gly gene cloning and expression and purification of glycinecin A, a bacteriocin produced by Xanthomonas campestris pv. glycines 8ra

Glycinecin A, a bacteriocin produced by Xanthomonas campestris pv. glycines, inhibits the growth of X. campestris pv. vesicatoria. We have cloned and expressed the genes encoding glycinecin A in Escherichia coli. Recombinant glycinecin A was purified from cell extracts by ammonium sulfate precipitation followed by chromatography on Q-Sepharose, Mono Q (ion exchange), and size exclusion columns. Purified glycinecin A is composed of two polypeptides, is active over a wide pH range (6 to 9), and is stable at temperatures up to 60 degrees C. Glycinecin A is a heterodimer consisting of 39- and 14-kDa subunits, as revealed through size exclusion chromatography and cross-linking analysis. Two genes, glyA and glyB, encoding the 39- and 14-kDa subunits, respectively, were identified based on the N-terminal sequences of the subunits. From the nucleotide sequences of glyA and glyB, we conclude that both genes are translated as bacteriocin precursors that include N-terminal leader sequences. When expressed in E. coli, recombinant glycinecin A was found primarily in cell extracts. In contrast, most glycinecin A from Xanthomonas was found in the culture media. E. coli transformed with either glyA or glyB separately did not show the bacteriocin activity.     

Evolution of the Archaea

Archaea, members of the third domain of life, are bacterial-looking prokaryotes that harbour many unique genotypic and phenotypic properties, testifying for their peculiar evolutionary status. The archaeal ancestor was probably a hyperthermophilic anaerobe. Two archaeal phyla are presently recognized, the Euryarchaeota and the Crenarchaeota. Methanogenesis was the main invention that occurred in the euryarchaeal phylum and is now shared by several archaeal groups. Adaptation to aerobic conditions occurred several times independently in both Euryarchaeota and Crenarchaeota. Recently, many new groups of Archaea that have not yet been cultured have been detected by PCR amplification of 16S ribosomal RNA from environmental samples. The phenotypic and genotypic characterization of these new groups is now a top priority for further studies on archaeal evolution.     

Identification of a new segment involved in cagA 3' region variation of Helicobacter pylori

The cagA 3' region shows marked variation among Helicobacter pylori strains. Two segments of 102 bp and 57 bp are reportedly responsible for this variation. We analysed the cagA 3' region in 70 H. pylori strains using polymerase chain reaction and sequencing. We found that another segment, namely beta segment, was also involved in the variation of this region. The beta segment was 105 bp long and located between the aforementioned two segments. Six genotypes were identified based on the structure of the cagA 3' region. No relationship was found between these genotypes and the clinical outcomes or vacA genotypes. The numbers of tyrosine phosphorylation sites within the cagA 3' region varied among strains, but this was not related to the cagA genotypes. Our data suggest that the cagA 3' region is significantly variable. It appears that the variation of the cagA 3' region might contribute to the modification of virulence.     

Genome-wide RNAi of C. elegans using the hypersensitive rrf-3 strain reveals novel gene functions

RNA-mediated interference (RNAi) is a method to inhibit gene function by introduction of double-stranded RNA (dsRNA). Recently, an RNAi library was constructed that consists of bacterial clones expressing dsRNA, corresponding to nearly 90% of the 19,427 predicted genes of C. elegans. Feeding of this RNAi library to the standard wild-type laboratory strain Bristol N2 detected phenotypes for approximately 10% of the corresponding genes. To increase the number of genes for which a loss-of-function phenotype can be detected, we undertook a genome-wide RNAi screen using the rrf-3 mutant strain, which we found to be hypersensitive to RNAi. Feeding of the RNAi library to rrf-3 mutants resulted in additional loss-of-function phenotypes for 393 genes, increasing the number of genes with a phenotype by 23%. These additional phenotypes are distributed over different phenotypic classes. We also studied interexperimental variability in RNAi results and found persistent levels of false negatives. In addition, we used the RNAi phenotypes obtained with the genome-wide screens to systematically clone seven existing genetic mutants with visible phenotypes. The genome-wide RNAi screen using rrf-3 significantly increased the functional data on the C. elegans genome. The resulting dataset will be valuable in conjunction with other functional genomics approaches, as well as in other model organisms.     

