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:3.2.1.31 (beta-glucuronidase)
7,680 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The in vitro and in vivo disposition of DPC 423 was investigated in mice, rats, dogs and humans and the metabolites characterized by LC/MS, LC/NMR and high field-NMR. The rodents produced several metabolites that included an aldehyde (M1), a carboxylic acid (M2), a benzyl alcohol (M3), glutamate conjugates (M4 and M5), an acyl glucuronide (M6) and its isomers; a carbamyl glucuronide (M7); a phenol (M8) and its glucuronide conjugate (M9), two glutathione adducts (M10 and M11), a sulfamate conjugate (M12), isomers of an oxime metabolite (M13), and an amide (M14). Humans and dogs produced less complex metabolite profiles than rats. While unchanged DPC 423 was the major component in plasma and urine samples, differences in the metabolic disposition of this compound among species were noted. M1 is believed to be rapidly oxidized to the carboxylic acid (M2), which forms the potentially reactive acyl glucuronide (M6). The formation of novel glutamate conjugates (M4 and M5) and their role in depleting endogenous glutathione have been described previously. The carbamyl glucuronide M7, found as the major metabolite in rats and in other species, was considered nonreactive and was easily hydrolyzed to the parent compound in the presence of beta-glucuronidase. The identification of GSH adducts M10 and M11 led us to postulate the existence of at least two reactive intermediates responsible for their formation, an epoxide and possibly a nitrile oxide, respectively. Although the formation of GSH adducts such as M10 from epoxides has been described before, there are no reports to date describing the existence of a GSH adduct (M11) of an oxime. The formation of a sulfamate conjugate (M12) formed by direct coupling of sulfate to the nitrogen of benzylamine is described. A mechanism is proposed for the formation of the oxime (M13) that involves sequential oxidation of the benzylamine to the corresponding hydroxylamine and nitroso intermediate. The rearrangement of the nitroso intermediate is believed to produce the oxime (M13). In vitro studies suggested that both the oxime (M13) and the aldehyde (M1) were precursors to the carboxylic acid (M2). This is the first demonstration of carboxylic acid formation via an oxime intermediate produced from an amine. The stability of DPC423 in plasma obtained from several species was studied. Significant species differences in the plasma stability of DPC 423 were observed. The formation of the aldehyde metabolite (M1) was found to be catalyzed by a semicarbazide-sensitive monoamine oxidase (SSAO) found in plasma of rabbits, dogs, and rhesus monkeys. Rat, chimpanzee, and human plasma did not form M1.
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PMID:Disposition of 1-[3-(aminomethyl)phenyl]-N-[3-fluoro-2'- (methylsulfonyl)-[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)- 1H-pyrazole-5-carboxamide (DPC 423) by novel metabolic pathways. Characterization of unusual metabolites by liquid chromatography/mass spectrometry and NMR. 1180 May 97

Expression of the fis1 gene from flax (Linum usitatissimum) is induced by a compatible rust (Melampsora lini) infection. Infection of transgenic plants containing a beta-glucuronidase (GUS) reporter gene under the control of the fis1 promoter showed that induction is highly localized to those leaf mesophyll cells within and immediately surrounding rust infection sites. The level of induction reflects the extent of fungal growth. In a strong resistance reaction, such as the hypersensitive fleck mediated by the L6 resistance gene, there is very little fungal growth and a microscopic level of GUS expression. Partially resistant flax leaves show levels of GUS expression that were intermediate to the level observed in the fully susceptible infection. Sequence and deletion analysis using both transient Agrobacterium tumefaciens expression and stable transformation assays have shown that the rust-inducible fis1 promoter is contained within a 580-bp fragment. Homologs of fis1 were identified in expressed sequence tag databases of a range of plant species including dicots, monocots, and a gymnosperm. Homologous genes isolated from maize (Zea mays; mis1), barley (Hordeum vulgare; bis1), wheat (Triticum aestivum; wis1), and Arabidopsis encode proteins that are highly similar (76%-82%) to the FIS1 protein. The Arabidopsis homologue has been reported to encode a delta1-pyrroline-5-carboxylate dehydrogenase that is involved in the catabolism of proline to glutamate. RNA-blot analysis showed that mis1 in maize and the bis1 homolog in barley are both up-regulated by a compatible infection with the corresponding species-specific rust. The rust-induced genes homologous to fis1 are present in many plants. The promoters of these genes have potential roles for the engineering of synthetic rust resistance genes by targeting transgene expression to the sites of rust infection.
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PMID:A plant gene up-regulated at rust infection sites. 1201 48

