The N-terminal region of the Escherichia coli WecA (Rfe) protein, containing three predicted transmembrane helices, is required for function but not for membrane insertion

The correct site for translation initiation for Escherichia coli WecA (Rfe), presumably involved in catalyzing the transfer of N-acetylglucosamine 1-phosphate to undecaprenylphosphate, was determined by using its FLAG-tagged derivatives. The N-terminal region containing three predicted transmembrane helices was found to be necessary for function but not for membrane localization of this protein.

The rfaE gene from Escherichia coli encodes a bifunctional protein involved in biosynthesis of the lipopolysaccharide core precursor ADP-L-glycero-D-manno-heptose

The intermediate steps in the biosynthesis of the ADP-L-glycero-D-manno-heptose precursor of inner core lipopolysaccharide (LPS) are not yet elucidated. We isolated a mini-Tn10 insertion that confers a heptoseless LPS phenotype in the chromosome of Escherichia coli K-12. The mutation was in a gene homologous to the previously reported rfaE gene from Haemophilus influenzae. The E. coli rfaE gene was cloned into an expression vector, and an in vitro transcription-translation experiment revealed a polypeptide of approximately 55 kDa in mass. Comparisons of the predicted amino acid sequence with other proteins in the database showed the presence of two clearly separate domains. Domain I (amino acids 1 to 318) shared structural features with members of the ribokinase family, while Domain II (amino acids 344 to 477) had conserved features of the cytidylyltransferase superfamily that includes the aut gene product of Ralstonia eutrophus. Each domain was expressed individually, demonstrating that only Domain I could complement the rfaE::Tn10 mutation in E. coli, as well as the rfaE543 mutation of Salmonella enterica SL1102. DNA sequencing of the rfaE543 gene revealed that Domain I had one amino acid substitution and a 12-bp in-frame deletion resulting in the loss of four amino acids, while Domain II remained intact. We also demonstrated that the aut::Tn5 mutation in R. eutrophus is associated with heptoseless LPS, and this phenotype was restored following the introduction of a plasmid expressing the E. coli Domain II. Thus, both domains of rfaE are functionally different and genetically separable confirming that the encoded protein is bifunctional. We propose that Domain I is involved in the synthesis of D-glycero-D-manno-heptose 1-phosphate, whereas Domain II catalyzes the ADP transfer to form ADP-D-glycero-D-manno-heptose.

The GalF protein of Escherichia coli is not a UDP-glucose pyrophosphorylase but interacts with the GalU protein possibly to regulate cellular levels of UDP-glucose

We report the functional characterization of the galF gene of strain VW187 (Escherichia coli O7:K1), which encodes a polypeptide displaying structural features common to bacterial UDP-glucose pyrophosphorylases, including the E. coli GalU protein. These enzymes catalyse a reversible reaction converting UTP and glucose-1-phosphate into UDP-glucose and PPi. We show that, although the GalF protein is expressed in vivo, GalF-expressing plasmids cannot complement the phenotype of a galU mutant and extracts from this mutant which only produces GalF are enzymatically inactive. In contrast, the presence of GalU and GalF proteins in the same cell-free extract caused a significant reduction in the rate of pyrophosphorolysis (conversion of UDP-glucose into glucose-1-phosphate) but no significant effect on the kinetics of synthesis of UDP-glucose. The presence of GalF also increased the thermal stability of the enzyme in vitro. The effect of GalF in the biochemical properties of the UDP-glucose pyrophosphorylase required the co-synthesis of GalF and GalU, suggesting that they could interact as components of the oligomeric enzyme. The physical interaction of GalU and GalF was demonstrated in vivo by the co-expression of both proteins as fusion products using a yeast two-hybrid system. Furthermore, using a pair of galF-/galU+ and galF/galU+ isogenic strains, we demonstrated that the presence of GalF is associated with an increased concentration of intracellular UDP-glucose as well as with an enhancement of the thermal stability of the UDP-glucose pyrophosphorylase in vivo. We propose that GalF is a non-catalytic subunit of the UDP-glucose pyrophosphorylase modulating the enzyme activity to increase the formation of UDP-glucose, and this function is important for bacterial adaptation to conditions of stress.

