Structural-functional analyses of textile dye degrading azoreductase, laccase and peroxidase: a comparative in silico study

Background: Textile industry not only plays a vital role in our daily life but also a prominent factor in improving global economy. One of the environmental concern is it releases huge quantities of toxic dyes in the water leading to severe environmental pollution. Bacterial laccase and azoreductase successfully oxidize complex chemical structure of nitrogen group-containing azo dyes. Additionally, the presence of textile dye infuriates bacterial peroxidase to act as a dye degrading enzyme. Our present study deals with three textile dye degrading enzymes laccase, azoreductase, and peroxidase through analyzing their structural and functional properties using standard computational tools. Result: According to the comparative analysis of physicochemical characteristics, it was clear that laccase was mostly made up of basic amino acids whereas azoreductase and peroxidase both comprised of acidic amino acids. Higher aliphatic index ascertained the thermostability of all these three enzymes. Negative GRAVY value of the enzymes confirmed better water interaction of the enzymes. Instability index depicted that compared to laccase and preoxidase, azoreductase was more stable in nature. It was also observed that the three model proteins had more than 90% of total amino acids in the favored region of Ramachandran plot. Functional analysis revealed laccase as multicopper oxidase type enzyme and azoreductase as FMN dependent enzyme, while peroxidase consisted of α-ß barrel with additional haem group. Conclusion: Present study aims to provide knowledge on industrial dye degrading enzymes, choosing the suitable enzyme for industrial set up and to help in understanding the experimental laboratory requirements as well.

Lactate-upregulation of lactate oxidation complex-related genes is blunted in left ventricle of myocardial infarcted rats

Lactate modulates the expression of lactate oxidation complex (LOC)-related genes and cardiac blood flow under physiological conditions, but its modulatory role remains to be elucidated regarding pathological cardiac stress. The present study evaluated the effect of lactate on LOC-related genes expression and hemodynamics of hearts submitted to myocardial infarction (MI). Four weeks after MI or sham operation, isolated hearts of male Wistar rats were perfused for 60 min with Na+-lactate (20 mM). As expected, MI reduced cardiac contractility and relaxation with no changes in perfusion. The impaired cardiac hemodynamics were associated with increased reactive oxygen species (ROS) levels (Sham: 19.3±0.5 vs MI: 23.8±0.3 µM), NADPH oxidase (NOX) activity (Sham: 42.2±1.3 vs MI: 60.5±1.5 nmol·h−1·mg−1) and monocarboxylate transporter 1 (mct1) mRNA levels (Sham: 1.0±0.06 vs MI: 1.7±0.2 a.u.), but no changes in superoxide dismutase (SOD), catalase, NADH oxidase (NADox), and xanthine oxidase activities. Lactate perfusion in MI hearts had no additional effect on ROS levels, NADox, and NOX activity, however, it partially reduced mct1 mRNA expression (MI-Lactate 1.3±0.08 a.u.). Interestingly, lactate significantly decreased SOD (MI-Lactate: 54.5±4.2 µmol·mg−1·min−1) and catalase (MI: 1.1±0.1 nmol·mg−1·min−1) activities in MI. Collectively, our data suggest that under pathological stress, lactate lacks its ability to modulate the expression of cardiac LOC-related genes and the perfused pressure in hearts submitted to chronic MI. Together, these data contribute to elucidate the mechanisms involved in the pathogenesis of heart failure induced by MI.

Keratinolytic abilities of Micrococcus luteus from poultry waste

Keratinolytic microorganisms have become the subject of scientific interest due to their ability to biosynthesize specific keratinases and their prospective application in keratinic waste management. Among several bacterial classes, actinobacteria remain one of the most important sources of keratin-degrading strains, however members of the Micrococcaceae family are rarely scrutinized in regard to their applicatory keratinolytic potential. The tested Micrococcus sp. B1pz isolate from poultry feather waste was identified as M. luteus. The strain, grown in the medium with 1–2% chicken feathers and a yeast extract supplement, produced keratinases of 32 KU and lower level of proteases, 6 PU. It was capable to effectively decompose feathers or “soft” keratin of stratum corneum, in contrast to other “hard” hair-type keratins. The produced keratinolytic enzymes were mainly a combination of alkaline serine or thiol proteases, active at the optimum pH 9.4, 55 °C. Four main protease fractions of 62, 185, 139 and 229 kDa were identified in the crude culture fluid. The research on the auxiliary role of reducing factors revealed that reducing sulfur compounds could be applied in keratinolysis enhancement during enzymatic digestion of keratin, rather than in culture conditions. The presented M. luteus isolate exhibits a significant keratinolytic potential, which determines its feasible applicatory capacity towards biodegradation of poultry by-products or formulation of keratin-based feed components.

