The copper resistance operon copAB from Xanthomonas axonopodis pathovar citri: gene inactivation results in copper sensitivity

Xanthomonas axonopodis pv. citri (Xac) causes citrus canker and the completion of the Xac genome sequence has opened up the possibility of investigating basic cellular mechanisms at the genomic level. Copper compounds have been extensively used in agriculture to control plant diseases. The copA and copB genes, identified by annotation of the Xac genome, encode homologues of proteins involved in copper resistance. A gene expression assay by Northern blotting revealed that copA and copB are expressed as a unique transcript specifically induced by copper. Synthesis of the gene products was also induced by copper, reaching a maximum level at 4 h after addition of copper to the culture medium. CopA was a cytosolic protein and CopB was detected in the cytoplasmic membrane. The gene encoding CopA was disrupted by the insertion of a transposon, leading to mutant strains that were unable to grow in culture medium containing copper, even at the lowest CUSO4 concentration tested (0.25 mM), whereas the wild-type strain was able to grow in the presence of 1 mM copper. Cell suspensions of the wild-type and mutant strains in different copper concentrations were inoculated in lemon leaves to analyse their ability to induce citrus canker symptoms. Cells of mutant strains showed higher sensitivity than the wild-type strain in the presence of copper, i.e. they were not able to induce citrus canker symptoms at high copper concentrations and exhibited a more retarded growth in planta.

Inactivation and disposal of by-products from Mycobacterium smegmatis by photoelectrocatalytic oxidation using Ti/TiO2-Ag nanotube electrodes

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of photodynamic therapy on the development of ligature-induced periodontitis in rats

Background: the purpose of this study was to evaluate, histologically and radiographically, the effect of photodynamic therapy on the progression of experimentally induced periodontal disease in rats.Methods: Ligatures were placed at the first mandibular molar in rats. The animals were divided into four groups: group 1 (C) received no treatment; group 2 was treated topically with methylene blue (MB; 100 mu g/ml); group 3 was treated with low-level laser therapy (LLLT); and group 4 was treated topically with methylene blue followed by LLLT (4.5 J/cm(2)) (photodynamic therapy; PDT). Rats were sacrificed 5, 15, or 30 days postoperatively. Standardized radiographs were taken to measure bone loss around the mesial root surface of the first molar. Data were analyzed statistically (analysis of variance and Tukey test; P < 0.05). A scoring system was used to evaluate the connective tissue, periodontal ligament, and alveolar bone histologically. Data were analyzed statistically (Kruskal-Wallis test; P < 0.05).Results: Radiographic examination showed that there was significantly less bone loss in Group PDT compared to Group C at 5 and 15 days postoperatively. There was no significant difference in bone loss at 30 days. At 15 days, the histologic results showed significant differences in the extent of inflammatory reaction in the gingival tissue, with a greater extent of chronic inflammatory reaction in Group LLLT.Conclusion: PDT transiently reduced the periodontal tissue destruction.

A new class of neurotoxin from wasp venom slows inactivation of sodium current

The effects of alpha-pompilidotoxin (alpha-PMTX), a new neurotoxin isolated from the venom of a solitary wasp, were studied on the neuromuscular synapses in lobster walking leg and the rat trigeminal ganglion (TG) neurons. Paired intracellular recordings from the presynaptic axon terminals and the innervating lobster leg muscles revealed that alpha-PMTX induced long bursts of action potentials in the presynaptic axon, which resulted in facilitated excitatory and inhibitory synaptic transmission. The action or alpha-PMTX was distinct from that of other known facilitatory presynaptic toxins, including sea anemone toxins and alpha-scorpion toxins, which modify the fast inactivation of Na+ current. We further characterized the action of alpha-PMTX on Na+ channels by whole-cell recordings from rat trigeminal neurons. We found that alpha-PMTX stowed the Na+ channels inactivation process without changing the peak current-voltage relationship or the activation time course of tetrodotoxin (TTX)-sensitive Na+ currents, and that alpha-PMTX had voltage-dependent effects on the rate of recovery from Na+ current inactivation and deactivating tail currents. The results suggest that alpha-PMTX slows or blocks conformational changes required for fast inactivation of the Na+ channels on the extracellular surface. The simple structure of alpha-PMTX, consisting of 13 amino acids, would be advantageous for understanding the functional architecture of Na+ channel protein.

