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Biomedical subjects

G Xie

Publications and source records attributed to G Xie.

At least 19 recordsLinked to original sources

In situ lifetimes and kinetics of a reductive whey barrier and an oxidative ORC barrier in the subsurface.

Permeable reactive barriers (PRB) are being used to engineer favorable field conditions for in-situ remediation efforts. Two redox adjustment barriers were installed to facilitate a 10-month research effort on the fate and transport of MTBE (methyl tert-butyl ether) at a site called the Michigan Integrated Remediation Technology Laboratory (MIRTL). Thirty kilograms of whey were injected as a slurry into an unconfined aquifer to establish an upgradient reductive zone to reduce O2 concentration in the vicinity of a contaminant injection source. To minimize the impact of contaminant release, 363 kg of oxygen release compound (ORC) were placed in the aquifer as a downgradient oxidative barrier. Dissolved oxygen and other chemical species were monitored in the field to evaluate the effectiveness of this technology. A transient one-dimensional advective-dispersive-reaction (ADR) model was proposed to simulate the dissolved oxygen transport. The equations were solved with commonly encountered PRB initial and constant/variable boundary conditions. No similar previous solution was found in the literature. The in-situ lifetimes, based on variable source loading, were estimated to be 1,661 and 514 days for the whey barrier and ORC barrier, respectively. Estimates based on either maximum O2 consumption/production or measured O2 curves were found to under- or overestimate the lifetime of the barriers. The pseudo-first-order rate constant of whey depletion was estimated to be 0.303/d with a dissolution rate of 0.04/d. The oxygen release rate constant in the ORC barrier was estimated to be 0.03/d. This paper provides a means to design and predict the performance of reactive redox barriers, especially when only limited field data are available.

Biodegradation, Environmental↗

The emerging periplasm-localized subclass of AroQ chorismate mutases, exemplified by those from Salmonella typhimurium and Pseudomonas aeruginosa.

BACKGROUND: Chorismate mutases of the AroQ homology class are widespread in the Bacteria and the Archaea. Many of these exist as domains that are fused with other aromatic-pathway catalytic domains. Among the monofunctional AroQ proteins, that from Erwinia herbicola was previously shown to have a cleavable signal peptide and located in the periplasmic compartment. Whether or not this might be unique to E. herbicola was unknown. RESULTS: The gene coding for the AroQ protein was cloned from Salmonella typhimurium, and the AroQ protein purified from both S. typhimurium and Pseudomonas aeruginosa was shown to have a periplasmic location. The periplasmic chorismate mutases (denoted *AroQ) are shown to be a distinct subclass of AroQ, being about twice the size of cytoplasmic AroQ proteins. The increased size is due to a carboxy-terminal extension of unknown function. In addition, a so-far novel aromatic aminotransferase was shown to be present in the periplasm of P. aeruginosa. CONCLUSIONS: Our analysis has detected a number of additional *aroQ genes. The joint presence of *AroQ, cyclohexadienyl dehydratase and aromatic aminotransferase in the periplasmic compartment of P. aeruginosa comprises a complete chorismate-to-phenylalanine pathway and accounts for the "hidden overflow pathway" to phenylalanine described previously.

Amino Acid Sequence↗

FMRFamide-related neuropeptides are agonists of the orphan G-protein-coupled receptor GPR54.

We have isolated and determined the coding sequences of human and mouse orthologs of the rat orphan G-protein-coupled receptor GPR54. Mouse and rat GPR54 are nearly 95% identical to each other, and both are approximately 85% identical to human GPR54 at the amino acid level. Screening of agonists for GPR54 identified several invertebrate neuropeptides of the RFamide and RWamide family that were able to activate GPR54 at microM range through the G(alpha)q pathway. Substitution analysis showed that the C-terminal optimal sequence of GPR54-activating peptides is Gly-Leu-Arg-Trp-NH2. Northern analysis of human GPR54 detected expression in several peripheral tissues and many regions of the central nervous system.

Amino Acid Sequence↗

Genome analysis with gene-indexing databases.

