Characterization of ‘Schizokinen’; a dihydroxamate-type siderophore produced by Rhizobium leguminosarum IARI 917

The Rhizobia comprise one of the most important groups of beneficial bacteria, which form nodules on the roots (rarely on the stems) of leguminous plants. They live within the nodules and reduce atmospheric nitrogen to ammonia, which is further assimilated by plants into required nitrogenous compounds. The Rhizobia in return obtain nutrition from the plant. Rhizobia are free-living soil bacteria and have to compete with other microorganisms for the limited available iron in the rhizosphere. In order to acquire iron Rhizobia have been shown to express siderophore-mediated iron transport systems. Rhizobium leguminosarum IARI 917 was investigated for its ability to produce siderophore. It was found to produce a dihydroxamate type siderophore under iron restricted conditions. The siderophore was purified and chemically characterized. The ESMS, MS/MS and NMR analysis indicate the dihydroxamate siderophore to be ‘schizokinen’, a siderophore reported to be produced by Bacillus megaterium that shares a similar structure to ‘rhizobactin 1021’ produced by Sinorhizobium meliloti 1021. This is the first report of production of schizokinen by a strain of R. leguminosarum, therefore it was carefully investigated to confirm that it is indeed ‘schizokinen’ and not a degradation product of ‘rhizobactin 1021’. Since ferric–siderophore complexes are transported across the outer membrane (OM) into the periplasm via an OM receptor protein, R. leguminosarum IARI 917 was investigated for the presence of an OM receptor for ‘ferric–schizokinen’. SDS PAGE analysis of whole cell pellet and extracted OM fractions indicate the presence of a possible iron-repressible OM receptor protein with the molecular weight (MW) of approximately 74 kDa.     

Evidence of a sex-dependent association between the MSX1 locus and nonsyndromic cleft lip with or without cleft palate in the Chilean population

Prior studies have implicated an involvement of the Msx1 homeobox gene in cleft palate in mice and its homolog in humans (called MSX1 in the HOX7 gene, located on chromosome 4). In this study we present evidence of a sex-dependent association between MSX1 and non-syndromic cleft lip/palate (NSCLP) in the Chilean population. The sample included 73 NSCLP cases, 37 from multiplex families (Mx), 36 from simplex families (Sx), and 87 controls. Polymerase chain reaction amplification of the MSX1 intragenic microsatellite (CA)n-sequence shows significant (p = 0.035) differences in the allele frequencies between NSCLP-Mx males and control males. These differences are mainly due to frequency differences in allele *2 (173 base pairs) among cases (21.9%) and controls (13.2%). When the NSCLP cases are subdivided by sex and positive family history (Mx versus Sx), the Mx males (27.8%) as well as the total NSCLP-Mx cases (25.7%) showed significantly higher frequencies of allele *2, compared to controls (11.4% and 13.2%, respectively). Analysis of the genotype data indicates that the relative risk for NSCLP is greater for persons carrying allele *2 (i.e., odds ratio [OR] larger than 1), reaching significance for all Mx cases (OR = 2.67; 95% confidence interval [CI], 1.10 to 6.52) and even more pronounced for Mx males (OR = 3.33; 95% CI, 1.08 to 10.32). Taken together, these findings support the hypothesis that the genetic variation at the MSX1 locus is a predisposing gene involved in sex-dependent susceptibility to clefting and that it also differentiates simplex from multiplex families.     

Bacterial biofilms and quorum sensing: fidelity in bioremediation technology

Increased contamination of the environment with toxic pollutants has paved the way for efficient strategies which can be implemented for environmental restoration. The major problem with conventional methods used for cleaning of pollutants is inefficiency and high economic costs. Bioremediation is a growing technology having advanced potential of cleaning pollutants. Biofilm formed by various micro-organisms potentially provide a suitable microenvironment for efficient bioremediation processes. High cell density and stress resistance properties of the biofilm environment provide opportunities for efficient metabolism of number of hydrophobic and toxic compounds. Bacterial biofilm formation is often regulated by quorum sensing (QS) which is a population density-based cell–cell communication process via signaling molecules. Numerous signaling molecules such as acyl homoserine lactones, peptides, autoinducer-2, diffusion signaling factors, and α-hydroxyketones have been studied in bacteria. Genetic alteration of QS machinery can be useful to modulate vital characters valuable for environmental applications such as biofilm formation, biosurfactant production, exopolysaccharide synthesis, horizontal gene transfer, catabolic gene expression, motility, and chemotaxis. These qualities are imperative for bacteria during degradation or detoxification of any pollutant. QS signals can be used for the fabrication of engineered biofilms with enhanced degradation kinetics. This review discusses the connection between QS and biofilm formation by bacteria in relation to bioremediation technology.     

