Molecular analysis of Ascochyta rabiei (Pass.) Labr., the pathogen of ascochyta blight in chickpea

Genetic diversity in Ascochyta rabiei (Pass.) Labr., the causative agent of ascochyta blight of chickpea, was determined using 37 Indian, five American (USA), three Syrian, and two Pakistani isolates. A total of 48 polymorphic RAPD markers were scored for each isolate and the data used for cluster analysis. Most of the isolates clustered in the dendrogram essentially according to geographic origin. Based on the two major clusters A and B, Indian isolates were grouped into two categories, type-A and type-B. Isolates of A. rabiei within the Punjab state were more diverse than isolates from other states in northwestern India. A DNA marker (ubc756_{1.6 kb}), specific to Indian isolates was identified. This is the first report of a molecular diversity analysis of Indian isolates of A. rabiei. The information may assist Indian chickpea breeders in the proper deployment of blight-resistant cultivars and in disease management. Received: 25 April 2000 / Accepted: 11 July 2000     

Recombinant canarypox vaccine-elicited CTL specific for dominant and subdominant simian immunodeficiency virus epitopes in rhesus monkeys

Since virus-specific CTL play a central role in containing HIV replication, a candidate AIDS vaccine should generate virus-specific CTL responses. In this study, the ability of a recombinant canarypox virus expressing SIV Gag-Pol-Env (ALVAC/SIV gag-pol-env) was assessed for its ability to elicit both dominant and subdominant epitope-specific CTL responses in rhesus monkeys. Following a series of five immunizations, memory CTL responses specific for a dominant Gag epitope could be demonstrated in the peripheral blood of vaccinated monkeys. Memory CTL responses to a subdominant Pol epitope were undetectable in these animals. Following challenge with SIVmac251, the experimentally vaccinated animals developed high frequency CTL responses specific for the dominant Gag epitope that emerged in temporal association with the early containment of viral replication. Interestingly, the experimentally vaccinated, but not the control vaccinated animals, developed CTL responses to the subdominant Pol epitope that were detectable only after containment of early viremia. Thus, recombinant canarypox vaccination elicited low frequency, but durable memory CTL populations. The temporal association of the emergence of the dominant epitope-specific response with early viral containment following challenge suggests that this immune response played a role in the accelerated clearing of early viremia in these animals. The later emerging CTL response specific for the subdominant epitope may contribute to the control of viral replication in the setting of chronic infection.     

Cationic oxyethylene lipids. Synthesis, aggregation, and transfection properties

Four cationic lipids (1-4) with oligo-oxyethylene units at the linkage region between the pseudoglyceryl backbone and the hydrocarbon chains have been synthesized. Two of these lipids (1 and 2) have an equal number of (CH(2)CH(2)O)(n)() units attached to both C-1 and C-2 positions of the pseudoglyceryl backbone, making their linkage regions similar, while the other two (3 and 4) are unsymmetrical in terms of the number of oxyethylene units in the linkage. Synthesis of lipids 1 and 2 involved the coupling of benzyl glycerol with the corresponding tosylates as a key step. Each of these lipids formed membranous aggregates when dispersed in water and exhibited clear thermotropic phase transitions typical of vesicular assemblies. The lipids 1-4 exhibited enhanced biological activities as gene transfer agents compared to their non-oxyethylene diether analogue, DHTMA. Transfection experiments using aqueous suspensions of these lipids and also their mixtures with cholesterol or dioleoyl phosphatidyl ethanolamine (DOPE) were performed on HeLa cells. The best transfection activity was demonstrated by unsymmetrical lipid 3, which had two oxyethylene units only at the C-1 position of the pseudoglycerylbackbone.     

Magnitude and diversity of cytotoxic-T-lymphocyte responses elicited by multiepitope DNA vaccination in rhesus monkeys

In an effort to develop an AIDS vaccine that elicits high-frequency cytotoxic-T-lymphocyte (CTL) responses with specificity for a diversity of viral epitopes, we explored two prototype multiepitope plasmid DNA vaccines in the simian-human immunodeficiency virus/rhesus monkey model to determine their efficiency in priming for such immune responses. While a simple multiepitope vaccine construct demonstrated limited immunogenicity in monkeys, this same multiepitope genetic sequence inserted into an immunogenic simian immunodeficiency virus gag DNA vaccine elicited high-frequency CTL responses specific for all of the epitopes included in the vaccine. Both multiepitope vaccine prototypes primed for robust epitope-specific CTL responses that developed following boosting with recombinant modified vaccinia virus Ankara vaccines expressing complete viral proteins. The natural hierarchy of immunodominance for these epitopes was clearly evident in the boosted monkeys. These studies suggest that multiepitope plasmid DNA vaccine-based prime-boost regimens can efficiently prime for CTL responses of increased breadth and magnitude, although they do not overcome predicted hierarchies of immunodominance.     

High cooperativity of the SV40 major capsid protein VP1 in virus assembly

SV40 is a small, non enveloped DNA virus with an icosahedral capsid of 45 nm. The outer shell is composed of pentamers of the major capsid protein, VP1, linked via their flexible carboxy-terminal arms. Its morphogenesis occurs by assembly of capsomers around the viral minichromosome. However the steps leading to the formation of mature virus are poorly understood. Intermediates of the assembly reaction could not be isolated from cells infected with wt SV40. Here we have used recombinant VP1 produced in insect cells for in vitro assembly studies around supercoiled heterologous plasmid DNA carrying a reporter gene. This strategy yields infective nanoparticles, affording a simple quantitative transduction assay. We show that VP1 assembles under physiological conditions into uniform nanoparticles of the same shape, size and CsCl density as the wild type virus. The stoichiometry is one DNA molecule per capsid. VP1 deleted in the C-arm, which is unable to assemble but can bind DNA, was inactive indicating genuine assembly rather than non-specific DNA-binding. The reaction requires host enzymatic activities, consistent with the participation of chaperones, as recently shown. Our results demonstrate dramatic cooperativity of VP1, with a Hill coefficient of approximately 6. These findings suggest that assembly may be a concerted reaction. We propose that concerted assembly is facilitated by simultaneous binding of multiple capsomers to a single DNA molecule, as we have recently reported, thus increasing their local concentration. Emerging principles of SV40 assembly may help understanding assembly of other complex systems. In addition, the SV40-based nanoparticles described here are potential gene therapy vectors that combine efficient gene delivery with safety and flexibility.     

