ArsP: a methylarsenite efflux permease

Trivalent organoarsenic compounds are far more toxic than either pentavalent organoarsenicals or inorganic arsenite. Many microbes methylate inorganic arsenite (As(III)) to more toxic and carcinogenic methylarsenite (MAs(III)). Additionally, monosodium methylarsenate (MSMA or MAs(V)) has been used widely as an herbicide and is reduced by microbial communities to MAs(III). Roxarsone (3‐nitro‐4‐hydroxybenzenearsonic acid) is a pentavalent aromatic arsenical that is used as antimicrobial growth promoter for poultry and swine, and its active form is the trivalent species Rox(III). A bacterial permease, ArsP, from Campylobacter jejuni, was recently shown to confer resistance to roxarsone. In this study, C. jejuni arsP was expressed in Escherichia coli and shown to confer resistance to MAs(III) and Rox(III) but not to inorganic As(III) or pentavalent organoarsenicals. Cells of E. coli expressing arsP did not accumulate trivalent organoarsenicals. Everted membrane vesicles from those cells accumulated MAs(III) > Rox(III) with energy supplied by NADH oxidation, reflecting efflux from cells. The vesicles did not transport As(III), MAs(V) or pentavalent roxarsone. Mutation or modification of the two conserved cysteine residues resulted in loss of transport activity, suggesting that they play a role in ArsP function. Thus, ArsP is the first identified efflux system specific for trivalent organoarsenicals.     

Overproduction of mouse estrogen receptor alpha-ligand binding domain decreases bacterial growth

Escherichia coli (E. coli) is the most widely used prokaryotic host system for the synthesis of recombinant proteins. The overproduction of recombinant proteins is sometimes lethal to the host cells. In the present study, we expressed the ligand binding domain (LBD) of mouse estrogen receptor alpha (mouse ERα) using an expression vector (pIVEX) in E. coli BL21(DE3) and examined the effect of production of this protein on bacterial growth. The expressed protein was immunologically detected as a 30 kD histidine-tagged protein in the soluble part of the bacterial lysate. The overproduction of mouse ERα-LBD, as reflected by total protein content and expression pattern, resulted in the decrease of bacterial growth.     

Effect of thymosin alpha 1 on the antitumor activity of tumor-associated macrophage-derived dendritic cells

We have previously suggested that thymosin ₁ (thy1), an immunomodulating thymic hormone, can activate tumor-associated macrophages to a tumoricidal state in a murine model bearing a transplantable T-cell lymphoma of spontaneous origin designated as Dalton's lymphoma (DL). Since tumor-infiltrating dendritic cells (DC) also play an important role in the host's antitumor response and are as such in an immunocompromised state in a tumor-bearing host, in the present investigation we studied if thy1 is able to influence the differentiation of tumor-associated macrophages (TAM) into DC with granulocyte macrophage colony stimulating factor (GM-CSF), interleukin (IL)-4 and tumor necrosis factor (TNF) and whether these TAM-derived DC show enhanced antitumor activity. It was observed that DC generated from thy1-administered tumor-bearing mice showed augmented antitumor activity in vitro. Adoptive immunotherapy using TAM-derived DC showed a significant delay in the tumor growth and a prolongation of the survival time in tumor-bearing mice. DC obtained from TAM of thy1-administered mice also produced an enhanced amount of cytokines like IL-1 and TNF-. This is the first study of its kind regarding the effect of thy1 on the differentiation of DC from TAM and the role of TAM-derived DC in tumor progression.     

Cooperative binding of monodisperse anionic amphiphiles to the i-face: phospholipase A2-paradigm for interfacial binding

Equilibrium parameters for the binding of monodisperse alkyl sulfate along the i-face (the interface binding surface) of pig pancreatic IB phospholipase A(2) (PLA2) to form the premicellar complexes (E(i)(#)) are characterized to discern the short-range specific interactions. Typically, E(i)(#) complexes are reversible on dilution. The triphasic binding isotherm, monitored as the fluorescence emission from the single tryptophan of PLA2, is interpreted as a cooperative equilibrium for the sequential formation of three premicellar complexes (E(i)(#), i = 1, 2, 3). In the presence of calcium, the dissociation constant K(1) for the E(1)(#) complex of PLA2 with decyl sulfate (CMC = 4500 microM) is 70 microM with a Hill coefficient n(1) = 2.1 +/- 0.2; K(2) for E(2)(#) is 750 microM with n(2) = 8 +/- 1, and K(3) for E(3)(#) is 4000 microM with an n(3) value of about 12. Controls show that (a) self-aggregation of decyl sulfate alone is not significant below the CMC; (b) occupancy of the active site is not necessary for the formation of E(i)(#); (c) K(i) and n(i) do not change significantly due to the absence of calcium, possibly because alkyl sulfate does not bind to the active site of PLA2; (d) the E(i)(#) complexes show a significant propensity for aggregation; and (e) PLA2 is not denatured in E(i)(#). The results are interpreted to elaborate the model for atomic level interactions along the i-face: The chain length dependence of the fit parameters suggests that short-range specific anion binding of the headgroup is accompanied by desolvation of the i-face of E(i)(#). We suggest that allosteric activation of PLA2 results from such specific interactions of the amphiplies and the desolvation of the i-face. The significance of these primary interfacial binding events and the coexistence of the E and E(i)(#) aggregates is discussed.     

