The bean polygalacturonase-inhibiting protein 2 (PvPGIP2) is highly conserved in common bean (Phaseolus vulgaris L.) germplasm and related species

Polygalacturonase-inhibiting proteins (PGIPs) are extracellular plant protein inhibitors of endo-polygalacturonases (PGs) that belong to the leucine-rich repeat (LRR) protein family. In bean, PGIP is encoded by a small gene family of four members among which Pvpgip2 encodes the most wide-spectrum and efficient inhibitor of fungal PGs. In order to evaluate the sequence polymorphism of Pvpgip2 and its functional significance, we have analyzed a number of wild and cultivated bean (P. vulgaris) accessions of Andean and Mesoamerican origin, and some genotypes from the related species P. coccineus, P. acutifolius, and P. lunatus. Our analyses indicate that the protein encoded by Pvpgip2 is highly conserved in the bean germplasm. The few detected polymorphic sites correspond to synonymous substitutions and only two wild genotypes contain a Pvpgip2 with a single non-synonymous replacement. Sequence comparison showed a slightly larger variation in the related bean species P. coccineus, P. acutifolius, and P. lunatus and confirmed the known phylogenetic relationships with P. vulgaris. The majority of the replacements were within the xxLxLxx region of the leucine rich repeat (LRR) domain and none of them affected residues contributing to structural features. The variant PGIPs were expressed in Nicotiana benthamiana using PVX as vector and their inhibitory activity compared to that of PvPPGIP2. All the variants were able to fully inhibit the four fungal PGs tested with minor differences. Taken together these results support the hypothesis that the overall sequence conservation of PGIP2 and minor variation at specific sites is necessary for high-affinity recognition of different fungal PGs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Micronucleus induction in mouse polychromatic erythrocytes by an X-ray contrast agent containing iodine

In this article, the authors report the results of in vivo studies on bone marrow polychromatic erythrocytes (PCE) from mice treated with Urografina?292 (a mixture of sodium amidotrizoate and meglumine amidotrizoate) and with purified sodium amidotrizoate and meglumine amidotrizoate separately or in combination at the same ratio and concentration as that of the highest dose of Urografina?292 used in the experiment. The results showed that Urografina?292 significantly increased the frequencies of micronucleated polychromatic erythrocytes (MNPCEs) in both male (p=0.0082 and p=0.0062) and female (p=0.0350 and p=0.0101) mice treated with doses of 14.3 and 20.0 ml/kg body weight, respectively. When lower doses were used (5.7 and 8.6 ml/kg body weight), the treated mice did not show any significant increase in the frequencies of MNPCEs compared with the negative control group. The same result was observed for both male and female animals treated with purified sodium amidotrizoate and meglumine amidotrizoate separately or in combination. In addition, there was a significant positive correlation between the Urografina?292 doses used and the frequency of micronuclei. These results supported the hypothesis that small amounts of aryl amines present in all X-ray contrast agents containing diatrizoate and closely related triiodobenzoates were responsible for genotoxicity. The frequencies of PCEs in treated animals were determined to estimate the toxicity of Urografina?292, sodium amidotrizoate, and meglumine amidotrizoate to bone marrow, and the results indicated that they did not show any significant difference compared with the negative control group. The fact that mutagenic agents are also generally carcinogenic contributes to the concern with regard to the possible long-term risks of these agents in case of patients who are exposed to iodine-containing X-ray contrast agents during radiodiagnostic procedures.     

The salmonid cathelicidins: A gene family with highly varied C-terminal antimicrobial domains

The cathelicidin family of host defence peptides is regarded as an important component of the host innate immune system. Its members have been found in mammals, birds, primitive vertebrate Atlantic hagfish and, most recently, also in ray-finned fish such as rainbow trout and Atlantic salmon. By using genomic PCR amplifications and RT-PCR tissue analyses we have here investigated and characterized the cathelicidin gene family in three salmonids: brown trout (Salmo trutta fario), brook trout (Salvelinus fontinalis) and grayling (Thymallus thymallus). One or two different genes were found in each species coding for almost identical cathelin-like domains and largely varied cationic C-terminal regions. Multiple alignment of the amino acid sequences let us recognize two distinctive hallmarks of these peptides: the presence of a high number of serine and glycine residues, which may collocate them in a new class of antimicrobial peptides, and of the six-amino-acid repeated sequence RPGGGS detected in a variable number of copies among different cathelicidins. The high variation in length and sequence of this region suggests the existence of a genetically unstable region similar to that found in some mammalian cathelicidins.     

