids increase both nitrogen balance and muscle accretion. We hypothesised that co-infusion of glucose ( to stimulate insulin) and essential amino acids (EAA) would act additively to improve nitrogen balance by decreasing muscle protein degradation in association with alterations in muscle expression of components of the ubiquitin-proteasome proteolytic pathway. Methods: We examined the effect of a 5 day intravenous infusions of saline, glucose, EAA and glucose + EAA, on urinary nitrogen excretion and muscle protein degradation. We carried out the study in 6 restrained calves since ruminants offer the advantage that muscle protein degradation can be assessed by excretion of 3 methyl-histidine and multiple muscle biopsies can be taken from the same animal. On the final day of infusion blood samples were taken for hormone and metabolite measurement and muscle biopsies for expression of ubiquitin, the 14-kDa E2 ubiquitin conjugating enzyme, and proteasome sub-units C2 and C8. Results: On day 5 of glucose infusion, plasma glucose, insulin and IGF-1 concentrations were increased while urea nitrogen excretion and myofibrillar protein degradation was decreased. Co-infusion of glucose + EAA prevented the loss of urinary nitrogen observed with EAA infusions alone and enhanced the increase in plasma IGF-1 concentration but there was no synergistic effect of glucose + EAA on the decrease in myofibrillar protein degradation. Muscle mRNA expression of the ubiquitin conjugating enzyme, 14-kDa E2 and proteasome sub-unit C2 were significantly decreased, after glucose but not amino acid infusions, and there was no further response to the combined infusions of glucose + EAA. Conclusion: Prolonged glucose infusion decreases myofibrillar protein degradation, prevents the excretion of infused EAA, and acts additively with EAA to increase plasma IGF-1 and improve net nitrogen balance. There was no evidence of synergistic effects between glucose + EAA infusion on muscle protein degradation or expression of components of the ubiquitin-proteasome proteolytic pathway.

Sequentially testing for a gene-drug interaction in a genomewide analysis

Assaying a large number of genetic markers from patients in clinical trials is now possible in order to tailor drugs with respect to efficacy. The statistical methodology for analysing such massive data sets is challenging. The most popular type of statistical analysis is to use a univariate test for each genetic marker, once all the data from a clinical study have been collected. This paper presents a sequential method for conducting an omnibus test for detecting gene-drug interactions across the genome, thus allowing informed decisions at the earliest opportunity and overcoming the multiple testing problems from conducting many univariate tests. We first propose an omnibus test for a fixed sample size. This test is based on combining F-statistics that test for an interaction between treatment and the individual single nucleotide polymorphism (SNP). As SNPs tend to be correlated, we use permutations to calculate a global p-value. We extend our omnibus test to the sequential case. In order to control the type I error rate, we propose a sequential method that uses permutations to obtain the stopping boundaries. The results of a simulation study show that the sequential permutation method is more powerful than alternative sequential methods that control the type I error rate, such as the inverse-normal method. The proposed method is flexible as we do not need to assume a mode of inheritance and can also adjust for confounding factors. An application to real clinical data illustrates that the method is computationally feasible for a large number of SNPs. Copyright (c) 2007 John Wiley & Sons, Ltd.

The evolution of mobile DNAs: when will transposons create phylogenies that look as if there is a master gene?

Some families of mammalian interspersed repetitive DNA, such as the Alu SINE sequence, appear to have evolved by the serial replacement of one active sequence with another, consistent with there being a single source of transposition: the "master gene." Alternative models, in which multiple source sequences are simultaneously active, have been called "transposon models." Transposon models differ in the proportion of elements that are active and in whether inactivation occurs at the moment of transposition or later. Here we examine the predictions of various types of transposon model regarding the patterns of sequence variation expected at an equilibrium between transposition, inactivation, and deletion. Under the master gene model, all bifurcations in the true tree of elements occur in a single lineage. We show that this property will also hold approximately for transposon models in which most elements are inactive and where at least some of the inactivation events occur after transposition. Such tree shapes are therefore not conclusive evidence for a single source of transposition.