Redução da água corporal de renais crônicos em diálise peritoneal se associa com melhor controle da hipertensão arterial / Total body water reduction in subjects with chronic kidney disease on peritoneal dialysis is associated with a better hypertension control

Introdução: A hipertensão arterial tem alta prevalência em renais crônicos, sendo a hipervolemia um de seus fatores causais. Objetivo: Avaliar a influência da redução da volemia no controle pressórico e em parâmetros ecocardiográficos de pacientes renais crônicos em diálise peritoneal contínua. Métodos: Doze renais crônicos sem sinais clínicos de hipervolemia foram submetidos à intensificação da diálise com o objetivo de reduzir o peso corporal em 5%. A volemia foi avaliada pela bioimpedância elétrica e pela ultrassonografia de veia cava inferior (VCI). Os voluntários foram submetidos à monitorização ambulatorial da pressão arterial e a exame ecocardiográfico no período basal e após 5 semanas de intervenção. Resultados: Após a intensificação da ultrafiltração, houve redução significativa do peso corporal, da água extracelular e do diâmetro inspiratório da VCI, enquanto o índice de colapsamento da VCI não alterou de modo significativo. A despeito da redução do número de anti-hipertensivos, a pressão sistólica do período de sono reduziu de 138,4 ± 18,6 para 126,7 ± 18,0 mmHg, o descenso pressórico do sono aumentou e o diâmetro sistólico final do ventrículo esquerdo reduziu significantemente. Conclusão: A redução da volemia de pacientes em diálise peritoneal, clinicamente euvolêmicos, se associou a melhor controle pressórico e à diminuição do diâmetro sistólico final do ventrículo esquerdo. .

Introduction: Hypertension is highly prevalent in patients with chronic kidney disease and hypervolemia is one of the principal causes. Objective: To evaluate the influence of the reduction of volemia on blood pressure as well as on echocardiographic parameters in patients on continuous ambulatory peritoneal dialysis. Methods: Twelve patients with no clinical evidence of hypervolemia were submitted to an increase in the rate of the dialysis with the purpose of reducing body weight by 5%. The volemia was evaluated by electrical bioimpedance and by ultrasound of the inferior cava vena (ICV). Blood pressure was measured by ambulatory blood pressure monitoring and cardiac function was evaluated by echocardiography both at baseline and 5 weeks after the intervention period. Results: After the increase in the ultrafiltration, body weight, extracellular water and the inspiratory diameter of the ICV decreased significantly in parallel with a non-significant increase in the collapsing ICV index. Despite the reduction of anti-hypertensive drugs, systolic blood pressure during the sleep period decreased from 138.4 ± 18.6 to 126.7 ± 18.0 mmHg, the nocturnal blood pressure drop increased and the final systolic left ventricular diameter decreased significantly. Conclusion: Reduction of the volemia of patients on peritoneal dialysis, with no signs of hypervolemia, was associated with a better blood pressure control and with a decrease of the final systolic left ventricular diameter. .

The Relationship between Leaf Rolling and Ascorbate-Glutathione Cycle Enzymes in Apoplastic and Symplastic Areas of Ctenanthe setosa Subjected to Drought Stress

The ascorbate-glutathione (ASC-GSH) cycle has an important role in defensive processes against oxidative damage generated by drought stress. In this study, the changes that take place in apoplastic and symplastic ASC-GSH cycle enzymes of the leaf and petiole were investigated under drought stress causing leaf rolling in Ctenanthe setosa (Rose.) Eichler (Marantaceae). Apoplastic and symplastic extractions of leaf and petiole were performed at different visual leaf rolling scores from 1 to 4 (1 is unrolled, 4 is tightly rolled and the others are intermediate forms). Glutathione reductase (GR), a key enzyme in the GSH regeneration cycle, and ascorbate (ASC) were present in apoplastic spaces of the leaf and petiole, whereas dehydroascorbate reductase (DHAR), which uses glutathione as reductant, monodehydroascorbate reductase (MDHAR), which uses NAD(P)H as reductant, and glutathione were absent. GR, DHAR and MDHAR activities increased in the symplastic and apoplastic areas of the leaf. Apoplastic and symplastic ASC and dehydroascorbate (DHA), the oxidized form of ascorbate, rose at all scores except score 4 of symplastic ASC in the leaf. On the other hand, while reduced glutathione (GSH) content was enhanced, oxidized glutathione (GSSG) content decreased in the leaf during rolling. As for the petiole, GR activity increased in the apoplastic area but decreased in the symplastic area. DHAR and MDHAR activities increased throughout all scores, but decreased to the score 1 level at score 4. The ASC content of the apoplast increased during leaf rolling. Conversely, symplastic ASC content increased at score 2, however decreased at the later scores. While the apoplastic DHA content declined, symplastic DHA rose at score 2, but later was down to the level of score 1. While GSH content enhanced during leaf rolling, GSSG content did not change except at score 2. As well, there were good correlations between leaf rolling and ASC-GSH cycle enzyme activities ...

