Association of the microsatellite in the 3' untranslated region of the CD154 gene with rheumatoid arthritis in females from a Spanish cohort: a case-control study

CD40-CD154 interaction is an important mediator of inflammation and has been implicated in T helper type 1-mediated autoimmune diseases including rheumatoid arthritis (RA). Linkage studies have shown association of markers in the proximity of the CD154 gene. In the present work we investigated whether specific allele variants of the microsatellite in the 3' UTR of the CD154 gene might modulate the risk of RA. The study, in a case-control setting, included 189 patients and 150 healthy controls from the Canary Islands, Spain. The 24CAs allele was less represented in female patients than in controls (0.444 in controls versus 0.307 in patients, P = 0.006, odds ratio (OR) 0.556, 95% confidence interval (CI) 0.372 to 0.831) but not in males (0.414 versus 0.408), and only when homozygous (P = 0.012; OR 0.35, 95% CI 0.16 to 0.77). We also verified that CD154 association with RA was independent of human leukocyte antigen (HLA) phenotype. A further functional study showed that after stimulation anti-CD3, CD154 mRNA was more stable in CD4+ T lymphocytes from patients with RA bearing the 24CAs allele (mRNA half-life 208 minutes) than in patients without the 24CAs allele (109 minutes, P = 0.009). However, a lower percentage of CD154+CD4+ T lymphocytes was seen in freshly isolated peripheral blood mononuclear cells from patients carrying 24CAs alleles (mean 4.28 versus 8.12; P = 0.033), and also in CD4+ T lymphocytes stimulated with anti-CD3 (median 29.40 versus 47.60; P = 0.025). These results were concordant with the smaller amounts of CD154 mRNA isolated from stimulated T lymphocytes with 24CAs alleles. The CD154 microsatellite therefore seems to affect the expression of the gene in a complex manner that implies not only mRNA stability. These data suggest that the CD154 microsatellite contributes to the regulation of mRNA and protein expression, although further studies will be necessary to elucidate its role in disease predisposition.     

Edible Myrciaria vexator fruits: bioactive phenolics for potential COPD therapy

The edible fruits of Myrciaria vexator McVaugh (Myrtaceae), from northern South America, are eaten in certain locales, either fresh or processed into jellies and drinks. Activity-guided fractionation of M. vexator resulted in identification of ellagic acid (1), cyanidin-3-O-glucoside (2), delphinidin-3-O-glucoside (3), 2-O-(3,4-dihydroxybenzoyl)-2,4,6-trihydroxyphenylacetic acid (4), and jaboticabin (5), and latter two compounds are being reported for the first time in this species. Ellagic acid was further examined, and found to inhibit cigarette smoke extract induced MMP-1 expression in vitro, and may be of significance in the treatment of chronic obstructive pulmonary (COPD). Other compounds identified for the first time from M. vexator include cyanidin-3-O-galactoside (6), cyanidin-3-O-arabinoside (7), cyanidin-3-O-rutionoside (8), petunidin (9), peonidin-3-O-galactoside (10) malvidin (11), hyperoside (12), querecetin-3-O-glucoside (13), and guajaverin (14), methyl protocatechuate (15), and protocatechuic acid (16).     

Study of the role played by NfsA,NfsB nitroreductase and NemA flavin reductase from <Emphasis Type="Italic">Escherichia coli</Emphasis> in the conversion of ethyl 2-(2′-nitrophenoxy)acetate to 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), a benzohydroxamic acid with interesting biological properties

Benzohydroxamic acids, such as 4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (D-DIBOA), exhibit interesting herbicidal, fungicidal and bactericidal properties. Recently, the chemical synthesis of D-DIBOA has been simplified to only two steps. In a previous paper, we demonstrated that the second step could be replaced by a biotransformation using Escherichia coli to reduce the nitro group of the precursor, ethyl 2-(2′-nitrophenoxy)acetate and obtain D-DIBOA. The NfsA and NfsB nitroreductases and the NemA xenobiotic reductase of E. coli have the capacity to reduce one or two nitro groups from a wide variety of nitroaromatic compounds, which are similar to the precursor. By this reason, we hypothesised that these three enzymes could be involved in this biotransformation. We have analysed the biotransformation yield (BY) of mutant strains in which one, two or three of these genes were knocked out, showing that only in the double nfsA/nfsB and in the triple nfsA/nfsB/nemA mutants, the BY was 0%. These results suggested that NfsA and NfsB are responsible for the biotransformation in the tested conditions. To confirm this, the nfsA and nfsB open reading frames were cloned into the pBAD expression vector and transformed into the nfsA and nfsB single mutants, respectively. In both cases, the biotransformation capacity of the strains was recovered (6.09 ± 0.06% as in the wild-type strain) and incremented considerably when NfsA and NfsB were overexpressed (40.33% ± 9.42% and 59.68% ± 2.0% respectively).     

