Histamine-producing pathway encoded on an unstable plasmid in Lactobacillus hilgardii 0006

Histamine production from histidine in fermented food products by lactic acid bacteria results in food spoilage and is harmful to consumers. We have isolated a histamine-producing lactic acid bacterium, Lactobacillus hilgardii strain IOEB 0006, which could retain or lose the ability to produce histamine depending on culture conditions. The hdcA gene, coding for the histidine decarboxylase of L. hilgardii IOEB 0006, was located on an 80-kb plasmid that proved to be unstable. Sequencing of the hdcA locus disclosed a four-gene cluster encoding the histidine decarboxylase, a protein of unknown function, a histidyl-tRNA synthetase, and a protein, which we named HdcP, showing similarities to integral membrane transporters driving substrate/product exchange. The gene coding for HdcP was cloned downstream of a sequence specifying a histidine tag and expressed in Lactococcus lactis. The recombinant HdcP could drive the uptake of histidine into the cell and the exchange of histidine and histamine. The combination of HdcP and the histidine decarboxylase forms a typical bacterial decarboxylation pathway that may generate metabolic energy or be involved in the acid stress response. Analyses of sequences present in databases suggest that the other two proteins have dispensable functions. These results describe for the first time the genes encoding a histamine-producing pathway and provide clues to the parsimonious distribution and the instability of histamine-producing lactic acid bacteria.     

Generation of a proton motive force by histidine decarboxylation and electrogenic histidine/histamine antiport in Lactobacillus buchneri.

Lactobacillus buchneri ST2A vigorously decarboxylates histidine to the biogenic amine histamine, which is excreted into the medium. Cells grown in the presence of histidine generate both a transmembrane pH gradient, inside alkaline, and an electrical potential (delta psi), inside negative, upon addition of histidine. Studies of the mechanism of histidine uptake and histamine excretion in membrane vesicles and proteoliposomes devoid of cytosolic histidine decarboxylase activity demonstrate that histidine uptake, histamine efflux, and histidine/histamine exchange are electrogenic processes. Histidine/histamine exchange is much faster than the unidirectional fluxes of these substrates, is inhibited by an inside-negative delta psi and is stimulated by an inside positive delta psi. These data suggest that the generation of metabolic energy from histidine decarboxylation results from an electrogenic histidine/histamine exchange and indirect proton extrusion due to the combined action of the decarboxylase and carrier-mediated exchange. The abundance of amino acid decarboxylation reactions among bacteria suggests that this mechanism of metabolic energy generation and/or pH regulation is widespread.     

Proteasome activity and autophagosome content in liver are reciprocally regulated by ethanol treatment

The proteasome and autophagy are two major intracellular protein degradation pathways and the regulation of each by ethanol metabolism affects cellular integrity. Using acute and chronic ethanol feeding to mice in vivo, and precision-cut rat liver slices (PCLS) ex vivo, we examined whether ethanol treatment altered these proteolytic pathways. In acute studies, we gave C57Bl/6 mice either ethanol or phosphate-buffered saline (PBS) by gastric intubation and sacrificed them 12h later. PCLS were exposed to 0 or 50mM ethanol for 12 and 24h with or without 4-methylpyrazole (4MP). In chronic studies we pair-fed control and ethanol liquid diets for 4-6 weeks to transgenic mice, expressing the green fluorescent protein (GFP) fused to the autophagic marker, microtubule associated protein-1 light chain 3 (LC3). Acute ethanol administration elevated autophagosomes (AVs), as judged by a 1.5-fold increase in LC3II content over PBS-gavaged control mice. Hepatic proteasome activity was unaffected by this treatment. Compared with controls, ethanol exposure for 12 and 24h to PCLS inhibited proteasome activity by 1.5- to 3-fold and simultaneously enhanced AVs by 2- to 5-fold. The decrease in proteasome activity and the rise in AVs both depended on ethanol oxidation as its inhibition by 4-methylpyrazole (4MP) blocked both proteasome inhibition and AV induction. Hepatocytes from mice chronically consuming ethanol exhibited a 1.6-fold decline in proteasome activity, and a 4-fold rise in GFP-LC3 puncta compared with pair-fed control mice. When we exposed hepatocytes from these animals to MG262, a proteasome inhibitor, LC3II puncta per cell further increased 2- to 5-fold over untreated cells. Conclusion: Our findings demonstrate that ethanol metabolism generates oxidants, the levels of which differentially influence the activities of the proteasome and autophagy.     