The mobile element IS1207 of Brevibacterium lactofermentum ATCC21086: isolation and use in the construction of Tn5531, a versatile transposon for insertional mutagenesis of Corynebacterium glutamicum

IS1207 is the insertion most frequently found among the spontaneous mutations that abolish the activity of an Escherichia coli phage lambda cI gene integrated in the Corynebacterium Brevibacterium lactofermentum ATCC21086 genome. We examined the transposition of transposon-like structures composed of a selective kanamycin resistance gene (aph3), and one or two IS1207 sequences. One of these, the Tn5531 transposon, transposed efficiently in Corynebacterium glutamicum. A replicative and a non-replicative Tn5531 delivery vector were used in Tn5531 mutagenesis. As IS1207, transposon Tn5531 shows a high frequency of transposition and mutagenesis, and a low target specificity. These features make of Tn5531 an adequate choice for gene identification and gene tagging experiments.     

Relationship between E-selectin L/F554 polymorphism and blood pressure in the Stanislas cohort

We investigated the relationship between polymorphisms of the E-selectin gene SELE (L/F554, S/R128 and 98G/T), a cell adhesion molecule, and interindividual variability in blood pressure and changes over time. The study population was extracted from the Stanislas cohort (1006 families), a cohort of nuclear families volunteering for a free health check-up and recruited by the Center of Preventive Medicine in Nancy (CMP) between 1993 and 1994. For this specific study 359 men and 337 women were selected from families who had already visited the CMP 11 years before recruitment of the Stanislas Cohort. Measurements of blood pressure 11 years before (t(-11)) and at the time of recruitment (t(0)), and all other measurements necessary for the analysis (body mass index, lipids, SELE genotypes) were available. Pregnant women or subjects taking antihypertensive, lipid-lowering, or anti-inflammatory medications were excluded from the study. During the follow-up period systolic and diastolic blood pressures were lower in SELE F554 allele carriers than in those with the L/L554 genotype (P<0.05), but longitudinal changes were not related to any SELE polymorphism. Multiple regression analysis showed that at t(-11) SELE L/F554 polymorphism was associated with both systolic and diastolic blood pressure levels (P<0.01 and P<0.05, respectively). However, these associations were no longer present at t(0). Our results suggest an age-specific effect of the SELE L/F554 polymorphism on blood pressure levels. If confirmed in other studies, these findings would suggest that assessment of common variation in an adhesion molecule could be useful in predicting blood pressure.     

Blood-Based Protein Biomarker Panel for the Detection of Colorectal Cancer

Background

The majority of colorectal cancer (CRC) cases are preventable by early detection and removal of precancerous polyps. Even though CRC is the second most common internal cancer in Australia, only 30 per cent of the population considered to have risk factors participate in stool-based test screening programs. Evidence indicates a robust, blood-based, diagnostic assay would increase screening compliance. A number of potential diagnostic blood-based protein biomarkers for CRC have been reported, but all lack sensitivity or specificity for use as a stand-alone diagnostic. The aim of this study was to identify and validate a panel of protein-based biomarkers in independent cohorts that could be translated to a reliable, non-invasive blood-based screening test.

Principal Findings

In two independent cohorts (n = 145 and n = 197), we evaluated seven single biomarkers in serum of CRC patients and age/gender matched controls that showed a significant difference between controls and CRC, but individually lack the sensitivity for diagnostic application. Using logistic regression strategies, we identified a panel of three biomarkers that discriminated between controls and CRC with 73% sensitivity at 95% specificity, when applied to either of the two cohorts. This panel comprised of Insulin like growth factor binding protein 2 (IGFBP2), Dickkopf-3 (DKK3), and Pyruvate kinase M2(PKM2).

Conclusions

Due to the heterogeneous nature of CRC, a single biomarker is unlikely to have sufficient sensitivity or specificity for use as a stand-alone diagnostic screening test and a panel of markers may be more effective. We have identified a 3 biomarker panel that has higher sensitivity and specificity for early stage (Stage I and -II) disease than the faecal occult blood test, raising the possibility for its use as a non-invasive blood diagnostic or screening test.     

