Genetic mapping of the bean golden yellow mosaic geminivirus resistance gene bgm-1 and linkage with potyvirus resistance in common bean (Phaseolus vulgaris L.)

Bean golden yellow mosaic virus (BGYMV) is a whitefly-transmitted geminivirus of the Begomovirus family that causes important yield losses to common beans grown in tropical and sub-tropical countries of Latin America and the Caribbean. A major resistance gene that has been widely deployed in this region is the recessive locus bgm-1 that prevents the development of severe yellowing typical of the disease. In this study, we developed a co-dominant sequence-characterized amplified region (SCAR) marker, SR2, based on a previously identified random amplified polymorphic DNA (RAPD) marker that is tightly linked to the bgm-1 resistance gene and identified the position of the locus in the common bean genome through comparative mapping using two genetic maps for the species. The SR2 marker was mapped relative to bgm-1 in a segregating population of recombinant inbred lines developed from the resistant × susceptible cross of DOR476 × SEL1309. Polymorphism was shown to be based on a 37 bp insertion event in the SR2 allele associated with susceptibility compared to the allele associated with resistance and the marker mapped at a distance of 7.8 cM from the resistance gene. The SR2 marker was significantly associated with overall disease symptoms and with three of the four symptoms associated with the disease (yellowing or chlorosis, flower abortion, foliar deformation) in a greenhouse trial in Colombia with the mechanically transmissible BGYMV–Guatemala strain. In both the DOR364 × G19833 and BAT93 × Jalo EEP558 mapping populations, SR2 was located near the end of linkage group b03 (chromosome 5) suggesting a sub-telomeric position. The position of the bgm-1 resistance gene was estimated to be close to that of bc-1, a strain-specific resistance gene for Bean common mosaic virus (BCMV), based on linkage of SR2 with the SCAR marker SBD5 in the DOR364 × G19833 mapping population. The implications of linkage between these two recessive resistance genes are discussed, as this is the first association between resistance genes against both a begomovirus and a potyvirus. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Nanosecond electric pulses penetrate the nucleus and enhance speckle formation

Nanosecond electric pulses generate nanopores in the interior membranes of cells and modulate cellular functions. Here, we used confocal microscopy and flow cytometry to observe Smith antigen antibody (Y12) binding to nuclear speckles, known as small nuclear ribonucleoprotein particles (snRNPs) or intrachromatin granule clusters (IGCs), in Jurkat cells following one or five 10 ns, 150 kV/cm pulses. Using confocal microscopy and flow cytometry, we observed changes in nuclear speckle labeling that suggested a disruption of pre-messenger RNA splicing mechanisms. Pulse exposure increased the nuclear speckled substructures by ∼2.5-fold above basal levels while the propidium iodide (PI) uptake in pulsed cells was unchanged. The resulting nuclear speckle changes were also cell cycle dependent. These findings suggest that 10 ns pulses directly influenced nuclear processes, such as the changes in the nuclear RNA-protein complexes.     

SAR and species/stereo-selective metabolism of the sorbitol dehydrogenase inhibitor,CP-470,711

SAR studies on the stereoisomers of CP-470,711 suggested that in vivo epimerization was taking place in rats. Further metabolism studies revealed that no epimerization was occurring in dogs, and that no epimerization was expected in humans. A mechanism for the in vivo epimerization is proposed involving an oxidation-reduction pathway of the secondary benzylic alcohol, in contrast to an acid/base-promoted epimerization of the same center during chemical synthesis.     

Synergistic effects of nanosecond pulsed electric fields combined with low concentration of gemcitabine on human oral squamous cell carcinoma in vitro

Treatment of cancer often involves uses of multiple therapeutic strategies with different mechanisms of action. In this study we investigated combinations of nanosecond pulsed electric fields (nsPEF) with low concentrations of gemcitabine on human oral cancer cells. Cells (Cal-27) were treated with pulse parameters (20 pulses, 100 ns in duration, intensities of 10, 30 and 60 kV/cm) and then cultured in medium with 0.01 μg/ml gemcitabine. Proliferation, apoptosis/necrosis, invasion and morphology of those cells were examined using MTT, flow cytometry, clonogenics, transwell migration and TEM assay. Results show that combination treatments of gemcitabine and nsPEFs exhibited significant synergistic activities versus individual treatments for inhibiting oral cancer cell proliferation and inducing apoptosis and necrosis. However, there was no apparent synergism for cell invasion. By this we demonstrated synergistic inhibition of Cal-27 cells in vitro by nsPEFs and gemcitabine. Synergistic behavior indicates that these two treatments have different sites of action and combination treatment allows reduced doses of gemcitabine and lower nsPEF conditions, which may provide better treatment for patients than either treatment alone while reducing systemic toxicities.     

