QUANTIFICATION OF TRYPTOPHAN IN PLASMA BY HIGH PERFORMANCE LIQUID CHROMATOGRAPHY

A simple, rapid and selective method using high-performance liquid chromatography with ultraviolet detection (267 nm) was applied for the determination of tryptophan in plasma. Separation was carried out on a C18 column (150 x 4.6 mm internal diameter) in 6 min. The mobile phase consisted of 5 mM the sodium acetate and acetonitrile (92:8, v/v). The method was shown to be precise and accurate, and good recovery of analyte was achieved, characterizing the method as efficient and reliable for use in laboratory analysis.

Hydrogen production and consumption of organic acids by a phototrophic microbial consortium

Alternative fuel sources have been extensively studied. Hydrogen gas has gained attention because its combustion releases only water, and it can be produced by microorganisms using organic acids as substrates. The aim of this study was to enrich a microbial consortium of photosynthetic purple non-sulfur bacteria from an Upflow Anaerobic Sludge Blanket reactor (UASB) using malate as carbon source. After the enrichment phase, other carbon sources were tested, such as acetate (30 mmol l(-1)), butyrate (17 mmol l(-1)), citrate (11 mmol l(-1)), lactate (23 mmol l(-1)) and malate (14.5 mmol l(-1)). The reactors were incubated at 30 degrees C under constant illumination by 3 fluorescent lamps (81 mu mol m(-2) s(-1)). The cumulative hydrogen production was 7.8, 9.0, 7.9, 5.6 and 13.9 mmol H-2 l(-1) culture for acetate, butyrate, citrate, lactate and malate, respectively. The maximum hydrogen yield was 0.6, 1.4, 0.7, 0.5 and 0.9 mmol H-2 mmol(-1) substrate for acetate, butyrate, citrate, lactate and malate, respectively. The consumption of substrates was 43% for acetate, 37% for butyrate, 100% for citrate, 49% for lactate and 100% for malate. Approximately 26% of the clones obtained from the Phototrophic Hydrogen-Producing Bacterial Consortium (PHPBC) were similar to Rhodobacter, Rhodospirillum and Rhodopseudomonas, which have been widely cited in studies of photobiological hydrogen production. Clones similar to the genus Sulfurospirillum (29% of the total) were also found in the microbial consortium. Copyright (C) 2012, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.

Solvation in aqueous binary mixtures: consequences of the hydrophobic character of the ionic liquids and the solvatochromic probes

Information on the solvation in mixtures of water, W, and the ionic liquids, ILs, 1-allyl-3-R-imidazolium chlorides; R = methyl, 1-butyl, and 1-hexyl, has been obtained from the responses of the following solvatochromic probes: 2,6-dibromo-4-[(E)-2-(1-R-pyridinium-4-yl)ethenyl] phenolate, R = methyl, MePMBr2; 1-octyl, OcPMBr(2), and the corresponding quinolinium derivative, MeQMBr(2). A model developed for solvation in binary mixtures of W and molecular solvents has been extended to the present mixtures. Our objective is to assess the relevance to solvation of hydrogen-bonding and the hydrophobic character of the IL and the solvatochromic probe. Plots of the medium empirical polarity, E-T(probe) versus its composition revealed non-ideal behavior, attributed to preferential solvation by the IL and, more efficiently, by the IL-W hydrogen-bonded complex. The deviation from linearity increases as a function of increasing number of carbon atoms in the alkyl group of the IL, and is larger than that observed for solvation by W plus molecular solvents (1-propanol and 2-(1-butoxy)ethanol) that are more hydrophobic than the ILs investigated. This enhanced deviation is attributed to the more organized structure of the ILs proper, which persists in their aqueous solutions. MeQMBr(2) is more susceptible to solvent lipophilicity than OcPMBr(2), although the former probe is less lipophilic. This enhanced susceptibility agrees with the important effect of annelation on the contributions of the quinonoid and zwitterionic limiting structures to the ground and excited states of the probe, hence on its response to both medium composition and lipophilicity of the IL.