Genetic analysis of the dTDP-rhamnose biosynthesis region of the Escherichia coli VW187 (O7:K1) rfb gene cluster:identification of functional homologs of rfbB and rfbA in the rff cluster and correct location of the rffE gene

The O-repeating unit of the Escherichia coli O7-specific lipopolysaccharide is made of galactose, mannose, rhamnose, 4-acetamido-4,6-dideoxyglucose, and N-acetyglucosamine. We have recently characterized the genes involved in the biosynthesis of the sugar precursor GDP-mannose occurring in the E. coli O7:K1 strain VW187 (C. L. Marolda and M. A. Valvano, J. Bacteriol. 175:148-158, 1993). In the present study, we identified and sequenced the rfbBDAC genes encoding the enzymes for the biosynthesis of another precursor, dTDP-rhamnose. These genes are localized on the upstream end of the rfbEcO7 region, and they are strongly conserved compared with similar genes found in various enteric and nonenteric bacteria. Upstream of rfbB we identified a DNA segment containing the rfb promoter and a highly conserved untranslated leader sequence also present in the promoter regions of other surface polysaccharide gene clusters. Also, we have determined that rfbB and rfbA have homologs, rffG (o355) and rffH (o292), respectively, located on the rff cluster, which is involved in the synthesis of enterobacterial common antigen. We provide biochemical evidence that rffG and rffH encode dTDP-glucose dehydratase and glucose-1-phosphate thymidylyltransferase activities, respectively, and we also show that rffG complemented the rfbB defect in the O7+ cosmid pJHCV32. We also demonstrate that rffG is distinct from rffE and map the rffE gene to the second gene of the rff cluster.

Galactose Metabolism in Saccharomyces cerevisiae

Galactose is metabolised to the more metabolically useful glucose 6-phosphate by the enzymes of the Leloir pathway. This pathway is necessary as the initial enzymes of glycolysis are unable to recognise galactose. In most organisms, including Saccharomyces cerevisiae, five enzymes are required to catalyse the conversion: galactose mutarotase, galactokinase, galactose 1-phosphate uridyltransferase, UDP-galactose 4-epimerase and phosphoglucomutase. The pathway has attracted interest in S. cerevisiae as it is under very strict genetic control and thus provides an excellent model for the study of gene expression in eukaryotes. In the presence of glucose the genes encoding the Leloir pathway enzymes (the GAL genes) are completely repressed through the action of a transcription factor Mig1p. Only in the presence of galactose and the absence of glucose do the concerted actions of Gal4p, Gal80p and Gal3p enable the rapid and high level activation of the GAL genes. The exact mechanism of action of these three proteins is controversial. Galactose metabolism in S. cerevisiae is also of interest because it can be exploited both in the laboratory (for high level expression of heterologous proteins and in the yeast two hybrid screen) and industrially (increasing flux through the Leloir pathway in order to make more efficient use of feedstocks with high galactose content). Recent work on the structures of the various proteins, their mechanisms of action and attempts to gain an integrated understanding of transcriptional and metabolic events will assist our understanding of both the fundamental biochemical processes and how these might be exploited commercially.

Purple sweet potato colour - a potential therapy for galactosemia?

Galactosemia is an inherited metabolic disease in which galactose is not properly metabolised. There are various theories to explain the molecular pathology, and recent experimental evidence strongly suggests that oxidative stress plays a key role. High galactose diets are damaging to experimental animals and oxidative stress also plays a role in this toxicity which can be alleviated by purple sweet potato colour (PSPC). This plant extract is rich in acetylated anthocyanins which have been shown to quench free radical production. The objective of this Commentary is to advance the hypothesis that PSPC, or compounds therefrom, may be a viable basis for a novel therapy for galactosemia.