Phenotypic and proteomic analysis of positively regulated gellan biosynthesis pathway in Sphingomonas elodea

Sphingomonas elodea is a Gram-negative bacterium capable of producing ‘gellan gum’ exopolysaccharide that is the most extensively studied expolysaccharides of microbial origin. In this study, we investigated the phenotypic and proteomic alterations in S. elodea by homogeneously expressing both gelA and gelN involved in positive regulation and extracellular secretion of metabolites in gellan biosynthesis, respectively. Expression of six histidine-tagged GelA and GelN was determined by Western blot analysis. Successful expression of GelA and GelN resulted in both morphological changes of colonies and enhanced secretion of gellan into the growth medium (GelA, 21.2% more and GelN, 48.3% more) overexpressed compared to the wile-type. Comparative two-dimensional gel electrophoresis analysis revealed a differential proteome expression in S. elodea overexpressing GelA and GelN. Proteins up- or down-regulated by GelA and GelN overexpression were found to be mainly sugar transportation proteins, two-component regulatory proteins, and proteins involved in secretion pathways. The results suggest that the effect of GelA and GelN overexpression on gellan biosynthesis might be mainly caused by increased transportation of sugar units or enhanced exportation of gellan.     

Opioid and Notch signaling pathways are reciprocally regulated through miR- 29a and miR-212 expression

Background

The abuse of opioids, such as morphine and phentanyl or other drugs as heroin is a social and health problem that affects an increasing number of people each year. The activation of the mu opioid receptor triggers several molecular changes that alter the expression of diverse genes, including miRNAs. The dysregulation of these molecules could explain some of the developmental alterations that are induced after drug intake. In addition, the Notch signaling cascade has also been related to alterations on these processes.

Proteins involved in platelet signaling are differentially regulated in acute coronary syndrome: a proteomic study

Background

Platelets play a fundamental role in pathological events underlying acute coronary syndrome (ACS). Because platelets do not have a nucleus, proteomics constitutes an optimal approach to follow platelet molecular events associated with the onset of the acute episode.

Methodology/Principal Findings

We performed the first high-resolution two-dimensional gel electrophoresis-based proteome analysis of circulating platelets from patients with non-ST segment elevation ACS (NSTE-ACS). Proteins were identified by mass spectrometry and validations were by western blotting. Forty protein features (corresponding to 22 unique genes) were found to be differentially regulated between NSTE-ACS patients and matched controls with chronic ischemic cardiopathy. The number of differences decreased at day 5 (28) and 6 months after the acute event (5). Interestingly, a systems biology approach demonstrated that 16 of the 22 differentially regulated proteins identified are interconnected as part of a common network related to cell assembly and organization and cell morphology, processes very related to platelet activation. Indeed, 14 of those proteins are either signaling or cytoskeletal, and nine of them are known to participate in platelet activation by αIIbβ3 and/or GPVI receptors. Several of the proteins identified participate in platelet activation through post-translational modifications, as shown here for ILK, Src and Talin. Interestingly, the platelet-secreted glycoprotein SPARC was down-regulated in NSTE-ACS patients compared to stable controls, which is consistent with a secretion process from activated platelets.

Conclusions/Significance

The present study provides novel information on platelet proteome changes associated with platelet activation in NSTE-ACS, highlighting the presence of proteins involved in platelet signaling. This investigation paves the way for future studies in the search for novel platelet-related biomarkers and drug targets in ACS.     

The arginine deiminase pathway in the wine lactic acid bacterium Lactobacillus hilgardii X1B: structural and functional study of the arcABC genes

The genes implicated in the catabolism of the amino acid arginine by Lactobacillus hilgardii X(1)B were investigated to assess the potential for formation of ethyl carbamate precursors in wine. L. hilgardii X(1)B can use arginine via the arginine deiminase pathway. The complete nucleotide sequence of the arc genes involved in this pathway has been determined. They are clustered in an operon-like structure in the order arcABC. No evidence was found for the presence of a homologue of the arcD gene, coding for the arginine/ornithine antiporter. The arc genes have been expressed in Escherichia coli resulting in arginine deiminase (ArcA), ornithine carbamoyltransfera (ArcB) and carbamate kinase (ArcC) activities. The results indicate the need for caution in the selection of lactic acid bacteria for conducting malolactic fermentation in wine since arginine degradation could result in high amounts of ethyl carbamate.     