The recent release of the draft sequence and the eventual completion of the human genome present the scientific community with a rich source of data to mine. Yet, these data are content poor in the absence of additional correlative information. Expressed sequence tag (EST) datasets and their associated gene indices have existed for many years, and represent the first attempt at understanding the complexity of the genome. These datasets remain extremely important as information sources and, in particular, as tools for analyzing the completed genomes. Here, we discuss the nature of ESTs and their associated tools and gene-indexing databases. In particular, we will compare three EST gene indices (UNIGENE, Merck Gene Index Version 2.0 and Doubletwist CAT), discuss how these gene indices are applied for both genome analysis and drug discovery, and demonstrate their importance as a complementary dataset to the annotated human genome.

Databases, Factual↗

PARM: a practical utility for drug design.

To accommodate situations in which the 3D structure of the target receptor is not available, we have developed the Pseudo Atomic Receptor Model (PARM) software package. In this article we describe PARM and illustrate its use with three examples: elemenes (potential anticancer drugs), angiotensin converting enzyme inhibitors, and human HIV-1 inhibitors TTD (1,1,3-trioxo-2H, 4H-thieno[3,4-e][1,2,4] thiadiazine derivatives). The results show that PARM can build models with favorable cross-validation statistics (Rcv2 values 0.7-0.9) and give helpful SAR insight. PARM has certain advantages: (a) it can be used for many systems, regardless of whether the 3D structure of the receptor is known; (b) PARM models were demonstrated to be highly statistically reliable; and (c) PARM analyses are robust and reproducible.

Angiotensin-Converting Enzyme Inhibitors↗

Intra-hepatic expression of scavenger receptor and CD14 and their relationship with local inflammatory responses in endotoxemia in mice.

Our objective was to investigate the expression of scavenger receptor (SR) and CD14 in the liver and their relationship with local anti-inflammatory and proinflammatory responses in endotoxemia in order to uncover the mechanism for the liver to turn into effector organ from defense one at the level of cell receptors in sepsis. Mouse models of endotoxemia of different severity were reproduced by injection of different doses of lipopolysaccharide (LPS) via tail vein. Expression of SR and CD14 in the liver was assayed by immunohistochemistry and was then analyzed with an image analysis system. The levels of TNFalpha, IL-6, IL-4, and IL-10 in liver tissue were determined with ELISA. Expression of SR in the liver in the high-dose group was markedly decreased 1 h after injection of LPS, and also in low- and medium-dose groups at 3 h. The expression of SR in the liver in the three groups was shown to be progressively decreased with the time prolonged. There was significant difference in average optical density (OD) values of SR among the three groups. The expression of CD14 in the liver in the three groups was shown to be significantly increased 1 h after injection of LPS, and much more with the time prolonged. But there was no significant difference in OD values of CD14 among the three groups. The contents of intrahepatic proinflammatory mediators TNFalpha and IL-6 and anti-inflammatory mediators IL-4 and IL-10 were successively significantly increased after injection of LPS. The release of anti-inflammatory mediators was shown to be later than that of proinflammatory mediators. Correlation analysis indicated that there was negative correlation between expression of SR and CD14, and that changes of TNFalpha, IL-6, IL-4, and IL-10 levels in liver tissues were correlated significantly positively with OD values of CD14 and negatively with OD values of SR. Expression of SR in the liver was shown to be progressively decreased, and that of CD14 increased in endotoxemia, which was closely related to the uncontrolled inflammatory response in liver. This might be an important mechanism for the liver to turn into effector organ from defense one in sepsis.

Animals↗

Association of protein kinase A with AKAP150 facilitates pepsinogen secretion from gastric chief cells.

