Primidone oxidation catalyzed by metalloporphyrins and Jacobsen catalyst

Primidone (PRM) oxidation by various oxidants such as iodosylbenzene (PhIO), tert-butyl hydroperoxide 70wt.% (t-BOOH), 3-chloroperoxybenzoic acid (m-CPBA) and hydrogen peroxide 30wt.%, mediated by either a salen complex or metalloporphyrins, was investigated. The catalytic systems led to phenylethyl-malondiamide (PEMA) and phenobarbital (FEND), the same metabolites obtained in vivo with P450 enzymes, although three other products were also detected. Product formation was highly dependent on the oxidant, co-catalyst (imidazole), pH and dioxygen. These biomimetic chemical models have potential application in the synthesis of drug metabolites. which should provide samples for pharmacological tests. They can also be employed in studies that pursue the elucidation of in vivo drug metabolism. (C) 2008 Elsevier B.V. All rights reserved.

The human sulfotransferase SULT1A1 gene is regulated in a synergistic manner by Sp1 and GA binding protein

Human sulfotransferase SULT1A1 is an important phase II xenobiotic metabolizing enzyme that is highly expressed in the liver and mediates the sulfonation of drugs, carcinogens, and steroids. Until this study, the transcriptional regulation of the SULT1A subfamily had been largely unexplored. Preliminary experiments in primary human hepatocytes showed that SULT1A mRNA levels were not changed in response to nuclear receptor activators, such as dexamethasone and 3-methylcolanthrene, unlike other metabolizing enzymes. Using HepG2 cells, the high activity of the TATA-less SULT1A1 promoter was shown to be dependent on the presence of Sp1 and Ets transcription factor binding sites (EBS), located within - 112 nucleotides from the transcriptional start site. The homologous promoter of the closely related SULT1A3 catecholamine sulfotransferase, which is expressed at negligible levels in the adult liver, displayed 70% less activity than SULT1A1. This was shown to be caused by a two-base pair difference in the EBS. The Ets transcription factor GA binding protein (GABP) was shown to bind the SULT1A1 EBS and could transactivate the SULT1A1 promoter in Drosophila melanogaster S2 cells. Cotransfection of Sp1 could synergistically enhance GABP-mediated activation by 10-fold. Although Sp1 and GABP alone could induce SULT1A3 promoter activity, the lack of the EBS on this promoter prevented a synergistic interaction between the two factors. This study reports the first insight into the transcriptional regulation of the SULT1A1 gene and identifies a crucial difference in regulation of the closely related SULT1A3 gene, which accounts for the two enzymes' differential expression patterns observed in the adult liver.

Development of nanostructured bioanodes containing dendrimers and dehydrogenases enzymes for application in ethanol biofuel cells

This paper describes the use of the electrostatic layer-by-layer (LbL) technique for the preparation of bioanodes with potential application in ethanol/O(2) biofuel cells. More specifically, the LbL technique was employed for immobilization of dehydrogenase enzymes and polyamidoamine (PAMAM) dendrimers onto carbon paper support. Both mono (anchoring only the enzyme alcohol dehydrogenase, ADH) and bienzymatic (anchoring both ADH and aldehyde dehydrogenase, AldDH) systems were tested. The amount of ADH deposited onto the Toray (R) paper was 95 ng cm(-2) per bilayer. Kinetic studies revealed that the LbL technique enables better control of enzyme disposition on the bioanode, as compared with the results obtained with the bioanodes prepared by the passive adsorption technique. The power density values achieved for the mono-enzymatic system as a function of the enzyme load ranged from 0.02 to 0.063 mW cm(-2) for the bioanode containing 36 ADH bilayers. The bioanodes containing a gas diffusion layer (GDL) displayed enhanced performance, but their mechanical stability must be improved. The bienzymatic system generated a power density of 0.12 mW cm(-2). In conclusion, the LbL technique is a very attractive approach for enzyme immobilization onto carbon platform, since it enables strict control of enzyme disposition on the bioanode surface with very low enzyme consumption. (C) 2010 Elsevier B.V. All rights reserved.

