Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A 6-month clinical trial of azithromycin (AZM) in American cystic fibrosis (CF) patients with chronic Pseudomonas aeruginosa infection showed clinical improvement without significant reduction in bacterial density. Sub-inhibitory AZM has been hypothesized to affect P. aeruginosa virulence, partly contributing to the mechanism of action of AZM. To correlate bacterial phenotypes of P. aeruginosa isolates with clinical response to AZM in CF patients. Pre-treatment P. aeruginosa isolates from subjects randomized to AZM in the US trial were characterized for bacterial phenotypes: AZM minimal inhibitory concentration (MIC), mucoidy, and baseline and AZM effects on twitching and swimming motility, and production of pyocyanin, protease and phospholipase C (PLC). Initial analyses of a subset of subjects identified phenotypes most strongly associated with FEV(1) response and pulmonary exacerbation. These phenotypes were subsequently characterized and tested in isolates from subjects of the complete AZM cohort. Exploratory analyses of the initial subset suggested that the MIC and in vitro change in PLC and swimming motility with AZM were the strongest candidates among the bacterial phenotypes. When tested, only the change in PLC was significantly correlated with the change in FEV(1) (P=0.05), and occurrence and time to pulmonary exacerbation (both P=0.02). In the complete cohort, change in PLC continued to show significant correlation with FEV(1) response (P=0.006), but not exacerbation. The in vitro effect of AZM on PLC correlates with FEV(1) response to AZM. This suggests that AZM anti-virulence effects may be predictive of clinical response and play a role in the mechanism of action of AZM in CF patients.
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PMID:Clinical response to azithromycin in cystic fibrosis correlates with in vitro effects on Pseudomonas aeruginosa phenotypes. 1746 54

Knowledge about the virulence mechanisms of species from the Burkholderia cepacia complex (BCC) is still limited. The genomovar heterogeneity and production of different virulence factors are likely to contribute to the variation in the clinical outcome observed in BCC-infected cystic fibrosis (CF) patients. Therefore, in this study we investigated the genetic polimorphism, the presence of genetic makers associated with virulence and transmissibility in BCC, and the profile of exoenzyme production of 59 BCC isolates obtained from 59 CF patients attending the reference CF centre in Rio de Janeiro, Brazil. The DNA sequence analyses of the recA gene allowed us to identify 40 of these 59 BCC species as being B. cenocepacia, 9 as B. vietnamiensis, 6 as B. multivorans and 4 as B. ambifaria. The assessment of the bacterial genetic polymorphism by PFGE revealed that B. cenocepacia and the B. multivorans isolates belonged to four and two different PFGE profiles with prevalence of two clones, A and B, respectively. All B. vietnamiensis and B. ambifaria belonged to only one PFGE profile (J and E, respectively). None of the isolates exhibited the genetic markers cblA and BCESM, assessed by polymerase chain reaction. In contrast, the profile of enzymatic activity, assessed by phenotypic methods, differed among the BCC species: protease activity was detected only in B. cenocepacia and B. ambifaria isolates, whereas only B. vietnamiensis isolates produced hemolysin. Although the phospholipase C activity was similar among the different species, the level of lipase activity produced by B. multivorans was higher than in the other species. We speculate that the differential characteristics of exoenzyme production may account for the differences in the pathogenic potentials of each BCC species.
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PMID:Burkholderia cenocepacia, B. multivorans, B. ambifaria and B. vietnamiensis isolates from cystic fibrosis patients have different profiles of exoenzyme production. 1750 97

Inositol 1,3,4-trisphosphate 5/6-kinase (ITPK1) is a reversible, poly-specific inositol phosphate kinase that has been implicated as a modifier gene in cystic fibrosis. Upon activation of phospholipase C at the plasma membrane, inositol 1,4,5-trisphosphate enters the cytosol and is inter-converted by an array of kinases and phosphatases into other inositol phosphates with diverse and critical cellular activities. In mammals it has been established that inositol 1,3,4-trisphosphate, produced from inositol 1,4,5-trisphosphate, lies in a branch of the metabolic pathway that is separate from inositol 3,4,5,6-tetrakisphosphate, which inhibits plasma membrane chloride channels. We have determined the molecular mechanism for communication between these two pathways, showing that phosphate is transferred between inositol phosphates via ITPK1-bound nucleotide. Intersubstrate phosphate transfer explains how competing substrates are able to stimulate each others' catalysis by ITPK1. We further show that these features occur in the human protein, but not in plant or protozoan homologues. The high resolution structure of human ITPK1 identifies novel secondary structural features able to impart substrate selectivity and enhance nucleotide binding, thereby promoting intersubstrate phosphate transfer. Our work describes a novel mode of substrate regulation and provides insight into the enzyme evolution of a signaling mechanism from a metabolic role.
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PMID:Integration of inositol phosphate signaling pathways via human ITPK1. 1761 25

