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R A Dean

Publications and source records attributed to R A Dean.

At least 37 records · Page 2Linked to original sources
PubMed1997

Purification and cloning of a broad substrate specificity human liver carboxylesterase that catalyzes the hydrolysis of cocaine and heroin.

A human liver carboxylesterase (hCE-2) that catalyzes the hydrolysis of the benzoyl group of cocaine and the acetyl groups of 4-methylumbelliferyl acetate, heroin, and 6-monoacetylmorphine was purified from human liver. The purified enzyme exhibited a single band on SDS-polyacrylamide gel electrophoresis with a subunit mass of approximately 60 kDa. The native enzyme was monomeric. The isoelectric point of hCE-2 was approximately 4.9. Treatment with endoglycosidase H caused an increase in electrophoretic mobility indicating that the liver carboxylesterase was a glycoprotein of the high mannose type. The complete cDNA nucleotide sequence was determined. The authenticity of the cDNA was confirmed by a perfect sequence match of 78 amino acids derived from the hCE-2 purified from human liver. The mature 533-amino acid enzyme encoded by this cDNA shared highest sequence identity with the rabbit liver carboxylesterase form 2 (73%) and the hamster liver carboxylesterase AT51p (67%). Carboxylesterases with high sequence identity to hCE-2 have not been reported in mouse and rat liver. hCE-2 exhibited different drug ester substrate specificity from the human liver carboxylesterase called hCE-1, which hydrolyzes the methyl ester of cocaine. hCE-2 had higher catalytic efficiencies for hydrolysis of 4-methylumbelliferyl acetate, heroin, and 6-monoacetylmorphine and greater inhibition by eserine than hCE-1. hCE-2 may play an important role in the degradation of cocaine and heroin in human tissues.

Amino Acid Sequence↗
PubMed1997

A large-insert (130 kbp) bacterial artificial chromosome library of the rice blast fungus Magnaporthe grisea: genome analysis, contig assembly, and gene cloning.

Magnaporthe grisea (Hebert) Barr causes rice blast, one of the most devastating diseases of rice (Oryza sativa) worldwide. This fungus is an ideal organism for studying a number of aspects of plant-pathogen interactions, including infection-related morphogenesis, avirulence, and pathogen evolution. To facilitate M. grisea genome analysis, physical mapping, and positional cloning, we have constructed a bacterial artificial chromosome (BAC) library from the rice infecting strain 70-15. A new method was developed for separation of partially digested large-molecular-weight DNA fragments that facilitated library construction with large inserts. The library contains 9216 clones, with an average insert size of 130 kbp (> 25 genome equivalents) stored in 384-well microtiter plates that can be double spotted robotically on to a single nylon membrane. Several unlinked single-copy DNA probes were used to screen 4608 clones in the library and an average of 13 (minimum of 6) overlapping BAC clones was found in each case. Hybridization of total genomic DNA to the library and analysis of individual clones indicated that approximately 26% of the clones contain single-copy DNA. Approximately 35% of BAC clones contained the retrotransposon MAGGY. The library was used to identify BAC clones containing a adenylate cyclase gene (mac1). In addition, a 550-kbp contig composed of 6 BAC clones was constructed that encompassed two adjacent RFLP markers on chromosome 2. These data show that the BAC library is suitable for genome analysis of M. grisea. Copies of colony hybridization membranes are available upon request.

Ascomycota↗
PubMed1997

G protein alpha subunit genes control growth, development, and pathogenicity of Magnaporthe grisea.

Three G protein alpha subunit genes have been cloned and characterized from Magnaporthe grisea: magA is very similar to CPG-2 of Cryphonectria parasitica; magB is virtually identical to CPG-1 of Cryphonectria parasitica, to gna1 of Neurospora crassa, and to fadA of Emericella nidulans; and magC is most similar to gna2 of Neurospora crassa. Homologous recombination resulting in targeted deletion of magA had no effect on vegetative growth, conidiation, or appressorium formation. Deletion of magC reduced conidiation, but did not affect vegetative growth or appressorium formation. However, disruption of magB significantly reduced vegetative growth, conidiation, and appressorium formation. magB- transformants, unlike magA- and magC- transformants, exhibited a reduced ability to infect and colonize susceptible rice leaves. G protein alpha subunit genes are required for M. grisea mating. magB- transformants failed to form perithecia, whereas magA- and magC- transformants did not produce mature asci. These results suggest that G protein alpha subunit genes are involved in signal transduction pathways in M. grisea that control vegetative growth, conidiation, conidium attachment, appressorium formation, mating, and pathogenicity.

