Transgenic tobacco and peanut plants expressing a mustard defensin show resistance to fungal pathogens

Defensins are small positively charged, antimicrobial peptides (~5 kDa in size) and some of them exhibit potent antifungal activity. We have cloned the complete cDNA containing an ORF of 243 bp of a defensin of mustard. The deduced amino acid sequence of the peptide showed more than 90% identity to the amino acid sequence of the well-characterized defensins, RsAFP-1 and RsAFP-2 of Raphanus sativus. We have generated and characterized transgenic tobacco and peanut plants constitutively expressing the mustard defensin. Transgenic tobacco plants were resistant to the fungal pathogens, Fusarium moniliforme and Phytophthora parasitica pv. nicotianae. Transgenic peanut plants showed enhanced resistance against the pathogens, Pheaoisariopsis personata and Cercospora arachidicola, which jointly cause serious late leaf spot disease. These observations indicate that the mustard defensin gene can be deployed for deriving fungal disease resistance in transgenic crops.     

Plastid omega3-fatty acid desaturase-dependent accumulation of a systemic acquired resistance inducing activity in petiole exudates of Arabidopsis thaliana is independent of jasmonic acid

Systemic acquired resistance (SAR) is an inducible defense mechanism that is activated throughout the plant, subsequent to localized inoculation with a pathogen. The establishment of SAR requires translocation of an unknown signal from the pathogen-inoculated leaf to the distal organs, where salicylic acid-dependent defenses are activated. We demonstrate here that petiole exudates (PeXs) collected from Arabidopsis leaves inoculated with an avirulent (Avr) Pseudomonas syringae strain promote resistance when applied to Arabidopsis, tomato ( Lycopersicum esculentum ) and wheat ( Triticum aestivum ). Arabidopsis FATTY ACID DESATURASE7 ( FAD7 ), SUPPRESSOR OF FATTY ACID DESATURASE DEFICIENCY1 ( SFD1 ) and SFD2 genes are required for accumulation of the SAR-inducing activity. In contrast to Avr PeX from wild-type plants, Avr PeXs from fad7 , sfd1 and sfd2 mutants were unable to activate SAR when applied to wild-type plants. However, the SAR-inducing activity was reconstituted by mixing Avr PeXs collected from fad7 and sfd1 with Avr PeX from the SAR-deficient dir1 mutant. Since FAD7 , SFD1 and SFD2 are involved in plastid glycerolipid biosynthesis and SAR is also compromised in the Arabidopsis monogalactosyldiacylglycerol synthase1 mutant we suggest that a plastid glycerolipid-dependent factor is required in Avr PeX along with the DIR1- encoded lipid transfer protein for long-distance signaling in SAR. FAD7 -synthesized lipids provide fatty acids for synthesis of jasmonic acid (JA). However, co-infiltration of JA and methylJA with Avr PeX from fad7 and sfd1 did not reconstitute the SAR-inducing activity. In addition, JA did not co-purify with the SAR-inducing activity confirming that JA is not the mobile signal in SAR.     

African HIV/AIDS trials are more likely to report adequate allocation concealment and random generation than North American trials

Background

Adherence to good methodological quality is necessary to minimise bias in randomised conrolled trials (RCTs). Specific trial characteristics are associated with better trial quality, but no studies to date are specific to HIV/AIDS or African trials. We postulated that location may negatively impact on trial quality in regions where resources are scarce.

Methods

1) To compare the methodological quality of all HIV/AIDS RCTs conducted in Africa with a random sample of similar trials conducted in North America; 2) To assess whether location is predictive of trial quality. We searched MEDLINE, EMBASE, CENTRAL and LILACS. Eligible trials were 1) randomized, 2) evaluations of preventive or treatment interventions for HIV/AIDS, 3) reported before 2004, and 4) conducted wholly or partly (if multi-centred) in Africa or North America. We assessed adequacy of random generation, allocation concealment and masking of assessors. Using univariate and multivariate logistic regression analyses we evaluated the association between location (Africa versus North America) and these domains.

Findings

The African search yielded 12,815 records, from which 80 trials were identified. The North American search yielded 13,158 records from which 785 trials were identified and a random sample of 114 selected for analysis. African trials were three times more likely than North American trials to report adequate allocation concealment (OR = 3.24; 95%CI: 1.59 to 6.59; p<0.01) and twice as likely to report adequate generation of the sequence (OR = 2.36; 95%CI: 1.20 to 4.67; p = 0.01), after adjusting for other confounding factors. Additional significant factors positively associated with quality were an a priori sample size power calculation, restricted randomization and inclusion of a flow diagram detailing attrition. We did not detect an association between location and outcome assessor masking.