Taste receptor cells (TRCs) are the sensory cells of taste transduction and are organized into taste buds embedded in the epithelium of the tongue, palate, pharynx, and larynx. Several studies have demonstrated that TRCs involved in sweet as well as bitter and umami responses express alpha-gustducin, an alpha-subunit of the G-protein complex. It has been further demonstrated that this typical taste protein is a potent marker of chemosensory cells located in several tissues, including gastric and pancreatic mucosa and the respiratory apparatus. We recently observed that alpha-gustducin and phospholipase C beta 2-immunoreactive cells were colocalized in the airways with cystic fibrosis transmembrane regulator (CFTR) and Clara cell-specific secretory protein of 10 (CC10) and 26 kDa (CC26). This finding suggests that TRCs might themselves express secretory markers. To test this hypothesis, we investigated the expression of CFTR, CC10, and CC26 in rat circumvallate papillae using reverse transcriptase-polymerase chain reaction analysis, immunohistochemistry, and confocal laser microscopy. The results showed that secretory markers such as CFTR, CC10, and CC26 are present in taste cells of rat circumvallate papillae, and their immunoreactivity is expressed, to a different extent, in subsets of taste cells that express alpha-gustducin. The presence of CFTR, CC10, and CC26 in taste bud cells and their coexpression pattern with alpha-gustducin confirms and extends our previous findings in airway epithelium, lending further credence to the notion that chemoreception and secretion may be related processes.
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PMID:Immunohistochemical localization of cystic fibrosis transmembrane regulator and clara cell secretory protein in taste receptor cells of rat circumvallate papillae. 1815 3

Cystic fibrosis (CF) is caused by mutations in the gene producing the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR functions as a Cl(-) channel. Its dysfunction limits Cl(-) secretion and enhances Na+ absorption, leading to viscous mucus in the airway. Ca2+-activated Cl(-) channels (CaCCs) are coexpressed with CFTR in the airway surface epithelia. Increases in cytosolic Ca(2+) activate the epithelial CaCCs, which provides an alternative Cl(-) secretory pathway in CF. We developed a screening assay and screened a library for compounds that could enhance cytoplasmic Ca2+, activate the CaCC, and increase Cl(-) secretion. We found that spiperone, a known antipsychotic drug, is a potent intracellular Ca2+ enhancer and demonstrated that it stimulates intracellular Ca2+, not by acting in its well-known role as an antagonist of serotonin 5-HT2 or dopamine D2 receptors, but through a protein tyrosine kinase-coupled phospholipase C-dependent pathway. Spiperone activates CaCCs, which stimulates Cl(-) secretion in polarized human non-CF and CF airway epithelial cell monolayers in vitro and in CFTR-knockout mice in vivo. In conclusion, we have identified spiperone as a new therapeutic platform for correction of defective Cl(-) secretion in CF via a pathway independent of CFTR.
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PMID:Spiperone, identified through compound screening, activates calcium-dependent chloride secretion in the airway. 1898 51