Nanosecond pulsed electric fields (nsPEFs) activate intrinsic caspase-dependent and caspase-independent cell death in Jurkat cells

NsPEF ablation induces apoptosis markers, but specific cell death pathways have not been fully defined. To identify nsPEF-activated cell death pathways, wildtype human Jurkat cells and clones with deficiencies in extrinsic and intrinsic apoptosis pathways were investigated. NsPEFs activated caspase isozymes and induced identical electric field-dependent cell death in clones deficient in FADD or caspase-8, indicating that extrinsic apoptosis pathways were not activated. This was confirmed when cytochrome c release was shown to be unaffected by the pan caspase inhibitor, z-VAD-fmk. NsPEF-treated APAF-1-silenced cells did not exhibit caspase-3/7 and -9 activities and corresponding electric field-dependent cell death in this clone was attenuated compared to its vector control at low, but not at high electric fields. These data demonstrate that nsPEFs induce intrinsic apoptosis activate by cytochrome c release from mitochondria through an APAF-1- and caspase-dependent pathway as well as through caspase-independent mechanisms that remain to be defined. Furthermore, the results establish that nsPEFs can overcome natural and oncogenic mechanisms that promote cell survival through inhibition of apoptosis and other cell death mechanisms.     

Nanosecond pulse electric field (nanopulse): a novel non-ligand agonist for platelet activation

Nanosecond pulse stimulation of a variety of cells produces a wide range of physiological responses (e.g., apoptosis, stimulation of calcium (Ca²⁺) fluxes, changes in membrane potential). In this study, we investigated the effect of nanosecond pulses, which generate intense electric fields (nsPEFs), on human platelet aggregation, intracellular free Ca²⁺ ion concentration ([Ca²⁺]_i) and platelet-derived growth factor release. When platelet rich plasma was pulsed with one 300 ns pulse with an electric field of 30 kV/cm, platelets aggregated and a platelet gel was produced. Platelet aggregation was observed with pulses as low as 7 kV/cm with maximum effects seen with approximately 30 kV/cm. The increases in intracellular Ca²⁺ release and Ca²⁺ influx were dose dependent on the electrical energy density and were maximally stimulated with approximately 30 kV/cm. The increases in [Ca²⁺]_i induced by nsPEF were similar to those seen with thapsigargin but not thrombin. We postulate that nsPEF caused Ca²⁺ to leak out of intracellular Ca²⁺ stores by a process involving the formation of nanopores in organelle membranes and also caused Ca²⁺ influx through plasma membrane nanopores. We conclude that nsPEFs dose-dependently cause platelets to rapidly aggregate, like other platelet agonists, and this is most likely initiated by the nsPEFs increasing [Ca²⁺]_i, however by a different mechanism.     

Integration of gene dosage and gene expression in non-small cell lung cancer, identification of HSP90 as potential target

Background

Lung cancer causes approximately 1.2 million deaths per year worldwide, and non-small cell lung cancer (NSCLC) represents 85% of all lung cancers. Understanding the molecular events in non-small cell lung cancer (NSCLC) is essential to improve early diagnosis and treatment for this disease.

Methodology and Principal Findings

In an attempt to identify novel NSCLC related genes, we performed a genome-wide screening of chromosomal copy number changes affecting gene expression using microarray based comparative genomic hybridization and gene expression arrays on 32 radically resected tumor samples from stage I and II NSCLC patients. An integrative analysis tool was applied to determine whether chromosomal copy number affects gene expression. We identified a deletion on 14q32.2-33 as a common alteration in NSCLC (44%), which significantly influenced gene expression for HSP90, residing on 14q32. This deletion was correlated with better overall survival (P = 0.008), survival was also longer in patients whose tumors had low expression levels of HSP90. We extended the analysis to three independent validation sets of NSCLC patients, and confirmed low HSP90 expression to be related with longer overall survival (P = 0.003, P = 0.07 and P = 0.04). Furthermore, in vitro treatment with an HSP90 inhibitor had potent antiproliferative activity in NSCLC cell lines.

Conclusions

We suggest that targeting HSP90 will have clinical impact for NSCLC patients.     