The molecular basis of galactosemia - Past, present and future

Galactosemia, an inborn error of galactose metabolism, was first described in the 1900s by von Ruess. The subsequent 100years has seen considerable progress in understanding the underlying genetics and biochemistry of this condition. Initial studies concentrated on increasing the understanding of the clinical manifestations of the disease. However, Leloir's discovery of the pathway of galactose catabolism in the 1940s and 1950s enabled other scientists, notably Kalckar, to link the disease to a specific enzymatic step in the pathway. Kalckar's work established that defects in galactose 1-phosphate uridylyltransferase (GALT) were responsible for the majority of cases of galactosemia. However, over the next three decades it became clear that there were two other forms of galactosemia: type II resulting from deficiencies in galactokinase (GALK1) and type III where the affected enzyme is UDP-galactose 4'-epimerase (GALE). From the 1970s, molecular biology approaches were applied to galactosemia. The chromosomal locations and DNA sequences of the three genes were determined. These studies enabled modern biochemical studies. Structures of the proteins have been determined and biochemical studies have shown that enzymatic impairment often results from misfolding and consequent protein instability. Cellular and model organism studies have demonstrated that reduced GALT or GALE activity results in increased oxidative stress. Thus, after a century of progress, it is possible to conceive of improved therapies including drugs to manipulate the pathway to reduce potentially toxic intermediates, antioxidants to reduce the oxidative stress of cells or use of "pharmacological chaperones" to stabilise the affected proteins.

Molecular features of hepatosplenic T-cell lymphoma unravels potential novel therapeutic targets.

The pathogenesis of hepatosplenic T-cell lymphoma (HSTL), a rare entity mostly derived from γδ T cells and usually with a fatal outcome, remains largely unknown. In this study, HSTL samples (7γδ and 2αβ) and the DERL2 HSTL cell line were subjected to combined gene-expression profiling and array-based comparative genomic hybridization. Compared with other T-cell lymphomas, HSTL had a distinct molecular signature irrespective of TCR cell lineage. Compared with peripheral T-cell lymphoma, not otherwise specified and normal γδ T cells, HSTL overexpressed genes encoding NK-cell-associated molecules, oncogenes (FOS and VAV3), the sphingosine-1-phosphatase receptor 5 involved in cell trafficking, and the tyrosine kinase SYK, whereas the tumor-suppressor gene AIM1 (absent in melanoma 1) was among the most down-expressed. We found highly methylated CpG islands of AIM1 in DERL2 cells, and decitabine treatment induced a significant increase in AIM1 transcripts. Syk was present in HSTL cells and DERL2 cells contained phosphorylated Syk and were sensitive to a Syk inhibitor in vitro. Genomic profiles confirmed recurrent isochromosome 7q (n = 6/9) without alterations at the SYK and AIM1 loci. Our results identify a distinct molecular signature for HSTL and highlight oncogenic pathways that offer rationale for exploring new therapeutic options such as Syk inhibitors and demethylating agents.

Electrochemical Reduction and Voltammetric Determination of Metronidazole Benzoate at Modified Carbon Paste Electrode

A metalloporphyrin incorporated carbon paste sensor has been developed for the determination of metronidazole benzoate (MTZB). Zn(II) complex of 5,10,15,20-tetrakis (3-methoxy-4-hydroxy phenyl) porphyrin (TMHPP) was used as the active material. The MTZB gave a well-defined reduction peak at - 0.713V in 0.1 mol l -1 phosphate buffer solution of pH around 7. Compared with bare carbon paste electrode (CPE), the TMHPP Zn(II) modified electrode significantly enhanced the reduction peak current of MTZB as well as lowered its reduction potential. Under optimum conditions the reduction peak current was proportional to MTZB concentration over the range 1×10-3 mol1-1 to 1×10-5mol1-1. The detection limit was found to be 4.36×10-6mol1-1 . This sensor has been successfully applied for the determination of MTZB in pharmaceutical formulations and urine samples.

Radical acetylation of 2`-deoxyguanosine and L-histidine coupled to the reaction of diacetyl with peroxynitrite in aerated medium