Refolding and purification of the human secreted group IID phospholipase A(2) expressed as inclusion bodies in Escherichia coli

The secreted phospholipases A(2) (sPLA(2)s) are water-soluble enzymes that bind to the surface of both artificial and biological lipid bilayers and hydrolyze the membrane phospholipids. The tissue expression pattern of the human group IID secretory phospholipase A(2) (hsPLA(2)-IID) suggests that the enzyme is involved in the regulation of the immune and inflammatory responses. With an aim to establish an expression system for the hsPLA(2)-IID in Escherichia coli, the DNA-coding sequence for hsPLA(2)-IID was subcloned into the vector pET3a, and expressed as inclusion bodies in E. coli (BL21). A protocol has been developed to refold the recombinant protein in the presence of guanidinium hydrochloride, using a size-exclusion chromatography matrix followed by dilution and dialysis to remove the excess denaturant. After purification by cation-exchange chromatography, far ultraviolet circular dichroism spectra of the recombinant hsPLA(2)-IID indicated protein secondary structure content similar to the homologous human group IIA secretory phospholipase A(2). The refolded recombinant hsPLA(2)-IID demonstrated Ca(2+)-dependent hydrolytic activity, as measuring the release free fatty acid from phospholipid liposomes. This protein expression and purification system may be useful for site-directed mutagenesis experiments of the hsPLA(2)-IID which will advance our understanding of the structure-function relationship and biological effects of the protein. (C) 2009 Elsevier Inc. All rights reserved.

Characterization of temperature dependent and substrate-binding cleft movements in Bacillus circulans family 11 xylanase: A molecular dynamics investigation

Background: Xylanases (EC 3.2.1.8) hydrolyze xylan, one of the most abundant plant polysaccharides found in nature, and have many potential applications in biotechnology. Methods: Molecular dynamics simulations were used to investigate the effects of temperature between 298 to 338 K and xylobiose binding on residues located in the substrate-binding cleft of the family 11 xylanase from Bacillus circulans (BcX). Results: In the absence of xylobiose the BcX exhibits temperature dependent movement of the thumb region which adopts an open conformation exposing the active site at the optimum catalytic temperature (328 K). In the presence of substrate, the thumb region restricts access to the active site at all temperatures, and this conformation is maintained by substrate/protein hydrogen bonds involving active site residues, including hydrogen bonds between Tyr69 and the 2` hydroxyl group of the substrate. Substrate access to the active site is regulated by temperature dependent motions that are restricted to the thumb region, and the BcX/substrate complex is stabilized by extensive intermolecular hydrogen bonding with residues in the active site. General significance: These results call for a revision of both the ""hinge-bending"" model for the activity of group 11 xylanases, and the role of Tyr69 in the catalytic mechanism. (C) 2009 Elsevier B.V. All rights reserved.

Root Secretion of Defense-related Proteins Is Development-dependent and Correlated with Flowering Time

Proteins found in the root exudates are thought to play a role in the interactions between plants and soil organisms. To gain a better understanding of protein secretion by roots, we conducted a systematic proteomic analysis of the root exudates of Arabidopsis thaliana at different plant developmental stages. In total, we identified 111 proteins secreted by roots, the majority of which were exuded constitutively during all stages of development. However, defense-related proteins such as chitinases, glucanases, myrosinases, and others showed enhanced secretion during flowering. Defense-impaired mutants npr1-1 and NahG showed lower levels of secretion of defense proteins at flowering compared with the wild type. The flowering-defective mutants fca-1, stm-4, and co-1 showed almost undetectable levels of defense proteins in their root exudates at similar time points. In contrast, root secretions of defense-enhanced cpr5-2 mutants showed higher levels of defense proteins. The proteomics data were positively correlated with enzymatic activity assays for defense proteins and with in silico gene expression analysis of genes specifically expressed in roots of Arabidopsis. In conclusion, our results show a clear correlation between defense-related proteins secreted by roots and flowering time.