The mechanism of the tyrosine transporter TyrP supports a proton motive tyrosine decarboxylation pathway in Lactobacillus brevis

The tyrosine decarboxylase operon of Lactobacillus brevis IOEB9809 contains, adjacent to the tyrosine decarboxylase gene, a gene for TyrP, a putative tyrosine transporter. The two genes potentially form a proton motive tyrosine decarboxylation pathway. The putative tyrosine transporter gene of L. brevis was expressed in Lactococcus lactis and functionally characterized using right-side-out membranes. The transporter very efficiently catalyzes homologous tyrosine-tyrosine exchange and heterologous exchange between tyrosine and its decarboxylation product tyramine. Tyrosine-tyramine exchange was shown to be electrogenic. In addition to the exchange mode, the transporter catalyzes tyrosine uniport but at a much lower rate. Analysis of the substrate specificity of the transporter by use of a set of 19 different tyrosine substrate analogues showed that the main interactions between the protein and the substrates involve the amino group and the phenyl ring with the para hydroxyl group. The carboxylate group that is removed in the decarboxylation reaction does not seem to contribute to the affinity of the protein for the substrates significantly. The properties of the TyrP protein are those typical for precursor-product exchangers that operate in proton motive decarboxylation pathways. It is proposed that tyrosine decarboxylation in L. brevis results in proton motive force generation by an indirect proton pumping mechanism.     

Hyperosmotic oxidation of glucose and decarboxylation of histidine in the gastric mucosa.

Paired slices of rat gastric mucosa were incubated with labeled glucose or histidine in isosomotic solution of 3-fold hyperosmotic solutions concentrated in NaCl, KCl, or ethanol. The rate of (1-14C)glucose oxidation to 14CO2 in isosmotic solution was reduced by 74% in hyperosmotic NaCl and by 28% in hyperosmotic KCl. The rate of (6-14C)glucose oxidation to 14CO2 in isosmotic solution was reduced by 64% in hyperosmotic NaCl and by 53% in hyperosmotic KCl. Reductions of glucose oxidation in hyperosmotic ethanol were not significant. The ratio of 14CO2 formed from (1-14C)glucose to that formed from (6-14C)glucose was not significantly changes by hyperosmotic NaCl or ethanol, but was significantly raised by hyperosmotic KCl. The rate of (carboxyl-14C)histidine decarboxylation in isosomotic solution was reduced significantly by 48% in hyperosmotic NaCl, by 30% in hyperosmotic KCl, and by 27% in hyperosmotic ethanol. We conclude that hyperosmotic solutions reduce glucose oxidation and histidine decarboxylation by rat gastric mucosa in the order of potency: NaCl greater than Kcl greater than or equal to ethanol. Thus hyperosmotic solutions inhibit the source of metabolic energy for stimulated acid secretion, the citric acid cycle, and the formation of the secretagogue histamine.     

High-fat-diet-induced obesity and heart dysfunction are regulated by the TOR pathway in Drosophila

High-fat-diet (HFD)-induced obesity is a major contributor to diabetes and cardiovascular disease, but the underlying genetic mechanisms are poorly understood. Here, we use Drosophila to test the hypothesis that HFD-induced obesity and associated cardiac complications have early evolutionary origins involving nutrient-sensing signal transduction pathways. We find that HFD-fed flies exhibit increased triglyceride (TG) fat and alterations in insulin/glucose homeostasis, similar to mammalian responses. A HFD also causes cardiac lipid accumulation, reduced cardiac contractility, conduction blocks, and severe structural pathologies, reminiscent of diabetic cardiomyopathies. Remarkably, these metabolic and cardiotoxic phenotypes elicited by HFD are blocked by inhibiting insulin-TOR signaling. Moreover, reducing insulin-TOR activity (by expressing TSC1-2, 4EBP or FOXO), or increasing lipase expression-only within the myocardium-suffices to efficiently alleviate cardiac fat accumulation and dysfunction induced by HFD. We conclude that deregulation of insulin-TOR signaling due to a HFD is responsible for mediating the detrimental effects on metabolism and heart function.     