A potent trypanocidal component from the fungus Lentinus strigosus inhibits trypanothione reductase and modulates PBMC proliferation

The fungus Lentinus strigosus (Pegler 1983) (Polyporaceae, basidiomycete) was selected in a screen for inhibitory activity on Trypanosoma cruzi trypanothione reductase (TR). The crude extract of L. strigosus was able to completely inhibit TR at 20 µg/ml. Two triquinane sesquiterpenoids (dihydrohypnophilin and hypnophilin), in addition to two panepoxydol derivatives (neopanepoxydol and panepoxydone), were isolated using a bioassay-guided fractionation protocol. Hypnophilin and panepoxydone displayed IC50 values of 0.8 and 38.9 µM in the TR assay, respectively, while the other two compounds were inactive. The activity of hypnophilin was confirmed in a secondary assay with the intracellular amastigote forms of T. cruzi, in which it presented an IC50 value of 2.5 µ M. Quantitative flow cytometry experiments demonstrated that hypnophilin at 4 µM also reduced the proliferation of human peripheral blood monocluear cells (PBMC) stimulated with phytohemaglutinin, without any apparent interference on the viability of lymphocytes and monocytes. As the host immune response plays a pivotal role in the adverse events triggered by antigen release during treatment with trypanocidal drugs, the ability of hypnophilin to kill the intracellular forms of T. cruzi while modulating human PBMC proliferation suggests that this terpenoid may be a promising prototype for the development of new chemotherapeutical agents for Chagas disease.

Glutathione and the redox control system trypanothione/trypanothione reductase are involved in the protection of Leishmania spp. against nitrosothiol-induced cytotoxicity

Glutathione is the major intracellular antioxidant thiol protecting mammalian cells against oxidative stress induced by oxygen- and nitrogen-derived reactive species. In trypanosomes and leishmanias, trypanothione plays a central role in parasite protection against mammalian host defence systems by recycling trypanothione disulphide by the enzyme trypanothione reductase. Although Kinetoplastida parasites lack glutathione reductase, they maintain significant levels of glutathione. The aim of this study was to use Leishmania donovani trypanothione reductase gene mutant clones and different Leishmania species to examine the role of these two individual thiol systems in the protection mechanism against S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a nitrogen-derived reactive species donor. We found that the resistance to SNAP of different species of Leishmania was inversely correlated with their glutathione concentration but not with their total low-molecular weight thiol content (about 0.18 nmol/10(7) parasites, regardless Leishmania species). The glutathione concentration in L. amazonensis, L. donovani, L. major, and L. braziliensis were 0.12, 0.10, 0.08, and 0.04 nmol/10(7) parasites, respectively. L. amazonensis, that have a higher level of glutathione, were less susceptible to SNAP (30 and 100 µM). The IC50 values of SNAP determined to L. amazonensis, L. donovani, L. major, and L. braziliensis were 207.8, 188.5, 160.9, and 83 µM, respectively. We also observed that L. donovani mutants carrying only one trypanothione reductase allele had a decreased capacity to survive (40 percent) in the presence of SNAP (30-150 µM). In conclusion, the present data suggest that both antioxidant systems, glutathione and trypanothione/trypanothione reductase, participate in protection of Leishmania against the toxic effect of nitrogen-derived reactive species.

Arylfurans as potential Trypanosoma cruzi trypanothione reductase inhibitors

The natural lignans veraguensin and grandisin have been reported to be active against Trypanosoma cruzi bloodstream forms. Aiming at the total synthesis of these and related compounds, we prepared three 2-arylfurans and eight 2,5-diarylfurans. They were evaluated for their potential as T. cruzi trypanothione reductase (TR) inhibitors as well against the parasite's intracellular (amastigote) and bloodstream (trypomastigote) forms. Compound 12 was the most effective against TR with an IC50 of 48.5 æM while 7 and 14 were active against amastigotes, inhibiting the parasite development by 60 percent at 20 æg/ml (59 and 90 æM, respectively). On the other hand, none of the compounds was significantly active against the parasite bloodstream forms even at 250 æg/ml (0.6-1.5 mM).