Existence results for partial neutral functional differential equations with state-dependent delay

In this paper we study the existence of mild solutions for a class of first order abstract partial neutral differential equations with state-dependent delay. (C) 2008 Elsevier Ltd. All rights reserved.

Production of fibrolytic enzymes by Aspergillus japonicus C03 using agro-industrial residues with potential application as additives in animal feed

Solid-state fermentation obtained from different and low-cost carbon sources was evaluated to endocellulases and endoxylanases production by Aspergillus japonicus C03. Regarding the enzymatic production the highest levels were observed at 30 degrees C, using soy bran added to crushed corncob or wheat bran added to sugarcane bagasse, humidified with salt solutions, and incubated for 3 days (xylanase) or 6 days (cellulase) with 70% relative humidity. Peptone improved the xylanase and cellulase activities in 12 and 29%, respectively. The optimum temperature corresponded to 60 degrees C and 50-55 degrees C for xylanase and cellulase, respectively, both having 4.0 as optimum pH. Xylanase was fully stable up to 40 degrees C, which is close to the rumen temperature. The enzymes were stable in pH 4.0-7.0. Cu(++) and Mn(++) increased xylanase and cellulase activities by 10 and 64%, respectively. A. japonicus C03 xylanase was greatly stable in goat rumen fluid for 4 h during in vivo and in vitro experiments.

Characterization of inflammatory responses to eccentric exercise in humans

Eccentric exercise commonly results in muscle damage. The primary sequence of events leading to exercise-induced muscle damage is believed to involve initial mechanical disruption of sarcomeres, followed by impaired excitation-contraction coupling and calcium signaling, and finally, activation of calcium-sensitive degradation pathways. Muscle damage is characterized by ultrastructural changes to muscle architecture, increased muscle proteins and enzymes in the bloodstream, loss of muscular strength and range of motion and muscle soreness. The inflammatory response to exercise-induced muscle damage is characterized by leukocyte infiltration and production of pro-inflammatory cytokines within damaged muscle tissue, systemic release of leukocytes and cytokines, in addition to alterations in leukocyte receptor expression and functional activity. Current evidence suggests that inflammatory responses to muscle damage are dependent on the type of eccentric exercise, previous eccentric loading (repeated bouts), age and gender. Circulating neutrophil counts and systemic cytokine responses are greater after eccentric exercise using a large muscle mass (e.g. downhill running, eccentric cycling) than after other types of eccentric exercise involving a smaller muscle mass. After an initial bout of eccentric exercise, circulating leukocyte counts and cell surface receptor expression are attenuated. Leukocyte and cytokine responses to eccentric exercise are impaired in elderly individuals, while cellular infiltration into skeletal muscle is greater in human females than males after eccentric exercise. Whether alterations in intracellular calcium homeostasis influence inflammatory responses to muscle damage is uncertain. Furthermore, the effects of antioxidant supplements are variable, and the limited data available indicates that anti-inflammatory drugs largely have no influence on inflammatory responses to eccentric exercise. In this review, we compare local versus systemic inflammatory responses, and discuss some of the possible mechanisms regulating the inflammatory responses to exercise-induced muscle damage in humans.

Age and Method-dependent Variability of Predicted Oxygen Consumption in Congenital Heart Disease

Background and Purpose. There has been a lot of debate about the use of predicted oxygen consumption to calculate pulmonary vascular resistance using the Fick principle. We therefore comparatively analyzed predicted oxygen consumption in infants and children in specific age groups, using different methods (formulas), as an attempt to better understand the usefulness and limitations of predictions. Methods and Results. Four models (LaFarge & Miettinen, Bergstra et al., Lindahl, and Lundell et al.) were used to predict oxygen consumption in 200 acyanotic patients with congenital cardiac defects aged 0-2.0, > 2.0-4.0, > 4.0-6.0, and > 6.0-8.75 years (median 2.04 years). Significant differences were observed between the age groups (P < .001) and between the methods (P < .001), not related to diagnoses. Differences between methods were more impressive in the first age group (P < .01). In patients aged 0-2.0 years, the lowest values of oxygen consumption (corresponding to the highest estimates of pulmonary vascular resistance) were obtained with the method of Lindahl; above this age, any method except that of Lundell et al. Conclusions. Although measuring oxygen consumption is always preferable, a rational use of predictions, using different methods, may be of help in situations where measurements are definitely not possible.