AREA (NIT2) is a general transcription factor involved in derepression of numerous genes responsible for nitrogen utilization in Gibberella fujikuroi and many other fungi. We have previously shown that the deletion of areA-GF resulted in mutants with significantly reduced gibberellin (GA) production. Here we demonstrate that the expression level of six of the seven GA biosynthesis genes is drastically reduced in mutants lacking areA. Furthermore, we show that, despite the fact that GAs are nitrogen-free diterpenoid compounds, which are not obviously involved in nitrogen metabolism, AREA binds directly to the promoters of the six N-regulated genes. The binding of AREA was analysed in more detail using the promoter of one of the GA-biosynthesis genes encoding the ent-kaurene oxidase (P450-4). Deletion/mutation analysis of the P450-4 promoter fused to the Escherichia coli uidA gene, which encodes beta-glucuronidase, allowed the in vivo identification of functional GATA motifs. We have also analysed the nmr gene of G. fujikuroi (nmr-GF) which has high similarity to the Neurospora crassa nmr-1 and Aspergillus nidulans nmrA genes, both involved in nitrogen metabolite repression. In contrast to our expectation, deletion of nmr-GF did not result in significant derepression of the GA biosynthesis genes in the presence of ammonium, glutamine or glutamate. Overexpression of the nmr-GF gene fused to the strong promoter of the G. fujikuroi glutamine synthetase (gs) gene revealed only a very slight repression of the nitrate reductase (niaD) gene, resulting in weak resistance to chlorate. Surprisingly, this effect was only observed in the presence of high amounts of glutamate; cultivation on ammonium failed to induce any resistance to chlorate. Despite the limited effect of gene replacement and overexpression of nmr-GF on the nitrogen metabolism of G. fujikuroi itself, the gene fully restored nitrogen metabolite repression in A. nidulans and N. crassa nmr mutants. Therefore, we postulate that, in contrast to A. nidulans and N. crassa, NMR does not function independently as the main modulator of AREA in G. fujikuroi.
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PMID:AREA directly mediates nitrogen regulation of gibberellin biosynthesis in Gibberella fujikuroi, but its activity is not affected by NMR. 1258 53

Previous reports suggest that age-related lysosomal disturbances contribute to Alzheimer-type accumulations of protein species, blockage of axonal/dendritic transport, and synaptic decline. Here, we tested the hypothesis that lysosomal enzymes are upregulated as a compensatory response to pathogenic protein accumulation. In the hippocampal slice model, tau deposits and amyloidogenic fragments induced by the lysosomal inhibitor chloroquine were accompanied by disrupted microtubule integrity and by corresponding declines in postsynaptic glutamate receptors and the presynaptic marker synaptophysin. In the same slices, cathepsins B, D, and L, beta-glucuronidase, and elastase were upregulated by 70% to 135%. To address whether this selective activation of the lysosomal system represents compensatory signaling, N-Cbz-L-phenylalanyl-L-alanyl-diazomethylketone (PADK) was used to enhance the lysosome response, generating 4- to 8-fold increases in lysosomal enzymes. PADK-mediated lysosomal modulation was stable for weeks while synaptic components remained normal. When PADK and chloroquine were co-infused, chloroquine no longer increased cellular tau levels. To assess pre-existing pathology, chloroquine was applied for 6 days after which its removal resulted in continued degeneration. In contrast, enhancing lysosomal activation by replacing chloroquine after 6 days with PADK led to clearance of accumulated protein species and restored microtubule integrity. Transport processes lost during chloroquine exposure were consequently re-established, resulting in marked recovery of synaptic components. These data indicate that compensatory activation of lysosomes follows protein accumulation events, and that lysosomal modulation represents a novel approach for treating Alzheimer disease and other protein deposition diseases.
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PMID:Lysosomal activation is a compensatory response against protein accumulation and associated synaptopathogenesis--an approach for slowing Alzheimer disease? 1276 85