Diacetyl, like other alpha-dicarbonyl compounds, is reportedly cytotoxic and genotoxic. A food and cigarette contaminant, it is related with alcohol hepatotoxicity and lung disease. Peroxynitrite is a potent oxidant formed in vivo by the diffusion-controlled reaction of the superoxide radical anion with nitric oxide, which is able to form adducts with carbon dioxide and carbonyl compounds. Here, we investigate the nucleophilic addition of peroxynitrite to diacetyl forming acetyl radicals, whose reaction with molecular oxygen leads to acetate. Peroxynitrite is shown to react with diacetyl in phosphate buffer (bell-shaped pH profile with maximum at 7.2) at a very high rate constant (k(2) = 1.0 X 10(4) M-1 s(-1)) when compared with monocarbonyl substrates (k(2) < 10(3) M-1 s(-1)). Phosphate ions (100-500 MM) do not affect the rate of spontaneous peroxynitrite decay, but the H2PO4- anion catalyzes the nucleophilic addition of the peroxynitrite anion to diacetyl. The intermediacy of acetyl radicals is suggested by a three-line spectrum (a(N) = a(H) = 0.83 mT) obtained by EPR spin trapping of the reaction mixture with 2-methyl-2-nitrosopropane. The peroxynitrite reaction is accompanied by concentration-dependent oxygen uptake. Stoichiometric amounts of acetate from millimolar amounts of peroxynitrite and diacetyl were obtained under nonlimiting conditions of dissolved oxygen. In the presence of either L-histidine or 2`-deoxyguanosine, the peroxynitrite/diacetyl system afforded the corresponding acetylated molecules identified by HPLC-MS"". These studies provide evidence that the peroxynitrite/diacetyl reaction yields acetyl radicals and raise the hypothesis that protein and DNA nonenzymatic acetylation may occur in cells and be implicated in aging and metabolic disorders in which oxygen and nitrogen reactive species are putatively involved.

Amperometric detection of benzoyl peroxide in pharmaceutical preparations using carbon paste electrodes with peroxidases naturally immobilized on coconut fibers

This paper describes the applications of anew carbon paste electrode containing fibers of coconut (Cocus nucifera L) fruit, which are very rich in peroxidase enzymes naturally immobilized on its structure. The new sensor was applied for the amperometric quantification of benzoyl peroxide in facial creams and dermatological shampoos. The amperometric measurements were performed in 0.1 mol L(-1) phosphate buffer (pH 5.2), at 0.0 V (versus Ag/AgCl). On these conditions, benzoyl peroxide was rapidly determined in the 5.0-55 mu mol L(-1), with a detection limit of 2.5 mu mol L(-1) (s/n = 3), response time of 4.1 s (90% of the steady state) and sensitivity limit of 0.33 A mol L(-1) cm(-2). The amperometric results are in good agreement with those obtained by spectrophotometric technique, used as a standard method. (C) 2009 Elsevier B.V. All rights reserved.

Fast batch injection analysis of H(2)O(2) using an array of Pt-modified gold microelectrodes obtained from split electronic chips

A fast and robust analytical method for amperometric determination of hydrogen peroxide (H(2)O(2)) based on batch injection analysis (BIA) on an array of gold microelectrodes modified with platinum is proposed. The gold microelectrode array (n = 14) was obtained from electronic chips developed for surface mounted device technology (SMD), whose size offers advantages to adapt them in batch cells. The effect of the dispensing rate, volume injected, distance between the platinum microelectrodes and the pipette tip, as well as the volume of solution in the cell on the analytical response were evaluated. The method allows the H(2)O(2) amperometric determination in the concentration range from 0.8 mu mol L(-1) to 100 mu mol L(-1). The analytical frequency can attain 300 determinations per hour and the detection limit was estimated in 0.34 mu mol L(-1) (3 sigma). The anodic current peaks obtained after a series of 23 successive injections of 50 mu L of 25 mu mol L(-1) H(2)O(2) showed an RSD < 0.9%. To ensure the good selectivity to detect H(2)O(2), its determination was performed in a differential mode, with selective destruction of the H(2)O(2) with catalase in 10 mmol L(-1) phosphate buffer solution. Practical application of the analytical procedure involved H(2)O(2) determination in rainwater of Sao Paulo City. A comparison of the results obtained by the proposed ampermetric method with another one which combines flow injection analysis (FIA) with spectrophotometric detection showed good agreement. (C) 2011 Elsevier B.V. All rights reserved.

