The development of simple sequence repeat markers for Magnaporthe grisea and their integration into an established genetic linkage map

Although microsatellite or simple sequence repeat (SSR) markers have several advantages, few have been developed in fungi. The goal of this study was to identify and characterize SSR-containing loci in the filamentous ascomycete Magnaporthe grisea, the causal agent of rice blast disease, and to add these markers to an integrated genetic map of this species [Theor. Appl. Genet. 95 (1997) 20]. We have constructed and screened a microsatellite-enriched small-insert genomic library as well as exploited both publicly available and one proprietary databases for identification of M. grisea SSR containing sequences. Twenty-four out of 49 primer pairs designed to amplify SSR, produced unambiguous polymorphic products in our test population of six isolates. The number of alleles at each locus ranged from two to six when assayed on 3% agarose gels. Twenty-three of the primer pairs amplified polymorphic products between Guy11 and 2539, the parents of a cross from which a genetic map for M. grisea has been established. Genetic analysis showed that all the markers segregated in the expected 1:1 ratio and map positions were determined for all 23 loci.     

Conformational spaces of Cinchona alkaloids

A systematic and comprehensive study of the conformational spaces of the Cinchona alkaloids quinine, quinidine, cinchonine, cinchonidine, epiquinine, epiquinidine, epicinchonine, and epicinchonidine using the semiempirical PM3 method is described. The results were analyzed in terms of syn/anti and open/closed/hindered and alpha/beta/gamma conformations. Special emphasis was given to the torsion angles T(1) (C(4a')-C(4')-C(9)-C(8)), T(2) (C(4')-C(9)-C(8)-N(1)) and T(3) (H-O(9)-C(9)-C(8)) that define the backbone and the hydroxy conformation, respectively. The results reveal the quasi-enantiomeric relationships between quinine and quinidine and between epiquinine and epiquinidine, and the main structural differences that exist between the therapeutically active Cinchona alkaloids, quinine and quinidine, and their inactive epimers, epiquinine and epiquinidine. The lowest energy conformation of quinine and quinidine is anti-closed-alpha. The lowest energy conformations of epiquinine and epiquinidine are anti-open-beta and anti-open-alpha, respectively. Low energy conformations with an intramolecular hydrogen bond (N(1.)H(.)O(9)) were found in epiquinine (the global minimum) and epiquinidine, but not in quinine and quinidine.     

Cardiomyocyte apoptosis triggered by RAFTK/pyk2 via Src kinase is antagonized by paxillin

Altered cellular adhesion and apoptotic signaling in cardiac remodeling requires coordinated regulation of multiple constituent proteins that comprise cytoskeletal focal adhesions. One such protein activated by cardiac remodeling is related adhesion focal tyrosine kinase (RAFTK, also known as pyk2). Adenoviral-mediated expression of RAFTK in neonatal rat cardiomyocytes involves concurrent increases in phosphorylation of Src, c-Jun N-terminal kinase, and p38 leading to characteristic apoptotic changes including cleavage of poly(ADP-ribose) polymerase, caspase-3 activation, and increased DNA laddering. DNA laddering was decreased by mutation of the Tyr(402) Src-binding site in RAFTK, suggesting a central role for Src activity in apoptotic cell death that was confirmed by adenoviral-mediated Src expression. Multiple apoptotic signaling cascades are recruited by RAFTK as demonstrated by prevention of apoptosis using caspase-3 inhibitor IV (caspase-3 specific inhibitor), PP2 (Src-specific kinase inhibitor), or Csk (cellular negative regulator for Src), as well as dominant negative constructs for p38beta or MKP-1. These RAFTK-mediated phenotypic characteristics are prevented by concurrent expression of wild-type or a phosphorylation-deficient paxillin mutated at Tyr(31) and Tyr(118). Wild-type or mutant paxillin protein accumulation in the cytoplasm has no overt effect upon cell structure, but paxillin accumulation prevents losses of myofibril organization as well as focal adhesion kinase, vinculin, and paxillin protein levels mediated by RAFTK. Apoptotic signaling cascade inhibition by paxillin indicates interruption of signaling proximal to but downstream of RAFTK activity. Chronic RAFTK activation in cardiac remodeling may represent a maladaptive reactive response that can be modulated by paxillin, opening up novel possibilities for inhibition of cardiomyocyte apoptosis and structural degeneration in heart failure.     

