Distinctively variable sequence-based nuclear DNA markers for multilocus phylogeography of the soybean- and rice-infecting fungal pathogen Rhizoctonia solani AG-1 IA

A series of multilocus sequence-based nuclear DNA markers was developed to infer the phylogeographical history of the Basidiomycetous fungal pathogen Rhizoctonia solani AG-1 IA infecting rice and soybean worldwide. The strategy was based on sequencing of cloned genomic DNA fragments (previously used as RFLP probes) and subsequent screening of fungal isolates to detect single nucleotide polymorphisms (SNPs). Ten primer pairs were designed based on these sequences, which resulted in PCR amplification of 200-320 bp size products and polymorphic sequences in all markers analyzed. By direct sequencing we identified both homokaryon and heterokaryon (i.e. dikaryon) isolates at each marker. Cloning the PCR products effectively estimated the allelic phase from heterokaryotic isolates. Information content varied among markers from 0.5 to 5.9 mutations per 100 bp. Thus, the former RFLP codominant probes were successfully converted into six distinctively variable sequence-based nuclear DNA markers. Rather than discarding low polymorphism loci, the combination of these distinctively variable anonymous nuclear markers would constitute an asset for the unbiased estimate of the phylogeographical parameters such as population sizes and divergent times, providing a more reliable species history that shaped the current population structure of R. solani AG-1 IA.     

No mutations found in exon 2 of gene p16CDKN2A during rat tongue carcinogenesis induced by 4-nitroquinoline-1-oxide

The medium-term tongue carcinogenesis assay is a useful model for studying oral squamous cell carcinomas phase by phase. The present study aimed to investigate mutations in exon 2 of gene p16CDKN2A during rat tongue carcinogenesis induced by 4-nitroquinoline 1-oxide (4NQO) using direct DNA-sequencing method. A total of 30 male Wistar rats were treated with 4-nitroquinoline 1-oxide (4NQO) in drinking water for 4, 12, and 20 weeks at 50 ppm dose. Ten animals were used as negative control. No histopathological changes in tongue epithelia were observed among controls or in the group treated for 4 weeks with 4NQO. Following 12-week treatment, hyperplasia and epithelial dysplasia were found in mild and moderate forms. At 20 weeks, the tongue presented moderate and/or severe oral dysplasia and squamous cell carcinoma, with squamous cell carcinoma in the majority of animals. No mutations were found in any experimental period evaluated that corresponded to normal oral mucosa, hyperplasia, dysplasia and squamous cell carcinomas. Taken together, our results suggest that p16CDKN2A mutations in exon 2 are not involved in the multistep tongue carcinogenesis of Wistar rats induced by 4NQO.     

Toxicity mechanisms of onion (Allium cepa) extracts and compounds in multidrug resistant erythroleukemic cell line

Onion (Allium cepa) is being studied as a potential anticancer agent, but little is known regarding its effect in multidrug resistance (MDR) cells. In this work, the cytotoxicity of crude onion extract (OE) and fractioned extract (aqueous, methanolic and ethyl acetate), as well as some onion compounds (quercetin and propyl disulfide) were evaluated in Lucena MDR human erythroleukemic and its K562 parental cell line. The capacity of OE to induce apoptosis and/or necrosis in these cells, the possible participation of oxidative stress and DNA damage were also assessed. Similar sensitivities were obtained for both tumoral cells, however only OE caused significant effects in the cells. In K562 cells, a significant increase of apoptosis was verified while the Lucena cells experienced a significant increase of necrosis. An antioxidant capacity was verified for OE discarding oxidative damage. However, OE provoked similar significant DNA damage in both cell lines. Thus, the OE capacity to overcome the MDR phenotype suggests anti-MDR action of OE.     

Serum cytokine levels as putative prognostic markers in the progression of chronic HCV hepatitis to cirrhosis

Hepatitis C virus (HCV) infection can present as an acute manifestation, and can lead to severe complications such as chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC). It represents a global health problem because there is no vaccine currently available. Cytokines play an important role in viral clearance, infection control, inflammation, regeneration and fibrosis, and also are implicated in the pathological processes occurring in the liver during viral infection. Immunological markers of chronic HCV hepatitis progression as compared to cirrhosis and HCC would be extremely useful, particularly for distinguishing between the molecules produced during HCV-induced chronic inflammation and those secreted during cirrhosis and HCC. In this work, we evaluated the serum levels of several cytokines, chemokines and growth factors in 30 patients affected by chronic HCV (HC), 30 patients affected by HCV-related cirrhosis (LC) and 20 healthy, control subjects. We used a multiplex biometric ELISA-based immunoassay in order to identify molecules that might be useful for monitoring the progression of HCV to liver cirrhosis and, possibly, to cancer. Our results show that some pro-inflammatory molecules are significantly up-regulated, and play a role as immunological markers in the intermediate steps towards liver cancer, and that hepatocyte growth factor (HGF) is a specific marker of liver cirrhosis. Finally, these data will be used to define a cytokinome profile, which might prove useful for studies involving the transition of chronic inflammation to neoplastic processes.     