A GlcNase (exo-beta-D-glucosaminidase) was purified from culture supernatant of Amycolatopsis orientalis subsp. orientalis grown in medium with chitosan. The enzyme hydrolysed the terminal GlcN (glucosamine) residues in oligomers of GlcN with transglycosylation observed at late reaction stages. 1H-NMR spectroscopy revealed that the enzyme is a retaining glycoside hydrolase. The GlcNase also behaved as an exochitosanase against high-molecular-mass chitosan with K(m) and kcat values of 0.16 mg/ml and 2832 min(-1). On the basis of partial amino acid sequences, PCR primers were designed and used to amplify a DNA fragment which then allowed the cloning of the GlcNase gene (csxA) associated with an open reading frame of 1032 residues. The GlcNase has been classified as a member of glycoside hydrolase family 2 (GH2). Sequence alignments identified a group of CsxA-related protein sequences forming a distinct GH2 subfamily. Most of them have been annotated in databases as putative beta-mannosidases. Among these, the SAV1223 protein from Streptomyces avermitilis has been purified following gene cloning and expression in a heterologous host and shown to be a GlcNase with no detectable beta-mannosidase activity. In CsxA and all relatives, a serine-aspartate doublet replaces an asparagine residue and a glutamate residue, which were strictly conserved in previously studied GH2 members with beta-galactosidase, beta-glucuronidase or beta-mannosidase activity and shown to be directly involved in various steps of the catalytic mechanism. Alignments of several other GH2 members allowed the identification of yet another putative subfamily, characterized by a novel, serine-glutamate doublet at these positions.
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PMID:Two exo-beta-D-glucosaminidases/exochitosanases from actinomycetes define a new subfamily within family 2 of glycoside hydrolases. 1631 14

A new type of protein was found in Arabidopsis thaliana, PCaP1, which is rich in glutamate and lysine residues. The protein bound (45)Ca(2+) even in the presence of a high concentration of Mg(2+). Real-time polymerase chain reaction and histochemical analysis of promoter-beta-glucuronidase fusions revealed that PCaP1 was expressed in most organs. The PCaP1 protein was detected immunochemically in these organs. Treatment of Arabidopsis seedlings with Cu(2+), sorbitol, or flagellin oligopeptide enhanced the transcription. On the other hand, other sugars, abscisic acid, gibberellic acid, dehydration, and low temperature had little or no effect on PCaP1 transcript abundance. The transient expression of PCaP1 fused to green fluorescent protein in Arabidopsis cells and the subcellular fractionation of tissue homogenate showed that PCaP1 protein is localized to the plasma membrane, although PCaP1 has no predicted transmembrane domain. PCaP1 was associated with the plasma membrane under natural conditions and was released from the membrane at high concentrations of Ca(2+) or Mg(2+) in vitro. These results suggest that the hydrophilic protein PCaP1 binds Ca(2+) and other cations and is stably associated with the plasma membrane.
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PMID:Molecular properties of a novel, hydrophilic cation-binding protein associated with the plasma membrane. 1726 65

gamma-Glutamyl transpeptidases (GGTs) are essential for hydrolysis of the tripeptide glutathione (gamma-glutamate-cysteine-glycine) and glutathione S-conjugates since they are the only enzymes known to cleave the amide bond linking the gamma-carboxylate of glutamate to cysteine. In Arabidopsis thaliana, four GGT genes have been identified based on homology with animal GGTs. They are designated GGT1 (At4g39640), GGT2 (At4g39650), GGT3 (At1g69820), and GGT4 (At4g29210). By analyzing the expression of each GGT in plants containing GGT:beta-glucuronidase fusions, the temporal and spatial pattern of degradation of glutathione and its metabolites was established, revealing appreciable overlap among GGTs. GGT2 exhibited narrow temporal and spatial expression primarily in immature trichomes, developing seeds, and pollen. GGT1 and GGT3 were coexpressed in most organs/tissues. Their expression was highest at sites of rapid growth including the rosette apex, floral stem apex, and seeds and might pinpoint locations where glutathione is delivered to sink tissues to supplement high demand for cysteine. In mature tissues, they were expressed only in vascular tissue. Knockout mutants of GGT2 and GGT4 showed no phenotype. The rosettes of GGT1 knockouts showed premature senescence after flowering. Knockouts of GGT3 showed reduced number of siliques and reduced seed yield. Knockouts were used to localize and assign catalytic activity to each GGT. In the standard GGT assay with gamma-glutamyl p-nitroanilide as substrate, GGT1 accounted for 80% to 99% of the activity in all tissues except seeds where GGT2 was 50% of the activity. Protoplasting experiments indicated that both GGT1 and GGT2 are localized extracellularly but have different physical or chemical associations.
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PMID:Localization of members of the gamma-glutamyl transpeptidase family identifies sites of glutathione and glutathione S-conjugate hydrolysis. 1754 9