Genome wide evolutionary analyses reveal serotype specific patterns of positive selection in selected Salmonella serotypes

Background  

The bacterium Salmonella enterica includes a diversity of serotypes that cause disease in humans and different animal species. Some Salmonella serotypes show a broad host range, some are host restricted and exclusively associated with one particular host, and some are associated with one particular host species, but able to cause disease in other host species and are thus considered "host adapted". Five Salmonella genome sequences, representing a broad host range serotype (Typhimurium), two host restricted serotypes (Typhi [two genomes] and Paratyphi) and one host adapted serotype (Choleraesuis) were used to identify core genome genes that show evidence for recombination and positive selection.     

AXIN2 and CDH1 polymorphisms, tooth agenesis, and oral clefts

BACKGROUND: AXIN2 and CDH1 genes play important roles during craniofacial morphogenesis. Mutations in these genes have been described in families presenting colorectal cancer and tooth agenesis, and gastric cancer and cleft lip/palate (CL/P). Oral clefts have been associated with tooth agenesis. We investigated if AXIN2 and CDH1 polymorphisms were associated with clefts or with any associated dental subphenotypes. METHODS: Markers in AXIN2 and CDH1 were genotyped using Taqman chemistry in a sample cohort comprised of 500 cleft individuals and 500 unrelated controls. RESULTS: Comparison between cleft and control groups showed a trend for association for AXIN2 with incomplete cleft palate (p = .006) and CDH1 with unilateral CL/P (p = .03 for left CL/P and p = .04 for right CL/P). Comparison of cleft subphenotypes with tooth agenesis and controls revealed borderline associations for CDH1 (p = .008) and AXIN2 (p = .01) with unilateral right CL/P with tooth agenesis. CONCLUSIONS: We observed only borderline results for the association of AXIN2 and CDH1 with CL/P with and without tooth agenesis. Nevertheless, implication of these genes in the simultaneous occurrence of CL/P and cancer, and in tooth agenesis and cancer, is rather intriguing and warrants further investigations with other geographic and ethnic populations. Birth Defects Research (Part A), 2009. © 2008 Wiley‐Liss, Inc.     

Mutagenesis and Chemical Cross-Linking Suggest that Wzz Dimer Stability and Oligomerization Affect Lipopolysaccharide O-Antigen Modal Chain Length Control