CD40 ligand and CTLA-4 are reciprocally regulated in the Th1 cell proliferative response sustained by CD8(+) dendritic cells

Subsets of murine dendritic cells (DCs) from the spleen differ in their ability to induce proliferative responses in both primary and secondary CD4(+) T cells. Recent evidence indicates that lymphoid-related CD8(+) DCs fail to provide appropriate signals to freshly isolated secondary CD4(+) T cells to sustain their proliferation in vitro. In the present study, we examined peptide-pulsed CD8(-) and CD8(+) DCs for ability to stimulate Th1 and Th2 cell clones with the same Ag specificity. Defective ability to induce proliferation was selectively shown by CD8(+) DCs presenting Ag to the Th1 clone. The deficiency in CD8(+) DCs was overcome by CD40 triggering before peptide pulsing. When exposed to CD8(+) DCs in the absence of CD40 activation, the Th1 clone expressed low levels of CD40 ligand and high levels of surface CTLA-4. Neutralization of CTLA-4 during the DC/T cell coculture resulted in increased CD40 ligand expression and proliferation of T cells. Remarkably, the activation of CD40 on DCs under conditions that would increase Th1 cell proliferation, also resulted in down-regulation of surface CTLA-4. These results confirm differential effects of CD8(+) and CD8(-) DCs in the stimulation of Ag-primed Th cells. In addition, they suggest that reciprocal regulation of CD40 ligand and CTLA-4 expression occurs in Th1 cells exposed to CD8(+) DCs.     

Genetic influences on oestrous cyclicity in mice: evidence that cycle length and frequency are differentially regulated.

Studies in C57BL/6J, DBA/2J and C3H/HeJ mice and in two F1 hybrid strains (B6D2F1 and B6C3HF1) 2-5 months old revealed marked genotypic differences among inbred strains. C57 mice had three times as many regular (3-6 days) cycles as DBA and C3H mice, due largely to fewer pseudopregnant-like (7-14 day) cycles. C57 had longer regular cycles than DBA and C3H mice. Although the frequencies of regular cycles of DBA and C3H mice were similar, the cycles of C3H mice were shorter than those of DBA mice. The results indicated that the genetic determinants of the frequency of regular cycles differ from those specifying cycle length. Frequency of regular cycles of F1 hybrids was either intermediate between the parent strains (B6D2F1) or similar to the C57 strain (B6C3HF1), suggesting that regular cycle frequency shows additive genetic variation in the former crosses, but mostly dominant variance in the latter background. Regular cycles were either shorter than in both parent strains (B6D2F1) or similar to one of them (B6C3HF1), indicating heterosis and dominance for genes specifying short cycles. Although the lack of reciprocal crosses meant that maternal effects and possible genomic imprinting effects could not be assessed, these results reveal marked genetic influences on cycle length and frequency and suggest that some of the genes specifying these two traits differ.     

Gold nanoparticles and bioconjugation: a pathway for proteomic applications

In the last few years gold nanoparticles (AuNPs) became extremely interesting materials due to their enhanced optical, chemical and electrical properties. With the intention of taking advantage of those properties, the use of AuNPs has spread into a wide variety of areas such as physics, chemistry, biology, industry and medicine. More interestingly, their ability to form robust conjugates with biomolecules has given proteomics a new tool to improve aspects where the current methods to study proteins and their interactions in living cells cannot achieve the success required. In this review we present some of the current methods for AuNPs synthesis, the tailoring of their surface with ligands to improve stability and strategies to conjugate with biomolecules. Lastly, we also discuss their application in proteomic methods and recent developments in clinical diagnosis.     