Bone demineralization mechanisms at level of free radicals and nanoscale subsystems of bone tissue

Influence of simulated microgravity on characteristics of rat bones was investigated by electron paramagnetic resonance (EPR). For simulation of microgravity condition the hanging of animal by tail was used. The main measurements were performed for diaphysis of femoral bones. The quantity of native radicals Rn, localized in an organic matrix, and carbonate radicals CO2-, localized on a surface of bioapatite nanocrystals, were determined. The decrease of CO2- radical quantity in bones of experimental animals have shown, that due to simulation of microgravity the decrease of "collagen-nanocrystals" interaction takes place. It is shown that the EPR method open possibilities to receive the unique information about bone demineralization processes at level of free radicals and nanoscale subsystems of bone tissue.     

Investigation of the Hepatoprotective Effects of Sesame (Sesamum indicum L.) in Carbon Tetrachloride-Induced Liver Toxicity

More than 600 chemicals can cause damage in liver, one of which is carbon tetrachloride (CCl₄). Hepatoprotective agents could prevent tissue damage and reduce morbidity and mortality rates; such agents may include alternative or folkloric treatments. We investigated sesame (Sesamum indicum L.) for its hepatoprotective effect in CCl₄-induced experimental liver damage. To this end, 0.8 mg/kg of sesame fixed oil was provided intraperitoneally to rats whose livers were damaged by CCl₄. Tissue and blood samples were taken at the end of the experiments and evaluated histologically and biochemically. Ballooning degenerations and an increase in lipid droplets in liver parenchyma and increases in serum alanine transaminase, aspartate transaminase, and bilirubin were found in the CCl₄ group. Biochemical and histopathological findings in the sesame fixed oil treated group were not significantly different from the CCl₄ group. Sesame did not show a hepatoprotective effect in CCl₄-induced liver toxicity.     

The role of apoptosis in the ameliorating effects of a CDR1-based peptide on lupus manifestations in a mouse model

Experimental systemic lupus erythematosus (SLE) can be induced in mice following immunization with an anti-DNA mAb expressing a major Id, 16/6Id. Treatment with a peptide, designated human CDR1 (hCDR1; Edratide), that is based on the sequence of CDR1 of the 16/6Id ameliorated disease manifestations. In the present study, we investigated the roles of apoptosis and related molecules in BALB/c mice with induced experimental SLE following treatment with hCDR1. A higher state of activation and increased rate of apoptosis were found in lymphocytes of SLE-afflicted mice as compared with healthy controls. The latter effects were associated with up-regulated caspase-8 and caspase-3, and down-regulated Bcl-x(L). The ameliorative effects of hCDR1 were associated with down-regulation of caspase-8 and caspase-3, up-regulation of Bcl-x(L), and a reduced rate of apoptosis. Treatment of diseased mice with an apoptosis-reducing compound that inhibited caspases down-regulated the secretion of the pathogenic cytokine IFN-gamma and lowered the intensity of glomerular immune complex deposits and the levels of proteinuria. Furthermore, coincubation of Bcl-x(L) inhibitors with hCDR1-treated cells abrogated the ability of hCDR1 to reduce the activation state of lymphocytes and to down-regulate the secretion of IL-10 and IFN-gamma. Moreover, the Bcl-x(L)-expressing CD4(+)CD25(+) cells from hCDR1-treated mice induced the expression of Bcl-x(L) in CFSE-labeled CD4(+)CD25(-) cells of the SLE-afflicted mice. Thus, the reduction of apoptosis and the up-regulation of Bcl-x(L), which plays an apparent role in tolerance induction, contribute to at least part of the beneficial effects of hCDR1 on lupus manifestations.     

M. tuberculosis and M. leprae translocate from the phagolysosome to the cytosol in myeloid cells

M. tuberculosis and M. leprae are considered to be prototypical intracellular pathogens that have evolved strategies to enable growth in the intracellular phagosomes. In contrast, we show that lysosomes rapidly fuse with the virulent M. tuberculosis- and M. leprae-containing phagosomes of human monocyte-derived dendritic cells and macrophages. After 2 days, M. tuberculosis progressively translocates from phagolysosomes into the cytosol in nonapoptotic cells. Cytosolic entry is also observed for M. leprae but not for vaccine strains such as M. bovis BCG or in heat-killed mycobacteria and is dependent upon secretion of the mycobacterial gene products CFP-10 and ESAT-6. The cytosolic bacterial localization and replication are pathogenic features of virulent mycobacteria, causing significant cell death within a week. This may also reveal a mechanism for MHC-based antigen presentation that is lacking in current vaccine strains.