The cytoplasmic phosphoproteome of the Gram-negative bacterium Campylobacter jejuni: evidence for modification by unidentified protein kinases

We have undertaken a comprehensive analysis of cytoplasmic protein phosphorylation in Campylobacter jejuni by mass spectrometric identification of phosphoproteins and localization of the sites of modification by phosphopeptide analyses. Cell extracts, enriched for phosphoproteins using Fe(III) IMAC or commercial phosphoprotein purification kits, were analyzed by 1-D and 2-D SDS-PAGE and subjected to mass fingerprinting by in-gel tryptic digestion and MALDI-TOF MS. Fifty-eight phosphopeptides were identified from 1-D gel bands by nano-LC-MS/MS and automated searching in a C. jejuni ORF database resulting in the unequivocal identification of 36 phosphoproteins of diverse function. In addition to elongation factors and chaperonins, which have been reported to be phosphorylated in other bacteria, the major phosphoproteins included bacterioferritin and superoxide dismutase. The sequences around the phosphorylated Ser and Thr residues are indicative of specific kinases being responsible for some of the modifications. However, many of the other identified proteins are enzymes that have phosphorylated substrates, including ATP, hence other modifications may arise from autophosphorylation. Comparative analyses of IMAC extracts from the Escherichia coli strain AD202 and Helicobacter pylori resulted in the identification of homologs of six of the C. jejuni phosphoproteins, though their overall phosphoproteome maps were distinctly different.     

Conformational differences between two amyloid β oligomers of similar size and dissimilar toxicity

Several protein conformational disorders (Parkinson and prion diseases) are linked to aberrant folding of proteins into prefibrillar oligomers and amyloid fibrils. Although prefibrillar oligomers are more toxic than their fibrillar counterparts, it is difficult to decouple the origin of their dissimilar toxicity because oligomers and fibrils differ both in terms of structure and size. Here we report the characterization of two oligomers of the 42-residue amyloid β (Aβ42) peptide associated with Alzheimer disease that possess similar size and dissimilar toxicity. We find that Aβ42 spontaneously forms prefibrillar oligomers at Aβ concentrations below 30 μm in the absence of agitation, whereas higher Aβ concentrations lead to rapid formation of fibrils. Interestingly, Aβ prefibrillar oligomers do not convert into fibrils under quiescent assembly conditions but instead convert into a second type of oligomer with size and morphology similar to those of Aβ prefibrillar oligomers. Strikingly, this alternative Aβ oligomer is non-toxic to mammalian cells relative to Aβ monomer. We find that two hydrophobic peptide segments within Aβ (residues 16-22 and 30-42) are more solvent-exposed in the more toxic Aβ oligomer. The less toxic oligomer is devoid of β-sheet structure, insoluble, and non-immunoreactive with oligomer- and fibril-specific antibodies. Moreover, the less toxic oligomer is incapable of disrupting lipid bilayers, in contrast to its more toxic oligomeric counterpart. Our results suggest that the ability of non-fibrillar Aβ oligomers to interact with and disrupt cellular membranes is linked to the degree of solvent exposure of their central and C-terminal hydrophobic peptide segments.     

Health effects of dietary Maillard reaction products: the results of ICARE and other studies

In food science the Maillard reaction is well known to cause degradation of amino acids and an overall decrease in the nutritional value of foods that have been subjected to heat in processing. There has been evidence more recently of the endogenous formation of some Maillard reaction products (MRPs) in biological systems and their association with pathophysiological conditions including diabetes, renal disease and cardiovascular disease. Several studies have suggested that dietary MRPs increase the in vivo pool of MRPs after intestinal absorption and contribute to the development of diabetes and related complications. This review focuses on the animal and human studies which have assessed the eventual implications of dietary MRPs on human health, highlighting the different diets tested, the experimental designs and the biomarkers selected to estimate the health effects. The results of these studies are compared to those of the recently published ICARE study. In this latter study an accurate determination of the MRP content of the diets was achieved, allowing the calculation of the contribution of individual food groups to daily MRP intakes in a regular western diet.     

Pregnancy complications among women born preterm

Background:

Adults who were born with low birth weights are at increased risk of cardiovascular and metabolic conditions, including pregnancy complications. Low birth weight can result from intrauterine growth restriction, preterm birth or both. We examined the relation between preterm birth and pregnancy complications later in life.

Methods:

We conducted a population-based cohort study in the province of Quebec involving 7405 women born preterm (554 < 32 weeks, 6851 at 32–36 weeks) and a matched cohort of 16 714 born at term between 1976 and 1995 who had a live birth or stillbirth between 1987 and 2008. The primary outcome measures were pregnancy complications (gestational diabetes, gestational hypertension, and preeclampsia or eclampsia).