Synthesis, Characterization and Biological Activities of a Bidentate Schiff Base Ligand: N,N '-Bis(1-phenylethylidene)ethane-1,2-diamine and its Transition Metals (II) Complexes

Schiff base ligand: N,N'-bis(1-phenylethylidene)ethane-1,2-diamine (L), was derived from acetophenone and ethylenediamine by condensation and its complexes (1-5) were prepared with Pb2+, Ni2+, Co2+, Cu2+ and Cd2+ metal ions. Their structures were characterized by FAB-MS, IR spectra, elemental analyses and molar conductance. The octahedral geometry of the complexes was proposed by electronic spectra and magnetic moment data. The conductivity data showed that the complexes have non-electrolytic nature. The complexes (1-5) have higher in vitro antimicrobial activity than the Schiff base ligand (L). In the nuclease activity, the complexes cleave DNA as compared to control DNA in the presence of H2O2.

Signaling Events During Cyclic Guanosine Monophosphate-Regulated Pigment Aggregation in Freshwater Shrimp Chromatophores

Crustacean color change results partly from granule aggregation induced by red pigment concentrating hormone (RPCH). In shrimp chromatophores, both the cyclic GMP (3', 5'-guanosine monophosphate) and Ca2+ cascades mediate pigment aggregation. However, the signaling elements upstream and downstream from cGMP synthesis by GC-S (cytosolic guanylyl cyclase) remain obscure. We investigate post-RPCH binding events in perfused red ovarian chromatophores to disclose the steps modulating cGMP concentration, which regulates granule translocation. The inhibition of calcium/calmodulin complex (Ca2+/CaM) by N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W7) induces spontaneous aggregation but inhibits RPCH-triggered aggregation, suggesting a role in pigment aggregation and dispersion. Nitric oxide synthase inhibition by N omega-nitro-L-arginine methyl ester hydrochloride (L-NAME) strongly diminishes RPCH-induced aggregation; protein kinase G inhibition (by rp-cGMPs-triethylamine) reduces RPCH-triggered aggregation and provokes spontaneous dispersion, disclosing NO/PKG participation in aggregation signaling. Myosin light chain phosphatase inhibition (by cantharidin) accelerates RPCH-triggered aggregation, whereas Rho-associated protein kinase inhibition (by Y-27632, H-11522) reduces RPCH-induced aggregation and accelerates dispersion. MLCP (myosin light chain kinase) and ROCK (Rho-associated protein kinase) may antagonistically regulate myosin light chain (MLC) dephosphorylation/phosphorylation during pigment dispersion/aggregation. We propose the following general hypothesis for the cGMP/Ca2+ cascades that regulate pigment aggregation in crustacean chromatophores: RPCH binding increases Ca2+ (int), activating the Ca2+/CaM complex, releasing NOS-produced nitric oxide, and causing GC-S to synthesize cGMP that activates PKG, which phosphorylates an MLC activation site. Myosin motor activity is initiated by phosphorylation of an MLC regulatory site by ROCK activity and terminated by MLCP-mediated dephosphorylation. Qualitative comparison reveals that this signaling pathway is conserved in vertebrate and invertebrate chromatophores alike.

Further monoterpene chromane esters from Peperomia obtusifolia: VCD determination of the absolute configuration of a new diastereomeric mixture

A reinvestigation of the monoterpene chromane ester enriched fraction from Peperomia obtusifolia using chiral chromatography led to the identification of a minor peak, which was elucidated by NMR and HRMS as fenchyl-3,4-dihydro-5-hydroxy-2,7-dimethyl-8-(3 ''-methyl-2 ''-butenyl)-2-(4'-methyl-1',3'-pentadienyl)-2H-1-benzopyran-6-carboxylate, the same structure assigned to two other fenchyl esters described previously, pointing out a stereoisomeric relationship among them. Further NMR analysis revealed that it was actually a mixture of two compounds, whose absolute configurations were determined by VCD measurements. Although, almost no vibrational transitions could be assigned to the chiral chromane, the experimental VCD spectrum was largely opposite to that obtained for the average experimental VCD [(2S,1'''R,2'''R,4'''S + 2R,1'''R,2'''R,4'''S)/2] for fenchol derivatives. These results allowed us to assign the putative compounds as a racemic mixture of the chiral chromane esterified with the monoterpene (1S,2S,4R)fenchol, which had not been identified in our early work. (C) 2012 Elsevier Ltd. All rights reserved.

One-Pot, Three-Step Reaction of 5-[(Trimethylsilyl)ethynyl]pyrrolidin-2-one with Azides Catalyzed by Cu-I: Synthesis of a Novel Class of 1,4-Disubstituted 1,2,3-Triazoles

A very fast, easy and efficient synthesis is described for a novel and biologically important class of 1,4-disubstituted-4-(5-pyrrolidin-2-one)-1,2,3-triazoles by an ultrasound-assisted one-pot, three-step click reaction sequence of 5-[(trimethylsilyl)ethynyl]pyrrolidin-2-one with organic azides mediated by catalytic Cu-I salts.