Amino Acid Sequence↗
PubMed1997

The adenylate cyclase gene MAC1 of Magnaporthe grisea controls appressorium formation and other aspects of growth and development.

Magnaporthe grisea, the causal agent of rice blast disease, differentiates a specialized infection structure called an appressorium that is crucial for host plant penetration. Previously, it was found that cAMP regulates appressorium formation. To further understand the cellular mechanisms involved in appressorium formation, we have cloned a gene (MAC1) encoding adenylate cyclase, a membrane-bound enzyme that catalyzes the production of cAMP from ATP, by using a polymerase chain reaction-based strategy. The entire gene was isolated and subcloned from a large insert bacterial artificial chromosome library. Sequence characterization showed that MAC1 has a high degree of identity with other adenylate cyclase genes from several filamentous fungi as well as yeasts. Gene deletion resulted in reduced vegetative growth, conidiation, and conidial germination. Transformants lacking MAC1 were unable to form appressoria on an inductive surface and were unable to penetrate susceptible rice leaves. mac1- transformants were also sterile and produced no perithecia. Appressorium formation was restored in the presence of exogenous cAMP derivatives. These results confirm that cell signaling involving cAMP plays a central role in the development and pathogenicity of M. grisea.

Amino Acid Sequence↗
PubMed1997

Molecular genetic evidence for the involvement of a specific polygalacturonase, P2c, in the invasion and spread of Aspergillus flavus in cotton bolls.

Isolates of Aspergillus flavus can be differentiated based on production of the polygalacturonase P2c. One group of isolates produces P2c, whereas the other group does not. In general, the group that produces P2c causes more damage and spreads to a greater extent in cotton bolls than those isolates that do not produce P2c. To determine whether P2c contributes to disease, the expression of pecA, the gene previously determined to encode P2c, was genetically altered. Adding the pecA gene to a strain previously lacking the gene resulted in the ability to cause significantly more damage to the intercarpellary membrane and the ability spread to a greater extent within the adjacent locule compared to the abilities of a control transformant. Conversely, eliminating the expression of pecA by targeted disruption caused a significant reduction in aggressiveness compared to that of a nondisrupted control transformant. These results provide direct evidence that P2c contributes to the invasion and spread of A. flavus during infection of cotton bolls. However, other factors not evaluated in this study also contribute to aggressiveness.

Aspergillus flavus↗
PubMed1997

Signal pathways and appressorium morphogenesis.

Fungal pathogens have evolved elaborate strategies to gain access to plant tissues. For many pathogens, following attachment of spores to the leaf surface, germ tubes emerge and grow across the surface, often in response to particular environmental cues and to a specific location. At an appropriate site, polar elongation of the germ tube ceases, the tip attaches to the surface and swells to form an appressorium, a uniquely organized infection structure. Following a period of maturation, a hypha then emerges at the plant interface and penetrates into the plant tissues. This chapter discusses recent developments that provide new insight into the molecular mechanism regulating induction and function of appressoria. Topics include attachment to the leaf surface; environmental cues that signal germ-tube growth and appressorium formation; mechanisms for sensing environmental cues; endogenous signaling pathways; and mechanisms of penetration from the appressorium.

Journal Article↗
PubMed1997

Human liver carboxylesterase hCE-1: binding specificity for cocaine, heroin, and their metabolites and analogs.