In Shigella flexneri, the polysaccharide copolymerase (PCP) protein Wzz_SF confers a modal length of 10 to 17 repeat units (RUs) to the O-antigen (Oag) component of lipopolysaccharide (LPS). PCPs form oligomeric structures believed to be related to their function. To identify functionally important regions within Wzz_SF, random in-frame linker mutagenesis was used to create mutants with 5-amino-acid insertions (termed Wzz_i proteins), and DNA sequencing was used to locate the insertions. Analysis of the resulting LPS conferred by Wzz_i proteins identified five mutant classes. The class I mutants were inactive, resulting in nonregulated LPS Oag chains, while classes II and III conferred shorter LPS Oag chains of 2 to 10 and 8 to 14 RUs, respectively. Class IV mutants retained near-wild-type function, and class V mutants increased the LPS Oag chain length to 16 to 25 RUs. In vivo formaldehyde cross-linking indicated class V mutants readily formed high-molecular-mass oligomers; however, class II and III Wzz_i mutants were not effectively cross-linked. Wzz dimer stability was also investigated by heating cross-linked oligomers at 100°C in the presence of SDS. Unlike the Wzz_SF wild type and class IV and V Wzz_i mutants, the class II and III mutant dimers were not detectable. The location of each insertion was mapped onto available PCP three-dimensional (3D) structures, revealing that class V mutations were most likely located within the inner cavity of the PCP oligomer. These data suggest that the ability to produce stable dimers may be important in determining Oag modal chain length.Lipopolysaccharide (LPS) of Shigella flexneri is an important virulence factor, providing protection against host defenses and affecting interaction with host cells. LPS is composed of three regions: the hydrophobic lipid A membrane anchor, the core sugar region, and the O-antigen (Oag) polysaccharide chain (18). The basic Oag repeat unit of S. flexneri is a tetrasaccharide consisting of three rhamnose sugars and one N-acetylglucosamine sugar (19). The contribution of Shigella Oag to establishing virulence has been extensively investigated, and results indicate that regulated Oag modal length is required for virulence (8, 23, 25). Loss of Oag modal chain length regulation affects virulence due to the masking of the outer membrane (OM) protein IcsA (8, 23), and the type III secretion system is also affected by Oag chain length (24).The current model for Oag biogenesis in S. flexneri involves the initiation of Oag repeat unit synthesis on the cytoplasmic face of the inner membrane (IM) and continues with a series of successive sugar transferase reactions. The repeat units are assembled on the lipid carrier undecaprenol phosphate (Und-P), and transported across the IM by the Wzx flippase to the periplasmic face of the IM. Polymerization of Oag repeat units is catalyzed by the Wzy polymerase, linking the individual oligosaccharide repeat units into a chain; the nascent chain is transferred from its lipid carrier to the nonreducing end of the newly flipped oligosaccharide repeat unit. The resulting chain is then ligated to the lipid A core by WaaL ligase (18, 26) to form LPS.The regulation of the chain length of the Oag polysaccharide is controlled by the Wzz protein, a member of the polysaccharide copolymerase 1a (PCP1a) family (13, 21). S. flexneri Wzz (Wzz_SF) confers an average chain modal length of 10 to 17 Oag repeat units. In addition to determining the Oag chain modal length, PCP proteins are involved in enterobacterial common antigen (ECA) modal chain length regulation and biosynthesis and in capsule polysaccharide (CPS) and exopolysaccharide (EPS) biosynthesis (13). The PCP1a proteins are located in the IM and have two transmembrane (TM) regions, TM1 and TM2 (14). TM1 is located close to the N-terminal end, and TM2 is located near the C-terminal end, while the hydrophilic region between TM regions is located in the periplasm (14). PCPs exhibit a conserved motif, proximal to and partly overlapping the TM2 region, rich in proline and glycine residues (2, 3, 13). Site-directed mutagenesis studies targeting a number of these conserved residues, singularly or in combination, indicate that changes to this region have a significant effect on the resulting Oag modal chain length (4). Many mutagenesis studies on residues throughout Wzz indicate that function may be an overall property of the protein and may not be limited to one particular region (4, 6, 21). Despite studies conducted to probe the Wzz structure function relationship, little is known about the mode of action in determining Oag modal chain length. Recently, the periplasmic domain structures of a collection of PCP proteins including Salmonella enterica serovar Typhimurium WzzB (Wzz_ST) and Escherichia coli O157 FepE and WzzE have been solved, and it has been deduced that these structures show marked similarities at the protomer and oligomer levels (21). These protomers are elongated and consist of two structural components: a trapezoidal α/β base domain close to the membrane and an extended α-helical hairpin containing an ∼100-Å-long helix forming anti-parallel coiled-coil interactions with two helices that fold back toward the membrane (21). The protomers self-assemble into bell-shaped oligomers displaying comparable structural features, with Wzz_ST forming pentameric oligomers, WzzE assembling into octameric oligomers, and FepE assembling into nonameric structures (21). In contrast, a recent study from Larue et al. reports that Wzz_ST, FepE, and Wzz_K40 favor hexameric structures (9). A previous study on the oligomeric status of S. flexneri WzzB (Wzz_SF) via in vivo cross-linking with formaldehyde indicated that Wzz_SF has the ability to form hexamers and high-order oligomers, suggesting that oligomerization is important in function (4). Related to this, Marolda et al. have shown that the ECA-associated Wzx can fully complement an LPS Oag-associated Wzx-deficient mutant if the remaining ECA gene cluster is deleted, providing genetic evidence that proteins involved in Oag/ECA biosynthesis and processing may function as a complex (11).Several models of the likely mechanisms of Oag chain regulation have been proposed. Bastin et al. initially suggested that Wzz acts as a molecular timer, allowing polymerization to occur to a particular point, hence increasing the number of repeat units added to the chain (1). An alternative model proposed by Morona et al. suggested that Wzz acts as a molecular chaperone, facilitating the interaction between Wzy and WaaL, and modal length is the result of the ratio of Wzy and WaaL (14). Published data indicated that the ratio of Wzy and Wzz was important in determining Oag modal chain length, which is supportive of the latter model (5). With recent developments in solving the PCP three-dimensional (3D) structure and oligomeric arrangement, a new model has been proposed by Tocilj et al. in which the Wzz oligomers act as molecular scaffolds for multiple Wzy polymerase molecules and the growing Oag chain is transferred from one Wzy to another Wzy molecule (21).In a previous study, site-directed mutagenesis analysis was conducted on Wzz_SF (4). Although mutational alterations targeting the TM regions caused dramatic changes in the resulting LPS Oag chain length, mutations targeting the periplasmic domain generally did not have an obvious effect on the resulting LPS Oag chain length. This was also shown for mutations in FepE (17). Hence, we decided that a more severe approach to Wzz_SF mutagenesis was needed to investigate the relationship between Wzz structure and function by increasing the likelihood of acquiring Wzz mutants displaying phenotypic changes. In this study, we have investigated the structure and function of Wzz_SF by constructing a library of in-frame linker mutants with 5-amino-acid (aa) insertions throughout the Wzz_SF protein. We have identified regions in Wzz_SF which alter the modal length in different ways and present biochemical evidence acquired by in vivo chemical cross-linking that indicates oligomeric differences exist between Wzz mutants and the wild type (WT). We also present evidence that suggests the dimeric form of Wzz_SF is important in establishing modal length.     