Influence of a natural and a synthetic inhibitor of factor XIIIa on fibrin clot rheology

We investigated the origins of greater clot rigidity associated with FXIIIa-dependent cross-linking. Fibrin clots were examined in which cross-linking was controlled through the use of two inhibitors: a highly specific active-center-directed synthetic inhibitor of FXIIIa, 1,3-dimethyl-4,5-diphenyl-2[2(oxopropyl)thio]imidazolium trifluoromethylsulfonate, and a patient-derived immunoglobulin directed mainly against the thrombin-activated catalytic A subunits of thrombin-activated FXIII. Cross-linked fibrin chains were identified and quantified by one- and two-dimensional gel electrophoresis and immunostaining with antibodies specific for the alpha- and gamma-chains of fibrin. Gamma-dimers, gamma-multimers, alpha(n)-polymers, and alpha(p)gamma(q)-hybrids were detected. The synthetic inhibitor was highly effective in preventing the production of all cross-linked species. In contrast, the autoimmune antibody of the patient caused primarily an inhibition of alpha-chain cross-linking. Clot rigidities (storage moduli, G') were measured with a cone and plate rheometer and correlated with the distributions of the various cross-linked species found in the clots. Our findings indicate that the FXIIIa-induced dimeric cross-linking of gamma-chains by itself is not sufficient to stiffen the fibrin networks. Instead, the augmentation of clot rigidity was more strongly correlated with the formation of gamma-multimers, alpha(n)-polymers, and alpha(p)gamma(q)-hybrid cross-links. A mechanism is proposed to explain how these cross-linked species may enhance clot rigidity.     

Neutralization of influenza virus by normal human sera: mechanisms involving antibody and complement

All normal human sera examined neutralized WS/33 H1N1 influenza virus efficiently by one of two antibody-dependent mechanisms. A minority of the sera contained moderate levels of IgG antibody directed against the viral hemagglutinin that had the ability to directly neutralize the virus. The majority of sera tested contained very low levels of IgG anti-hemagglutinin antibody, which was detectable with a specific ELISA but not by conventional HAI assays. Such IgG antibody was unable to directly neutralize the virus. Studies with agammaglobulinemic serum and with sera depleted of and reconstituted with complement components established essential roles for IgG and the components of the classical complement pathway through C3 for neutralization. The components of the alternative and membrane attack pathways were not needed for neutralization. As anticipated from the requirement for IgG and exclusive mediation of neutralization by the classical pathway, the virus-IgG immune complex activated purified C1. Binding of C3 and C4 to the virus was demonstrated, as was classical pathway-mediated triggering of the alternative pathway, with recruitment of properdin. In addition, the H1N1 influenza virus also directly activated the alternative complement pathway in human serum, leading to C3 and properdin deposition on the viral envelope. Such direct alternative pathway activation also required immunoglobulin. However, the alternative pathway alone was unable to neutralize the virus. Thus, most normal sera examined contain low levels of IgG anti-hemagglutinin antibody, which activate the classical pathway of the complement system and neutralize WS/33 influenza virus by deposition of C3 and C4 on the viral envelope.     

Lipids of Branhamella catarrhalis and Neisseria gonorrhoeae.

Three strains of Branhamella catarrhalis and three strains of Neisseria gonorrhoeae were analyzed with regard to their phospholipid and neutral lipid composition. B. catarrhalis (ATCC 23246) contained 5.12 +/- 0.34% lipid, determined gravimetrically, compared to 8.56 +/- 0.15% and 9.73 +/- 0.06% for two strains of N. gonorrhoeae. Cardiolipin, phosphatidylglycerol, and phosphatidyl-ethanolamine were identified in extracts of both species. In addition, B. catarrhalis contained small amounts of phosphatidylcholine, and N. gonorrhoeae contained small amounts of lyso-phosphatidylethanolamine, which accumulated with autolysis accompanying late cell culture growth. The kinetics of change of relative amounts of phospholipids in both species were measured and found to differ substantially. Neutral lipid accounted for 30.4% of the total lipid of B. catarrhalis (ATCC 23246) and 7.6% of the total lipid of N. gonorrhoeae NYH 002. Hydrocarbons, triglycerides, free fatty acids, coenzyme Q, diglycerides, and free hydroxy fatty acids were identified in the neutral lipid fraction of both species. The three strains of N. gonorrhoeae, sensitive, intermediate, and resistant to penicillin, exhibited no significant difference in the composition or metabolism of phospholipid.