Chemoselective screening for the reduction of a chiral functionalised (+/-)-2-(phenylthio)cyclohexanone by whole cells of Brazilian micro-organisms

The use of whole cells of micro-organisms to bring about the biotransformation of an organic compound offers a number of advantages, but problems caused by enzymatic Promiscuity may be encountered upon With Substrates hearing more than one functional group. A one-pot screening method, in which whole fungal cells were incubated with a Mixture of 4-rnethylcyclohexanone I and phenyl methyl Sulfide 2, has been employed to determine the chemoselectivity of various biocatalysts. The hyphomycetes, Aspergillus terreus CCT 3320 and A. terreus URM 3571, catalysed the oxidation of 2 accompanied by the reduction of I to 4-methylcyclohexanol 1a and, for strain A. terreus CCT 3320, the Baeyer-Villiger oxidation of 1. The Basidomycetes, Trametes versicolor CCB 202, Pycnoporus sanguineus CCB 501 and Trichaptum byssogenum CCB 203, catalysed the oxidation of 2 and the reduction 1, but no Baeyer-Villiger reaction products were detected. In contrast. Trametes rigida CCB 285 catalysed the biotransformation of 1 to 1a, exclusively, in the absence of any detectable Sulfide oxidation reactions. The chemoselective reduction Of (+/-)-2-(phenylthio)cyclohexanone 3 by T. rigida CCB 285 afforded exclusively the (+)-cis-(1R,2S) and (+)-trans-(1S,2S) diastereoisomers of 2-(phenylthio)cyclohexan-1-ol 3a in moderate yields (13% and 27%, respectively) and high enantiomeric excesses (>98%). Chemoselective screening for the reduction of a ketone and/or the oxidation Of a Sulfide group in one pot by whole cells of micro-organisms represents an attractive technique with applications in the development of synthesis of complex molecule hearing different functional groups. (C) 2008 Published by Elsevier Ltd.

Bioreduction of alpha-chloroacetophenone by whole cells of marine fungi

The asymmetric reduction of 2-chloro-1-phenylethanone (1) by seven strains of marine fungi was evaluated and afforded (S)-(-)-2-chloro-1-phenylethanol with, in the best case, an enantiomeric excess of 50% and an isolated yield of 60%. The ability of marine fungi to catalyse the reduction was directly dependent on growth in artificial sea water-based medium containing a high concentration of Cl(-) (1.2 M). When fungi were grown in the absence of artificial sea water, no reduction of 1 by whole cells was observed. The biocatalytic reduction of 1 was more efficient at neutral rather than acidic pH values and in the absence of glucose as co-substrate.

Thermal Phase Behavior of DMPG Bilayers in Aqueous Dispersions as Revealed by (2)H- and (31)P-NMR

The synthetic lipid 1,2-dimyristoyl-sn-3-phosphoglycerol (DMPG), when dispersed in water/NaCl exhibits a complex phase behavior caused by its almost unlimited swelling in excess water. Using deuterium ((2)H)- and phosphorus ((31)P)-NMR we have studied the molecular properties of DMPG/water/NaCl dispersions as a function of lipid and NaCl concentration. We have measured the order profile of the hydrophobic part of the lipid bilayer with deuterated DMPG while the orientation of the phosphoglycerol headgroup was deduced from the (31)P NMR chemical shielding anisotropy. At temperatures > 30 degrees C we observe well-resolved (2)H- and (31)P NMR spectra not much different from other liquid crystalline bilayers. From the order profiles it is possible to deduce the average length of the flexible fatty acyl chain. Unusual spectra are obtained in the temperature interval of 20-25 degrees C, indicating one or several phase transitions. The most dramatic changes are seen at low lipid concentration and low ionic strength. Under these conditions and at 25 degrees C, the phosphoglycerol headgroup rotates into the hydrocarbon layer and the hydrocarbon chains show larger flexing motions than at higher temperatures. The orientation of the phosphoglycerol headgroup depends on the bilayer surface charge and correlates with the degree of dissociation of DMPG-Na(+). The larger the negative surface charge, the more the headgroup rotates toward the nonpolar region.