CXCR4/CCR5 down-modulation and chemotaxis are regulated by the proteasome pathway

Chemokines and their receptors play a critical role in host immune surveillance and are important mediators of human immunodeficiency virus (HIV) pathogenesis and inflammatory response. The chemokine receptors CCR5 and CXCR4, which act as co-receptors along with CD4 for HIV docking and entry, are down-modulated by their respective ligands, MIP-1beta/SDF-1alpha or by the HIV envelope protein, gp120. We have studied the role of the proteasome pathway in the down-regulation of these receptors. Using the yeast and mammalian two-hybrid systems, we observed that the CCR5 receptor is constitutively associated with the zeta subunit of proteasome. Immunoprecipitation studies in CCR5 L1.2 cells revealed that this association was increased with MIP-1beta stimulation. The proteasome inhibitors, lactacystin and epoxomicin, attenuated MIP-1beta induced CCR5 down-modulation as detected by fluorescence-activated cell sorter analysis and confocal microscopy. The proteasome inhibitors also inhibited the SDF-1alpha and gp120 protein-induced down-modulation of the CXCR4 receptor in Jurkat cells. However, the inhibitors had no significant effect on the gp120-induced internalization of the CD4 receptor. These inhibitors also blocked cognate ligand-mediated chemotaxis but had no effect on SDF-1alpha-induced p44/42 MAP kinase or MIP-1beta-induced p38 kinase activities, thus indicating differential effects of the inhibitors on signaling mediated by these receptors. These results indicate that the CCR5 and CXCR4 receptor down-modulation mechanism and chemotaxis mediated by these receptors are dependent upon proteasome activity.     

Increased hexosamine pathway flux and high fat feeding are not additive in inducing insulin resistance: evidence for a shared pathway

Excess fatty acids and carbohydrates have both been implicated in the pathogenesis of type 2 diabetes, and both can reproduce essential features of the disease including insulin resistance and beta cell failure. It has been proposed that both nutrients may regulate metabolism through a common fuel sensing mechanism, namely hexosamine synthesis. We have previously shown that transgenic overexpression of the rate-limiting enzyme for hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase (GFA), targeted to muscle and fat, leads to insulin resistance mediated by increased O-linked glycosylation of nuclear and cytosolic proteins. We report here that hexosamine-induced insulin resistance is not additive with that induced by high fat feeding. In control mice fed a high fat diet, glucose disposal rates during euglycemic hyperinsulinemia were decreased by 37% (p < 0.02) compared to mice on a low fat diet. Transgenic mice overexpressing GFA and fed a low fat diet exhibited a 51% decrease in glucose disposal compared to controls on a low fat diet (p < 0.001), but no further decrease was evident in the transgenic mice fed a high fat diet. Decreased glucose disposal rates were mirrored by increases in skeletal muscle levels of the principal end product of the hexosamine pathway, UDP-N-acetyl glucosamine. Serum leptin levels, which are modulated both by feeding and hexosamine flux, also show no additivity in their stimulation by GFA overexpression and high fat feeding. These data are consistent with a shared nutrient sensing pathway for high fat and carbohydrate fluxes and a common pathway by which glucose and lipids induce insulin resistance.     

Arginine dihydrolase pathway in Lactobacillus buchneri: a review

The arginine dihydrolase system was studied in homo- and hetero-fermentative lactic acid bacteria. This system is widely distributed in Betabacteria lactobacilli subgroup (group II in Bergey's Manual). It is generally absent in the Thermobacterium lactobacilli subgroup (group IA in Bergey's Manual) and also in the Streptobacterium subgroup (group IB in Bergey's Manual). It is present in some species of the genus Streptococcus (groups II, III and IV in Bergey's Manual). In Lactobacillus buchneri NCDO110 the 3 enzymes of the arginine dihydrolase pathway, arginine deiminase, ornithine transcarbamylase and carbamate kinase, were purified and characterized. Arginine deiminase was partially purified (68-fold); ornithine transcarbamylase was also partially purified (14-fold), while carbamate kinase was purified to homogeneity. The apparent molecular weight of the enzymes was 199,000, 162,000 and 97,000 for arginine deiminase, ornithine transcarbamylase and carbamate kinase respectively. For arginine deiminase, maximum enzymatic activity was observed at 50 degrees C and pH 6; for ornithine transcarbamylase it was observed at 35 degrees C and pH 8.5, and for carbamate kinase at 30 degrees C and pH 5.4. The activation energy of the reactions was determined. For arginine deiminase, delta G* values were: 8,700 cal mol-1 below 50 degrees C and 380 cal mol-1 above 50 degrees C; for ornithine transcarbamylase, the values were: 9,100 cal mol-1 below 35 degrees C and 4,300 cal mol-1 above 35 degrees C; for carbamate kinase, the activation energy was: 4,078 cal mol-1 for the reaction with Mn2+ and 3,059 cal mol-1 for the reaction with Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prolactin and insulin are targeted to the regulated pathway in GH4C1 cells, but their storage is differentially regulated