Phenylalanine-Rich Peptides Potently Bind ESAT6, a Virulence Determinant of Mycobacterium tuberculosis,and Concurrently Affect the Pathogen's Growth

Background

The secretory proteins of Mycobacterium tuberculosis (M. tuberculosis) have been known to be involved in the virulence, pathogenesis as well as proliferation of the pathogen. Among this set, many proteins have been hypothesized to play a critical role at the genesis of the onset of infection, the primary site of which is invariably the human lung.

Methodology/Principal Findings

During our efforts to isolate potential binding partners of key secretory proteins of M. tuberculosis from a human lung protein library, we isolated peptides that strongly bound the virulence determinant protein Esat6. All peptides were less than fifty amino acids in length and the binding was confirmed by in vivo as well as in vitro studies. Curiously, we found all three binders to be unusually rich in phenylalanine, with one of the three peptides a short fragment of the human cytochrome c oxidase-3 (Cox-3). The most accessible of the three binders, named Hcl1, was shown also to bind to the Mycobacterium smegmatis (M. smegmatis) Esat6 homologue. Expression of hcl1 in M. tuberculosis H37Rv led to considerable reduction in growth. Microarray analysis showed that Hcl1 affects a host of key cellular pathways in M. tuberculosis. In a macrophage infection model, the sets expressing hcl1 were shown to clear off M. tuberculosis in much greater numbers than those infected macrophages wherein the M. tuberculosis was not expressing the peptide. Transmission electron microscopy studies of hcl1 expressing M. tuberculosis showed prominent expulsion of cellular material into the matrix, hinting at cell wall damage.

Conclusions/Significance

While the debilitating effects of Hcl1 on M. tuberculosis are unrelated and not because of the peptide''s binding to Esat6–as the latter is not an essential protein of M. tuberculosis–nonetheless, further studies with this peptide, as well as a closer inspection of the microarray data may shed important light on the suitability of such small phenylalanine-rich peptides as potential drug-like molecules against this pathogen.     

Selenium availability in berseem (Trifolium alexandrinum) as affected by selenium and phosphorus application

Summary The effect of selenium and phosphorus application on Berseem (Trifolium alexandrium) was studied in the green house. Each dose of selenium from O to 16 ppm decreased the dry matter yield of Berseem significantly at all the levels of applied phosphorus. Phosphorus stimulated the dry matter yield at all levels of selenium. Yields in the second cutting were of a higher order than in the first cut. Selenium application increased selenium and phosphorus content in Berseem, but at 8 and 16 ppm of selenium the phosphorus content decreased significantly. Sulphur concentration decreased with selenium application but application of phosphorus increased sulphur concentration showing synergetic effect.Studies on residual effect of selenium revealed that the application of 50 ppm of phosphorus increased the yield while that of 100 ppm decreased it. Selenium concentration which increased with increasing phosphorus was many fold higher in the first cutting than in the second cutting and similar was the trend with phosphorus and sulphur.     

Auxin: a master regulator in plant root development

The demand for increased crop productivity and the predicted challenges related to plant survival under adverse environmental conditions have renewed the interest in research in root biology. Various physiological and genetic studies have provided ample evidence in support of the role of plant growth regulators in root development. The biosynthesis and transport of auxin and its signaling play a crucial role in controlling root growth and development. The univocal role of auxin in root development has established it as a master regulator. Other plant hormones, such as cytokinins, brassinosteroids, ethylene, abscisic acid, gibberellins, jasmonic acid, polyamines and strigolactones interact either synergistically or antagonistically with auxin to trigger cascades of events leading to root morphogenesis and development. In recent years, the availability of biological resources, development of modern tools and experimental approaches have led to the advancement of knowledge in root development. Research in the areas of hormone signal perception, understanding network of events involved in hormone action and the transport of plant hormones has added a new dimension to root biology. The present review highlights some of the important conceptual developments in the interplay of auxin and other plant hormones and associated downstream events affecting root development.     

A bioinformatic filter for improved base-call accuracy and polymorphism detection using the Affymetrix GeneChip® whole-genome resequencing platform

DNA resequencing arrays enable rapid acquisition of high-quality sequence data. This technology represents a promising platform for rapid high-resolution genotyping of microorganisms. Traditional array-based resequencing methods have relied on the use of specific PCR-amplified fragments from the query samples as hybridization targets. While this specificity in the target DNA population reduces the potential for artifacts caused by cross-hybridization, the subsampling of the query genome limits the sequence coverage that can be obtained and therefore reduces the technique's resolution as a genotyping method. We have developed and validated an Affymetrix Inc. GeneChip® array-based, whole-genome resequencing platform for Francisella tularensis, the causative agent of tularemia. A set of bioinformatic filters that targeted systematic base-calling errors caused by cross-hybridization between the whole-genome sample and the array probes and by deletions in the sample DNA relative to the chip reference sequence were developed. Our approach eliminated 91% of the false-positive single-nucleotide polymorphism calls identified in the SCHU S4 query sample, at the cost of 10.7% of the true positives, yielding a total base-calling accuracy of 99.992%.