Expression of Pancreatic Endocrine Markers by Mesenchymal Stem Cells From Human Umbilical Cord Vein

Background. Mesenchymal stem cells (MSCs) from human umbilical cord vein have great potential for use in cell therapy because of their ease of isolation, expansion, and differentiation, in addition to their relative acceptance from the ethical point of view. Obtaining the umbilical cord at birth does not present any risk to either mother or child. Objective. To isolate and promote in vitro expansion and differentiation of MSCs from human umbilical cord vein into cells with a pancreatic endocrine phenotype. Methods. Mesenchymal stem cells obtained from human umbilical cord vein via collagenase digestion were characterized at cytochemistry and fluorescent-activated cell sorting, and expanded in vitro. Differentiation of MSCs into an endocrine phenotype was induced using high-glucose (23 mmol/L) medium containing nicotinamide, exendin-4, and 2-mercaptoethanol. Expression of insulin, somatostatin, glucagon, and pancreatic and duodenal homeobox 1 was analyzed using immunofluorescence. Results. Cells isolated from the umbilical cord vein were MSCs as confirmed at cytochemistry and fluorescent-activated cell sorting. Expression of somatostatin, glucagon, and pancreatic and duodenal homeobox 1 by differentiated cells was demonstrated using immunofluorescence. Insulin was not expressed. Conclusions. The MSC differentiation protocol used in the present study induced expression of some endocrine markers. Insulin was not produced by these cells, probably because of incomplete induction of differentiation.

ATP pulse and calcium homeostasis in cells from hepatopancreas of Dilocarcinus pagei, a freshwater crab

Calcium (Ca) is critical for crustaceans due to their molting cycle and its presence in the carapace as calcium carbonate, apart from the usual functions of Ca, such as cell signalling. Ca transport in Dilocarcinus pagei, a freshwater crab, was studied in isolated cells from hepatopancreas to further characterize Ca transport mechanisms in these crabs. Cells were isolated and loaded with Fluo-3, a calcium fluorescent dye. Three different cell treatments were performed: Group 1 cells were Ca free during cell dissociation, and calcium was present (at 1mM) for fluorescence cell loading and transport experiments (FC); Group 2 cells were calcium free during cell dissociation and for transport experiments, but not during cell loading (LC); and Group 3 cells were Ca free during cell dissociation, cell loading and transport experiments (WC). Intracellular Ca was recorded through time after ATP was added to the cells and ATP caused an increase in Ca efflux within 30s in all cells. WC cells showed the smallest Ca efflux compared to the other cells, probably because it was intracellularly Ca ""depleted"". Vanadate and amiloride decreased the Ca efflux when ATP was added to the cells, while verapamil did not cause any effect in Ca efflux, confirming the presence of a Ca(2+)-ATPase sensitive to vanadate in hepatopancreas of D. pagei. In a different set of experiments, cells were also exposed to a Ca pulse of 1 and 10mM during 180s. 10mM Ca increased intracellular Ca compared to 1mM, and the increase was not recovered during the experimental time. Additionally, Ca influx was reduced by verapamil and amiloride, but not completely. The results suggest that Ca influx probably occurs through an undefined exchanger, apart from Ca channels (verapamil sensitive) and electrogenic 1Na(+)(1H(+))/1 Ca(2+) exchanger (amiloride-sensitive). Similarities between freshwater and seawater crabs, lobsters and crayfish in relation to plasma membrane Ca transporters, although the environment where they live is quite diverse, suggest that universal mechanisms for Ca homeostasis are widespread among crustaceans. (C) 2010 Elsevier Inc. All rights reserved.