Acerola's pectin methylesterase: studies of heat inactivation

The effect of temperature on the activity of acerola's pectin methylesterase (PME) was studied to determine its heat-inactivation. The acerola's pectin methylesterase (PME; EC: 3.1.1.11) is very stable at 50 degrees C (10% loss of activity in 100 min) and needed 110 min for its inactivation at 98 degrees C. These values are much higher than the ones required for inactivation of the citrus PME, that has been reported as being equal to 1 min at 90 degrees C. Heat-inactivation of PME was shown to be nonlinear, suggesting the presence of fractions of PME with differing heat-stabilities. The times to inactive the enzyme at 98, 102 and 106 degrees C were 110, 10 and 2.17 min, respectively. The Z value (the rise in temperature necessary to observe a ten times faster heat-inactivation) was 4.71 degrees C. (C) 2000 Elsevier B.V. Ltd. All rights reserved.

Cellular and tissue effects induced by photogemA (R) and red LED in photodynamic therapy

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Inactivation of COX-2, HMLH1 and CDKN2A Gene by Promoter Methylation in Gastric Cancer: Relationship with Histological Subtype, Tumor Location and Helicobacter pylori Genotype

Objective: We aimed to evaluate the inactivation of COX-2, HMLH1 and CDKN2A by promoter methylation and its relationship with the infection by different Helicobacter pylori strains in gastric cancer. Methods: DNA extracted from 76 H. pylori-positive gastric tumor samples was available for promoter methylation identification by methylation-specific PCR and H. pylori subtyping by PCR. Immunohistochemistry was used to determine COX-2, p16(INK4A) and HMLH1 expression. Results: A strong negative correlation was found between the expression of these markers and the presence of promoter methylation in their genes. Among cardia tumors, negativity of p16(INK4A) was a significant finding. on the other hand, in noncardia tumors, the histological subtypes had different gene expression patterns. In the intestinal subtype, a significant finding was HMLH1 inactivation by methylation, while in the diffuse subtype, CDKN2A inactivation by methylation was the significant finding. Tumors with methylated COX-2 and HMLH1 genes were associated with H. pylori vac A s1 (p = 0.025 and 0.047, respectively), and the nonmethylated tumors were associated with the presence of the gene flaA. Conclusions: These data suggest that the inactivation of these genes by methylation occurs by distinct pathways according to the histological subtype and tumor location and depends on the H. pylori genotype. Copyright (C) 2011 S. Karger AG, Basel

Photodynamic therapy of oral candidiasis hi hiv-positive patients

The conventional treatments for Candidiasis include therapies that promote serious side effects to patients. Recent research indicates the use of red emission laser associated with a blue photosensitizer as a current method for microbial reduction. This study aimed to evaluate the effectiveness of Photodynamic Therapy in the treatment of oral candidiasis in HIV patients. The response to treatment by the photodynamic therapy has been demonstrated successfully in 100% of the total sample, as in the 7th and 21st days, confirmed he complete absence of clinical and cytological lesions. This therapy is enhanced by its easy applicability and no adverse side effects making it an alternative method of effective recommended treatment.

Antimicrobial photodynamic action on dentin using a light-emitting diode light source

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Heat treatment and inactivation of trypsin-chymotrypsin inhibitors and lectins from beans (Phaseolus vulgaris L.)