Results:

Overall, 19.9% of women born at less than 32 weeks, 13.2% born at 32–36 weeks and 11.7% born at term had at least 1 pregnancy complication at least once during the study period (p < 0.001). Women born small for gestational age (both term and preterm) had increased odds of having at least 1 pregnancy complication compared with women born at term and at appropriate weight for gestational age. After adjustment for various factors, including birth weight for gestational age, the odds of pregnancy complications associated with preterm birth was elevated by 1.95-fold (95% confidence interval [CI] 1.54–2.47) among women born before 32 weeks’ gestation and 1.14-fold (95% CI 1.03–1.25) among those born at 32–36 weeks’ gestation relative to women born at term.

Interpretation:

Being born preterm, in addition to, and independent of, being small for gestational age, was associated with a significantly increased risk of later having pregnancy complications.Numerous studies examining cohorts born mostly in the first half of the 20th century have emphasized the inverse relation between low birth weight and incidence later in life of cardiovascular and metabolic conditions, such as hypertension and type 2 diabetes.¹ Epidemiologic studies seldom consider the effects of preterm birth and intrauterine growth restriction separately when studying the relation of these factors to low birth weight. Studies have suggested that adolescents and young adults born preterm have higher incidence of risk factors for metabolic (insulin resistance) and cardiovascular (higher blood pressure) dysfunctions.^2–4 With the increased survival of preterm newborns over the past 30 years, a substantially greater proportion of young adults are born before 37 or even 32 weeks’ gestation and thus may represent a growing population at risk for conditions related to metabolic syndrome as they get older.Pregnancy can be considered a stress test for future cardiovascular and metabolic health. Women with a history of gestational diabetes, gestational hypertension or preeclampsia are at increased risk of metabolic syndrome later in life.^5–7 Furthermore, studies have shown that women born with low birth weights are at increased risk of gestational hypertension, preeclampsia and gestational diabetes.^8–10 However, many of these studies either have not taken into account gestational age or have a number of shortcomings, such as small sample, young population (mostly teenagers), degree of prematurity not specified or study population consisting mostly of late preterm births.^6,11,12In the province of Quebec, weight and gestational age have been recorded in a registry for all births since 1976, and data on all hospital-based diagnoses have been collected since 1987. The aim of our study was to examine the relation between preterm birth and later pregnancy complications, independently of intrauterine growth restriction, among women born preterm in Quebec between 1976 and 1995 who delivered at least 1 newborn between 1987 and 2008. We also examined whether this association represents a dose–response relation, namely, whether the more prematurely born a woman is, the greater her risk of gestational diabetes, gestational hypertension, preeclampsia or eclampsia.     

Quantitative methylation-sensitive arbitrarily primed PCR method to determine differential genomic DNA methylation in Down Syndrome

Relative levels of DNA hypermethylation were quantified in DS individuals using a new method based on a combination of methylation-sensitive arbitrarily primed polymerase chain reaction (MS-AP-PCR) and quantification of DNA fragments with the Agilent 2100 bioanalyzer. Four of the DS individuals had low plasma total homocysteine (tHcy) level (4.3 +/- 0.3 micromol/l) and 4 other had high-tHcy level (14.1 +/- 0.9 micromol/l). Eight healthy control individuals were matched to the DS cases for age, sex, and tHcy levels. We have identified and quantified six hypermethylated fragments. Their sizes ranged from 230-bp to 700-bp. In cases and controls, low-tHcy did not affect methylation level of identified fragments, mean methylation values were 68.0 +/- 39.7% and 52.1 +/- 40.3%, respectively. DNA methylation in DS individuals did not change significantly (59.7+/-34.5%) in response to high-tHcy level in contrast to controls (23.4 +/- 17.7%, P = 0.02). Further, the quantitative MS-AP-PCR using this microfludic system is a useful method for determining differential genomic DNA methylation.     

Endopolygalacturonases reveal molecular features for processivity pattern and tolerance towards acetylated pectin