Oxidative stress in Lactococcus lactis

Lactococcus lactis, the most extensively characterized lactic acid bacterium, is a mesophilic- and microaerophilic-fermenting microorganism widely used for the production of fermented food products. During industrial processes, L. lactis is often exposed to multiple environmental stresses (low and high temperature, low pH, high osmotic pressure, nutrient starvation and oxidation) that can cause loss or reduction of bacterial viability, reproducibility, as well as organoleptic and/or fermentative qualities. Among these stress factors, oxidation can be considered one of the most deleterious to the cell, causing cellular damage at both molecular and metabolic levels. During the last two decades, considerable efforts have been made to improve our knowledge of oxidative stress in L. lactis. Many genes involved with both oxidative stress resistance and control mechanisms have been identified; functionally they seem to overlap. The finding of new genes, and a better understanding of the molecular mechanisms of stress resistance in L. lactis and other lactic acid bacterium, will lead to the construction and isolation of stress-resistant strains. Such strains could be exploited for both traditional and probiotic uses

Inactivación de la tripanotiona reductasa de Trypanosoma cruzi por radicales libres catiónicos de fenotiazinas / Inactivation of Trypanosoma cruzi trypanothione reductase by phenothiazine cationic free radicals

Peroxidase/H2O2/phenothiazine systems produced irreversible inhibition (inactivation) of Trypanosoma cruzi trypanothione reductase (TR). The enzyme inactivation depended on (a) the incubation time of TR with the peroxidase/H2O2/phenothiazine system; (b) the peroxidase nature and (c) the phenothiazine structure. With the more effective peroxidase/H2O2/phenothiazine systems, TR inactivation kinetics presented a relatively fast initial phase, lasting for about 10 min, in which most of the enzyme activity disappeared. This phase was followed by a slower one and, after 30 min incubation, TR was totally inactivated. Three peroxidases were assayed as catalysts of TR inactivation: the horseradish peroxidase (HRP), leukocyte myeloperoxidase (MPO) and modified myoglobin (Mb). Under comparable experimental conditions, the peroxidase system activity decreased in the given order. With HRP systems, 10 microM Thioridazine (TRDZ), Promazine (PZ), Trimeprazine (TMPZ), Prochlorperazine (PCZ), Propionylpromazine (PPZ), Chlorpromazine (CPZ) and Perphenazine (PFZ), produced 95-100 inactivation of TR. With the MPO/H2O2 systems, PZ. TRDZ and TMPZ were the most effective. Under similar experimental condition, the Mb/H2O2/PZ,/TMPZ, /TRDZ and CPZ systems effectively inactivated TR. The presence of alkylamino, piperazinyl, or piperidinyl groups in PTZ N atom (position 10) and -Cl, -CF3, -SCH3, COCH2CH3 and -CN in position C2 exerted significant influence on phenothiazine activity. Glutathione (GSH) prevented TR inactivation by the HRP/H2O2/PZ and MPO/H2O2/PZ systems. The HRP/H2O2 and MPO/H2O2/phenothiazines systems generated the corresponding cationic radicals (FTZ.+) the stability of which was limited by their conversion into phenothiazine-sulfoxides (PTZ-SO). The latter ones were inactive on TR. GSH rapidly reacted with PTZ+.; thus producing cation radical detoxication. These reactions fit in well with GSH protection of TR against the peroxidase/H2O2/phenothiazine system, as well as with the FTZ.+ role in phenothiazine cytotoxicity.

Inactivación de la tripanotiona reductasa del Trypanosoma Cruzi por radicales libres catiónicos de fenotiazinas / Inactivation of Trypanosoma Cruzi, trypanothione reductase by peroxidase/H202/phenothiazine systems