Oxidation of lysergic acid diethylamide (LSD) by peroxidases: a new metabolic pathway

Lysergic acid diethylamide (LSD) is a potent hallucinogen that is primarily metabolized to 2-oxo-3-hydroxy-LSD (O-H-LSD) and N-desmethyl-LSD (nor-LSD) by cytochrome P450 complex liver enzymes. Due to its extensive metabolism, there still is an interest in the identification of new metabolites and new routes of its metabolism in humans. In the present study, we investigated whether LSD could be a substrate for horseradish peroxidase or myeloperoxidase (MPO). Using liquid chromatography coupled to UV detection and electrospray ionization mass spectrometry (LC-UV-ESI-MS), we found that both peroxidases were capable of metabolizing LSD to the same compounds that have been observed in vivo (i.e., O-H-LSD and nor-LSD). In addition, we found another major metabolite, N,N-diethyl-7-formamido-4-methyl-6-oxo-2,3,4,4a,5,6-hexahydrobenzo[f]quinoline-2-carboxamide (FOMBK), which is an opened indolic ring compound. Hydrolysis of FOMBK led to the deformylated compound 7-amino-N,N-diethyl-4-methyl-6-oxo-2,3,4,4a,5,6-hexahydrobenzo[f]quinoline-2-carboxamide. The reactions of LSD with the peroxidases were chemiluminescent and sensitive to inhibition by reactive oxygen scavengers, which indicated that the classic peroxidase cycle is involved in this new alternative metabolic pathway. Considering that MPO is abundant in immune cells and also present in the central nervous system, the degradation pathway described in this study suggests a possible route of LSD metabolism that may occur concurrently with the in vivo reaction catalyzed by the cytochrome P450 system.

Removal from the plasma of the free and esterified forms of cholesterol and transfer of lipids to HDL in type 2 diabetes mellitus patients

Background: The aim was to investigate new markers for type 2 diabetes (T2DM) dyslipidemia related with LDL and HDL metabolism. Removal from plasma of free and esterified cholesterol transported in LDL and the transfer of lipids to HDL are important aspects of the lipoprotein intravascular metabolism. The plasma kinetics (fractional clearance rate, FCR) and transfers of lipids to HDL were explored in T2DM patients and controls, using as tool a nanoemulsion that mimics LDL lipid structure (LDE). Results: C-14- cholesteryl ester FCR of the nanoemulsion was greater in T2DM than in controls (0.07 +/- 0.02 vs. 0.05 +/- 0.01 h(-1), p = 0.02) indicating that LDE was removed faster, but FCR H-3- cholesterol was equal in both groups. Esterification rates of LDE free-cholesterol were equal. Cholesteryl ester and triglyceride transfer from LDE to HDL was greater in T2DM (4.2 +/- 0.8 vs. 3.5 +/- 0.7%, p = 0.03 and 6.8 +/- 1.6% vs. 5.0 +/- 1.1, p = 0.03, respectively). Phospholipid and free cholesterol transfers were not different. Conclusions: The kinetics of free and esterified cholesterol tended to be independent in T2DM patients and the lipid transfers to HDL were also disturbed. These novel findings may be related with pathophysiological mechanisms of diabetic macrovascular disease.

Expression of laminin-5 and integrins in actinic cheilitis and superficially invasive squamous cell carcinomas of the lip

The progression of carcinogenesis entails the detachment of cells, invasion and migration of neoplastic cells. Alterations in epithelial adhesion and basement membrane proteins might mediate the early stages of carcinogenesis. This study investigated the expression of adhesion molecules and the basement membrane protein laminin-5 in actinic cheilitis (AC) and incipient squamous cell carcinoma of the lower lip to understand early photocarcinogenesis. Ln-5 gamma 2 chain as well as alpha 3, beta 1 subunits of alpha 3 beta 1 heterodimer and beta 4 subunit of integrin alpha 6 beta 4 were evaluated by immunohistochemistry in 16 cases of AC and 16 cases of superficially invasive squamous cell carcinoma (SISCC). Most AC cases showed reduced expression of beta 1, beta 4 and alpha 3 integrins, and SISCCs lacked beta 1, beta 4 and alpha 3 integrins in the invasive front. AC cases were negative for the Ln-5 gamma 2 chain. Five cases of SISCC (31%) showed heterogeneous Ln-5 gamma 2 chain expression in the invasive front of the tumor. Integrin beta 1, beta 4 and alpha 3 expression is lost during the early stages of lip carcinogenesis. Expression of Ln-5 gamma 2 in the invasive front in cases and its correlation with tumor progression suggest that it mediates the acquisition of the migrating and invading epithelial cell phenotype. (C) 2012 Elsevier GmbH. All rights reserved.