Cross talk between signal transduction pathways augments pepsinogen secretion from gastric chief cells. A-kinase anchoring proteins (AKAPs) associate with regulatory subunits of protein kinase A (PKA), protein kinase C (PKC), and protein phosphatase 2B (PP2B) and localize this protein complex to specific cell compartments. We determined whether an AKAP-signaling protein complex exists in chief cells and whether this modulates secretion. In Western blots, we identified AKAP150, a rodent homologue of human AKAP79 that coimmunoprecipitates with PKA, PKC, and actin. The association of PKA and PP2B was demonstrated by affinity chromatography. Confocal microscopy revealed colocalized staining at the cell periphery for AKAP150 and PKC. Ht31, a peptide that competitively displaces PKA from the AKAP complex, but not Ht31P, a control peptide, inhibited 8-Br-cAMP-induced pepsinogen secretion. Ht31 did not inhibit secretion that was stimulated by agents whose actions are mediated by PKC and/or calcium. However, Ht31, but not Ht31P, inhibited carbachol- and A23187-stimulated augmentation of secretion from cells preincubated with cholera toxin. These data suggest the existence in chief cells of a protein complex that includes AKAP150, PKA, PKC, and PP2B. Disruption of the AKAP-PKA linkage impairs cAMP-mediated pepsinogen secretion and cross talk between signaling pathways.

A Kinase Anchor Proteins↗

[Effect of lead exposure during pregnancy on hippocampal long-term potentiation and expression of NMDAR-2A mRNA in the off-spring rats].

Chronic lead exposure during brain development is known to affect cognitive and behavioral functions in children and animals. The lead exposure on pregnant rats was used as a model to examine the effects of lead on the long-term potentiation(LTP) in hippocampal dentate gyrus(DG) region in vivo and the expression of NMDAR-2A mRNA by in situ hybridization in the off-springs. Female rats were exposed to 0.5 g/L or 2 g/L lead acetate in drinking water since 10 days before mating to weaning. The filial rats of 70-90 days old with blood lead levels below 100 micrograms/L were prepared for recording their DG-evoked population spike(PS) in the hippocampal DG area in vivo. The results showed that the range of LTP were (136 +/- 31)% in low lead group, (145 +/- 30)% in high lead group, and (319 +/- 114)% in control group. The reduction of NMDAR-2A mRNA expression 34%-44% in the granule and pyramidal cell layers caused by lead were seen in 21 days old rats. It indicated that the cognitive deficits induced by low-level lead exposure in early stage of life may persist to adulthood, and the modified NMDAR gene expression may play a key role in the cognitive deficits associated with lead exposure during development.

Animals↗

[Relationship between the dynamic expression of scavenger receptor on Kupffer cells and endotoxin-induced hepatic injury].

OBJECTIVE: To observe the change of scavenger receptor (SR) expression on Kupffer cells and study its role in pathogenesis of liver injury in mice. METHODS: 1mg/kg or 10 mg/kg of E.Coli 026: B6 LPS was bolus injected via i.a.to reproduce an animal model for this experiment. The change of SR on Kupffer cells was determined with immunohistochemistry. TNF-alpha and IL-6 level in hepatic tissue were assayed using ELISA. RESULTS: The expression of SR on Kupffer cells was aggressively downregulated in a LPS does dependent fashion. TNF-alpha, IL-6 level in hepatic tissue and ALT, TBIL in plasma were significantly negatively correlated with the expression of SR. CONCLUSIONS: With the down-regulation of SR expression on Kupffer cells, the clearance of endotoxin by Kupffer cells is decreased and the activation of Kupffer cells by endotoxin is accordingly enhanced. SR expression is closely related to hepatic injury.

Animals↗

[The relationship between bronchial hyperresponsiveness and chronic obstructive pulmonary disease].