Purified human liver carboxylesterase (hCE-1) catalyzes the hydrolysis of cocaine to form benzoylecgonine, the deacetylation of heroin to form 6-acetylmorphine, and the ethanol-dependent transesterification of cocaine to form cocaethylene. In this study, the binding affinities of cocaine, cocaine metabolites and analogs, heroin, morphine, and 6-acetylmorphine for hCE-1 were evaluated by measuring their kinetic inhibition constants with 4-methylumbelliferyl acetate in a rapid spectrophotometric assay. The naturally occurring (R)-(-)-cocaine isomer displayed the highest affinity of all cocaine and heroin analogs or metabolites. The pseudo- or allopseudococaine isomers of cocaine exhibited lower affinity indicating that binding to the enzyme is stereoselective. The methyl ester, benzoyl, and N-methyl groups of cocaine play important roles in binding because removal of these groups increased K(i) values substantially. Compounds containing a variety of hydrophobic substitutions at the benzoyl group of cocaine bound to the enzyme with high affinity. The high K(i) value obtained for cocaethylene relative to cocaine is consistent with weaker binding to the esterase and a longer elimination half-life reported for the metabolite. The spectrophotometric competitive inhibition assay used here represents an effective method to identify drug or environmental esters metabolized by carboxylesterases like hCE-1.

Carboxylesterase↗
PubMed1996

Serum ethylene glycol by high-performance liquid chromatography.

A high-performance liquid chromatography procedure for detection and quantitation of ethylene glycol in serum is described. Ethylene glycol and internal standard are derivatized with benzoyl chloride under alkaline conditions, purified by solid-phase extraction and analyzed by HPLC with UV detection. Analytical recovery of ethylene glycol ranges between 96 and 103%. The calibration curve is linear from 20 to 2000 mg/l. The limits of detection and quantitation are 10 and 20 mg/l, respectively. Assay imprecision is 4.8% or less. The assay is free from common interferences and provides increased sensitivity, improved precision and extended linearity.

Animals↗
PubMed1996

Gender-specific differences in activity and protein levels of cocaine carboxylesterase in rat tissues.

The gender-specific differences in the content of cocaine methyl esterase and ethyl transferase activities are examined in rat tissues and related to differences in hydrolase A protein in rat liver, lung, and kidney reported previously. The rat hydrolase A catalyzes the conversion of cocaine to benzoylecgonine and the ethyl transesterification of cocaine to form cocaethylene. An HPLC assay was used to quantitate and compare cocaine esterase activities in male and female rat tissues. The cocaine methyl esterase and ethyl transferase activities are 1.4 to 2.5 fold greater in male than in female liver and slightly greater in female than in male lung. No gender-specific differences were detected in the kidney. Gel electrophoresis was used to separate three non-specific carboxylesterases (hydrolases A, B, and C) in rat tissues and the isoenzymes were visualized with a hydrolase activity stain using 4-methylumbelliferyl acetate as substrate. The activity of cocaine methyl esterase and content of hydrolase A protein are not consistently different in the lung or the kidney of male versus female rats. Activity of hydrolase A in gels of male liver is greater than in female liver. Similarly, the content of the corresponding hydrolase A immunoreactive protein in male liver is 1.6 fold greater than in female liver. In contrast to hydrolase A, hydrolase C activity is greater in gels of female than male liver extracts. The greater content of cocaine methyl esterase and ethyl transferase activity in male versus female rat livers suggests that there may be gender-specific differences in pharmacokinetics of cocaine metabolism and extent of cocaine-induced hepatotoxicity in rats.

Animals↗
PubMed1996

Simultaneous measurement of ethanol and ethyl-d5 alcohol by stable isotope gas chromatography-mass spectrometry.

Efforts to define gender- and ethnic-dependent differences in ethanol first-pass metabolism by gastric mucosa and liver have been limited by a lack of analytical tools that distinguish ethanol concurrently administered by oral and intravenous routes. A stable isotope gas chromatography-mass spectrometry method for simultaneous measurement of ethanol and ethyl-d5 alcohol in serum was developed to meet this need. The assay was linear from 1 to 30 mmol/L. The limit of quantification was 1 mmol/L. Analytical imprecision (CV) was <10%. Analytical recovery was >90%. Specificity was based on retention time and reproducibility of ion ratios. The assay was free from interference by other volatile alcohols. Simultaneous oral administration of ethanol and ethyl-d5 alcohol produced nearly identical pharmacokinetic profiles. Simultaneous oral ingestion of ethanol and intravenous infusion of ethyl-d5 alcohol, adjusted for gastric emptying time, revealed decreased bioavailability of ethanol by the oral route. The method described is sufficient to study the first-pass metabolism of ethanol.

