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H Xiang

Publications and source records attributed to H Xiang.

At least 19 recordsLinked to original sources
PubMed2000

hTERT can function with rabbit telomerase RNA: regulation of gene expression and attenuation of apoptosis.

Telomerase is a specialized DNA polymerase that adds telomeric sequences onto chromosome ends. The functional telomerase complex contains a telomerase reverse transcriptase (TERT) and also a telomerase RNA (TR). Although it is well established that the human telomerase reverse transcriptase (hTERT) can function well in different human cell lines, it has not been shown whether it is compatible with telomerase template RNA from other species. Here we report that the expressed hTERT is functionally compatible with rabbit telomerase template RNA (rTR) as demonstrated by TRAP assay. The direct interaction between hTERT and rTR is further confirmed by immunoprecipitation-linked RT-PCR in which rTR is detected from the complex immunoprecipitated by an anti-hTERT antibody. The hTERT expressed in rabbit lens epithelial cells demonstrates two major functions: modulation of expression of other genes and attenuation of apoptosis. Thus, telomerase has a variety of functions besides telomere synthesis, and the template RNA is functionally conserved between human and rabbit.

Animals↗
PubMed2000

An A245T mutation conveys on cytochrome P450eryF the ability to oxidize alternative substrates.

Cytochrome P450(eryF) (CYP107A1), which hydroxylates deoxyerythronolide B in erythromycin biosynthesis, lacks the otherwise highly conserved threonine that is thought to promote O-O bond scission. The role of this threonine is satisfied in P450(eryF) by a substrate hydroxyl group, making deoxyerythronolide B the only acceptable substrate. As shown here, replacement of Ala(245) by a threonine enables the oxidation of alternative substrates using either H(2)O(2) or O(2)/spinach ferredoxin/ferredoxin reductase as the source of oxidizing equivalents. Testosterone is oxidized to 1-, 11alpha-, 12-, and 16alpha-hydroxytestosterone. A kinetic solvent isotope effect of 2.2 indicates that the A245T mutation facilitates dioxygen bond cleavage. This gain-of-function evidence confirms the role of the conserved threonine in P450 catalysis. Furthermore, a Hill coefficient of 1.3 and dependence of the product distribution on the testosterone concentration suggest that two testosterone molecules bind in the active site, in accord with a published structure of the P450(eryF)-androstenedione complex. P450(eryF) is thus a structurally defined model for the catalytic turnover of multiply bound substrates proposed to occur with CYP3A4. In view of its large active site and defined structure, catalytically active P450(eryF) mutants are also attractive templates for the engineering of novel P450 activities.

Amino Acid Substitution↗
PubMed2000

The active-site residue Cys-29 is responsible for the neutral-pH inactivation and the refolding barrier of human cathepsin B.

Human cathepsin B, the most abundant lysosomal cysteine protease, has been implicated in a variety of important physiological and pathological processes. It has been known for a long time that like other lysosomal cysteine proteases, cathepsin B becomes inactivated and undergoes irreversible denaturation at neutral or alkaline pH. However, the mechanism of this denaturation process remains mostly unknown up to this day. In the present work, nuclear magnetic resonance spectroscopy was used to characterize the molecular origin of the neutral-pH inactivation and the refolding barrier of human cathepsin B. Two forms of human cathepsin B, the native form with Cys-29 at the active site and a mutant with Cys-29 replaced by Ala, were shown to have well-folded structures at the active and slightly acidic condition of pH 5. Surprisingly, while the native cathepsin B irreversibly unfolds at pH 7.5, the C29A mutant was found to maintain a stable three-dimensional structure at neutral pH conditions. In addition, replacement of Cys-29 by Ala renders the process of the urea denaturation of human cathepsin B completely reversible, in contrast to the opposite behavior of the wild-type cathepsin B. These results are very surprising in that replacement of one single residue, the active-site Cys-29, can eliminate the neutral-pH denaturation and the refolding barrier. We speculate that this finding may have important implications in understanding the process of pH-triggered inactivation commonly observed for most lysosomal cysteine proteases.

Cathepsin B↗
PubMed2000

A geographic information assessment of birth weight and crop production patterns around mother's residence.

A landscape approach using remote sensing (RS) and geographic information system (GIS) technologies was developed to identify the proximity of maternal residence to agricultural areas, and the association between crop production patterns around mothers residences and low birth weight was evaluated. Satellite data obtained for Weld County, Colorado, in 1991 and 1993 were digitally processed to generate crop location maps for this study. GIS procedures were then used to determine crop types within 300- and 500-m circular zones around mothers' homes for 125 births that occurred from 1991 to 1993 in the study area. The relationships between different crop patterns around the mothers' residences and the birth weight of babies were investigated while controlling for potential confounding variables. The results indicated that low birth weight was associated with total crop production area within a 300-m buffer zone around mothers' residences (P=0.058). When each of the study crops was considered separately, low birth weight was associated with sugar beet production (P=0.05) and corn production (P=0.1) within both the 300- and the 500-m buffer zones. This RS/GIS approach provides clues to the potential relationships between birth weight and crop production near the residence.

