Inactivation of formyltransferase (wbkC) gene generates a Brucella abortus rough strain that is attenuated in macrophages and in mice

Rough mutants of Brucella abortus were generated by disruption of wbkC gene which encodes the formyltransferase enzyme involved in LPS biosynthesis. In bone marrow-derived macrophages the B. abortus Delta wbkC mutants were attenuated, could not reach a replicative niche and induced higher levels of IL-12 and TNF-alpha when compared to parental smooth strains. Additionally, mutants exhibited attenuation in vivo in C57BL/6 and interferon regulatory factor-1 knockout mice. Delta wbkC mutant strains induced lower protective immunity in C56BL/6 than smooth vaccine S19 but similar to rough vaccine RB51. Finally, we demonstrated that Brucella wbkC is critical for LPS biosynthesis and full bacterial virulence. (C) 2010 Elsevier Ltd. All rights reserved.

Basic oxygen furnace slag as a treatment material for pathogens: Contribution of inactivation and attachment in virus attenuation

Basic oxygen furnace (BOF) slag media were studied as a potential treatment material in on-site sanitation systems. Batch and column studies were conducted to evaluate attenuation of the bacteriophage PR772 and 0.190 mu m diameter microspheres by BOF media, and to delineate the relative contributions of two principle processes of virus attenuation: inactivation and attachment. In the batch studies, conducted at 4 degrees C, substantial inactivation of PR772 did not occur in the pH 7.6 and 9.5 suspensions. At pH 11.4, bimodal inactivation of PR772 was observed, at an initial rate of 2.1 log C/C(0) day(-1) for the first two days, followed by a much slower rate of 0.124 log C/C(0) day(-1) over the following 10 days. Two column studies were conducted at 4 degrees C at a flow rate of 1 pore volume day(-1) using two slag sources (Stelco, Ontario; Tubarao, Brazil) combined with sand and pea gravel. In both column experiments, the effluent microsphere concentration approached input concentrations over time (reductions of 0.1-0.2 log C/C(0)), suggesting attachment processes for microspheres were negligible. Removal of PR772 virus was more pronounced both during the early stages of the experiments, but also after longer transport times (0.5-1.0 log C/C(0)). PR772 reduction appeared to be primarily as a result of virus inactivation in response to the elevated pH conditions generated by the BOF mixture (10.6-11.4). On-site sanitation systems using BOF media should be designed to maintain sufficient contact time between the BOF media and the wastewater to allow sufficient residence time of pathogens at elevated pH conditions. (C) 2009 Published by Elsevier Ltd.

INSECT CHYMOTRYPSINS: CHLOROMETHYL KETONE INACTIVATION AND SUBSTRATE SPECIFICITY RELATIVE TO POSSIBLE COEVOLUTIONAL ADAPTATION OF INSECTS AND PLANTS

Insect digestive chymotrypsins are present in a large variety of insect orders but their substrate specificity still remains unclear. Ewer insect chymotrypsins from 3 different insect orders (Dictyoptera, Coleoptera and two Lepidoptera) were isolated using affinity chromatography. Enzymes presented molecular masses in the range of 20 to 31 kDa and pH optima in the range of 7.5 to 10.0. Kinetic characterization. using different, colorimetric and fluorescent substrates indicated that insect chymotrypsins differ from, bovine chymotrypsin in their primary specificity toward small substrates (like N-benzoyl-L-Tyr p-nitroanilide) rather than on their preference for large substrates (exemplified by Succynil-Ala-Ala-Pro-Phe P-nitroanilide). Chloromethyl ketones (TPCK, N-alpha-tosyl-L-Phe chloromethyl ketone and Z-GGF-CK, N-carbobenzoxy-Gly-Gly-phe-CK) inactivated all chymotrypsins legated. Inactivation rates follow apparent first-order kinetics with variable second order rates (TPCK, 42 to 130 M(-1)s(-1); Z-GGF-CK, 150 to 450 M(-1)s(-1) that may be remarkably low for S. frugiperda chymotrypsin (TPCK, 6 M(-1)s(-1); Z-GGF-CK, 6.1 M(-1) s(-1)). Homology modelling and sequence alignment showed that. in lepidopteran chymotrypsins, differences in the amino acid residues in the neighborhood of the catalytic His 57 may affect its pKa, value. This is Proposed as the cause of the decrease in His 57 reactivity toward chloromethyl ketones. Such amino acid replacement in the active site is proposed. to be an adaptation to the presence of dietary ketones. (C) 2009 Wiley Periodicals, Inc.