Human bronchial epithelial (HBE) cells exhibit constitutive anion secretion that is absent in cells from cystic fibrosis (CF) patients. The identity of this conductance is unknown, but SLC26A9, a member of the SLC26 family of CF transmembrane conductance regulator (CFTR)-interacting transporters, is found in the human airway and exhibits chloride channel behavior. We sought differences in the properties of SLC26A9 and CFTR expressed in HEK 293 (HEK) cells as a fingerprint to identify HBE apical anion conductances. HEK cells expressing SLC26A9 displayed a constitutive chloride current that was inhibited by the CFTR blocker GlyH-101 (71 +/- 4%, 50 microM) and exhibited a near-linear current-voltage (I-V) relation during block, while GlyH-101-inhibited wild-type (wt)CFTR exhibited a strong inward-rectified (IR) I-V relation. We tested polarized HBE cells endogenously expressing either wt or DeltaF508-CFTR for similar activity. After electrical isolation of the apical membrane using basolateral alpha-toxin permeabilization, wtCFTR monolayers displayed constitutive chloride currents that were inhibited by GlyH-101 (68 +/- 6%) while maintaining a near-linear I-V relation. In the absence of blocker, the addition of forskolin stimulated a current increase having a linear I-V; GlyH-101 blocked 69 +/- 7% of the current and shifted the I-V relation IR, consistent with CFTR activation. HEK cells coexpressing SLC26A9 and wtCFTR displayed similar properties, as well as forskolin-stimulated currents that exceeded the sum of those in cells separately expressing SLC26A9 or wtCFTR, and an I-V relation during GlyH-101 inhibition that was moderately IR, indicating that SLC26A9 contributed to the stimulated current. HBE cells from CF patients expressed SLC26A9 mRNA, but no constitutive chloride currents. HEK cells coexpressing SLC26A9 with DeltaF508-CFTR also failed to exhibit SLC26A9 current. We conclude that SLC26A9 functions as an anion conductance in the apical membranes of HBE cells, it contributes to transepithelial chloride currents under basal and cAMP/protein kinase A-stimulated conditions, and its activity in HBE cells requires functional CFTR.
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PMID:SLC26A9 is a constitutively active, CFTR-regulated anion conductance in human bronchial epithelia. 1928 74

The chemical composition of the luminal content is now accepted to have a profound influence on the performance of chemosensory receptors. Gustatory and intestinal chemoreceptors have in common their expression of molecules involved in taste sensing and signal transduction pathways. The recent finding that enterocytes of the duodenal epithelium are capable of expressing luminal pancreatic amylase suggests that taste cells of the gustatory epithelium might, in the same way, express salivary amylase in the oral cavity. Therefore, we investigated amylase expression in rat circumvallate papillae by using analyses involving immunohistochemistry, Western blot, and reverse transcription with the polymerase chain reaction. In addition, we used double-labeling confocal laser microscopy to compare amylase immunolabeling with that of the following markers: protein gene product 9.5 (PGP 9.5) and chromogranin A (CgA) for endocrine cells, alpha-gustducin and phospholipase C beta 2 (PLC beta 2) as taste-signaling molecules, and cystic fibrosis transmembrane regulator (CFTR) and Clara-cell-specific secretory protein of 10-kDa (CC10) as secretory markers. The results showed that amylase was present in some taste bud cells; its immunoreactivity was observed in subsets of cells that expressed CgA, alpha-gustducin, PLC beta 2, CFTR, or CC10. PGP 9.5 immunoreactivity was never colocalized with amylase. The data suggest that amylase-positive cells constitute an additional subset of taste receptor cells also associated with chemoreceptorial and/or secretory molecules, confirming the occurrence of various pathways in taste buds.
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PMID:Amylase expression in taste receptor cells of rat circumvallate papillae. 1940 14

Mucous hypersecretion is a serious feature of chronic airway diseases such as asthma, chronic obstructive pulmonary disease (COPD), and cystic fibrosis. Although mucins are produced via activation of an EGF receptor (EGFR) signaling cascade, the mechanisms leading to exaggerated mucin production in mucous hypersecretory diseases are unknown. Because expression of ICAM-1 and of the ICAM-1 ligand fibrinogen is increased in the airways of subjects with mucous hypersecretory diseases, we hypothesized that fibrinogen binding to ICAM-1 could increase EGFR-dependent mucin production in human airway (NCI-H292) epithelial cells. Consistent with this hypothesis, we found that an ICAM-1 neutralizing antibody and an ICAM-1(8-22) peptide that binds fibrinogen decreased mucin production induced by the EGFR ligand transforming growth factor (TGF)-alpha dose-dependently. Exogenous fibrinogen and a fibrinogen(117-133) peptide that binds ICAM-1 rescued mucin production in cells treated with the ICAM-1(8-22) peptide. Surprisingly, the ICAM-1(8-22) peptide increased EGFR phosphotyrosine and phospho-ERK1/2 in cells treated with TGF-alpha. The ICAM-1(8-22) peptide-induced increases in EGFR phosphotyrosine and phospho-ERK1/2 were prevented by exogenous fibrinogen, by the fibrinogen(117-133) peptide, and by selective inhibitors of phospholipase C (PLC), protein kinase C (PKC)-alpha/beta, and metalloproteases. These results suggest that fibrinogen binding to ICAM-1 promotes mucin production by decreasing TGF-alpha-induced EGFR and ERK1/2 activation and that the fibrinogen-ICAM-1-dependent decrease in EGFR and ERK1/2 activation occurs via inhibition of an early positive feedback pathway involving PLC- and PKC-alpha/beta-dependent metalloprotease activation and subsequent metalloprotease-dependent EGFR reactivation.
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PMID:Fibrinogen binding to ICAM-1 promotes EGFR-dependent mucin production in human airway epithelial cells. 1942 76