BRCA1 Is Required for Maintenance of Phospho-Chk1 and G2/M Arrest during DNA Cross-Link Repair in DT40 Cells

The Fanconi anemia DNA repair pathway is pivotal for the efficient repair of DNA interstrand cross-links. Here, we show that FA-defective (Fancc⁻) DT40 cells arrest in G₂ phase following cross-link damage and trigger apoptosis. Strikingly, cell death was reduced in Fancc⁻ cells by additional deletion of the BRCA1 tumor suppressor, resulting in elevated clonogenic survival. Increased resistance to cross-link damage was not due to loss of toxic BRCA1-mediated homologous recombination but rather through the loss of a G₂ checkpoint. This proapoptotic role also required the BRCA1-A complex member ABRAXAS (FAM175A). Finally, we show that BRCA1 promotes G₂ arrest and cell death by prolonging phosphorylation of Chk1 on serine 345 after DNA damage to sustain arrest. Our data imply that DNA-induced cross-link death in cells defective in the FA pathway is dependent on the ability of BRCA1 to prolong cell cycle arrest in G₂ phase.     

Transcriptional profiling of peripheral blood mononuclear cells in pancreatic cancer patients identifies novel genes with potential diagnostic utility

Background

It is well known that many malignancies, including pancreatic cancer (PC), possess the ability to evade the immune system by indirectly downregulating the mononuclear cell machinery necessary to launch an effective immune response. This knowledge, in conjunction with the fact that the trancriptome of peripheral blood mononuclear cells has been shown to be altered in the context of many diseases, including renal cell carcinoma, lead us to study if any such alteration in gene expression exists in PC as it may have diagnostic utility.

Methods and Findings

PBMC samples from 26 PC patients and 33 matched healthy controls were analyzed by whole genome cDNA microarray. Three hundred eighty-three genes were found to be significantly different between PC and healthy controls, with 65 having at least a 1.5 fold change in expression. Pathway analysis revealed that many of these genes fell into pathways responsible for hematopoietic differentiation, cytokine signaling, and natural killer (NK) cell and CD8+ T-cell cytotoxic response. Unsupervised hierarchical clustering analysis identified an eight-gene predictor set, consisting of SSBP2, Ube2b-rs1, CA5B, F5, TBC1D8, ANXA3, ARG1, and ADAMTS20, that could distinguish PC patients from healthy controls with an accuracy of 79% in a blinded subset of samples from treatment naïve patients, giving a sensitivity of 83% and a specificity of 75%.

Conclusions

In summary, we report the first in-depth comparison of global gene expression profiles of PBMCs between PC patients and healthy controls. We have also identified a gene predictor set that can potentially be developed further for use in diagnostic algorithms in PC. Future directions of this research should include analysis of PBMC expression profiles in patients with chronic pancreatitis as well as increasing the number of early-stage patients to assess the utility of PBMCs in the early diagnosis of PC.     

Detection of cross-reactive antigens shared byFusarium oxysporum andGlycine max by indirect ELISA and their cellular location in root tissues

Pathogenicity test ofFusarium oxysporum on ten cultivars of soybean revealed Soymax and Punjab-1 to be most resistant while JS-2 and UPSM-19 were most susceptible. Antigens were prepared from the roots of all the ten varieties of soybean and the mycelium ofF. oxysporum. Polyclonal antisera were raised against the mycelial suspension ofF. oxysporum and the root antigen of the susceptible cultivar UPSM-19. Cross reactive antigens shared by the host and the pathogen were detected first by immunodiffusion. The immunoglobulin fraction of the antiserum was purified by ammonium sulfate precipitation and DEAE-Sephadex column chromatography. The immunoglobulin fractions were used for detection of cross-reactive antigens by enzyme-linked immunosorbent assay. In enzyme-linked immunosorbent assay, antigens of susceptible cultivars showed higher absorbance values when tested against the purified anti-F. oxysporum antiserum. Antiserum produced against UPSM-19 showed cross-reactivity with the antigens of other cultivars. Indirect staining of antibodies using fluorescein isothiocyanate indicated that in cross-sections of roots of susceptible cultivar (UPSM-19) cross-reactive antigens were concentrated around xylem elements, endodermis and epidermal cells, while in the resistant variety, fluorescence was concentrated mainly around epidermal cells and distributed in the cortical tissues. CRAs were also present in microconidia, macroconidia and chlamydospores of the fungus.     