PRL storage in GH4C1 cells, rat pituitary tumor cells, can be induced by treatment with a combination of estradiol, epidermal growth factor, and bovine insulin. This increase in storage is characterized by a preferential increase in intracellular PRL compared with secreted PRL and a 50-fold increase in the number of secretory granules. Treatment with the combined hormones stimulates PRL synthesis approximately 6-fold, but this effect is not sufficient to increase PRL storage, because epidermal growth factor alone increases PRL synthesis to the same extent without affecting storage. The cDNA for human proinsulin down-stream of the RSV-LTR promoter was transfected into GH4C1 cells to determine whether storage of another protein known to be targeted to the regulated pathway would also increase with hormone treatment. Proinsulin and PRL release were stimulated over the same time course and to the same peak height, compared to basal release, by both KCl and TRH, indicating that proinsulin is targeted to the regulated pathway in GH4C1 cells. There was little intracellular processing of proinsulin to insulin. Proinsulin synthesis increased 3.9-fold with the combined treatment, assessed by accumulation of proinsulin immunoreactivity in the medium and increases at the mRNA level. Treatment with the combined hormones did not cause the preferential increase in intracellular proinsulin that occurred with PRL; the increase in intracellular proinsulin could be accounted for primarily by effects on synthesis. These results suggest that storage of the two hormones can be differentially regulated.     

Expression and characterization of Lactobacillus 30a histidine decarboxylase in Escherichia coli

The genes coding for histidine decarboxylase from a wild-type strain and an autoactivation mutant strain of Lactobacillus 30a have been cloned and expressed in Escherichia coli. The mutant protein, G58D, has a single Asp for Gly substitution at position 58. The cloned genes were placed under control of the beta-galactosidase promoter and the products are natural length, not fusion proteins. The enzyme kinetics of the proteins isolated from E. coli are comparable to those isolated from Lactobacillus 30a. At pH 4.8 the Km of wild-type enzyme is 0.4 mM and the kcat = 2800 min-1; the corresponding values for G58D are 0.5 mM and 2750 min-1. The wild-type and G58D have autoactivation half-times of 21 and 9 h respectively under pseudophysiological conditions of 150 mM K+ and pH 7.0. At pH 7.6 and 0.8 M K+ the half-times are 4.9 and 2.9 h. The relatively slow rate of autoactivation for purified protein and the differences in cellular and non-cellular activation rates, coupled with the fact that wild-type protein is readily activated in wild-type Lactobacillus 30a but poorly activated in E. coli, suggest that wild-type Lactobacillus 30a contains a factor, possibly an enzyme, that enhances the activation rate.     

Effect of inactivation of ccpA and aerobic growth in Lactobacillus plantarum: A proteomic perspective

Lactobacillus plantarum is a facultative heterofermentative lactic acid bacterium widely used in the production of most fermented food due to its ability to thrive in several environmental niches, including the human gut. In order to cope with different growth conditions, it has developed complex molecular response mechanisms, characterized by the induction of a large set of proteins mainly regulated by HrcA and CtsR repressors as well as by global regulators such as carbon catabolite control protein A (CcpA). In this study, the role of CcpA in the regulation of growth under anaerobiosis and aerobiosis, and the adaptation to aeration in L. plantarum WCFS1 were comprehensively investigated by differential proteomics. The inactivation of ccpA, in both growth conditions, significantly changed the expression level of 76 proteins, mainly associated with carbohydrate and energy metabolism, membrane transport, nucleotide metabolism, protein biosynthesis and folding. The role of CcpA as pleiotropic regulator was particularly evident at the shift from homolactic fermentation to mixed fermentation. Proteomic results also indicated that the mutant strain was more responsive to aerobic growth condition.