Molecular Mechanisms by Which Saturated Fatty Acids Modulate TNF-alpha Expression in Mouse Macrophage Lineage

Many macrophage functions are modulated by fatty acids (FAs), including cytokine release, such as tumor necrosis factor-alpha (TNF-alpha). TNF-alpha is of great interest due to its role in the inflammation process observed in several diseases such as rheumatoid arthritis, atherosclerosis, and obesity. However, the mechanisms by which FA effects occur have not been completely elucidated yet. In this study, we used a mouse monocyte lineage (J774 cells) to evaluate the effect of 50 and 100 mu M of saturated (palmitic and stearic acids), monounsaturated (oleic acid) and polyunsaturated (linoleic acid) FAs on TNF-alpha production. Alterations in gene expression, poly(A) tail length and activation of transcription factors were evaluated. Oleic and linoleic acids, usually known as neutral or pro-inflammatory FA, inhibited LPS-induced TNF-alpha secretion by the cells. Saturated FAs were potent inducers of TNF-alpha expression and secretion under basal and inflammatory conditions (in the presence of LPS). Although the effect of the saturated FA was similar, the mechanism involved in each case seem to be distinct, as palmitic acid increased EGR-1 and CREB binding activity and stearic acid increased mRNA poly(A) tail. These results may contribute to the understanding of the molecular mechanisms by which saturated FAs modulate the inflammatory response and may lead to design of associations of dietary and pharmacological strategies to counteract the pathological effects of TNF-alpha.

Acid-base transport by the renal distal nephron

The functional versatility of the distal nephron is mainly due to the large cytological heterogeneity of the segment. Part of Na(+) uptake by distal tubules is dependent on Na(+)/H(+). exchanger 2 (NHE2), implicating a role of distal convoluted cells also in acid-base homeostasis. In addition, intercalated (IC) cells expressed in distal convoluted tubules, connecting tubules and collecting ducts are involved in the final regulation of acid-base excretion. IC cells regulate acid-base handling by 2 main transport proteins, a V-type H(+)-ATPase and a Cl/HCO(3)(-) exchanger, localized at different membrane domains. Type A IC cells are characterized by a luminal H(+)-ATPase in series with a basolateral Cl/HCO(3)(-) exchanger, the anion exchanger AE1. Type B IC cells mediate HCO(3)(-) secretion through the apical Cl(-)/HCO(3)(-) exchanger pendrin in series with a H(+)-ATPase at the basolateral membrane. Alternatively, H(+)/K(+)-ATPases have also been found in several distal tubule cells, particularly in type A and B IC cells. All of these mechanisms are finely regulated, and mutations of 1 or more proteins ultimately lead to expressive disorders of acid-base balance.

Expression of a dendritic cell maturation marker CD83 on tumor cells from lung cancer patients and several human tumor cell lines: is there a biological meaning behind it?

The present paper shows, for the first time, the membrane expression of the dendritic cell maturation marker CD83 on tumor cells from lung cancer patients. CD83 was also detected on freshly cultured fibroblast-like cells from these tissues and on several adherent human tumor cell lines (lung adenocarcinomas P9, A459 and A549, melanomas A375 and C81-61, breast adenocarcinomas SKBR-3 and MCF-7 and colon carcinoma AR42-J), but not in the non-adherent MOT leukemia cell line. CD83 may have immunosuppressive properties and its expression by cancer cells could have a role in facilitating tumor growth.

Lung inflammation is induced by renal ischemia and reperfusion injury as part of the systemic inflammatory syndrome