The role of disulfide bonds and N‐terminus in the structural properties of hepcidins: Insights from molecular dynamics simulations

The main purpose of this work is to analyse, by means of molecular dynamics (MD) simulations both structural and mechanical‐dynamical differences between Hepcidin‐20 and Hepcidin‐25 in both oxidized and reduced states in aqueous solution. Results indicate that the presence of disulfide bonds is essential, in both peptides, for maintaining their β‐hairpin motif. As a matter of fact, the lack of this intra‐peptide covalent interactions produces an almost immediate deviation from the oxidized, plausibly active, structure in both the systems. Interestingly, reduced Hepcidin‐25 turns out to be characterized by a highly fluctuating structure which is found to rapidly span a large number of configurations at equilibrium. On the other hand, loss of disulfide bonds in the shorter peptide, results in a more compact and relatively rigid double‐turn structure. Comparison of mechanical–dynamical properties and sidechains–sidechains interactions in oxidized Hepcidin‐20 and Hepcidin‐25 strongly suggest also the key role of N‐terminus in the aggregation tendency of Hepcidin‐25. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 917–926, 2010.     

Social Networks Shape the Transmission Dynamics of Hepatitis C Virus

Hepatitis C virus (HCV) infects 170 million people worldwide, and is a major public health problem in Brazil, where over 1% of the population may be infected and where multiple viral genotypes co-circulate. Chronically infected individuals are both the source of transmission to others and are at risk for HCV-related diseases, such as liver cancer and cirrhosis. Before the adoption of anti-HCV control measures in blood banks, this virus was mainly transmitted via blood transfusion. Today, needle sharing among injecting drug users is the most common form of HCV transmission. Of particular importance is that HCV prevalence is growing in non-risk groups. Since there is no vaccine against HCV, it is important to determine the factors that control viral transmission in order to develop more efficient control measures. However, despite the health costs associated with HCV, the factors that determine the spread of virus at the epidemiological scale are often poorly understood. Here, we sequenced partial NS5b gene sequences sampled from blood samples collected from 591 patients in São Paulo state, Brazil. We show that different viral genotypes entered São Paulo at different times, grew at different rates, and are associated with different age groups and risk behaviors. In particular, subtype 1b is older and grew more slowly than subtypes 1a and 3a, and is associated with multiple age classes. In contrast, subtypes 1a and 3b are associated with younger people infected more recently, possibly with higher rates of sexual transmission. The transmission dynamics of HCV in São Paulo therefore vary by subtype and are determined by a combination of age, risk exposure and underlying social network. We conclude that social factors may play a key role in determining the rate and pattern of HCV spread, and should influence future intervention policies.     

The horizontal flow of the plasmid resistome: clues from inter‐generic similarity networks

By integrating sequence similarity data of plasmid‐encoded antibiotic resistance determinants with those coming from a less transferred molecular marker, we constructed a network in which all the sequences that most likely underwent horizontal gene transfer (HGT) were linked together. The analysis of this network revealed that either geographical barriers or taxonomical distance can often be overcome since phylogenetically unrelated bacteria, and/or those inhabiting distinct environments, were found to share common antibiotic resistance determinants, probably as a result of (one or multiple) HGT event(s). Data obtained also revealed that bacteria viable through multiple environments (ubiquitous) are likely to give a crucial contribution to the spreading of bacterial resistance towards antimicrobial compounds. These analyses represent a first attempt to give an almost global picture of the horizontal flow of antibiotic resistance determinants at the whole bacterial community level, also underlining the power of HGT among bacteria and how this ‘horizontal flow’ is poorly affected by both taxonomy and physical distance. Finally, data presented may be useful in the infections control procedures, suggesting which bacterial species are more likely acting as vectors of antibiotic resistance determinants.