Lysyl Hydroxylase 3 Localizes to Epidermal Basement Membrane and Is Reduced in Patients with Recessive Dystrophic Epidermolysis Bullosa

Recessive dystrophic epidermolysis bullosa (RDEB) is caused by mutations in COL7A1 resulting in reduced or absent type VII collagen, aberrant anchoring fibril formation and subsequent dermal-epidermal fragility. Here, we identify a significant decrease in PLOD3 expression and its encoded protein, the collagen modifying enzyme lysyl hydroxylase 3 (LH3), in RDEB. We show abundant LH3 localising to the basement membrane in normal skin which is severely depleted in RDEB patient skin. We demonstrate expression is in-part regulated by endogenous type VII collagen and that, in agreement with previous studies, even small reductions in LH3 expression lead to significantly less secreted LH3 protein. Exogenous type VII collagen did not alter LH3 expression in cultured RDEB keratinocytes and we show that RDEB patients receiving bone marrow transplantation who demonstrate significant increase in type VII collagen do not show increased levels of LH3 at the basement membrane. Our data report a direct link between LH3 and endogenous type VII collagen expression concluding that reduction of LH3 at the basement membrane in patients with RDEB will likely have significant implications for disease progression and therapeutic intervention.     

Dynamics of a Kalahari long-lived mega-lake system: hydromorphological and limnological changes in the Makgadikgadi Basin (Botswana) during the terminal 50 ka

The Kalahari features a long-lived lacustrine system which may exist since the Early Pleistocene. The emergence of an extant cichlid fish radiation from this (palaeo-) lake during the Middle Pleistocene indicates an ancient lake character. The early history of the system remains speculative, but it is established that lake extensions matching modern Lake Victoria in size have occurred during the Late Pleistocene. It has been assumed that the hydrographical dynamics chiefly depended on the inflow from the Okavango River and thus on ITCZ-controlled precipitation. Our studies, which focused the hydromorphological and palaeolimnological development of the Makgadikgadi Basin during the last 50 ka, suggest that from c. 46–16 ka it did not receive water from the Okavango River but from palaeo-rivers located in the northern and south-western catchment. A northward shift of the winter rainfall zone during the Last Glacial Maximum sustained a high lake level for a period of c. 6 ka. During Heinrich Event 1 (17–16 ka) the lake probably desiccated abruptly and completely. Higher lake levels, controlled by water from the Okavango river system, were reached again during the Holocene before the lake dried up in the middle of the last millennium.     

Variety matters: adaptive genetic diversity and parasite load in two mouse opossums from the Brazilian Atlantic forest

The adaptive potential of a species to a changing environment and in disease defence is primarily based on genetic variation. Immune genes, such as genes of the major histocompatibility complex (MHC), may thereby be of particular importance. In marsupials, however, there is very little knowledge about natural levels and functional importance of MHC polymorphism, despite their key role in the mammalian evolution. In a previous study, we discovered remarkable differences in the MHC class II diversity between two species of mouse opossums (Gracilinanus microtarsus, Marmosops incanus) from the Brazilian Atlantic forest, which is one of the most endangered hotspots for biodiversity conservation. Since the main forces in generating MHC diversity are assumed to be pathogens, we investigated in this study gastrointestinal parasite burden and functional associations between the individual MHC constitution and parasite load. We tested two contrasting scenarios, which might explain differences in MHC diversity between species. We predicted that a species with low MHC diversity would either be under relaxed selection pressure by low parasite diversity (‘Evolutionary equilibrium’ scenario), or there was a recent loss in MHC diversity leading to a lack of resistance alleles and increased parasite burden (‘Unbalanced situation’ scenario). In both species it became apparent that the MHC class II is functionally important in defence against gastrointestinal helminths, which was shown here for the first time in marsupials. On the population level, parasite diversity did not markedly differ between the two host species. However, we did observe considerable differences in the individual parasite load (parasite prevalence and infection intensity): while M. incanus revealed low MHC DAB diversity and high parasite load, G. microtarsus showed a tenfold higher population wide MHC DAB diversity and lower parasite burden. These results support the second scenario of an unbalanced situation.