Adult↗
PubMed2000

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Journal Article↗
PubMed2000

Agricultural work-related injuries among farmers in Hubei, People's Republic of China.

OBJECTIVES: This population-based study evaluated patterns of and risk factors for, agricultural injuries among farmers in the People's Republic of China. METHODS: A multistage sample of 1500 Chinese farmers was selected from 14 villages. Face-to-face interviews with 1358 farmers were conducted between July 1997 and September 1997 (response rate = 91%). Agricultural work-related injuries that occurred in the previous 24 months and the associated factors were evaluated. RESULTS: A total of 33% of the farmers reported at least 1 work-related injury in the 24 months before the survey. Major external causes of the injuries were hand tools (50%), falls (26%), and heavy falling objects (10%). The statistically significant risk factors for injury were low family income, 1 to 6 school years of education, self-reported pesticide exposure, tension in relationships with neighbors, and stress in life. The most notable result was the relation between self-reported pesticide exposure and injury, with farmers with greater pesticide exposure at significantly greater risk for injury. CONCLUSIONS: The results of this study indicated that injuries occurring among Chinese farmers may have unique patterns and potential risk factors.

Accidents, Occupational↗
PubMed2000

[Cloning and expression of human Cu/Zn superoxide dismutase gene in Lactococcus lactis].

The cDNA encoding human Cu/Zn SOD was amplified by RT-PCR using the total RNA of human liver as the template, and was cloned into an E. coli expression vector pET23b. After the DNA sequence was determined, the recombinant plasmid pET23bsod was introduced into E. coli BL21(DE3)/pLysS. SDS-PAGE analysis revealed that the recombinant E. coli expressed the predicted 19 kDa human Cu/Zn SOD, and its amount was over 50% of total proteins. The Cu/Zn SOD cDNA was then subcloned into a lactococcal expression vector pMG36e, and resulting pMG36esod was introduced into L. lactis MG1363 by electroporation. The human Cu/Zn-SOD was expressed up to 5% of the soluble proteins, and the enzymatic activity was also observed by SOD activity dying.

Cloning, Molecular↗
PubMed1999

Interchange of catalytic activity within the 2-enoyl-coenzyme A hydratase/isomerase superfamily based on a common active site template.

The structures and chemical pathways associated with the members of the 2-enoyl-CoA hydratase/isomerase enzyme superfamily are compared to show that a common active site design provides the members of this family with a CoA binding site, an expandable acyl binding pocket, an oxyanion hole for binding/polarizing the thioester C=O, and multiple active site stations for the positioning of acidic and basic amino acid side chains for use in proton shuttling. It is hypothesized that this active site template can be tailored to catalyze a wide range of chemical transformations through strategic positioning of acid/base residues among the active site stations. To test this hypothesis, the active site of one member of the 2-enoyl-CoA hydratase/isomerase family, 4-chlorobenzoyl-CoA dehalogenase, was altered by site-directed mutagenesis to include the two glutamate residues functioning in acid/base catalysis in a second family member, crotonase. Catalysis of the syn hydration of crotonyl-CoA, absent in the wild-type 4-chlorobenzoyl-CoA dehalogenase, was shown to occur with the structurally modified 4-chlorobenzoyl-CoA dehalogenase at kcat = 0.06 s-1 and Km = 50 microM.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1999

Conformation of a Cdc42/Rac interactive binding peptide in complex with Cdc42 and analysis of the binding interface.

Most of the putative effectors for the Rho-family small GTPases Cdc42 and Rac share a common sequence motif referred to as the Cdc42/Rac interactive binding (CRIB) motif. This sequence, with a consensus of I-S-x-P-(x)2-4-F-x-H-x-x-H-V-G [Burbelo, P. D., et al. (1995) J. Biol. Chem. 270, 29071-29074], has been shown to be essential for the functional interactions between these effector proteins and Cdc42. We have characterized the interactions of a 22-residue CRIB peptide derived from human PAK2 [PAK2(71-92)] with Cdc42 using proton and heteronuclear NMR spectroscopy. This CRIB peptide binds to GTP-gammaS-loaded Cdc42 in a saturable manner, with an apparent Kd of 0.6 microM, as determined by fluorescence titration using sNBD-labeled Cdc42. Interaction of the 22-residue peptide PAK2(71-92) with GTP-gammaS-loaded Cdc42 causes resonance perturbations in the 1H-15N HSQC spectrum of Cdc42 that are similar to those observed for a longer (46-amino acid) CRIB-containing protein fragment [Guo, W., et al. (1998) Biochemistry 37, 14030-14037]. Proton NMR studies of PAK2(71-92) demonstrate structuring of PAK2(71-92) in the presence of GTP-gammaS-loaded Cdc42, through the observation of many nonsequential transferred NOEs. Structure calculations based on the observed transferred NOEs show that the central portion of the Cdc42-bound CRIB peptide assumes a loop conformation in which the side chains of consensus residues Phe80, His82, Ile84, His85, and Val86 are brought into proximity. The CRIB motif may therefore represent a minimal interfacial region in the complexes between Cdc42 and its effector proteins.