La incubación de la tripanotiona reductasa (TR) de Trypanosoma cruzi con sistemas peroxidasa/H2O2/fenotiazina (FTZ) produjo la inhibición irreversible (inactivación) de TR. El grado de inactivación dependió de: (a) el tiempo de incubación de TR con el sistema peroxidasa/H2O2/FTZ; (b) la naturaleza de la peroxidasa y (c) la estructura de la FTZ. Con las FTZ más activas, la cinética de la inactivación presentó una fase inicial no mayor de 10 minutos, durante la cual TR perdió cerca del 90 por ciento de su actividad. Esa fase fue seguida por otra más lenta, y después de 30 minutos de incubación, TR fue completamente inactiva. Se ensayaron tres peroxidasas, a saber: la peroxidasa de rábano (HRP), la mieloperoxidasa de leucocitos (MPO) y la mioglobina modificada (Mb). En condiciones experimentales comparables, la actividad de las peroxidasas como componentes de los sistemas ensayados decreció en el orden indicado. Con el sistema HRP/H2O2/FTZ, la inactivación final de TR fue de 95-100 por ciento con Tioridazina (TRDZ), Promazina (PZ), Trimeprazina (TMPZ), Proclorpromazina (PCP), Propionilpromazina (PPZ) y Perfenazina (PFZ), todas en concentración 10 µM. Con los sistemas MPO/H2O2/FTZ, las FTZ más activas fueron PZ, TRDZ, TMPZ, PCP y Clorpromazina (CPZ). En iguales condiciones, los sistemas Mb/H2O2/FTZ también inactivaron a TR, utilizando PZ, TMPZ, TRDZ y CPZ. Grupos alquilamino, piperazinilo o piperidilo en la posición 10 (el N) y átomos o grupos -CI, -CF3, -SCH3, COCH2CH3 y -CN en la posición C2 de FTZ afectaron significativamente la actividad de las FTZs. El glutatión (GSH) previno la inactivación de TR por los sistemas HRP/H2O2/PZ y MPO/H2O2/PZ. Los sistemas HRP/H2O2/FTZ y MPO/H2O2/FTZ generaron los radicales catiónicos FTZú+, con estabilidad limitada por su conversión en fenotiazina-sulfoxidos (FTZ-SO), aparentemente inactivos sobre TR. El GSH reaccionó con los radicales catiónicos, regenerando las FTZ originales, lo que concuerda con la protección de TR por GSH frente a los sistemas peroxidasa/H2O2/PZ y, por lo tanto, con la intervención de los radicales catiónicos en la inactivación de TR por los mismos sistemas.

Papel do PMN na falencia de multiplos orgaos e sistemas / Role of the polymorphonuclear neutrophil in the failure of multiple organs and systems

Os autores analisam o papel do neutrofilo polimorfonuclear (PMN) na FMOS pos-trauma e cirurgia, papel este atribuido quer a ativacao para producao de radicais superoxidos e enzimas, quer a depressao funcional dos PMNs. Destacam: 1) a investigacao da sequencia de transmissao intracelular de sinais entre receptores de membrana do PMN e a resposta efetora final; 2) a estrutura e funcao do sistema NADPH do PMN; 3) os estados funcionais do PMN (quiescente, sensibilizado, ativado ou responsivo) em termos de ativacao do sistema NADPH; 4) o mecanismo de lesao tecidual pelo PMN. Discutem investigacoes clinicas sobre o estado de ativacao do PMN, e abordam as perspectivas terapeuticas apontadas pelas pesquisas recentes

Identificación de bacilos gramnegativos, anaerobios facultativos, oxidasas positivos / Identification of Gram-negative, facultatively anaerobie, oxidase-positive bacilli

Se realizó la identificación de 100 cepas de bacilos gramnegativos, anaerobios facultativos, oxidasas positivos, aisladas de diferentes productos patológicos humanos por métodos bioquímicos convencionales. Del total de las cepas estudiadas 1 correspondió al genero Vibrio, 66 al género Aeromonas y 33 al género Plesiomonas. Se destaca la importancia de establecer estudios de vigilancia de estos microorganismos para investigar su circulación en nuestro medio

Reacciones inflamatorias neutrofilo-dependientes en lupus eritematoso sistemico / Neutrophil-dependent inflammatory reactions in systemic lupus erythematosus

Para estudiar el posible rol de la agregación de polimorfonucleares neutrófilos (PMNs) en el Lupus Eritematoso Sistémico (LES), se investigó la actividad agregante de neutrófilos (AAN) de sueros de 32 pacientes lúpicos. En 24 pacientes con LES en actividad se detectaron AAN con un valor medio significativamente mayor que el de los 8 restantes, no activos, y los controles. Particularmente, en 4 pacientes con compromiso del sistema nervioso central la característica fue el hallazgo de altos niveles de AAN. Esto sugiere que la formación intravascular de leucoagregados puede contribuir a la morbidad del LES. Los PMNs normales aumentan la producción de anión superóxido O2- cuando son estimulados con sueros lúpicos. Los PMNs de pacientes lúpicos muestran un aumento del 100% en la producción de 02- in vitro al ser estimulados con su propio suero. Cuando el estímulo es N formil metionil leucil fenilalanina FMLP los PMNs lúpicos producen 5 veces más superóxido que los normales. Factores séricos y celulares contribuyen a un aumento en la producción de O2- por PMNs lúpicos proporcionando las condiciones requeridas para el daño inmune tisular