Conclusions

The higher quality of reporting of methodology in African trials is noteworthy. Most African trials are externally funded, and it is possible that stricter agency requirements when leading trials in other countries and greater experience and training of principal investigators of an international stature, may account for this difference.     

Phylogenetic relationships among Oryza species revealed by AFLP markers

 The genus Oryza to which cultivated rice belongs has 22 wild species. Seventy-seven accessions of 23 Oryza species, five related genera, and three outgroup taxa were fingerprinted using amplified fragment length polymorphism (AFLP). A total of 1191 polymorphic markers were obtained using five AFLP primer combinations. AFLP data were analyzed to study species relationships using different clustering algorithms, and the resulting phenograms were tested for stability and robustness. The findings suggest a common ancestry to the genus Oryza. Moreover, the results demonstrate that: (1) evolution in Oryza has followed a polyphyletic path wherein multiple lineages underwent independent divergence after separation early in the evolution from a common ancestor/pool of related taxa; (2) newly assigned genomes, GG for O. meyeriana and HHJJ for O. ridleyi complexes, are among the most diverged in the genus; (3) CCDD tetraploids have a relatively ancient origin among the Officinalis complex; (4) O. malampuzhaensis, O. indandamanica, O. alta, and O. grandiglumis are diverged enough to deserve species status; (5) O. officinalis and O. eichingeri (CC) are putative progenitors of O. minuta ^* O. malampuzhaensis and tetraploid O. punctata, respectively, (6) O. brachyantha is most diverged species in the genus. AFLP is reliable molecular technique and provides one of the most informative approaches to ascertain genetic relationships in Oryza, which may also be true for other related species/organisms. Received: 1 July 1998 / Accepted: 2 November 1998     

Osteopontin: it's role in regulation of cell motility and nuclear factor kappa B-mediated urokinase type plasminogen activator expression

Cancer progression depends on an accumulation of metastasis supporting cell signaling molecules that target signal transduction pathways and ultimately gene expression. Osteopontin (OPN) is one such chemokine like metastasis gene which plays a key signaling event in regulating the oncogenic potential of various cancers by controlling cell motility, invasiveness and tumor growth. We have reported that OPN stimulates tumor growth and nuclear factor kappaB (NFkappaB)-mediated promatrix metalloproteinase-2 (pro-MMP-2) activation through IkappaBalpha/IKK (IkappaBalpha kinase) signaling pathway in melanoma cells. Urokinase type plasminogen activator (uPA), a widely acting serine protease degrades the ECM components and plays a pivotal role in cancer progression. However, the molecular mechanism by which upstream kinases regulate the OPN-induced NFkappaB activation and uPA secretion in human breast cancer cells is not well defined. Here we report that OPN induces the phosphatidylinositol 3'-kinase (PI 3'-kinase) activity and phosphorylation of Akt/PKB (protein kinase B) in highly invasive (MDA-MB-231) and low invasive (MCF-7) breast cancer cells. The OPN-induced Akt phosphorylation was inhibited when cells were transfected with dominant negative mutant of p85 domain of PI 3'-kinase (Deltap85) indicating that PI 3'-kinase is involved in Akt phosphorylation. OPN enhances the interaction between IkappaBalpha kinase (IKK) and phosphorylated Akt. OPN also induces NFkappaB activation through phosphorylation and degradation of IkappaBalpha by inducing the IKK activity. OPN also enhances uPA secretion, cell motility and ECM-invasion. Furthermore, cells transfected with Deltap85 or super-repressor form of IkappaBalpha suppressed the OPN-induced uPA secretion and cell motility. Pretreatment of cells with PI 3'-kinase inhibitors or NFkappaB inhibitory peptide (SN50) reduced the OPN-induced uPA secretion, cell motility and ECM-invasion. Taken together, OPN induces NFkappaB activity and uPA secretion by activating PI 3'-kinase/Akt/IKK-mediated signaling pathways and further demonstrates a functional molecular link between OPN induced PI 3'-kinase dependent Akt phosphorylation and NFkappaB-mediated uPA secretion, and all of these ultimately control the motility and invasiveness of breast cancer cells.     

Study of embryo rescue in floribunda rose

In the past few decades, breeders have faced a lot of problems in rose improvement due to low sexual reproduction and poor germination because of embryo abortion. Immature embryos may be recovered in vitro. An efficient protocol for embryo rescue in two floribunda roses (Arunima and Shocking Blue) was developed. The germination of immature embryos was achieved by manipulating the growth media, growth hormones and culture conditions. The embryos (rescued) germinated and grew considerably on Murashige and Skoog basal medium supplemented with 2.5 mg l^–l BA (6-benzylaminopurine), 0.5 mg l^–l GA₃ (gibberellic acid) and 3 (w/v) sucrose under 16-h photoperiod. A higher rate of germination was observed in cultures incubated 2 weeks in dark and subsequently transferred to 2 weeks in light at 16-h photoperiod. The embryo derived plantlets were successfully transferred to greenhouse and produced flowers. Embryo rescue technique in floribunda roses has great potential in floriculture industry.     