OBJECTIVE: To explore the relationship between bronchial hyperresponsiveness and chronic obstructive pulmonary disease (COPD). METHODS: 154 smokers with COPD (FEV1/FVC < 70%) served as a study group and 154 smokers without COPD as a control group and 23 never-smokers as a healthy control group from 23 villages in Fangshan Distract, Beijing in June, 1996. The patients with self-reported and doctor-diagnosed asthma were excluded from participants. Those subjects were divided into a positive group and a negative group based on methacholine bronchial responsiveness. All subjects were worked up in May, 2000. RESULTS: (1) Baseline case-control study: The positive rate of bronchial hyperresponsiveness was 78% in the study group, PC20 = (1.4 +/- 1.6) g/L; however 28% of the matched group were positive, PC20 = (2.7 +/- 2.3) g/L, (P < 0.001); (2) Prospective study: Of 154 pairs case-controls 241 subjects were followed up in May 2000. The rate of following up was 78.2%. The incidence of COPD was 23.7% in 38 subjects without COPD with positive hyperresponsiveness, however incidence of COPD was 9.5% in 84 subjects without COPD with negative hyperresponsiveness (P = 0.036), the relative risk (RR) = 2.5. The rate of FEV1/FVC > or = 70% was 12.9% in 93 patients with COPD and positive hyperresponsiveness, however it was 30.8% in 26 patients with COPD and negative hyperresponsiveness (P = 0.031). CONCLUSION: In smoking population, the bronchial hyperresponsiveness lead to more chronic obstructive pulmonary disease and it may be one of the susceptible risk factors for COPD.

Bronchial Hyperreactivity↗

A core-promoter region functions bi-directionally for human opioid-receptor-like gene ORL1 and its 5'-adjacent gene GAIP.

We report the genomic structure and functional activities of the promoter regions of the human opioid-receptor-like gene ORL1 and its 5'-adjacent gene GAIP (G alpha interacting protein). The transcription and alternative splicing of human ORL1 are controlled by two alternate promoters, located approximately 10 kb apart. The two promoter regions lack a TATA-box and are GC rich. Promoter 1A initiates, from a single transcription start point (TSP), two transcripts: one consisting of exons 1A, 1B, 2, etc., the other without exon 1B. A potential ATG codon upstream of the initiation codon of ORL1 starts a new open-reading frame encoding a theoretical polypeptide of 205 amino acid residues. The promoter 1B transcribes, from multiple TSPs, only one mRNA starting with exon 1B. Two different repeat sequence polymorphisms are found in the ORL1 promoter regions. Luciferase reporter gene assays with promoter regions and a series of deletion mutants have mapped the core-promoter 1A and 1B within two short fragments. DNA sequencing and a database search reveal that the human GAIP gene is located upstream of ORL1 and is oriented in the opposite direction. The transcription and alternative splicing of GAIP are also under the control of alternate promoters. The first exons of ORL1 and GAIP are separated by only 83 bp. This 83 bp fragment, together with short surrounding sequences from both first exons, functions bi-directionally as a core-promoter for both genes. The transcription and alternative splicing of human ORL1 and GAIP are cell-type specific. While GAIP is expressed in both NT2 precursor cells and differentiated NT2 neuronal cells, ORL1 is only expressed in differentiated NT2 neurons. Since ORL1 is a G protein-coupled receptor and GAIP interacts with G protein alpha subunits, their physical linkage in the genome and co-operative transcriptional regulation may play a significant role in ORL1 receptor signal transduction.

Alternative Splicing↗

NMR evidence for syn-anti interconversion of a trans opened (10R)-dA adduct of benzo[a]pyrene (7S,8R)-diol (9R,10S)-epoxide in a DNA duplex.