This work investigates some factors affecting the inactivation of common bean trypsin inhibitor and phytohemagglutin. Trypsin inhibitor activity was totally stable to heat treatment (30 min, 97C) in the total protein extract, albumin or globulin fraction. Heat treatment of the whole beans easily inactivated the inhibitor. Heat resistance of trypsin inhibitor was intermediate in the bean flour which received the same heat treatment. Independent of sample, the inhibitor was very stable to heat treatment at neutral and acidic pH and labile under strong alkaline conditions. Heating for 30 min in boiling water at pH 12 resulted in complete inactivation of the trypsin inhibitor. Autoclaving (121C) soaked whole beans and flour for 5 min inactivated 55% of the trypsin inhibitor activity in the soaked flour and 75% in the whole beans. After autoclaving 20 min, inactivation of trypsin inhibitor was about 65% in the flour and 80% in the whole beans. The phytohemagglutinin (lectin) activity was totally destroyed in the autoclaved beans after 5 min and in the flour after 15 min.

Thermal inactivation of Phytophthora nicotianae

Phytophthora nicotianae was added to pasteurized soil at the rate of 500 laboratory-produced chlamydospores per gram of soil and exposed to temperatures ranging from 35 to 53°C for 20 days. The time required to reduce soil populations to residual levels (0.2 propagule per gram of soil or less) decreased with increasing temperatures. Addition of cabbage residue to the soil reduced the time required to inactivate chlamydo spores. Temperature regimes were established to simulate daily temperature changes observed in the field, with a high temperature of 47°C for 3 h/day, and were good estimators of the efficacy of soil solarization for the control of P. nicotianae in soil. Cabbage amendment reduced the time required to inactivate chlamydospores of P. nicotianae and its effect was more pronounced at lower temperature regimes.

Inactivation of vesicular stomatitis virus through inhibition of membrane fusion by chemical modification of the viral glycoprotein

Membrane fusion is an essential step in the entry of enveloped viruses into their host cells triggered by conformational changes in viral glycoproteins. We have demonstrated previously that modification of vesicular stomatitis virus (VSV) with diethylpyrocarbonate (DEPC) abolished conformational changes on VSV glycoprotein and the fusion reaction catalyzed by the virus. In the present study, we evaluated whether treatment with DEPC was able to inactivate the virus. Infectivity and viral replication were abolished by viral treatment with 0.5 mM DEPC. Mortality profile and inflammatory response in the central nervous system indicated that G protein modification with DEPC eliminates the ability of the virus to cause disease. In addition, DEPC treatment did not alter the conformational integrity of surface proteins of inactivated VSV as demonstrated by transmission electron microscopy and competitive ELISA. Taken together, our results suggest a potential use of histidine (His) modification to the development of a new process of viral inactivation based on fusion inhibition. © 2006 Elsevier B.V. All rights reserved.

Candida albicans inactivation and cell membrane integrity damage by microwave irradiation

In indicating the microwave irradiation for disinfecting dentures it is necessary to see how this procedure influences Candida albicans integrity and viability. The aim of this study was to evaluate the ability of microwaves to inactivate C. albicans and damage cell membrane integrity. Two 200-ml C. albicans (ATCC 10231) suspensions were obtained. A sterile denture was placed in a beaker containing the Experimental (ES) or the Control suspension (CS). ES was microwaved at 650 W for 6 min. Suspensions were optically counted using methylene blue dye uptake as indicative of membrane-damaged cells; spread on Agar Sabouraud dextrose (ASD) for viability assay; or spectrophotometrically measured at 550 nm. Cell-free solutions were submitted to content analyses of protein (Bradford and Pyrogallol red methods); Ca++ (Cresolftaleine complexone method); DNA (spectrophotometer measurements at 260 nm) and K + (selective electrode technique). Data were analysed by Student's t- or Wilcoxon z-tests (α = 0.05). All ES cells demonstrated cell membrane damage. Viable cells were non-existent in the ES ASD plates. No significant difference in optical density between ES and CS was observed (P = 0.272). ES cells released significantly high protein (P < 0.001, Bradford; P = 0.005, Pyrogallol red), K+ (P < 0.001), Ca++ (P = 0.012) and DNA (P = 0.046) contents. Microwaves inactivated C. albicans and damaged cell membrane integrity. © 2007 The Authors.