We cloned the isovaleryl-CoA dehydrogenase (IVD)-encoding gene from Aspergillus oryzae. The promoter of ivdA was subjected to beta-glucuronidase (GUS) reporter assays in which certain amino acids were used as a major carbon source. L-leucine most strongly induced GUS-activity, while in the case of L-glutamate, significantly low activity was found, indicating that ivdA transcription was strongly repressed by glutamic acid.
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PMID:The promoter activity of isovaleryl-CoA dehydrogenase-encoding gene (ivdA) from Aspergillus oryzae is strictly repressed by glutamic acid. 1758 91

While polyamines (PAs) have been suggested to protect cells against Reactive Oxygen Species (ROS), their catabolism is known to generate ROS. We compared the activities of several enzymes and cellular metabolites involved in the ROS scavenging pathways in two isogenic cell lines of poplar (Populus nigraxmaximowiczii) differing in their PA contents. Whereas the control cell line was transformed with beta-glucuronidase (GUS), the other, called HP (High Putrescine), was transformed with a mouse ornithine decarboxylase (mODC) gene. The expression of mODC resulted in several-fold increased production of putrescine as well its enhanced catabolism. The two cell lines followed a similar trend of growth over the seven-day culture cycle, but the HP cells had elevated levels of soluble proteins. Accumulation of H(2)O(2) was higher in the HP cells than the control cells, and so were the activities of glutathione reductase and monodehydroascorbate reductase; the activity of ascorbate peroxidase was lower in the former. The contents of reduced glutathione and glutamate were significantly lower in the HP cells but proline was higher on some days of analysis. There was a small difference in mitochondrial activity between the two cell lines, and the HP cells showed increased membrane damage. In the HP cells, increased accumulation of Ca was concomitant with lower accumulation of K. We conclude that, while increased putrescine accumulation may have a protective role against ROS in plants, enhanced turnover of putrescine actually can make them vulnerable to increased oxidative damage.
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PMID:Putrescine overproduction negatively impacts the oxidative state of poplar cells in culture. 1913 66

We found a new hydrophilic protein in Arabidopsis thaliana. Real-time PCR demonstrated that the protein was expressed in roots. Histochemical analysis of promoter-beta-glucuronidase fusions demonstrated its extensive expression in root hairs. The protein is rich in proline, glutamate, valine and lysine residues (PEVK-rich domain), and bound Ca(2+) even in the presence of Mg(2+) and K(+) when examined by the (45)Ca overlay assay. Treatment of seedlings with K(+), Mn(2+), Zn(2+), Na(+), ABA and gibberellic acid, and cold and drought stresses enhanced the transcription. Expression of the protein linked to green fluorescent protein in A. thaliana showed its plasma membrane localization and cell-specific expression in the epidermal cells including root hairs and the elongating pollen tubes. Therefore, we named the protein PCaP2 (plasma membrane-associated Ca(2+)-binding protein-2). The substitution of glycine at position 2 with alanine resulted in cytoplasmic localization of PCaP2. These results and the N-terminal characteristic motif suggest that PCaP2 is N-myristoylated at Gly2. We examined the capacity for binding to phosphatidylinositol phosphates (PtdInsPs), and found that PCaP2 interacts strongly with PtdIns(3,5)P(2), PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3), and weakly with PtdIns(3,4)P(2). Furthermore, calmodulin was associated with PCaP2 in a Ca(2+)-dependent manner, and its association weakened the interaction of PCaP2 with PtdInsPs. These results indicate that PCaP2 is involved in intracellular signaling through interaction with PtdInsPs and calmodulin in growing root hairs. PCaP2 was previously reported as microtubule-associated protein-18. We discuss the physiological roles of PCaP2 in relation to microtubules in cells.
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PMID:An Arabidopsis hydrophilic Ca2(+) -binding protein with a PEVK-rich domain, PCaP2, is associated with the plasma membrane and interacts with calmodulin and phosphatidylinositol phosphates. 2044 67


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