Multiple domestications and their taxonomic consequences: the example of Phaseolus vulgaris

The evolutionary history of P. vulgaris is important to those working on its genetic resources, but is not reflected in its infraspecific taxonomy. Genetic isolation of wild populations between and also within Middle and South America has resulted in morphological and molecular differentiation. Populations from northern and southern ends of the range are assigned to different gene pools, though intermediates occur in intervening areas. Chloroplast haplotypes suggest three distinct lineages of wild beans and several intercontinental dispersals. The species was domesticated independently in both Middle and South America, probably several times in Middle America. This, together with further differentiation under human selection, has produced distinct races among domesticated beans. The informal categories of wild versus domesticated, gene pool, and race convey the evolutionary picture more clearly than the formal categories provided by the Codes of Nomenclature for wild or cultivated plants.

Flavonoids and brain health: multiple effects underpinned by common mechanisms

The neuroprotective actions of dietary flavonoids involve a number of effects within the brain, including a potential to protect neurons against injury induced by neurotoxins, an ability to suppress neuroinflammation, and the potential to promote memory, learning and cognitive function. This multiplicity of effects appears to be underpinned by two processes. Firstly, they interact with important neuronal signalling cascades leading to an inhibition of apoptosis triggered by neurotoxic species and to a promotion of neuronal survival and differentiation. These interactions include selective actions on a number of protein kinase and lipid kinase signalling cascades, most notably the PI3K/Akt and MAP kinase pathways which regulate pro-survival transcription factors and gene expression. Secondly, they induce peripheral and cerebral vascular blood flow in a manner which may lead to the induction of angiogenesis, and new nerve cell growth in the hippocampus. Therefore, the consumption of flavonoid-rich foods, such as berries and cocoa, throughout life holds a potential to limit the neurodegeneration associated with a variety of neurological disorders and to prevent or reverse normal or abnormal deteriorations in cognitive performance.

Variation in the FFAR1 gene modifies BMI, body composition and plasma lipids but not plasma insulin or Beta-cell function in overweight subjects

Background FFAR1 receptor is a long chain fatty acid G-protein coupled receptor which is expressed widely, but found in high density in the pancreas and central nervous system. It has been suggested that FFAR1 may play a role in insulin sensitivity, lipotoxicity and is associated with type 2 diabetes. Here we investigate the effect of three common SNPs of FFAR1 (rs2301151; rs16970264; rs1573611) on pancreatic function, BMI, body composition and plasma lipids. Methodology/Principal Findings For this enquiry we used the baseline RISCK data, which provides a cohort of overweight subjects at increased cardiometabolic risk with detailed phenotyping. The key findings were SNPs of the FFAR1 gene region were associated with differences in body composition and lipids, and the effects of the 3 SNPs combined were cumulative on BMI, body composition and total cholesterol. The effects on BMI and body fat were predominantly mediated by rs1573611 (1.06 kg/m2 higher (P = 0.009) BMI and 1.53% higher (P = 0.002) body fat per C allele). Differences in plasma lipids were also associated with the BMI-increasing allele of rs2301151 including higher total cholesterol (0.2 mmol/L per G allele, P = 0.01) and with the variant A allele of rs16970264 associated with lower total (0.3 mmol/L, P = 0.02) and LDL (0.2 mmol/L, P<0.05) cholesterol, but also with lower HDL-cholesterol (0.09 mmol/L, P<0.05) although the difference was not apparent when controlling for multiple testing. There were no statistically significant effects of the three SNPs on insulin sensitivity or beta cell function. However accumulated risk allele showed a lower beta cell function on increasing plasma fatty acids with a carbon chain greater than six. Conclusions/Significance Differences in body composition and lipids associated with common SNPs in the FFAR1 gene were apparently not mediated by changes in insulin sensitivity or beta-cell function.