Association between tumoral GH-releasing peptide receptor type 1a mRNA expression and in vivo response to GH-releasing peptide-6 in ACTH-dependent Cushing`s syndrome patients

Objective: GH secretagogues (GHS) produce exaggerated ACTH and cortisol responses in Cushing`s disease (CD) patients, attributable to their direct action on GH-releasing peptide receptor type la (GHSR-1a). However, there are no studies correlating the ill vivo response to GHS and GHSR-1a mRNA expression in ACTH-dependent Cushing`s syndrome (CS) patients. The aim of this study is to correlate the patterns of ACTH and cortisol response to GH-releasing peptide-6 (GHRP-6) to GHSR-1a expression in ACTH-dependent CS patients Design: Prospective study in a tertiary referral hospital center. Fifteen CD patients and two ectopic ACTH syndrome (EAS) patients were studied. Methods: Tumor fragments were submitted to RNA extraction, and GHSR-1a expression was studied through real-time qPCR and compared with normal tissue samples. The patients were also submitted to desmopressin test and vasopressin receptor type 1B (AVPR1B) mRNA analysis by qPCR. Results: GHSR-1a expression was similar in normal pituitary samples and in corticotrophic tumor samples. GHSR-1a expression was higher in patients (CD and EAS) presenting ill vivo response to GHRP-6. Higher expression of AVPR1B was observed in the EAS patients responsive to desmopressin, as well as in corticotrophic tumors, as compared with normal pituitary samples, but no correlation between AVPR1B expression and response to desmopressin was observed in the CD patients. Conclusions: Our results revealed a higher expression of GHSR-1a in the ACTH-dependent CS patients responsive to GHRP-6, suggesting an association between receptor gene expression and ill vivo response to the secretagogue in both the CD and the EAS patients.

An optimized model for rat liver perfusion studies

Conditions which influence the viability, integrity, and extraction efficiency of the isolated perfused rat liver were examined to establish optimal conditions for subsequent work in reperfusion injury studies including the choice of buffer, use of oncotic agents, hematocrit, perfusion flow rate, and pressure. Rat livers were perfused with MOPS-buffered Ringer solution with or without erythrocytes. Perfusates were collected and analyzed for blood gases, electrolytes, enzymes, radioactivity in MID studies, and lignocaine in extraction studies. Liver tissue was sampled for histological examinations, and wet:dry weight of the liver was also determined. MOPS-buffered Ringer solution was found to be superior to Krebs bicarbonate buffer, in terms of pH control and buffering capacity, especially during any prolonged period of liver perfusion. A pH of 7.2 is chosen for perfusion since this is the physiological pH of the portal blood. The presence of albumin was important as an oncotic agent, particularly when erythrocytes were used in the perfusate. Perfusion pressure, resistance, and vascular volume are how-dependent and the inclusion of erythrocytes in the perfusate substantially altered the flow characteristics for perfusion pressure and resistance but not vascular volume. Lignocaine extraction was relatively flow-independent. Perfusion injury as defined by enzyme release and tissue fine structure was closely related to the supply of O-2. The optimal conditions for liver perfusion depend upon an adequate supply of oxygen. This can be achieved by using either erythrocyte-free perfusate at a how rate greater than 6 ml/min/g liver or a 20% erythrocyte-containing perfusate at 2 ml/min/g. (C) 1996 Academic Press, Inc.

