Mucosal reactive oxygen species decrease virulence by disrupting Campylobacter jejuni phosphotyrosine signaling

Reactive oxygen species (ROS) play key roles in mucosal defense, yet how they are induced and the consequences for pathogens are unclear. We report that ROS generated by epithelial NADPH oxidases (Nox1/Duox2) during Campylobacter jejuni infection impair bacterial capsule formation and virulence by altering bacterial signal transduction. Upon C. jejuni invasion, ROS released from the intestinal mucosa inhibit the bacterial phosphotyrosine network that is regulated by the outer-membrane tyrosine kinase Cjtk (Cj1170/OMP50). ROS-mediated Cjtk inactivation results in an overall decrease in the phosphorylation of C. jejuni outer-membrane/periplasmic proteins, including UDP-GlcNAc/Glc 4-epimerase (Gne), an enzyme required for N-glycosylation and capsule formation. Cjtk positively regulates Gne by phosphorylating an active site tyrosine, while loss of Cjtk or ROS treatment inhibits Gne activity, causing altered polysaccharide synthesis. Thus, epithelial NADPH oxidases are an early antibacterial defense system in the intestinal mucosa that modifies virulence by disrupting bacterial signaling.     

Remarkable genetic diversity detected at river buffalo prolactin receptor (PRLR) gene and association studies with milk fatty acid composition

Prolactin is an anterior pituitary peptide hormone involved in many different endocrine activities and is essential for reproductive performance. This action is mediated by its receptor, the prolactin receptor, encoded by the PRLR gene. In this study, we sequenced and characterized the Mediterranean river buffalo PRLR gene (from exon 3 to 10), and we found remarkable genetic diversity. In particular, we found 24 intronic polymorphisms and 13 exonic SNPs, seven of which were non‐synonymous. Furthermore, the polymorphisms identified in the 3′‐UTR were investigated to establish their possible influence on microRNA binding sites. Considering all the amino acid changes and the observed allelic combinations, it is possible to deduce at least six different translations of the buffalo prolactin receptor and, consequently, the presence at the PRLR gene of at least six alleles. Furthermore, we identified a deletion of a CACTACC heptamer between nucleotides 1102 and 1103 of exon 10 (3′‐UTR), and we developed an allele‐specific PCR to identify the carriers of this genetic marker. Finally, the SNP g.11188A>G, detected in exon 10 and responsible for the amino acid replacement p.His328Arg, was genotyped in 308 Italian Mediterranean river buffaloes, and an association study with milk fat traits was carried out. The statistical analysis showed a tendency that approached significance for the AA genotype with higher contents of odd branched‐chain fatty acids. Thus, our results suggest that the PRLR gene is a good candidate for gene association studies with qualitative traits related to buffalo milk production.     

Spoiling the Whole Bunch: Quality Control Aimed at Preserving the Integrity of High-Throughput Genotyping

False-positive or false-negative results attributable to undetected genotyping errors and confounding factors present a constant challenge for genome-wide association studies (GWAS) given the low signals associated with complex phenotypes and the noise associated with high-throughput genotyping. In the context of the genetics of kidneys in diabetes (GoKinD) study, we identify a source of error in genotype calling and demonstrate that a standard battery of quality-control (QC) measures is not sufficient to detect and/or correct it. We show that, if genotyping and calling are done by plate (batch), even a few DNA samples of marginally acceptable quality can profoundly alter the allele calls for other samples on the plate. In turn, this leads to significant differential bias in estimates of allele frequency between plates and, potentially, to false-positive associations, particularly when case and control samples are not sufficiently randomized to plates. This problem may become widespread as investigators tap into existing public databases for GWAS control samples. We describe how to detect and correct this bias by utilizing additional sources of information, including raw signal-intensity data.     

Mice lacking the extracellular matrix adaptor protein matrilin-2 develop without obvious abnormalities.

Matrilins are putative adaptor proteins of the extracellular matrix (ECM) which can form both collagen-dependent and collagen-independent filamentous networks. While all known matrilins (matrilin-1, -2, -3, and -4) are expressed in cartilage, only matrilin-2 and matrilin-4 are abundant in non-skeletal tissues. To clarify the biological role of matrilin-2, we have developed a matrilin-2-deficient mouse strain. Matrilin-2 null mice show no gross abnormalities during embryonic or adult development, are fertile, and have a normal lifespan. Histological and ultrastructural analyses indicate apparently normal structure of all organs and tissues where matrilin-2 is expressed. Although matrilin-2 co-localizes with matrilin-4 in many tissues, Northern hybridization, semiquantitative RT-PCR, immunohistochemistry and biochemical analysis reveal no significant alteration in the steady-state level of matrilin-4 expression in homozygous mutant mice. Immunostaining of wild-type and mutant skin samples indicate no detectable differences in the expression and deposition of matrilin-2 binding partners including collagen I, laminin-nidogen complexes, fibrillin-2 and fibronectin. In addition, electron microscopy reveals an intact basement membrane at the epidermal-dermal junction and normal organization of the dermal collagen fibrils in mutant skin. These data suggest that either matrilin-2 and matrilin-2-mediated matrix-matrix interactions are dispensable for proper ECM assembly and function, or that they are efficiently compensated by other matrix components including wild-type levels of matrilin-4.     