Induction of the ferritin gene (ftnA) of Escherichia coli by Fe2+–Fur is mediated by reversal of H-NS silencing and is RyhB independent

FtnA is the major iron-storage protein of Escherichia coli accounting for < or = 50% of total cellular iron. The FtnA gene (ftnA) is induced by iron in an Fe(2+)-Fur-dependent fashion. This effect is reportedly mediated by RyhB, the Fe(2+)-Fur-repressed, small, regulatory RNA. However, results presented here show that ftnA iron induction is independent of RyhB and instead involves direct interaction of Fe(2+)-Fur with an 'extended' Fur binding site (containing five tandem Fur boxes) located upstream (-83) of the ftnA promoter. In addition, H-NS acts as a direct repressor of ftnA transcription by binding at multiple sites (I-VI) within, and upstream of, the ftnA promoter. Fur directly competes with H-NS binding at upstream sites (II-IV) and consequently displaces H-NS from the ftnA promoter (sites V-VI) which in turn leads to derepression of ftnA transcription. It is proposed that H-NS binding within the ftnA promoter is facilitated by H-NS occupation of the upstream sites through H-NS oligomerization-induced DNA looping. Consequently, Fur displacement of H-NS from the upstream sites prevents cooperative H-NS binding at the downstream sites within the promoter, thus allowing access to RNA polymerase. This direct activation of ftnA transcription by Fe(2+)-Fur through H-NS antisilencing represents a new mechanism for iron-induced gene expression.

Evolutionary genomics of the Fox genes: origin of gene families and the ancestry of gene clusters

Over the past decade genomic approaches have begun to revolutionise the study of animal diversity. In particular, genome sequencing programmes have spread beyond the traditional model species to encompass an increasing diversity of animals from many different phyla, as well as unicellular eukaryotes that are closely related to the animals. Whole genome sequences allow researchers to establish, with reasonable confidence, the full complement of any particular family of genes in a genome. Comparison of gene complements from appropriate genomes can reveal the evolutionary history of gene families, indicating when both gene diversification and gene loss have occurred. More than that, however, assembled genomes allow the genomic environment in which individual genes are found to be analysed and compared between species. This can reveal how gene diversification occurred. Here, we focus on the Fox genes, drawing from multiple animal genomes to develop an evolutionary framework explaining the timing and mechanism of origin of the diversity of animal Fox genes. Ancient linkages between genes are a prominent feature of the Fox genes, depicting a history of gene clusters, some of which may be relevant to understanding Fox gene function.

Pathogenic LRRK2 mutations do not alter gene expression in cell model systems or human brain tissue

Point mutations in LRRK2 cause autosomal dominant Parkinson's disease. Despite extensive efforts to determine the mechanism of cell death in patients with LRRK2 mutations, the aetiology of LRRK2 PD is not well understood. To examine possible alterations in gene expression linked to the presence of LRRK2 mutations, we carried out a case versus control analysis of global gene expression in three systems: fibroblasts isolated from LRRK2 mutation carriers and healthy, non-mutation carrying controls; brain tissue from G2019S mutation carriers and controls; and HEK293 inducible LRRK2 wild type and mutant cell lines. No significant alteration in gene expression was found in these systems following correction for multiple testing. These data suggest that any alterations in basal gene expression in fibroblasts or cell lines containing mutations in LRRK2 are likely to be quantitatively small. This work suggests that LRRK2 is unlikely to play a direct role in modulation of gene expression, although it remains possible that this protein can influence mRNA expression under pathogenic cicumstances.

Sequence heterogeneity of an mpb70 gene analogue in Mycobacterium kansasii

The protein antigen MPB70 is a major component of culture supernatants of Mycobacterium bovis and Is an active ingredient of bovine PPD used for skin-testing cattle for tuberculosis. we have shown that Mycobacterium kansasii possesses a similar gene that cross-reacts in a PCR test for M. bovis. Single strand conformational polymorphism analysis, and the DNA sequence of the PCR product, shows differences between M. kansasii strains, supporting the suggestion that M. kansasii is not a homogeneous species.