Ischemia and reperfusion injury (IRI) are mainly caused by leukocyte activation, endothelial dysfunction and production of reactive oxygen species. Moreover, IRI can lead to a systemic response affecting distant organs, such as the lungs. The objective was to study the pulmonary inflammatory systemic response after renal IRI. Male C57Bl/6 mice were subjected to 45 min of bilateral renal ischemia, followed by 4, 6, 12, 24 and 48 h of reperfusion. Blood was collected to measure serum creatinine and cytokine concentrations. Bronchoalveolar lavage fluid (BALF) was collected to determine the number of cells and PGE(2) concentration. Expressions of iNOS and COX-2 in lung were determined by Western blot. Gene analyses were quantified by real time PCR. Serum creatinine increased in the IRI group compared to sham mainly at 24 h after IRI (2.57 +/- A 0.16 vs. 0.43 +/- A 0.07, p < 0.01). The total number of cells in BAL fluid was higher in the IRI group in comparison with sham, 12 h (100 x 10(4) +/- A 15.63 vs. 18.1x10(4) +/- A 10.5, p < 0.05) 24 h (124 x 10(4) +/- A 8.94 vs. 23.2x10(4) +/- A 3.5, p < 0.05) and 48 h (79 x 10(4) +/- A 15.72 vs. 22.2 x 10(4) +/- A 4.2, p < 0.05), mainly by mononuclear cells and neutrophils. Pulmonary COX-2 and iNOS were up-regulated in the IRI group. TNF-alpha, IL-1 beta, MCP-1, KC and IL-6 mRNA expression were up-regulated in kidney and lungs 24 h after renal IRI. ICAM-1 mRNA was up-regulated in lungs 24 h after renal IRI. Serum TNF-alpha, IL-1 beta and MCP-1 and BALF PGE(2) concentrations were increased 24 h after renal IRI. Renal IRI induces an increase of cellular infiltration, up-regulation of COX-2, iNOS and ICAM-1, enhanced chemokine expression and a Th1 cytokine profile in lung demonstrating that the inflammatory response is indeed systemic, possibly leading to an amplification of renal injury.

Continuous and High-Level In Vivo Delivery of Endostatin From Recombinant Cells Encapsulated in TheraCyte (R) Immunoisolation Devices

Endostatin (ES) is a potent inhibitor of angiogenesis and tumor growth. Continuous ES delivery of ES improves the efficacy and potency of the antitumoral therapy. The TheraCyte (R) system is a polytetrafluoroethylene (PTFE) semipermeable membrane macroencapsulation system for implantation of genetically engineered cells specially designed for the in vivo delivery of therapeutic proteins, such as ES, which circumvents the problem of limited half-life and variation in circulating levels. In order to enable neovascularization at the tissues adjacent to the devices prior to ES secretion by the cells inside them, we designed a scheme in which empty TheraCyte (R) devices were preimplanted SC into immunodeficient mice. Only after healing (17 days later) were Chinese hamster ovary cells expressing ES injected into the preimplanted devices. In another model for device implantation, the cells expressing ES where loaded into the immunoisolation devices prior to implantation into the animals, and the TheraCyte (R) were then immediately implanted SC into the mice. Throughout the 2-month study, constant high ES levels of up to 3.7 mu g/ml were detected in the plasma of the mice preimplanted with the devices, while lower but also constant levels of ES (up to 2.1 mu g/ml plasma) were detected in the mice that had received devices preloaded with the ES-expressing cells. Immunohistochemistry using anti-ES antibody showed reaction within the device and outside it, demonstrating that ES, secreted by the confined recombinant cells, permeated through the membrane and reached the surrounding tissues.

MicroRNA expression is differentially altered by xenobiotic drugs in different human cell lines

Several noncoding microRNAs (miR or miRNA) have been shown to regulate the expression of drug-metabolizing enzymes and transporters. Xenobiotic drug-induced changes in enzyme and transporter expression may be associated with the alteration of miRNA expression. Therefore, this study investigated the impact of 19 xenobiotic drugs (e. g. dexamethasone, vinblastine, bilobalide and cocaine) on the expression of ten miRNAs (miR-18a, -27a, -27b, -124a, -148a, -324-3p, -328, -451, -519c and -1291) in MCF-7, Caco-2, SH-SY5Y and BE(2)-M17 cell systems. The data revealed that miRNAs were differentially expressed in human cell lines and the change in miRNA expression was dependent on the drug, as well as the type of cells investigated. Notably, treatment with bilobalide led to a 10-fold increase of miR-27a and a 2-fold decrease of miR-148a in Caco-2 cells, but no change of miR-27a and a 2-fold increase of miR-148a in MCF-7 cells. Neuronal miR-124a was generally down-regulated by psychoactive drugs (e. g. cocaine, methadone and fluoxetine) in BE(2)-M17 and SH-SY5Y cells. Dexamethasone and vinblastine, inducers of drug-metabolizing enzymes and transporters, suppressed the expression of miR-27b, -148a and -451 that down-regulate the enzymes and transporters. These findings should provide increased understanding of the altered gene expression underlying drug disposition, multidrug resistance, drug-drug interactions and neuroplasticity. Copyright (C) 2011 John Wiley & Sons, Ltd.