In silico-initiated cloning and molecular characterization of a novel human member of the L1 gene family of neural cell adhesion molecules

To discover genes contributing to mental retardation in 3p^- syndrome patients we have used in silico searches for neural genes in NCBI databases (dbEST and UniGene). An EST with strong homology to the rat CAM L1 gene subsequently mapped to 3p26 was used to isolate a full-length cDNA. Molecular analysis of this cDNA, referred to as CALL (cell adhesion L1-like), showed that it is encoded by a chromosome 3p26 locus and is a novel member of the L1 gene family of neural cell adhesion molecules. Multiple lines of evidence suggest CALL is likely the human ortholog of the murine gene CHL1: it is 84% identical on the protein level, has the same domain structure, same membrane topology, and a similar expression pattern. The orthology of CALL and CHL1 was confirmed by phylogenetic analysis. By in situ hybridization, CALL is shown to be expressed regionally in a timely fashion in the central nervous system, spinal cord, and peripheral nervous system during rat development. Northern analysis and EST representation reveal that it is expressed in the brain and also outside the nervous system in some adult human tissues and tumor cell lines. The cytoplasmic domain of CALL is conserved among other members of the L1 subfamily and features sequence motifs that may involve CALL in signal transduction pathways. Received: 14 April 1998 / Accepted: 18 June 1998     

Structural resolution of the complex between a fungal polygalacturonase and a plant polygalacturonase-inhibiting protein by small-angle X-ray scattering

We report here the low-resolution structure of the complex formed by the endo-polygalacturonase from Fusarium phyllophilum and one of the polygalacturonase-inhibiting protein from Phaseolus vulgaris after chemical cross-linking as determined by small-angle x-ray scattering analysis. The inhibitor engages its concave surface of the leucine-rich repeat domain with the enzyme. Both sides of the enzyme active site cleft interact with the inhibitor, accounting for the competitive mechanism of inhibition observed. The structure is in agreement with previous site-directed mutagenesis data and has been further validated with structure-guided mutations and subsequent assay of the inhibitory activity. The structure of the complex may help the design of inhibitors with improved or new recognition capabilities to be used for crop protection.     

Sedimentary nitrogen uptake and assimilation in the temperate zooxanthellate sea anemone Anthopleura aureoradiata

The sea anemone Anthopleura aureoradiata (Carlgren), which harbours symbiotic dinoflagellates (zooxanthellae), is abundant on mudflats and rocky shores around New Zealand. We measured the potential for particulate nitrogen uptake from sediment by A. aureoradiata and the subsequent consequences of this uptake on the nitrogen status of its zooxanthellae. Sediment was rinsed, labelled with (¹⁵NH₄)₂SO₄, and provided to anemones at low (0.23 g ml^− 1) and high (1.33 g ml^− 1) sediment loads for 6 h. Both anemone tissues and zooxanthellae became enriched with ¹⁵N. Enrichment of anemone tissues was similar at both high and low sediment loads, but the zooxanthellae became more enriched at the lower load. This was presumably because the uptake of ammonium, arising from host catabolism, by zooxanthellae is light driven and because the anemones at the lower load were able to extend their tentacles into the light while those at the higher load were not. The influence of sediment uptake on the nitrogen status of the zooxanthellae was determined by measuring the extent to which 20 μM NH₄⁺ enhanced the rate of zooxanthellar dark carbon fixation above that seen in filtered seawater (FSW) alone; the ammonium enhancement ratio (AER) was expressed as [dark NH₄⁺ rate/dark FSW rate], where ‘rate’ refers to C fixation and a ratio of 1.0 or less indicates nitrogen sufficiency. When anemones were starved with and without rinsed sediment in nitrogen-free artificial seawater for 8 weeks, zooxanthellar nitrogen deficiency became apparent at 2-4 weeks and reached similar levels in both treatments (AER = ~ 2). In contrast, anemones fed 5 times per week for 8 weeks with Artemia nauplii were nitrogen sufficient (AER = 1.03). In the field, zooxanthellae from mudflat anemones were largely nitrogen sufficient (AER = 1.26), while nitrogen deficient zooxanthellae were present in anemones from a rocky intertidal site (AER = 2.93). These results suggest that, while there was evidence for particulate nitrogen uptake, dissolved inorganic nitrogen (especially ammonium) in interstitial pore water may be a more important source of nitrogen for the zooxanthellae in mudflat anemones, and may explain the marked difference in nitrogen status between the mudflat and rocky shore populations.     

No reduction in genetic diversity of Swiss stone pine (Pinus cembra L.) in Tatra Mountains despite high fragmentation and small population size

In Europe, most of the alpine timberline ecotone has been altered by human activities and climate change. Hence, mountain forests are of the highest conservation interest. Here, we screened 25 populations of Swiss stone pine (Pinus cembra L.) from the Carpathians and the Alps, using a set of ten microsatellite primers to assess the relative conservation value of populations sampled in Polish and Slovak Tatra National Parks, where potential extinction risk is the highest within the Carpathian range. Although endangered, with small and fragmented populations, P. cembra in the Tatra Mts. shows high levels of allelic richness (AR = 5.0) and observed heterozygosity (H _o  = 0.554). Our results suggest that anthropogenic habitat fragmentation has had little impact on DNA variation of Swiss stone pine in the Tatra Mts. However, the effects of changing conditions on the genetic structure may occur with a substantial time delay due to the long life span of P. cembra. Moreover, inbreeding depression may occur in the next generations, since we found inbreeding (F _IS  = 0.063) and elevated coancestry coefficient (θ = 0.062) in all populations. Also a shallow pattern of genetic differentiation between populations was found, indicating recent fragmentation of a common gene pool that formerly occupied a larger range. Therefore, the Tatra Mts. can be considered as a single conservation unit. Based on our results, we suggest possible conservation activities for Swiss stone pine both in Poland and Slovakia.