Inactivation kinetics of polyphenol oxidase and peroxidase in green coconut water by microwave processing

The inactivation kinetics of enzymes polyphenol oxidase (PPO) and peroxidase (POD) was studied for the batch (discontinuous) microwave treatment of green coconut water. Inactivation of commercial PPO and POD added to sterile coconut water was also investigated. The complete time-temperature profiles of the experimental runs were used for determination of the kinetic parameters D-value and z-value: PPO (D(92.20 degrees C) = 52 s and z = 17.6 degrees C); POD (D(92.92 degrees C) = 16 s and z = 11.5 degrees C); PPO/sterile coconut water: (D(84.45 degrees C) = 43 s and z = 39.5 degrees C) and POD/sterile coconut water: (D(86.54 degrees C) = 20 s and z = 19.3 degrees C). All data were well fitted by a first order kinetic model. The enzymes naturally present in coconut water showed a higher resistance when compared to those added to the sterilized medium or other simulated solutions reported in the literature. The thermal inactivation of PPO and POD during microwave processing of green coconut water was significantly faster in comparison with conventional processes reported in the literature. (C) 2008 Elsevier Ltd. All rights reserved.

Expression of SMAD proteins, TGF-beta/activin signaling mediators, in human thyroid tissues

OBJECTIVE: To investigate the expression of SMAD proteins in human thyroid tissues since the inactivation of TGF-β/activin signaling components is reported in several types of cancer. Phosphorylated SMAD 2 and SMAD3 (pSMAD2/3) associated with the SMAD4 induce the signal transduction generated by TGF-β and activin, while SMAD7 inhibits this intracellular signaling. Although TGF-β and activin exert antiproliferative roles in thyroid follicular cells, thyroid tumors express high levels of these proteins. MATERIALS AND METHODS: The protein expression of SMADs was evaluated in multinodular goiter, follicular adenoma, papillary and follicular carcinomas by immunohistochemistry. RESULTS: The expression of pSMAD2/3, SMAD4 and SMAD7 was observed in both benign and malignant thyroid tumors. Although pSMAD2/3, SMAD4 and SMAD7 exhibited high cytoplasmic staining in carcinomas, the nuclear staining of pSMAD2/3 was not different between benign and malignant lesions. CONCLUSIONS: The finding of SMADs expression in thyroid cells and the presence of pSMAD2/3 and SMAD4 proteins in the nucleus of tumor cells indicates propagation of TGF-β/activin signaling. However, the high expression of the inhibitory SMAD7, mostly in malignant tumors, could contribute to the attenuation of the SMADs antiproliferative signaling in thyroid carcinomas.

Dosimetria esporular: Bacillus subtilis TKJ6312 como biossensor de radiação solar biologicamente ativa

Since 2000, spore dosimetry and spectral photometry have been performed in parallel at the Southern Space Observatory, São Martinho da Serra (Southern Brazil). A comparative study involving data from Punta Arenas - Chile (53.2º S), São Martinho da Serra (29.5º S), Padang - Indonesia (0.9ºS), Brussels - Belgium (50.9º N) and Kiyotake - Japan (31.9º N) from 2000 to 2006 is presented. The Spore Inactivation Doses presented the higher values in summer (973 ± 73 for Punta Arenas and 4,369 ± 202 for São Martinho da Serra, as well 1,402 ± 170 and 3,400 ± 1,674 for Brussels and Kiyotake, respectively). The simplicity, robustness and high resistance of bacterial spores makes the biosensor an potential biological tool for UV-B monitoring.