Administration, Oral↗
PubMed1996

Metabolism of cocaine and heroin is catalyzed by the same human liver carboxylesterases.

Concomitant i.v. use of cocaine and heroin ("speedballing") is prevalent among drug-abusing populations. Heroin is rapidly metabolized by sequential deacetylation of two separate ester bonds to yield 6-monoacetylmorphine and morphine. Hydrolysis of heroin to 6-monoacetylmorphine is catalyzed by pseudocholinesterase. The pathway for hydrolysis of 6-monoacetylmorphine to morphine in vivo has yet to be established. Pseudocholinesterase and two human liver carboxylesterases [human liver carboxylesterase form 1 (hCE-1) and human liver carboxylesterase form 2 (hCE-2)] catalyze the rapid hydrolysis of ester linkages in cocaine. This investigation examined the relative catalytic efficiencies of hCE-1, hCE-2 and pseudocholinesterase for heroin metabolism and compared them with cocaine hydrolysis. Enzymatic formation of 6-monoacetylmorphine and morphine was determined by reverse-phase high-performance liquid chromatography. All three enzymes rapidly catalyzed hydrolysis of heroin to 6-monoacetylmorphine (hCE-1 kcat = 439 min-1, hCE-2 kcat = 2186 min-1 and pseudocholinesterase kcat = 13 min-1). The catalytic efficiency, under first-order conditions, for hCE-2-catalyzed formation of 6-monoacetylmorphine (314 min-1 mM-1) was much greater than that for either hCE-1 or pseudocholinesterase (69 and 4 min-1 mM-1, respectively). Similarly, the catalytic efficiency for hydrolysis of 6-monoacetylmorphine to morphine by hCE-2 (22 min-1 mM-1) was substantially greater than that for hCE-1 (0.024 min-1 mM-1). Cocaine competitively inhibited hCE-1-, hCE-2- and pseudocholinesterase-catalyzed hydrolysis of heroin to 6-monoacetylmorphine (Ki = 530, 460 and 130 microM, respectively) and 6-monoacetylmorphine hydrolysis to morphine (Ki = 710, 220 and 830 microM, respectively). These data demonstrate that metabolism of cocaine and heroin in humans is mediated by common metabolic pathways. The role of hepatic hCE-2 is particularly important for the hydrolysis of heroin to 6-monoacetylmorphine and of 6-monoacetylmorphine to morphine.

Butyrylcholinesterase↗
PubMed1995

Cloning and characterization of a novel polygalacturonase-encoding gene from Aspergillus parasiticus.

Pectinases produced by Aspergillus flavus and A. parasiticus are believed to play a significant role in the ability of these fungi to spread in cotton bolls and other crops. Utilizing a DNA probe, generated by PCR, of the Aspergillus niger pgaII gene, we have isolated a novel, constitutively expressed polygalacturonase (PG)-encoding gene (pecA) from an A. parasiticus cDNA library. DNA sequence analysis and the deduced amino acid (aa) sequence of pecA demonstrated significant identity at the nucleotide and aa levels with other PG of fungal origin. Northern blot analysis of RNA isolated from A. parasiticus grown on either glucose or pectin as the sole carbon source showed that pecA was expressed during growth in both media.

Amino Acid Sequence↗
PubMed1995

Sequence analysis of the Aspergillus nidulans pectate lyase pelA gene and evidence for binding of promoter regions to CREA, a regulator of carbon catabolite repression.