Molecular analysis of maltotriose active transport and fermentation by Saccharomyces cerevisiae reveals a determinant role for the AGT1 permease

Incomplete and/or sluggish maltotriose fermentation causes both quality and economic problems in the ale-brewing industry. Although it has been proposed previously that the sugar uptake must be responsible for these undesirable phenotypes, there have been conflicting reports on whether all the known alpha-glucoside transporters in Saccharomyces cerevisiae (MALx1, AGT1, and MPH2 and MPH3 transporters) allow efficient maltotriose utilization by yeast cells. We characterized the kinetics of yeast cell growth, sugar consumption, and ethanol production during maltose or maltotriose utilization by several S. cerevisiae yeast strains (both MAL constitutive and AM inducible) and by their isogenic counterparts with specific deletions of the AGT1 gene. Our results clearly showed that yeast strains carrying functional permeases encoded by the MAL21, MAL31, and/or MAL41 gene in their plasma membranes were unable to utilize maltotriose. While both high-and low-affinity transport activities were responsible for maltose uptake from the medium, in the case of maltotriose, the only low-affinity (K-m, 36 +/- 2 mM) transport activity was mediated by the AGT1 permease. In conclusion, the AGT1 transporter is required for efficient maltotriose fermentation by S. cerevisiae yeasts, highlighting the importance of this permease for breeding and/or selection programs aimed at improving sluggish maltotriose fermentations.

Effects of the antimicrobial peptide gomesin on the global gene expression profile, virulence and biofilm formation of Xylella fastidiosa

In the xylem vessels of susceptible hosts, such as citrus trees, Xylella fastidiosa forms biofilm-like colonies that can block water transport, which appears to correlate to disease symptoms. Besides aiding host colonization, bacterial biofilms play an important role in resistance against antimicrobial agents, for instance antimicrobial peptides (AMPs). Here, we show that gomesin, a potent AMP from a tarantula spider, modulates X. fastidiosa gene expression profile upon 60 min of treatment with a sublethal concentration. DNA microarray hybridizations revealed that among the upregulated coding sequences, some are related to biofilm production. In addition, we show that the biofilm formed by gomesin-treated bacteria is thicker than that formed by nontreated cells or cells exposed to streptomycin. We have also observed that the treatment of X. fastidiosa with a sublethal concentration of gomesin before inoculation in tobacco plants correlates with a reduction in foliar symptoms, an effect possibly due to the trapping of bacterial cells to fewer xylem vessels, given the enhancement in biofilm production. These results warrant further investigation of how X. fastidiosa would respond to the AMPs produced by citrus endophytes and by the insect vector, leading to a better understanding of the mechanism of action of these molecules on bacterial virulence.

Three-Dimensional Quantitative Structure-Activity Relationship Study of Antitumor 2-Formylpyridine Thiosemicarbazones Derivatives as Inhibitors of Ribonucleotide Reductase