Polymorphisms and DNA methylation of gene TP53 associated with extra-axial brain tumors

The p53 tumor suppressor gene is the most frequently mutated gene in human cancer; this gene is mutated in up to 50% of human tumors. It has a critical role in the cell cycle, apoptosis and cell senescence, and it participates in many crucial physiological and pathological processes. Polymorphisms of p53 have been suggested to be associated with genetically determined susceptibility in various types of cancer. Another process involved with the development and progression of tumors is DNA hypermethylation. Aberrant methylation of the promoter is an alternative epigenetic change in genetic mechanisms, leading to tumor suppressor gene inactivation. In the present study, we examined the TP53 Arg72Pro and Pro47Ser polymorphisms using PCR-RFLP and the pattern of methylation of the p53 gene by methylation-specific PCR in 90 extra-axial brain tumor samples. Patients who had the allele Pro of the TP53 Arg72Pro polymorphism had an increased risk of tumor development ( odds ratio, OR = 3.23; confidence interval at 95%, 95% CI = 1.71-6.08; P = 0.003), as did the allele Ser of TP53 Pro47Ser polymorphism (OR = 1.28; 95% CI = 0.03-2.10; P = 0.01). Comparison of overall survival of patients did not show significant differences. In the analysis of DNA methylation, we observed that 37.5% of meningiomas, 30% of schwannomas and 52.6% of metastases were hypermethylated, suggesting that methylation is important for tumor progression. We suggest that TP53 Pro47Ser and Arg72Pro polymorphisms and DNA hypermethylation are involved in susceptibility for developing extra-axial brain tumors.

Photodynamic Therapy Can Be Effective as a Treatment for Herpes Simplex Labialis

Background Data and Objective: Herpes is a common infectious disease that is caused by human herpesviruses. Several treatments have been proposed, but none of them prevent reactivation of the virus. This article describes the use of photodynamic therapy (PDT) as a treatment for herpes lesions, and reports on four cases. Materials and Methods: PDT was used as an adjuvant therapy for the treatment of herpes labialis in four patients. A special type of 0.01% (m/V) of methylene blue solution was applied to the vesicular stage of herpesviral disease and the lesions were irradiated with laser energy (wavelength 660 nm, energy density 120 J/cm(2), output power of 40 mW, 2 min per point, 4.8 J of energy/point, at four points). After 24 h the patients returned and phototherapy was repeated with the same equipment, this time with 3.8 J/cm(2) and 15 mW, for a total dose of 0.6 J. The same procedure was repeated 72 h and 1 wk later. Results: Treatment with low-level laser therapy can be considered as an option in the treatment of herpes labialis, and decreases the frequency of vesicle recurrence and provides comfort for patients. No significant acute side effects were noted and the lesions healed rapidly. Conclusion: Treatment of herpes labialis with PDT was effective, had no side effects, and when associated with laser phototherapy, accelerated the healing process.

High-Intensity Laser and Photodynamic Therapy as a Treatment for Recurrent Herpes Labialis

Objective: The aim of this study was to report the treatment of recurrent herpes labialis (RHL) using a high-intensity laser or methylene blue (MB)-mediated photodynamic therapy (PDT) in combination with low-level laser therapy (LLLT). Materials and Methods: Four clinical cases of patients diagnosed with RHL are described in this report. Two patients were subjected to high-intensity laser therapy (HILT) followed by LLLT, and two patients received MB-mediated PDT, again followed by LLLT. LLLT was conducted at 24, 48, 72 h, and 7 d after HILT or PDT. Patients were followed up after 6 mo. Results: Throughout the follow-up period, all patients reported pain relief and did not show any signs or symptoms of RHL. A favorable healing process was observed in all cases. None of the patients reported pain as a consequence of the treatment. Conclusion: These results suggest that HILT and MB-mediated PDT, in combination with LLLT, may constitute a benefit when treating vesicles in RHL.