Mycobacterium abscessus is an emerging rapidly growing mycobacterium (RGM) causing a pseudotuberculous lung disease to which patients with cystic fibrosis (CF) are particularly susceptible. We report here its complete genome sequence. The genome of M. abscessus (CIP 104536T) consists of a 5,067,172-bp circular chromosome including 4920 predicted coding sequences (CDS), an 81-kb full-length prophage and 5 IS elements, and a 23-kb mercury resistance plasmid almost identical to pMM23 from Mycobacterium marinum. The chromosome encodes many virulence proteins and virulence protein families absent or present in only small numbers in the model RGM species Mycobacterium smegmatis. Many of these proteins are encoded by genes belonging to a "mycobacterial" gene pool (e.g. PE and PPE proteins, MCE and YrbE proteins, lipoprotein LpqH precursors). However, many others (e.g. phospholipase C, MgtC, MsrA, ABC Fe(3+) transporter) appear to have been horizontally acquired from distantly related environmental bacteria with a high G+C content, mostly actinobacteria (e.g. Rhodococcus sp., Streptomyces sp.) and pseudomonads. We also identified several metabolic regions acquired from actinobacteria and pseudomonads (relating to phenazine biosynthesis, homogentisate catabolism, phenylacetic acid degradation, DNA degradation) not present in the M. smegmatis genome. Many of the "non mycobacterial" factors detected in M. abscessus are also present in two of the pathogens most frequently isolated from CF patients, Pseudomonas aeruginosa and Burkholderia cepacia. This study elucidates the genetic basis of the unique pathogenicity of M. abscessus among RGM, and raises the question of similar mechanisms of pathogenicity shared by unrelated organisms in CF patients.
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PMID:Non mycobacterial virulence genes in the genome of the emerging pathogen Mycobacterium abscessus. 1954 27

Intestinal Cl- secretion is stimulated by cyclic AMP (cAMP) and intracellular calcium ([Ca2+]i). Recent studies show that protein kinase A (PKA) and the exchange protein directly activated by cAMP (Epac) are downstream targets of cAMP. Therefore, we tested whether both PKA and Epac are involved in forskolin (FSK)/cAMP-stimulated Cl- secretion. Human intestinal T84 cells and mouse small intestine were used for short circuit current (I(sc)) measurement in response to agonist-stimulated Cl- secretion. FSK-stimulated Cl- secretion was completely inhibited by the additive effects of the PKA inhibitor, H89 (1 microM), and the [Ca2+]i chelator, 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM; 25 microM). Both FSK and the Epac activator 8-pCPT-2'-O-Me-cAMP (50 microM) elevated [Ca2+]i, activated Ras-related protein 2, and induced Cl- secretion in intact or basolateral membrane-permeabilized T84 cells and mouse ileal sheets. The effects of 8-pCPT-2'-O-Me-cAMP were completely abolished by BAPTA-AM, but not by H89. In contrast, T84 cells with silenced Epac1 had a reduced I(sc) response to FSK, and this response was completely inhibited by H89, but not by the phospholipase C inhibitor U73122 or BAPTA-AM. The stimulatory effect of 8-pCPT-2'-O-Me-cAMP on Cl- secretion was not abolished by cystic fibrosis transmembrane conductance (CFTR) inhibitor 172 or glibenclamide, suggesting that CFTR channels are not involved. This was confirmed by lack of effect of 8-pCPT-2'-O-Me-cAMP on whole cell patch clamp recordings of CFTR currents in Chinese hamster ovary cells transiently expressing the human CFTR channel. Furthermore, biophysical characterization of the Epac1-dependent Cl- conductance of T84 cells mounted in Ussing chambers suggested that this conductance was hyperpolarization activated, inwardly rectifying, and displayed a Cl->Br->I- permeability sequence. These results led us to conclude that the Epac-Rap-PLC-[Ca2+]i signaling pathway is involved in cAMP-stimulated Cl- secretion, which is carried by a novel, previously undescribed Cl- channel.
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PMID:Epac1 mediates protein kinase A-independent mechanism of forskolin-activated intestinal chloride secretion. 2003 25


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