In order to extend previous SAR and QSAR studies, 3D-QSAR analysis has been performed using CoMFA and CoMSIA approaches applied to a set of 39 alpha-(N)-heterocyclic carboxaldehydes thiosemicarbazones with their inhibitory activity values (IC(50)) evaluated against ribonucleotide reductase (RNR) of H.Ep.-2 cells (human epidermoid carcinoma), taken from selected literature. Both rigid and field alignment methods, taking the unsubstituted 2-formylpyridine thiosemicarbazone in its syn conformation as template, have been used to generate multiple predictive CoMFA and CoMSIA models derived from training sets and validated with the corresponding test sets. Acceptable predictive correlation coefficients (Q(cv)(2) from 0.360 to 0.609 for CoMFA and Q(cv)(2) from 0.394 to 0.580 for CoMSIA models) with high fitted correlation coefficients (r` from 0.881 to 0.981 for CoMFA and r(2) from 0.938 to 0.993 for CoMSIA models) and low standard errors (s from 0.135 to 0.383 for CoMFA and s from 0.098 to 0.240 for CoMSIA models) were obtained. More precise CoMFA and CoMSIA models have been derived considering the subset of thiosemicarbazones (TSC) substituted only at 5-position of the pyridine ring (n=22). Reasonable predictive correlation coefficients (Q(cv)(2) from 0.486 to 0.683 for CoMFA and Q(cv)(2) from 0.565 to 0.791 for CoMSIA models) with high fitted correlation coefficients (r(2) from 0.896 to 0.997 for CoMFA and r(2) from 0.991 to 0.998 for CoMSIA models) and very low standard errors (s from 0.040 to 0.179 for CoMFA and s from 0.029 to 0.068 for CoMSIA models) were obtained. The stability of each CoMFA and CoMSIA models was further assessed by performing bootstrapping analysis. For the two sets the generated CoMSIA models showed, in general, better statistics than the corresponding CoMFA models. The analysis of CoMFA and CoMSIA contour maps suggest that a hydrogen bond acceptor near the nitrogen of the pyridine ring can enhance inhibitory activity values. This observation agrees with literature data, which suggests that the nitrogen pyridine lone pairs can complex with the iron ion leading to species that inhibits RNR. The derived CoMFA and CoMSIA models contribute to understand the structural features of this class of TSC as antitumor agents in terms of steric, electrostatic, hydrophobic and hydrogen bond donor and hydrogen bond acceptor fields as well as to the rational design of this key enzyme inhibitors.

The isatin-Schiff base copper(II) complex Cu(isaepy)(2) acts as delocalized lipophilic cation, yields widespread mitochondrial oxidative damage and induces AMP-activated protein kinase-dependent apoptosis

We previously demonstrated that Bis[(2-oxindol-3-ylimino)-2-(2-aminoethyl) pyridine-N, N`] copper(II) [Cu(isaepy)(2)] was an efficient inducer of the apoptotic mitochondrial pathway. Here, we deeply dissect the mechanisms underlying the ability of Cu(isaepy)(2) to cause mitochondriotoxicity. In particular, we demonstrate that Cu(isaepy)(2) increases NADH-dependent oxygen consumption of isolated mitochondria and that this phenomenon is associated with oxy-radical production and insensitive to adenosine diphosphate. These data indicate that Cu(isaepy)(2) behaves as an uncoupler and this property is also confirmed in cell systems. Particularly, SH-SY5Y cells show: (i) an early loss of mitochondrial transmembrane potential; (ii) a decrease in the expression levels of respiratory complex components and (iii) a significant adenosine triphosphate (ATP) decrement. The causative energetic impairment mediated by Cu(isaepy)(2) in apoptosis is confirmed by experiments carried out with rho(0) cells, or by glucose supplementation, where cell death is significantly inhibited. Moreover, gastric and cervix carcinoma AGS and HeLa cells, which rely most of their ATP production on oxidative phosphorylation, show a marked sensitivity toward Cu(isaepy)(2). Adenosine monophosphate-activated protein kinase (AMPK), which is activated by events increasing the adenosine monophosphate: ATP ratio, is deeply involved in the apoptotic process because the overexpression of its dominant/negative form completely abolishes cell death. Upon glucose supplementation, AMPK is not activated, confirming its role as fuel-sensing enzyme that positively responds to Cu(isaepy)(2)-mediated energetic impairment by committing cells to apoptosis. Overall, data obtained indicate that Cu(isaepy)(2) behaves as delocalized lipophilic cation and induces mitochondrial-sited reactive oxygen species production. This event results in mitochondrial dysfunction and ATP decrease, which in turn triggers AMPK-dependent apoptosis.