Gene silencing during development of in vitro-produced female bovine embryos

In early development, female embryos (XX) produce twice the transcripts of X-linked genes compared with male embryos (XY). During the course of development, inactivation of the X chromosome equilibrates gene dosage, making the development of female embryos viable. Moreover, the biotechnologies used for producing embryos in vitro seem to work better with male embryos, making it easier for them to reach the blastocyst stage and allow for complete gestation. We investigated the expression of three X-linked genes that are involved in development, XIST, G6PD, and HPRT, and of the transcript interferon-tau, in male and female bovine blastocysts produced by nuclear transfer (NT) and by in vitro fertilization (IVF). Oocytes that had been matured in vitro were enucleated and reconstructed with somatic cells from adult animals at 18 h post-maturation. After fusion (two pulses of 2.25 kv/cm) and chemical activation (5.0 mu M ionomycin for 5 min and 2.0 mM 6-DMAP for 3 h), the oocytesomatic cell units were cultivated in CR2 with a monolayer of granulosa cells at 38.8 degrees C, in a humidified 5% CO(2) atmosphere. IVF embryos were inseminated, after centrifugation in a Percoll gradient, with 2 x 10(6) sperm/mL TALP medium supplemented with BSA and PHE and cultivated under the same conditions as the cloned embryos. We used real-time PCR to analyze the gene expression of individual blastocysts compared to expression of the housekeeping gene, GAPDH. The gene XIST was expressed in female embryos and not in male embryos produced by IVF, though it was expressed at low levels in male embryos produced by NT. Unlike previous reports, we found lower levels of the transcript of G6PD in females than in males, suggesting double silencing or other mechanisms of control of this gene. Female embryos produced by IVF expressed the HPRT gene at a higher level than female embryos produced by NT, suggesting that gene silencing proceeds faster in NT-produced female embryos due to ""inactivation memory"" from the nucleus donor. In conclusion, male and female embryos express different levels of X-chromosome genes and failures of these genes that are essential for development could reduce the viability of females. Nuclear transfer can modify this relation, possibly due to epigenetic memory, leading to frequent failures in nuclear reprogramming.

Antimicrobial mechanisms behind photodynamic effect in the presence of hydrogen peroxide

This study describes the use of methylene blue (MB) plus light (photodynamic inactivation, PDI) in the presence of hydrogen peroxide (H(2)O(2)) to kill Staphylococcus aureus, Escherichia coli, and Candida albicans. When H(2)O(2) was added to MB plus light there was an increased antimicrobial effect, which could be due to a change in the type of ROS generated or increased microbial uptake of MB. To clarify the mechanism, the production of ROS was investigated in the presence and absence of H(2)O(2). It was observed that ROS production was almost inhibited by the presence of H(2)O(2) when cells were not present. In addition, experiments using different sequence combinations of MB and H(2)O(2) were performed and MB optical properties inside the cell were analyzed. Spectroscopy experiments suggested that the amount of MB was higher inside the cells when H(2)O(2) was used before or simultaneously with PDI, and ROS formation inside C. albicans cells confirmed that ROS production is higher in the presence of H(2)O(2). Moreover enzymatic reduction of MB by E. coli during photosensitizer uptake to the photochemically inactive leucoMB could be reversed by the oxidative effects of hydrogen peroxide, increasing ROS formation inside the microorganism. Therefore, the combination of a photosensitizer such as MB and H(2)O(2) is an interesting approach to improve PDI efficiency.