The nucleic acid and deduced amino-acid sequences of the pectate lyase gene (pelA) from Aspergillus nidulans are presented. The pelA gene contains two short introns, 68 and 49 bp in length, and encodes a peptide of 326 amino acids. Five transcriptional start sites are clustered between 65 and 79 bp upstream of the start codon as determined by primer extension. Comparison of the amino-acid sequences of pectate or pectin lyases from bacteria, fungi and plants revealed less than 30% overall identity. However, five regions within these enzymes, in particular domains associated with the active site, are highly conserved with amino-acid similarities greater than 50%. Phylogenetic analysis using the principle of parsimony (PAUP 3.1.1) showed that pelA is most closely related to pectate lyases from plants rather than pectin lyases from other fungi. Previously, pelA was shown to be induced by polygalacturonic acid and repressed in the presence of preferred carbon sources, such as glucose. Gel mobility shift analysis indicates that a PstI-SphI fragment from the pelA promoter binds to a fusion protein composed of the N-terminal part of CREA, a protein involved in carbon catabolite repression, and glutathione-S-transferase. This result suggests CREA may contribute to the regulation of pelA expression.

Amino Acid Sequence↗
PubMed1995

The cAMP-dependent protein kinase catalytic subunit is required for appressorium formation and pathogenesis by the rice blast pathogen Magnaporthe grisea.

Magnaporthe grisea, the causal agent of rice blast disease, differentiates a specialized infection cell, an appressorium, that is required for infection of its host. Previously, cAMP was implicated in the endogenous signaling pathway leading to appressorium formation. To obtain direct evidence for the role of cAMP in appressorium formation, the gene encoding the catalytic subunit of the cAMP-dependent protein kinase (cpkA) was cloned, sequenced, and disrupted. Polymerase chain reaction primers designed after highly conserved regions in the same gene from other organisms were used to amplify genomic DNA fragments. The cloned amplification products were used to identify genomic clones. DNA blot analysis indicated that cpkA is present as a single copy in the genome. cpkA consists of 1894 bp, including three short introns sufficient to encode a protein of 539 amino acids with a predicted molecular mass of 60.7 kD. The deduced peptide shares > 45% identity with other catalytic subunits and contains all functional motifs and residues with the addition of a glutamine-rich region at the N terminus. Two transformants, L5 and T-182, in which cpkA had been replaced with a hygromycin resistance gene cassette, were unable to produce appressoria, could not be induced to form appressoria by cAMP, and were nonpathogenic on susceptible rice, even when leaves were abraded. These results were confirmed by analysis of 57 progeny from a cross between transformant L5 and the wild-type laboratory strain 70-6. Other aspects of growth and development, including vegetative growth as well as asexual and sexual competence, were unaffected when measured in vitro. These results provide direct evidence that the cAMP-dependent protein kinase is necessary for infection-related morphogenesis and pathogenesis in a phytopathogenic fungus.

Amino Acid Sequence↗
PubMed1995

Isolation and characterization of polygalacturonase genes (pecA and pecB) from Aspergillus flavus.

Two genes, pecA and pecB, encoding endopolyglacturonases were cloned from a highly aggressive strain of Aspergillus flavus. The pecA gene consisted of 1,228 bp encoding a protein of 363 amino acids with a predicted molecular mass of 37.6 kDa, interrupted by two introns of 58 and 81 bp in length. Accumulation of pecA mRNA in both pectin- or glucose-grown mycelia in the highly aggressive strain matched the activity profile of a pectinase previously identified as P2c. Transformants of a weakly aggressive strain containing a functional copy of the pecA gene produced P2c in vitro, confirming that pecA encodes P2c. The coding region of pecB was determined to be 1,217 bp in length interrupted by two introns of 65 and 54 bp in length. The predicted protein of 366 amino acids had an estimated molecular mass of 38 kDa. Transcripts of this gene accumulated in mycelia grown in medium containing pectin alone, never in mycelia grown in glucose-containing medium, for both highly and weakly aggressive strains. Thus, pecB encodes the activity previously identified as P1 or P3. pecA and pecB share a high degree of sequence identity with polygalacturonase genes from Aspergillus parasiticus and Aspergillus oryzae, further establishing the close relationships between members of the A. flavus group. Conservation of intron positions in these genes also indicates that they share a common ancestor with genes encoding endopolyglacturonases of Aspergillus niger.

Amino Acid Sequence↗