Opioidergic, GABAergic and serotonergic neurotransmission in the dorsal raphe nucleus modulates tonic immobility in guinea pigs

Tonic immobility (TI) is an innate defensive behavior that can be elicited by physical restriction and postural inversion and is characterized by a profound and temporary state of akinesis. Our previous studies demonstrated that the stimulation of serotonin receptors in the dorsal raphe nucleus (DRN) appears to be biphasic during TI responses in guinea pigs (Cavia porcellus). Serotonin released by the DRN modulates behavioral responses and its release can occur through the action of different neurotransmitter systems, including the opioidergic and GABAergic systems. This study examines the role of opioidergic, GABAergic and serotonergic signaling in the DRN in TI defensive behavioral responses in guinea pigs. Microinjection of morphine (1.1 nmol) or bicuculline (0.5 nmol) into the DRN increased the duration of TI. The effect of morphine (1.1 nmol) was antagonized by pretreatment with naloxone (0.7 nmol), suggesting that the activation of pi opioid receptors in the DRN facilitates the TI response. By contrast, microinjection of muscimol (0.5 nmol) into the DRN decreased the duration of TI. However, a dose of muscimol (0.26 nmol) that alone did not affect TI, was sufficient to inhibit the effect of morphine (1.1 nmol) on TI, indicating that GABAergic and enkephalinergic neurons interact in the DRN. Microinjection of alpha-methyl-5-HT (1.6 nmol), a 5-HT2 agonist, into the DRN also increased TI. This effect was inhibited by the prior administration of naloxone (0.7 nmol). Microinjection of 8-OH-DPAT (1.3 nmol) also blocked the increase of TI promoted by morphine (1.1 nmol). Our results indicate that the opioidergic, GABAergic and serotonergic systems in the DRN are important for modulation of defensive behavioral responses of TI. Therefore, we suggest that opioid inhibition of GABAergic neurons results in disinhibition of serotonergic neurons and this is the mechanism by which opioids could enhance TI. Conversely, a decrease in TI could occur through the activation of GABAergic interneurons. (C) 2012 Elsevier Inc. All rights reserved.

Purinergic and glutamatergic interactions in the hypothalamic paraventricular nucleus modulate sympathetic outflow

P2X receptors are expressed on ventrolateral medulla projecting paraventricular nucleus (PVN) neurons. Here, we investigate the role of adenosine 5′-triphosphate (ATP) in modulating sympathetic nerve activity (SNA) at the level of the PVN. We used an in situ arterially perfused rat preparation to determine the effect of P2 receptor activation and the putative interaction between purinergic and glutamatergic neurotransmitter systems within the PVN on lumbar SNA (LSNA). Unilateral microinjection of ATP into the PVN induced a dose-related increase in the LSNA (1 nmol: 38 ± 6 %, 2.5 nmol: 72 ± 7 %, 5 nmol: 96 ± 13 %). This increase was significantly attenuated by blockade of P2 receptors (pyridoxalphosphate-6-azophenyl-20,40-disulphonic acid, PPADS) and glutamate receptors (kynurenic acid, KYN) or a combination of both. The increase in LSNA elicited by L-glutamate microinjection into the PVN was not affected by a previous injection of PPADS. Selective blockade of non-N-methyl-D-aspartate receptors (6-cyano-7-nitroquinoxaline-2,3-dione disodium salt, CNQX), but not N-methyl-D-aspartate receptors (NMDA) receptors (DL-2-amino-5-phosphonopentanoic acid, AP5), attenuated the ATP-induced sympathoexcitatory effects at the PVN level. Taken together, our data show that purinergic neurotransmission within the PVN is involved in the control of SNA via P2 receptor activation. Moreover, we show an interaction between P2 receptors and non-NMDA glutamate receptors in the PVN suggesting that these functional interactions might be important in the regulation of sympathetic outflow