ATP from subplasmalemmal mitochondria controls Ca2+-dependent inactivation of CRAC channels

A sustained Ca2+ entry is the primary signal for T lymphocyte activation after antigen recognition. This Ca2+ entry mainly occurs through store-operated Ca2+ channels responsible for a highly selective Ca2+ current known as I(CRAC). Ca2+ ions act as negative feedback regulators of I(CRAC), promoting its inactivation. Mitochondria, which act as intracellular Ca2+ buffers, have been proposed to control all stages of CRAC current and, hence, intracellular Ca2+ signaling in several types of non-excitable cells. Using the whole-cell configuration of the patch clamp technique, which allows control of the intracellular environment, we report here that respiring mitochondria located close to CRAC channels can regulate slow Ca2+-dependent inactivation of I(CRAC) by increasing the Ca2+-buffering capacity beneath the plasma membrane, mainly through the release of ATP.     

PI3K and ERK 1-2 regulate early stages during head regeneration in hydra

Different signaling systems coordinate and regulate the development of a multicellular organism. In hydra, the canonical Wnt pathway and the signal transduction pathways mediated by PKC and Src regulate early stages of head formation. In this paper, we present evidence for the participation of a third pathway, the PI3K-PKB pathway, involved in this process. The data presented here are consistent with the participation of ERK 1-2 as a point of convergence for the transduction pathways mediated by PKC, Src and PI3K for the regulation of the regeneration of the head in hydra. The specific developmental point regulated by them appears to be the commitment of tissue at the apical end of the regenerate to form the head organizer.     

Mutations in the tight-junction gene claudin 19 (CLDN19) are associated with renal magnesium wasting, renal failure, and severe ocular involvement

Claudins are major components of tight junctions and contribute to the epithelial-barrier function by restricting free diffusion of solutes through the paracellular pathway. We have mapped a new locus for recessive renal magnesium loss on chromosome 1p34.2 and have identified mutations in CLDN19, a member of the claudin multigene family, in patients affected by hypomagnesemia, renal failure, and severe ocular abnormalities. CLDN19 encodes the tight-junction protein claudin-19, and we demonstrate high expression of CLDN19 in renal tubules and the retina. The identified mutations interfere severely with either cell-membrane trafficking or the assembly of the claudin-19 protein. The identification of CLDN19 mutations in patients with chronic renal failure and severe visual impairment supports the fundamental role of claudin-19 for normal renal tubular function and undisturbed organization and development of the retina.     

In vitro characterization of the cysteine-rich capping domains in a plant leucine rich repeat protein

Plant leucine rich repeat (LRR) proteins have diverse functions and cellular locations. An important unresolved question involves the role of the cysteine-rich capping domains which flank the LRR domain. Such studies have been hampered by difficulties in producing recombinant LRR proteins in yields sufficient for biochemical analysis. We have used Escherichia coli to overproduce Leucine Rich Protein (LRP), a small model LRR protein from tomato containing approximately five LRRs. The LRP capping domain sequences resemble those from plant disease resistance proteins and receptor-like protein kinases. LRP was purified as a soluble, crystallizable, monomeric protein by renaturation of a GST-fusion protein. The four cysteine residues in LRP were found to form two disulfide bonds, one each in the N- and C-terminal LRR-capping domains, the presence of which is necessary to protect the LRR domain from proteolysis in vitro. Fluorescence and CD spectroscopies together with molecular modelling revealed that structural features of the N-capping domain may be destabilised on reduction. These include a tryptophan stacking interaction and a long alpha-helix of residues 30-44. LRP deletion mutants lacking the capping domains showed a propensity to aggregate and increased proteolytic sensitivity. These results have important implications for future structure-function studies of plant LRR proteins.     

Molecular Taxonomy of Phytopathogenic Fungi: A Case Study in Peronospora

Background

Inappropriate taxon definitions may have severe consequences in many areas. For instance, biologically sensible species delimitation of plant pathogens is crucial for measures such as plant protection or biological control and for comparative studies involving model organisms. However, delimiting species is challenging in the case of organisms for which often only molecular data are available, such as prokaryotes, fungi, and many unicellular eukaryotes. Even in the case of organisms with well-established morphological characteristics, molecular taxonomy is often necessary to emend current taxonomic concepts and to analyze DNA sequences directly sampled from the environment. Typically, for this purpose clustering approaches to delineate molecular operational taxonomic units have been applied using arbitrary choices regarding the distance threshold values, and the clustering algorithms.

Methodology

Here, we report on a clustering optimization method to establish a molecular taxonomy of Peronospora based on ITS nrDNA sequences. Peronospora is the largest genus within the downy mildews, which are obligate parasites of higher plants, and includes various economically important pathogens. The method determines the distance function and clustering setting that result in an optimal agreement with selected reference data. Optimization was based on both taxonomy-based and host-based reference information, yielding the same outcome. Resampling and permutation methods indicate that the method is robust regarding taxon sampling and errors in the reference data. Tests with newly obtained ITS sequences demonstrate the use of the re-classified dataset in molecular identification of downy mildews.

Conclusions

A corrected taxonomy is provided for all Peronospora ITS sequences contained in public databases. Clustering optimization appears to be broadly applicable in automated, sequence-based taxonomy. The method connects traditional and modern taxonomic disciplines by specifically addressing the issue of how to optimally account for both traditional species concepts and genetic divergence.