Endopolygalacturonases (EndoPGs) hydrolyse the 1-4 linkages between two alpha-d-galacturonic acids (GalA) of the smooth homogalacturonan regions of pectin. GalA may be methyl-esterified on the carboxylic group and acetyl-esterified on the hydroxylic groups. EndoPG activity most often decreases with such increasing degree of substitution. In this paper, we used bioinformatics and molecular modelling technics to explain the tolerance profile at the molecular scale and processivity scheme of three endoPGs with respect to acetylated pectin substrate; the first two enzymes originate from Aspergillus niger (AnPGI and AnPGII) and the third from Fusarium moniliforme (FmPG). Partly acetylated and methylated homogalacturonan fragments in complex with the three PGs were successively modelled in silico. The amino acid residues involved in substrate binding were identified for each enzyme. Similarly, the docking pattern of the differently decorated oligomers in the catalytic groove was individually characterized for each enzyme. This work shows full agreement with our previous extensive mass spectrometry analysis of the hydrolytic products that established distinct tolerance profiles for the three endoPGs and earlier work that ascertained processivity, specifically for AnPGI. In our previous work, AnPGI was shown to be the most powerful enzyme among the three enzymes with an enhanced tolerance towards O2- and O3-acetylated substrates. We report here amino acids of AnPGI that are unique in binding the pectin backbone and that are identified as possibly crucial for its specificity, namely S191(An)(PGI)/D240(An)(PGI). Similarly, topologically equivalent residues in AnPGII and FmPG were identified that could impede such binding; S234(An)(PGII)/S91(An)(PGII) and S245(Fm)(PG)/V89(Fm)(PG). In addition, we report here, from normal mode analysis computed on AnPG1, a shear bending motion of 15 A of amplitude that fully accredits the processive action pattern for this enzyme, with D240(An)(PGI) and R96(An)(PGI) working as crampons to favour the sliding of the substrate. Conversely, the same method clearly evidences a hinge binding motion for AnPGII and FmPG that should only authorize one hydrolytic event per enzyme/substrate encounter.     

Infectious salmon anaemia virus (ISAV) isolated from the ISA disease outbreaks in Chile diverged from ISAV isolates from Norway around 1996 and was disseminated around 2005, based on surface glycoprotein gene sequences

Background

All viruses in the family Bunyaviridae possess a tripartite genome, consisting of a small, a medium, and a large RNA segment. Bunyaviruses therefore possess considerable evolutionary potential, attributable to both intramolecular changes and to genome segment reassortment. Hantaviruses (family Bunyaviridae, genus Hantavirus) are known to cause human hemorrhagic fever with renal syndrome or hantavirus pulmonary syndrome. The primary reservoir host of Sin Nombre virus is the deer mouse (Peromyscus maniculatus), which is widely distributed in North America. We investigated the prevalence of intramolecular changes and of genomic reassortment among Sin Nombre viruses detected in deer mice in three western states.

Methods

Portions of the Sin Nombre virus small (S) and medium (M) RNA segments were amplified by RT-PCR from kidney, lung, liver and spleen of seropositive peromyscine rodents, principally deer mice, collected in Colorado, New Mexico and Montana from 1995 to 2007. Both a 142 nucleotide (nt) amplicon of the M segment, encoding a portion of the G2 transmembrane glycoprotein, and a 751 nt amplicon of the S segment, encoding part of the nucleocapsid protein, were cloned and sequenced from 19 deer mice and from one brush mouse (P. boylii), S RNA but not M RNA from one deer mouse, and M RNA but not S RNA from another deer mouse.

Results

Two of 20 viruses were found to be reassortants. Within virus sequences from different rodents, the average rate of synonymous substitutions among all pair-wise comparisons (π_s) was 0.378 in the M segment and 0.312 in the S segment sequences. The replacement substitution rate (π_a) was 7.0 × 10^-4 in the M segment and 17.3 × 10^-4 in the S segment sequences. The low π_a relative to π_s suggests strong purifying selection and this was confirmed by a Fu and Li analysis. The absolute rate of molecular evolution of the M segment was 6.76 × 10^-3 substitutions/site/year. The absolute age of the M segment tree was estimated to be 37 years. In the S segment the rate of molecular evolution was 1.93 × 10^-3 substitutions/site/year and the absolute age of the tree was 106 years. Assuming that mice were infected with a single Sin Nombre virus genotype, phylogenetic analyses revealed that 10% (2/20) of viruses were reassortants, similar to the 14% (6/43) found in a previous report.

Conclusion

Age estimates from both segments suggest that Sin Nombre virus has evolved within the past 37–106 years. The rates of evolutionary changes reported here suggest that Sin Nombre virus M and S segment reassortment occurs frequently in nature.     

Integrating genes and phenotype: a wheat–Arabidopsis–rice glycosyltransferase database for candidate gene analyses

Glycosyltransferases (GTs) constitute a very large multi-gene superfamily, containing several thousand members identified in sequenced organisms especially in plants. GTs are key enzymes involved in various biological processes such as cell wall formation, storage polysaccharides biosynthesis, and glycosylation of various metabolites. GTs have been identified in rice (Oryza sativa) and Arabidopsis thaliana, but their precise function has been demonstrated biochemically for only a few. In this work we have established a repertoire of virtually all the wheat (Triticum aestivum) GT sequences, using the large publicly available banks of expressed sequences. Based on sequence similarity with Arabidopsis and rice GTs compiled in the carbohydrate active enzyme database (CAZY), we have identified and classified these wheat sequences. The results were used to feed a searchable database available on the web () that can be used for initiating an exhaustive candidate gene survey in wheat applied to a particular biological process. This is illustrated through the identification of GT families which are expressed during cell wall formation in wheat grain maturation. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was funded by a grant of the French ministry of research.