DNA fragmentation

Pseudomonas aeruginosa, a common agent of septicemia, enters into human endothelial cellsin vitro but the effects of bacterial infection have not been addressed properly. In this study, human umbilical vein endothelial cells (HUVEC) were infected by the noninvasive PA103 and ...

Our aim was to evaluate the effect of neat semen vitrification on human sperm vital parameters and DNA integrity in men with normal and abnormal sperm parameters. Semen samples were 17 normozoospermic samples and 17 specimens with abnormal sperm parameters. Semen analysis was performed according to World Health Organization (WHO) criteria. Then, the smear was provided from each sample and fixed for terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining. Vitrification of neat semen was done by plunging cryoloops directly into liquid nitrogen and preserved for 7 days. The samples were warmed and re-evaluated for sperm parameters as well as DNA integrity. Besides, the correlation between sperm parameters and DNA fragmentation was assessed pre- and post vitrification. Cryopreserved spermatozoa showed significant decrease in sperm motility, viability and normal morphology after thawing in both normal and abnormal semen. Also, the rate of sperm DNA fragmentation was...

Abstract:  The pancreas is highly susceptible to the oxidative stress induced by ischemia/reperfusion (IR) injury leading to the generation of acute pancreatitis. Melatonin has been shown to be useful in the prevention of the damage by ischemia-reperfusion in liver, brain, myocardium, gut and kidney. The aim of the study was to evaluate the cytoprotective properties of melatonin against injury induced by IR in pancreas. The obstruction of gastro-duodenal and inferior splenic arteries induced pancreatic IR in male Wistar rats. Melatonin was intraperitoneally administered before or/and after IR injury. The animals were killed at 24 and 48 hr after reperfusion and there were evaluated parameters of oxidative stress (lipoperoxides, superoxide dismutase, catalase, glutathione peroxidase and reduced glutathione), glandular endocrine and exocrine function (lipase, amylase, insulin) and cell injury (apoptosis and necrosis). The IR induced a marked enhancement of oxidative stress and impaired pancreatic function. The histological analysis showed that IR induced acute pancreatitis with the accumulation of inflammatory infiltrate, disruption of tissue structure, cell necrosis and hemorrhage. Melatonin administration before or after pancreatic IR prevented all tissue markers of oxidative stress, biochemical and histological signs of apoptosis and necrosis, and restored glandular function. No histological signs of pancreatitis were observed 48 hr after reperfusion in 80% of the animals treated with melatonin, with only a mild edematous pancreatitis being observed in the remaining rats. Preventive or therapeutic administration of melatonin protected against the induction of oxidative stress and tissue injury, and restored cell function in experimental pancreatic IR in rats.

The utility of polymorphism analysis was determined for differentiation of the following subspecies of the Gram-positive plant pathogenic bacterium, Clavibacter michiganensis: C. m. subsp. michiganensis, C. m. subsp. sepedonicus, C. m. subsp. insidiosus C. m. subsp. nebraskensis, and C. m. subsp. tessellarius. Specific primers designed for amplification of the housekeeping genes recA, rpoB, and rpoD generated 827-, 1037-, and 862-bp DNA fragments, respectively. PCR products obtained from 40 C. michiganensis strains were analysed using RFLP with four restriction endonucleases, and those PCR products with specific RFLP patterns were sequenced. The genotypes discriminated after PCR–RFLP were specific for each subspecies and also allowed for differentiation of C. m. subsp. michiganensis strains. Sequence analysis of the recA, rpoB, and rpoD gene fragments also distinguished C. michiganensis subspecies and was useful for phylogenetic analysis of all subspecies. For rapid, inexpensive, and effective differentiation of the five subspecies in this research, we recommend the amplification of recA and/or rpoD gene fragments and digestion of the PCR products with the restriction endonuclease FnuDII.

To investigate whether apoptosis is involved in the fate of oligodendrocytes in chronic multiple sclerosis lesions, the pro-apoptotic molecules fas and tumor necrosis factor receptors and the anti-apoptotic molecule bcl-2 were examined by immunohistochemistry, and DNA fragmentation was assessed by an end labeling technique. Fas and both tumor necrosis factor receptors were preferentially expressed on oligodendrocytes in multiple sclerosis lesions, this phenotype being more evident at the lesion edge. The ligand for fasL, was constitutively present at high levels on microglia. The anti-apoptotic molecule bcl-2 was selectively expressed on oligodendrocytes in silent lesions and on astrocytes in active lesions. These molecules were also detected in control material, albeit at lower levels. In chronic active lesions, a few inflammatory cells displayed fas reactivity, whereas the majority expressed bcl-2. DNA fragmentation was found in a number of infiltrating cells and some microglia, whereas, with one possible exception, oligodendrocytes showed no evidence of apoptosis. Thus, while apoptosis is involved in the elimination of infiltrating cells, it plays little or no role in oligodendrocyte depletion in multiple sclerosis, a process that may be related to a lytic pathway. In addition, microglia constitutively displayed the ligand for fas, and appeared to be the major effector cell population in the central nervous system.

Yellow mosaic virus, YMV, causes one of the most severe of biotic stresses in Vignas, an important group of pulse crops. The viral disease is transmitted through the white fly, Bemicia tabaci, and the yield of the plants is affected drastically. YMV-tolerant lines, generated from a single YMV-tolerant plant identified in the field within a large population of the susceptible cultivar T-9, were crossed with T-9, and F1, F2 and F3 progenies raised. The different generations were phenotyped for YMV-reaction by forced inoculation using viruliferous white flies. A monogenic recessive control of YMV-tolerance was revealed from the F2 segregation ratio of 3:1 (susceptible: tolerant), which was confirmed by the segregation ratio of the F3 families. Of 24 pairs of resistance gene analog (RGA) primers screened, only one pair, RGA 1F-CG/RGA 1R, was found to be polymorphic among the parents. Selected F2 individuals and F3 families were genotyped with the polymorphic RGA primer pair and the polymorphism was found to be linked with YMV-reaction. This primer pair amplified a 445bp DNA fragment only from homozygous tolerant and the heterozygous lines. The 445bp marker band was sequenced and named ‘VMYR1’. The predicted amino acid sequence showed highly significant homology with the NB-ARC domain present in several gene products involved in plant disease resistance, nematode cell death and human apoptotic signaling. To the best of our knowledge, this is the first report of YMV-resistance linked DNA marker development in any crop species using segregating populations. This YMV-resistance linked marker is of potential commercial importance in resistance breeding of plants.

Fusarium oxysporum f. sp. cubense is the causal agent of Panama disease of banana. A rapid and reliable diagnosis is the foundation of integrated disease management practices in commodity crops. For this diagnostic purpose, we have developed a reliable molecular method to detect Foc race 4 isolates in Taiwan. By PCR amplification, the primer set Foc-1/Foc-2 derived from the sequence

To date, no polymerase chain reaction diagnostic technique exists to directly identify mammalian blood meals from mosquitoes by sized DNA fragments following agarose gel electrophoresis. We have developed a vertebrate-specific multiplexed primer set based on mitochondrial cytochrome b to identify the mammalian blood hosts of field-collected mosquitoes. Although designed for the study of African malaria vectors, the application of this tool is not restricted to this disease system. Validation of this diagnostic technique on dried anopheline and culicine field specimens collected in Zambia and Mali demonstrated that blood meals could be identified 2-7 months after collection. Time course experiments showed that host DNA was detectable in frozen mosquito abdomens 24-30 hours post-feeding. Additionally, multiple blood meals from different mammals could be detected in a single mosquito. This diagnostic assay will be a valuable tool for identifying the blood meals of field-collected mosqu...