2D NMR has been used to examine the structure and dynamics of a 12-mer DNA duplex, d(T(1)A(2)G(3)T(4)C(5)A(6)A(7)G(8)G(9)G(10)C(11)A(12))-d(T(13)G(14)C( 15)C(16)C(17)T(18)T(19)G(20)A(21)C(22)T(23)A(24)), containing a 10R adduct at dA(7) that corresponds to trans addition of the N(6)-amino group of dA(7) to (-)-(7S,8R,9R,10S)-7,8-dihydroxy-9, 10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene [(-)-(S,R,R,S)-BP DE-2]. This DNA duplex contains the base sequence for the major dA mutational hot spot in the HPRT gene when Chinese hamster V79 cells are given low doses of the highly carcinogenic (+)-(R,S,S,R)-BP DE-2 enantiomer. NOE data indicate that the hydrocarbon is intercalated on the 5'-side of the modified base as has been seen previously for other oligonucleotides containing BP DE-2 (10R)-dA adducts. 2D chemical exchange-only experiments indicate dynamic behavior near the intercalation site especially at the 10R adducted dA, such that this base interconverts between the normal anti conformation and a less populated syn conformation. Ab initio molecular orbital chemical shift calculations of nucleotide and dinucleotide fragments in the syn and anti conformations support these conclusions. Although this DNA duplex containing a 10R dA adduct exhibits conformational flexibility as described, it is nevertheless more conformationally stable than the corresponding 10S adducted duplex corresponding to trans opening of the carcinogenic isomer (+)-(R,S,S, R)-BP DE-2, which was too dynamic to permit NMR structure determination. UV and imino proton NMR spectral observations indicated pronounced differences between these two diastereomeric 12-mer duplexes, consistent with conformational disorder at the adduct site and/or an equilibrium with a nonintercalated orientation of the hydrocarbon in the duplex containing the 10S adduct. The existence of conformational flexibility around adducts may be related to the occurrence of multiple mutagenic outcomes resulting from a single DE adduct.

7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗

An alternatively spliced transcript of the rat nociceptin receptor ORL1 gene encodes a truncated receptor.

Opioid receptor-like protein ORL1, the receptor for the neuropeptide nociceptin (also named orphanin FQ), has two alternatively spliced isoforms in the rat. This alternative splicing event is generated by retaining of intron 3, 81 bases in length, in the mRNA region encoding the second extracellular loop of ORL1. A full-length rat ORL1 receptor has 367 amino acid residues. However, as revealed by sequencing of rat ORL1 genomic DNA and cDNA, the insertion of the unspliced intron 3 brings in an in-frame stop codon and, therefore, creates a truncated open-reading frame encoding only the N-terminal half of ORL1 (from the N-terminus to an alternate extracellular tail C-terminal to the fourth transmembrane domain). The two alternatively spliced transcripts are differentially expressed in tissues. In transfected mammalian cells, the full-length ORL1 displays high-affinity and selective binding for nociceptin, and inhibits the production of cyclic AMP. In contrast, the truncated ORL1 binds nociceptin and other opioid peptides very poorly and non-selectively (affinity in micromolar range), and it does not mediate any inhibitory effects on cyclic AMP production. Apparently, this truncated ORL1 does not function as a receptor for nociceptin or other ligands tested. Such alternative splicing to create a truncated ORL1 receptor might be an endogenous mechanism to negatively regulate nociceptin/ORL1 functions.

Alternative Splicing↗

Cyclohexadienyl dehydrogenase from Pseudomonas stutzeri exemplifies a widespread type of tyrosine-pathway dehydrogenase in the TyrA protein family.

The uni-domain cyclohexadienyl dehydrogenases are able to use the alternative intermediates of tyrosine biosynthesis, prephenate or L-arogenate, as substrates. Members of this TyrA protein family have been generally considered to fall into two classes: sensitive or insensitive to feedback inhibition by L-tyrosine. A gene (tyrA(c)) encoding a cyclohexadienyl dehydrogenase from Pseudomonas stutzeri JM300 was cloned, sequenced, and expressed at a high level in Escherichia coli. This is the first molecular-genetic and biochemical characterization of a purified protein representing the feedback-sensitive type of cyclohexadienyl dehydrogenase. The catalytic-efficiency constant k(cat)/K(m) for prephenate (7.0x10(7) M/s) was much better than that of L-arogenate (5.7x10(6) M/s). TyrA(c) was sensitive to feedback inhibition by either L-tyrosine or 4-hydroxyphenylpyruvate, competitively with respect to either prephenate or L-arogenate and non-competitively with respect to NAD(+). A variety of related compounds were tested as inhibitors, and the minimal inhibitor structure was found to require only the aromatic ring and a hydroxyl substituent. Analysis by multiple alignment was used to compare 17 protein sequences representing TyrA family members having catalytic domains that are independent or fused to other catalytic domains, that exhibit broad substrate specificity or narrow substrate specificity, and that possess or lack sensitivity to endproduct inhibitors. We propose that the entire TyrA protein family lacks a discrete allosteric domain and that inhibitors act competitively at the catalytic site of different family members which exhibit individuality in the range and extent of molecules recognized as substrate or inhibitor.