Chronic treatment with bark infusion from Croton cajucara lowers plasma triglyceride levels in genetic hyperlipidemic mice

Aqueous infusion and preparations containing dehydrocrotonin (DHC) and essential oil from Croton cajucara bark were tested for plasma lipid-lowering effects in genetically modified hyperlipidemic mice. Two mouse models were tested: 1) primary hypercholesterolemia resulting from the LDL-receptor gene knockout, and 2) combined hyperlipidemia resulting from crosses of LDL-receptor knockout mice with transgenic mice overexpressing apolipo protein (apo) CIII and cholesteryl ester-transfer protein. Mice treated with bark infusion, DHC, essential oil, or placebos for 25 days showed no signals of toxicity as judged by biochemical tests for liver and kidney functions. The bark infusion reduced triglyceride plasma levels by 40%, while essential oil and DHC had no significant effects on plasma lipid levels. The bark infusion treatment promoted a redistribution of cholesterol among the lipoprotein fractions in combined hyperlipidemic mice. There was a marked reduction in the VLDL fraction and an increase in the HDL fraction, in such a way that the (VLDL + LDL)/HDL ratio was reduced by half. The bark infusion treatment did not modify cholesterol distribution in hypercholesterolemic mice. In conclusion, C. cajucara bark infusion reduced plasma triglycerides levels and promoted a redistribution of cholesterol among lipoproteins in genetically combined hyperlipidemic mice. These changes modify risk factors for the development of atherosclerotic diseases.     

Growth optimization procedures for the phytopathogen Xylella fastidiosa

For the first time, growth curves are shown for the phytopathogen Xylella fastidiosa on traditional growth media such as PW (periwinkle wilt), BCYE (buffered charcoal yeast extract), and on new ones such as GYE (glutamate yeast extract) and PYE (phosphate yeast extract) that were developed in this work. The optimal growth conditions on solid and liquid media as well as their measurements are presented, by using total protein content and turbidity determinations. The results demonstrated that yeast extract provided sufficient nutrients for X. fastidiosa, since the cells grew well on PYE medium.     

Humoral immune response in aspergillosis: an immunodominant glycoprotein of 35 kDa from Aspergillus flavus

A glycoprotein preparation containing 70% carbohydrates and 30% proteins was isolated from the mycelium of two strains of Aspergillus flavus and fractionated by ConA-Sepharose affinity chromatography. An immunodominant 35-kDa antigen was detected in a ConA-bound fraction (B fraction). It contained mannose and galactose in a 1.4:1.0 ratio. This antigen seems to be able to elicit an antibody response in patients with aspergillosis and in rabbits immunized with A. flavus whole cells. The carbohydrate units of the BF fraction appeared to be responsible for the antigenicity, since treatment with periodate removed most of the antibody binding capacity.     

Exercise restores beta-adrenergic vasorelaxation in aged rat carotid arteries

Aging is associated with alterations in beta-adrenergic receptor (beta-AR) signaling and reduction in cardiovascular responses to beta-AR stimulation. Because exercise can attenuate age-related impairment in myocardial beta-AR signaling and function, we tested whether training could also exert favorable effects on vascular beta-AR responses. We evaluated common carotid artery responsiveness in isolated vessel ring preparations from 8 aged male Wistar-Kyoto (WKY) rats trained for 6 wk in a 5 days/wk swimming protocol, 10 untrained age-matched rats, and 10 young WKY rats. Vessels were preconstricted with phenylephrine (10-6 M), and vasodilation was assessed in response to the beta-AR agonist isoproterenol (10-10-3 x 10-8 M), the alpha2-AR agonist UK-14304 (10-9-10-6 M), the muscarinic receptor agonist ACh (10-9-10-6 M), and nitroprusside (10-8-10-5 M). beta-AR density and cytoplasmic beta-AR kinase (beta-ARK) activity were tested on pooled carotid arteries. beta-ARK expression was assessed in two endothelial cell lines from bovine aorta and aorta isolated from a 12-wk WKY rat. beta-AR, alpha2-AR, and muscarinic responses, but not that to nitroprusside, were depressed in untrained aged vs. young animals. Exercise training restored beta-AR and muscarinic responses but did not affect vasodilation induced by UK-14304 and nitroprusside. Aged carotid arteries showed reduced beta-AR number and increased beta-ARK activity. Training counterbalanced these phenomena and restored beta-AR density and beta-ARK activity to levels observed in young rat carotids. Our data indicate that age impairs beta-AR vasorelaxation in rat carotid arteries through beta-AR downregulation and desensitization. Exercise restores this response and reverts age-related modification in beta-ARs and beta-ARK. Our data support an important role for beta-ARK in vascular beta-AR vasorelaxation.     

Antioxidant enzymes and thiol/disulfide status in the digestive gland of the brown mussel Perna perna exposed to lead and paraquat

Lead (Pb) and paraquat (PQ) have different toxic mechanisms associated with cell damage. Pb may induce alterations in zinc containing proteins, including the known inhibitory effect on the enzyme delta-aminolevulinic acid dehydratase, disrupting the heme-synthesis pathway. During PQ biotransformation, redox cycle reactions enhance oxyradical production, which may lead to pro-oxidative conditions. In this study, we analyzed the effects of Pb and PQ on antioxidant enzymes and thiol status, using the digestive glands of the mussel Perna perna collected in a mussel farm on Santa Catarina Island. Mussels were exposed to Pb (1 ppm) and PQ (10 ppm), either separately or concomitantly, for 48 h. We were unable to detect an effect of Pb treatment on the enzymes, catalase, glucose 6-phosphate dehydrogenase (G6PDH), glutathione S-transferase (GST) and glutathione reductase (GSSG-reductase), which contrasts to the effect of PQ, increasing GSSG-reductase and G6PDH, but decreasing GST activity. The thiol status showed a pro-oxidative trend, observed mainly through a decrease in the reduced/oxidized glutathione ratio, despite the total-glutathione increase. Protein-mixed disulfides and protein thiols did not change by the treatments. The observed effects of PQ and Pb were consistent with literature. Pb had a suppressive effect on the enzymatic changes elicited by PQ, while the changes in the thiol/disulfide parameters were retained.