An Integrated Global Analysis of Compartmentalized HRAS Signaling

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Arsenic Toxicity is Counteracted by Exogenous Application of Melatonin to Different Extents in Arsenic-susceptible and Arsenic-tolerant Rice Cultivars

The current study established the protective effects of exogenous melatonin in ameliorating arsenic toxicity in Khitish (arsenic-sensitive) and Muktashri (arsenic-tolerant) rice cultivars. Melatonin highly improved the overall growth performance of arsenic-treated seedlings, more prominently in the sensitive variety, Khitish. Although the level of arsenic increased in both the cultivars, Khitish accumulated comparatively higher arsenic level. However, melatonin supplementation reduced arsenic bioaccumulation and restored physiological growth attributes, as supported by lowering of electrolyte leakage, chlorophyll loss (by inducing RuBisCo), protein carbonylation, malondialdehyde accumulation, lipoxygenase (LOX), NADPH oxidase (NOX) and protease activity, and improvement of membrane stability index. Isoforms of LOX and NOX showed varietal differences during arsenic stress, both in the presence and absence of melatonin. Melatonin reduced methylglyoxal content during arsenic stress, concomitant with down-regulated gene expression and enzyme activity for glyoxalases. The nitrogen assimilation was improved via induced nitrate reductase (NR) activity and NR expression. The variable accumulation of osmolytes like proline, glycine betaine and total amino acids, concomitant with suppressed P5CS and BADH1 expression, and induced PDH was noteworthy. Antioxidant metabolites like anthocyanins, flavonoids, carotenes, xanthophylls and total phenolics were accumulated upon supplementation of melatonin in arsenic-stressed Khitish, supported by the activation of ANS and PSY genes. Melatonin lowered the ascorbic-acid oxidase activity and restored the ascorbate sink in arsenic-affected seedlings. Overall, the study revealed the potential role of exogenous melatonin in mitigating arsenic-induced injuries by strengthening osmolytes and antioxidative machinery, leading to the restoration of growth and metabolism in rice, especially in the susceptible cultivar.

     

Radiological and Pathological Features Associated with IDH1-R132H Mutation Status and Early Mortality in Newly Diagnosed Anaplastic Astrocytic Tumours

Background

Glioblastoma can occur either de novo or by the transformation of a low grade tumour; the majority of which harbor a mutation in isocitrate dehydrogenase (IDH1). Anaplastic tumours are high-grade gliomas that may represent the final step in the evolution of a secondary glioblastoma or the initial presentation of an early primary glioblastoma. We sought to determine whether pathological and/or radiological variables exist that can reliably distinguish IDH1-R132H-positive from IDH1-R132H-negative tumours and to identify variables associated with early mortality.

Methods

Patients diagnosed with anaplastic astrocytic tumours were included. Magnetic resonance imaging was performed and immunohistochemistry was used to identify tumours with the IDH1-R132H mutation. Survival was assessed 12 months after diagnosis. Variables associated with IDH1-R132H status were identified by univariate and ROC analysis.

Results

37 gliomas were studied; 18 were positive for the IDH1-R132H mutation. No tumours demonstrated a combined loss of chromosomes 1p/19q. Patients with IDH1-R132H-positive tumours were less likely to die within 12 months of diagnosis (17% vs. 47%; p=0.046), more likely to have tumours located in the frontal lobe (55% vs. 16%; p=0.015), and have a higher minimum apparent diffusion coefficient (1.115 x 10^-3 mm²/sec vs. 0.838 x 10^-3 mm²/sec; p=0.016), however, these variables demonstrated only moderate strength for predicting the IDH1-R132H mutation status (AUC=0.735 and 0.711, respectively). The Ki-67 index was significantly lower in IDH1-R132H-positive tumours (0.13 vs. 0.21; p=0.034). An increased risk of death was associated with contrast-enhancement ≥ 5 cm³ in patients with IDH1-R132H-positive tumours while edema ≥ 1 cm beyond the tumour margin and < 5 mitoses/mm² were associated with an increased risk of death in patients with IDH1-R132H-negative tumours.

Conclusions

IDH1-R132H-positive and -negative anaplastic tumours demonstrate unique features. Factors associated with early mortality are also dependent on IDH1-R132H status and can be used to identify patients at high risk for death.     

Isolation of acdc28 mutation that abrogates the dependence of S phase on completion of M phase of the budding yeast cell cycle

We have isolated a mutation in the budding yeastSaccharomyces cerevisisae CDC28 gene that allowscdc13 cells, carrying damaged DNA, to continue with the cell division cycle. Whilecdc13 mutant cells are arrested as largebudded cells at the nonpermissive temperature 37‡C, thecdc13 cdc28 double mutant culture showed cells with one or more buds, most of which showed apical growth. The additional buds emerged without the intervening steps of nuclear division and cell separation. We suggest that thecdc28 mutation abrogates a checkpoint function and allows cells with damaged or incompletely replicated DNA an entry to another round of cell cycle and bypasses the mitotic phase of the cell cycle.