Amino Acid Sequence↗

Storing biological sequence databases in relational form.

SUMMARY: We have created a set of applications using Perl and Java in combination with XML technology to install biological sequence databases into an Oracle RDBMS. An easy-to-use interface using Java has been created for database query and other tools developed to integrate with our in-house bioinformatics applications. AVAILIBILITY: The database schema, DTD file, and source codes are available from the authors via email. CONTACT: guochun_ xie@merck. com

Amino Acid Sequence↗

Secretory response of endothelin-1 in cultured human glomerular microvascular endothelial cells to shear stress.

The shear-induced secretory response of endothelin-1 (ET-1) by human microvascular endothelial cells was studied using paired human glomerular microvascular endothelial cell (HGMEC) cultured monolayers exposed to steady-state laminar shear stress for up to 10 hours. The first cell monolayer was subjected to a shear stress of 0.65 N m-2 and the second, 1.3 N m-2. ET-1 secretion was determined by radioimmunoassay. Over 10 hours of shear, the total cumulative secretion of ET-1 was 237.4 pg/cm2 for the monolayer exposed to 1.3 N m-2 and 143.6 pg/cm2 for the monolayer exposed to 0.65 N m-2. The average ET-1 secretion rate was 20.90 +/- 2.15 and 12.45 +/- 1.05 pg/cm2.h at 0.65 N m-2 and 1.3 N m-2, respectively. The results showed that ET-1 secretion varied with the time of shear in a nonlinear fashion. Although the level of shear stress affected the absolute value of ET-1 cumulative secretion and secretion rate, the major secretion period for both monolayers occurred between 2.0 and 8.0 hours, with the peak secretion rate occurring at approximately 5 hours. Thus, the response of cultured human microvascular endothelial cells to shear stress differed from that of large vessel endothelial cell cultures in terms of ET-1 secretion. In addition to the level of shear stress, the time of shear was also an important determinant of ET-1 secretion. Consequently, the heterogeneity of vascular endothelial cells and the time of shear should both be considered in future research on the secretion of vascular endothelial cell cultures.

Capillaries↗

DNA-dependent protein kinase-independent activation of p53 in response to DNA damage.

Phosphorylation at serine 15 of the human p53 tumor suppressor protein is induced by DNA damage and correlates with accumulation of p53 and its activation as a transcription factor. The DNA-dependent protein kinase (DNA-PK) can phosphorylate serine 15 of human p53 and the homologous serine 18 of murine p53 in vitro. Contradictory reports exist about the requirement for DNA-PK in vivo for p53 activation and cell cycle arrest in response to ionizing radiation. While primary SCID (severe combined immunodeficiency) cells, that have defective DNA-PK, show normal p53 activation and cell cycle arrest, a transcriptionally inert form of p53 is induced in the SCID cell line SCGR11. In order to unambiguously define the role of the DNA-PK catalytic subunit (DNA-PKcs) in p53 activation, we examined p53 phosphorylation in mouse embryonic fibroblasts (MEFs) from DNA-PKcs-null mice. We found a similar pattern of serine 18 phosphorylation and accumulation of p53 in response to irradiation in both control and DNA-PKcs-null MEFs. The induced p53 was capable of sequence-specific DNA binding even in the absence of DNA-PKcs. Transactivation of the cyclin-dependent-kinase inhibitor p21, a downstream target of p53, and the G1 cell cycle checkpoint were also found to be normal in the DNA-PKcs -/- MEFs. Our results demonstrate that DNA-PKcs, unlike the related ATM protein, is not essential for the activation of p53 and G1 cell cycle arrest in response to ionizing radiation.

Animals↗