Implementing stepwise solvent elution in sequential injection chromatography for fluorimetric determination of intracellular free amino acids in the microalgae Tetraselmis gracilis

The concept of sequential injection chromatography (SIC) was exploited to automate the fluorimetric determination of amino acids after pre-column derivatization with ophthaldialdehyde (OPA) in presence of 2-mercaptoethanol (2MCE) using a reverse phase monolithic C(18) stationary phase. The method is low-priced and based on five steps of isocratic elutions. The first step employs the mixture methanol: tetrahydrofuran: 10 mmol L(-1) phosphate buffer (pH 7.2) at the volumetric ratio of 8:1:91; the other steps use methanol: 10 mmol L-1 phosphate buffer (pH 7.2) at volumetric ratios of 20:80, 35:65, SO:SO and 65:35. At a flow rate of 10 mu L s(-1) a 25 mm long-column was able to separate aspartic acid (Asp), glutamic acid (Glu), asparagine (Asn), serine (Ser), glutamine (Gln), glycine (Gly), threonine (Thr), citruline (Ctr), arginine (Arg), alanine (Ala), tyrosine (Tyr), phenylalanine (Phe), ornithine (Orn) and lysine (Lys) with resolution >1.2 as well as methionine (Met) and valine (Val) with resolution of 0.6. Under these conditions isoleucine (Ile) and leucine (Leu) co-eluted. The entire cycle of amino acids derivatization, chromatographic separation and column conditioning at the end of separation lasted 25 min. At a flow rate of 40 mu L s(-1) such time was reduced to 10 min at the cost of resolution worsening for the pairs Ctr/Arg and Orn/Lys. The detection limits varied from 0.092 mu mol L(-1) for Tyr to 0.51 mu mol L(-1) for Orn. The method was successfully applied to the determination of intracellular free amino acids in the green alga Tetraselmis gracilis during a period of seven days of cultivation. Samples spiked with known amounts of amino acids resulted in recoveries between 94 and 112%. (C) 2008 Elsevier B.V. All rights reserved.

Electrocatalytic behavior of glassy carbon electrodes modified with multiwalled carbon nanotubes and cobalt phthalocyanine for selective analysis of dopamine in presence of ascorbic acid

This work describes the development, electrochemical characterization and utilization of a cobalt phthalocyanine modified carbon nanotube electrode for the quantitative determination of dopamine in 0.2 mol L-1 phosphate buffer contaminated with high concentration of ascorbic acid. The electrode surface was analyzed by cyclic voltammetry and electrochemical impedance spectroscopy which showed a modified surface presenting a charge transfer resistance of 500 Omega, against the 16.46 k Omega value found for the bare glassy carbon surface. A pseudo rate constant value of 5.4 x 10(-4) cm s(-1) for dopamine oxidation was calculated. Voltammetric experiments showed a shift of the peak potential of DA oxidation to less positive value at 390 mV as compared with that of a bare GC electrode at 570 mV. The electrochemical determination of dopamine, in presence of ascorbic acid in concentrations up to 0.1 mol L-1 by differential pulse voltarnmetry, yielded a detection limit as low as 2.56 x 10(-7) mol L-1.

Determination of carbaryl in tomato ""in natura"" using an amperometric biosensor based on the inhibition of acetylcholinesterase activity

This work reports the utilization of two methodologies for carbaryl determination in tomatoes. The measurements were carried out using an amperometric biosensor technique based on the inhibition of acetylcholinesterase activity due to carbaryl adsorption and a HPLC procedure. The electrochemical experiments were performed in 0.1 mol L-1 phosphate buffer solutions at pH 7.4 with an incubation time of 8 min. The analytical curve obtained in pure solutions showed excellent linearity in the 5.0 x 10(-5) to 75 x 10(-5) mol L-1 range, with the limit of detection at 0.4 x 10(-3) gL(-1). The application of such a methodology in tomato samples involved solely liquidising the samples, which were spiked with 6.0 x 10(-6) and 5.0 x 10(-5) mol L-1 carbaryl. Recovery in such samples presented values of 99.0 and 92.4%, respectively. In order to obtain a comparison, HPLC experiments were also conducted under similar conditions. However, the tomato samples have to be manipulated by an extraction procedure (MSPD), which yielded much lower recovery values (78.3 and 84.8%, respectively). On the other hand, the detection limit obtained was much lower than that for the biosensor, i.e., 3.2 x 10(-6) g L-1. Finally, the biosensor methodology was employed to analyze carbaryl directly inside the tomato, without any previous manipulation. In this case, the biosensor was immersed in the tomato pulp, which had previously been spiked with the pesticide for 8 min, removed and inserted in the electrochemical cell. A recovery of 83.4% was obtained, showing very low interference of the matrix constituents. (C) 2007 Elsevier B.V. All rights reserved.