Molecular mapping of sorghum genes expressing tolerance to damage by greenbug (Homoptera: Aphididae)

Genetic linkage maps are fundamental for the localization of genes conferring tolerance to greenbug, Schizaphis graminum (Rondani), feeding damage in sorghum, Sorghum bicolor (L.) Moench. Thirteen linkage groups (LGs) containing 60 simple sequence repeat (SSR) loci were mapped by using a set of sorghum recombinant inbred lines (RILs) obtained from the cross '96-4121' (greenbug-tolerant parent) x Redlan (greenbug-susceptible parent). The LG spanned a distance of 603.5 cM, with the number of loci per LG varying from 2 to 14. Seventeen additional SSR loci were unlinked at a log of odds value of 3.0. Based on chlorophyll loss occurring after greenbug feeding, visual damage ratings, and soil plant analysis development (SPAD), chlorophyll-loss indices were recorded for each RIL and for the parents used in the cross. Composite-interval mapping identified three quantitative trait loci (QTLs) associated with biotype I and five QTLs associated with biotype K. The amount of phenotypic variation explained by these QTLs ranged from 9 to 19.6%. The identification of QTLs that influence greenbug tolerance will not only facilitate the use of marker-assisted selection in sorghum breeding programs but also will provide a solid foundation for detailed characterization of individual loci implicated in greenbug tolerance in sorghum.     

Dynamics of dendritic cell phenotype and interactions with CD4+ T cells in airway inflammation and tolerance

An emerging concept is that different types of dendritic cells (DCs) initiate different immune outcomes, such as tolerance vs inflammation. In this study, we have characterized the DCs from the lung draining lymph nodes of mice immunized for allergic airway inflammation or tolerance and examined their interactions with CD4(+) T cells. The DC population derived from tolerized mice was predominantly CD11c(+), B220(+), Gr-1(+), CD11b(-), and MHC class II(low), which resembled plasmacytoid-type DCs whereas DCs from the inflammatory condition were largely CD11c(+), B220(-), Gr-1(-), CD11b(+), and MHC class II(high) resembling myeloid-type DCs. The DCs from the tolerogenic condition were poor inducers of T cell proliferation. DCs from both conditions induced T cell IL-4 production but the T cells cultured with tolerogenic DCs were unresponsive to IL-4 as indicated by inhibition of STAT6 activation and expression of growth factor-independent 1, which has been recently shown to be important for STAT6-activated Th2 cell expansion. Our data suggest that airway tolerance vs inflammation is determined by the DC phenotype in lung draining lymph nodes.     

HIV-specific CD8+ lymphocytes in semen are not associated with reduced HIV shedding

Sexual contact with HIV-infected semen is a major driving force behind the global HIV pandemic. Little is known regarding the immune correlates of virus shedding in this compartment, although HIV-1-specific CD8+ T cells are present in semen. We collected blood and semen from 27 chronically HIV-infected, therapy-naive men without common sexually transmitted infections or urethral inflammation and measured HIV-1 RNA viral load and cytokine/chemokine levels in both compartments. HIV-1 RNA levels were 10-fold higher in blood than semen, but discordantly high semen shedding was associated with higher semen levels of the proinflammatory cytokines IL-6, IL-8, IL-12, and IFN-gamma. Virus-specific CD8+ T cell epitopes were mapped in blood by IFN-gamma ELISPOT, using an overlapping HIV-1 clade B peptide matrix, and blood and semen CD8+ T cell responses were then assayed ex vivo using intracellular IFN-gamma staining. HIV-specific CD8+ responses were detected in 70% of semen samples, and their frequency was similar to or higher than blood. There was no correlation between the presence of virus-specific CD8+ T cells in semen and levels of HIV-1 RNA shedding. Among participants with detectable CD8+ IFN-gamma semen responses, their relative frequency was not associated with reduced HIV-1 RNA shedding, and their absolute number was correlated with higher levels of HIV-1 RNA semen shedding (r = 0.6; p = 0.03) and of several proinflammatory cytokines. Neither the presence nor the frequency of semen HIV-specific CD8+ T cell IFN-gamma responses in semen correlated with reduced levels of HIV RNA in semen.