Interesterification of milkfat with soybean oil catalysed by Rhizopus oryzae lipase immobilised on SiO(2)-PVA on packed bed reactor

The potential of the lipase from Rhizopus oryzae immobilised on SiO(2)-PVA to catalyse the interesterification of the milkfat with soybean oil in a packed bed reactor running on continuous mode was evaluated. The reactor operated continuously for 35 days at 45 degrees C, and during 12 days, no significant decrease in the initial lipase activity was verified. Interesterification yields were in the range from 35 to 38% wt, which gave an interesterified product having 59% lower consistency in relation to non-interesterified blend. Results showed the potential of the lipase from Rhizopus oryzae to mediate the interesterification of milkfat with soybean oil in packed bed reactor, attaining a more spreadable product under a cool temperature. The biocatalyst operational stability was assessed and an inactivation profile was found to follow the Arrhenius model, revealing values of 34 days and 0.034 day(-1), for half-life and a deactivation coefficient, respectively.

Extraction of manganese peroxidase produced by Lentinula edodes

Lentinula edodes, commonly called shiitake, is considered a choice edible mushroom with exotic taste and medicinal quality. L. edodes grows very well and produces a range of enzymes when cultivated on eucalyptus residues. Development of appropriate experimental procedures for recovery and determination of enzymes became a widely important cash crop. In this work, enzymes produced by L. edodes were extracted using different pH buffer and determined regarding peroxidases and proteases. Lignin peroxidase (LiP) was not detected in the extracts based on veratryl alcohol or azure B oxidation. Proteases were very low while Mn-peroxidases (MnP) predominated. The optimal pH for MnP recovery was 5.0, under agitation at 25 degrees C. The oxidation of phenol red decreased after dark-colored small compounds or ions were eliminated by dialysis. The extract of L. edodes contained components of high molecular weight, such as proteases or high polyphenol, that could be involved in the LiP inactivation. L. edodes sample previously submitted to dialysis was also joined to UP of Phanerochaete chrysosporium and a total inhibition of UP was observed. (C) 2007 Elsevier Ltd. All rights reserved.

Extending the kinetic solution of the classic Michaelis-Menten model of enzyme action

The principal aim of studies of enzyme-mediated reactions has been to provide comparative and quantitative information on enzyme-catalyzed reactions under distinct conditions. The classic Michaelis-Menten model (Biochem Zeit 49:333, 1913) for enzyme kinetic has been widely used to determine important parameters involved in enzyme catalysis, particularly the Michaelis-Menten constant (K (M) ) and the maximum velocity of reaction (V (max) ). Subsequently, a detailed treatment of the mechanisms of enzyme catalysis was undertaken by Briggs-Haldane (Biochem J 19:338, 1925). These authors proposed the steady-state treatment, since its applicability was constrained to this condition. The present work describes an extending solution of the Michaelis-Menten model without the need for such a steady-state restriction. We provide the first analysis of all of the individual reaction constants calculated analytically. Using this approach, it is possible to accurately predict the results under new experimental conditions and to characterize and optimize industrial processes in the fields of chemical and food engineering, pharmaceuticals and biotechnology.

Packed-Bed Reactor Running on Babassu Oil and Glycerol to Produce Monoglycerides by Enzymatic Route using Immobilized Burkholderia cepacia Lipase

The aim of this study was the glycerolysis of babassu oil catalyzed by immobilized lipase from Burkholderia cepacia, in a continuous packed-bed reactor. The best reaction conditions were previously established in batchwise via response surface methodology as a function of glycerol-to-oil molar ratio and reaction temperature. The reactor operated continuously for 22 days at 50 A degrees C, and during the first 6 days, no significant decrease on the initial lipase activity was observed. Monoglycerides concentration was in the range from 25 to 33 wt.%. Subsequently, a progressive decrease in the activity was detected, and an inactivation profile described by Arrhenius model estimated values of 50 days and 1.37 x 10(-2) h(-1), for the half-life and deactivation coefficient, respectively.