Gut ischemia/reperfusion induced acute lung injury is an alveolar macrophage dependent event

Background:. Although the role of the lung alveolar macrophage (AM) as a mediator of acute lung injury (ALI) after lung ischemia/reperfusion (I/R) has been suggested by animal experiments, it has not been determined whether AMs mediate ALI after intestinal I/R. The objective of this study was to determine the effect of AM elimination on ALI after intestinal I/R in rats. Mwthods: Male Wistar rats (n = 90) were randomly divided into three groups: the clodronate-liposomes (CLOD-LIP) group received intratracheal treatment with CLOD-LIP; the liposomes (LIP) group received intratracheal treatment with LIP; and the nontreated (UNTREAT) group received no treatment. Twenty-four hours later each group was randomly divided into three subgroups: the intestinal I/R subgroup was subjected to 45-minute intestinal ischemia and 2-hour reperfusion; the laparotomy (LAP) subgroup was subjected to LAP and sham procedures; the control (CTR) subgroup received no treatment. At the end of reperfusion, ALI was quantitated in all the animals by the Evans blue dye (EBD) method. Results: ALI values are expressed as EBD lung leakage (mu g EBD/g dry lung weight). EBD lung leakage values in the CLOD-LIP group were 32.59 +/- 12.74 for I/R, 27.74 +/- 7.99 for LAP, and 33.52 +/- 10.17 for CTR. In the LIP group, lung leakage values were 58.02 +/- 18.04 for I/R, 31.90 +/- 8.72 for LAP, and 27.17 +/- 11.48 for CTR. In the UNTREAT group, lung leakage values were 55.60 +/- 10.96 for I/R, 35.99 +/- 6.89 for LAP, and 30.83 +/- 8.41 for CTR. Within each group, LAP values did not differ from CTR values. However, in the LIP and UNTREAT groups, values for both the LAP and CTR subgroups were lower than values for the I/R subgroup (p < 0.001). The CLOD-LIP I/R subgroup value was less (p < 0.001) than the I/R subgroup values in the LIP and UNTREAT groups. These results indicated that I/R provokes ALI that can be prevented by CLOD-LIP treatment, and further suggested that AMs are essential for ALI occurrence induced by intestinal I/R in rats.

Protein disulfide isomerase redox-dependent association with p47(phox): evidence for an organizer role in leukocyte NADPH oxidase activation

Mechanisms of leukocyte NADPH oxidase regulation remain actively investigated. We showed previously that vascular and macrophage oxidase complexes are regulated by the associated redox chaperone PDI. Here, we investigated the occurrence and possible underlying mechanisms of PDI-mediated regulation of neutrophil NADPH oxidase. In a semirecombinant cell-free system, PDI inhibitors scrRNase (100 mu g/mL) or bacitracin (1 mM) near totally suppressed superoxide generation. Exogenously incubated, oxidized PDI increased (by similar to 40%), whereas PDIred diminished (by similar to 60%) superoxide generation. No change occurred after incubation with PDI serine-mutated in all four redox cysteines. Moreover, a mimetic CxxC PDI inhibited superoxide production by similar to 70%. Thus, oxidized PDI supports, whereas reduced PDI down-regulates, intrinsic membrane NADPH oxidase complex activity. In whole neutrophils, immunoprecipitation and colocalization experiments demonstrated PDI association with membrane complex subunits and prominent thiol-mediated interaction with p47(phox) in the cytosol fraction. Upon PMA stimulation, PDI was mobilized from azurophilic granules to cytosol but did not further accumulate in membranes, contrarily to p47(phox). PDI-p47(phox) association in cytosol increased concomitantly to opposite redox switches of both proteins; there was marked reductive shift of cytosol PDI and maintainance of predominantly oxidized PDI in the membrane. Pulldown assays further indicated predominant association between PDIred and p47(phox) in cytosol. Incubation of purified PDI (> 80% reduced) and p47(phox) in vitro promoted their arachidonate-dependent association. Such PDI behavior is consistent with a novel cytosolic regulatory loop for oxidase complex (re) cycling. Altogether, PDI seems to exhibit a supportive effect on NADPH oxidase activity by acting as a redox-dependent enzyme complex organizer. J. Leukoc. Biol. 90: 799-810; 2011.