Novel screening protocol for precise phenotyping of salt-tolerance at reproductive stage in rice

The present study reports an unequivocal and improved protocol for efficient screening of salt tolerance at flowering stage in rice, which can aid phenotyping of population for subsequent identification of QTLs associated with salinity stress, particularly at reproductive stage. To validate the new method, the selection criteria, level and time of imposition of stress; plant growth medium were standardized using three rice genotypes. The setup was established with a piezometer placed in a perforated pot for continuous monitoring of soil EC and pH throughout the period of study. Further, fertilizer enriched soil was partially substituted by gravels for stabilization and maintaining the uniformity of soil EC in pots without hindering its buffering capacity. The protocol including modified medium (Soil:Stone, 4:1) at 8 dS m^?1 salinity level was validated using seven different genotypes possessing differential salt sensitivity. Based on the important selection traits such as high stability index for plant yield, harvest index and number of grains/panicle and also high K⁺ concentration and low Na⁺– K⁺ ratio in flag leaf at grain filling stage were validated and employed in the evaluation of a mapping population in the modified screening medium. The method was found significantly efficient for easy maintenance of desired level of soil salinity and identification of genotypes tolerant to salinity at reproductive stage.     

Centriole is the pivot co-ordinating dynamic signaling for cell proliferation and organization during early development in the vertebrates

Vertebrates have an elaborate and functionally segmented body. It evolves from a single cell by systematic cell proliferation but attains a complex body structure with exquisite precision. This development requires two cellular events: cell cycle and ciliogenesis. For these events, the dynamic molecular signaling is converged at the centriole. The cell cycle helps in cell proliferation and growth of the body and is a highly regulated and integrated process. Its errors cause malignancies and developmental disorders. The cells newly proliferated are organized during organogenesis. For a cellular organization, dedicated signaling hubs are developed in the cells, and most often cilia are utilized. The cilium is generated from one of the centrioles involved in cell proliferation. The developmental signaling pathways hosted in cilia are essential for the elaboration of the body plan. The cilium's compartmental seclusion is ideal for noise-free molecular signaling and is essential for the precision of the body layout. The dysfunctional centrioles and primary cilia distort the development of body layout that manifest as serious developmental disorders. Thus, centriole has a dual role in the growth and cellular organization. It organizes dynamically expressed molecules of cell cycle and ciliogenesis and plays a balancing act to generate new cells and organize them during development. A putative master molecule may regulate and co-ordinate the dynamic gene expression at the centrioles. The convergence of many critical signaling components at the centriole reiterates the idea that centriole is a major molecular workstation involved in elaborating the structural design and complexity in vertebrates.     

A new S4-ligated zinc-peptide 1:2 complex for the hydrolytic cleavage of DNA

A new sulfur-ligated Zn-peptide 1:2 complex, [Zn(II)(Boc-NH-Cys-Gly-Cys-OMe)2]2- (2), was prepared, characterized, and tested for its DNA-binding and -cleavage properties. Complex 2 was found to cleave DNA hydrolytically. The negative charge in 2 reduces the affinity of the complex for DNA, and enhances its binding specificity.     

Integration of clinical and gene expression endpoints to explore furan-mediated hepatotoxicity

Molecular techniques, such as cDNA microarrays, are being used to aid in the elucidation of the mechanisms of toxicity of a variety of compounds. In this study, we evaluate the molecular effects of furan in the rat liver. Sprague-Dawley rats were exposed to 4 or 40 mg/kg furan for up to 14 days. Furan induced an initial degenerative and necrotic phenotype that was followed by inflammation and fibrosis, consistent with previous observations for this compound. RNA was harvested from each lobe of the liver at several time points to observe whether lobe-specific gene expression effects occurred. Similar gene expression changes were observed in all lobes, however the magnitude of gene expression change was more pronounced in the right lobe. Finally, to help determine the correlation between gene expression changes and liver pathology, we applied traditional microarray visualization tools to the assessment of clinical chemistry and pathology parameters.