Amino Acid Sequence↗
PubMed1999

Contribution of p53-dependent caspase activation to neuronal cell death declines with neuronal maturation.

Caspases play a pivotal role in neuronal cell death during development and after trophic factor withdrawal. However, the mechanisms regulating caspase activity and the role played by caspase activation in response to neuronal injury is poorly understood. The tumor suppressor gene p53 has been implicated in the loss of neuronal viability caused by excitotoxic and DNA damaging agents. In the present study we determined if p53-mediated neuronal cell death required caspase activation. DNA damage increased caspase activity in both cultured embryonic telencephalic and postnatal cortical neurons in a p53-dependent manner. Caspase inhibitors protected embryonic telencephalic neurons, but not postnatal cortical neurons, from DNA damage-induced cell death as measured by direct cell counting and annexin V staining. In marked contrast to the caspase inhibitors, an inhibitor of the DNA repair enzyme, poly(ADP-ribose) polymerase, conferred significant protection from genotoxic and excitotoxic cell death on postnatal cortical neurons but had no effect on embryonic neurons. Glutamate-mediated excitotoxicity in postnatal neurons was not associated with measurable changes in caspase activity, consistent with the failure of caspase inhibitors to prevent cell death under these conditions. Moreover, adenovirus-mediated overexpression of p53 killed embryonic and postnatal neurons without activating caspases. Thus, p53-mediated neuronal cell death may occur via both caspase-dependent and caspase-independent pathways. These results demonstrate that p53 is required for caspase activation in response to some forms of neuronal injury. However, the relative importance of caspase activation in neurons depends on the developmental status of the cell and the specific nature of the death stimulus.

Adenoviridae↗
PubMed1999

Modulating electron density in the bound product, 4-hydroxybenzoyl-CoA, by mutations in 4-chlorobenzoyl-CoA dehalogenase near the 4-hydroxy group.

The enzyme 4-chlorobenzoyl-CoA dehalogenase hydrolyzes 4-chlorobenzoyl-CoA (4-CBA-CoA) to 4-hydroxybenzoyl-CoA (4-HBA-CoA). Biochemical and crystallographic studies have identified a critical role for the dehalogenase residue Asp 145 in close proximity to the ligand's 4-hydroxy group in the structure of the product-enzyme complex. In the present study the effects of site selective mutations at Asp 145 on the product complex are explored by Raman spectroscopy. The spectral signatures of the WT-product complex, the large red shift in lambdamax, and the complete reorganization of the benzoyl ring modes in Raman data are absent for the D145E complex. The major spectral perturbations in the WT complex are brought about by strong electron "pull" at the benzoyl carbonyl and electron "push" by the side chain of Asp 145 near the 4-OH group. Acting in concert, these factors polarize the benzoyl's pi-electrons. Since the Raman data show that very strong electron pull occurs at the benzoyl's carbonyl in the D145E complex, it is apparent that the needed electron push near the benzoyl's 4-OH group is missing. Thus, very precise positioning of Asp 145's side chain near the benzoyl's 4-position is needed to bring about the dramatic electron reorganization seen in the WT complex, and this criterion cannot be met by the glutamate side chain with its additional CH2 group. For two other Asp145 mutants D145A and D145S that lack catalytic activity, Raman difference spectroscopic data for product complexes demonstrate the presence of a population of ionized product (i.e., 4-O-) in the active sites. The presence of the ionized phenolate form explains the observation that these complexes have highly red-shifted absorbance maxima with lambdamaxs near 400 nm. For the WT complex only the 4-OH form is seen, ionization being energetically expensive with the presence of the proximal negative charge on the Asp 145 side chain. Semiquantitative estimates of the pKa for the bound product in D145S and D145A indicate that this ionization lies in the pH 6.5-7.0 range. This is approximately 2 pH units below the pKa for the free product. The Raman spectrum of 4-dimethylaminobenzoyl-CoA undergoes major changes upon binding to dehalogenase. The bound form has two features near 1562 and 1529 cm-1 and therefore closely resembles the spectrum of product bound to wild-type enzyme, which underlines the quinonoid nature in these complexes. The use of a newly developed Raman system allowed us to obtain normal (nonresonance) Raman data for the dehalogenase complexes in the 100-300 microM range and heralds an important advance in the application of Raman spectroscopy to dilute solutions of macromolecules.

Acyl Coenzyme A↗
PubMed1999

Product catalyzes the deamidation of D145N dehalogenase to produce the wild-type enzyme.

Aspartate 145 plays an essential role in the active site of 4-chlorobenzoyl-CoA dehalogenase, forming a transient covalent link at the 4-position of the benzoate during the conversion of the substrate to 4-hydroxybenzoyl-CoA. Replacement of Asp 145 by residues such as alanine or serine results in total inactivation, and stable complexes can be formed with either substrate or product. The Raman spectroscopic characterization of some of the latter is described in the preceding publication (Dong et al.). The present work investigates complexes formed by D145N dehalogenase and substrate or product. Time-resolved absorption and Raman difference spectroscopic data show that these systems evolve rapidly with time. For the substrate complex, initially the absorption and Raman spectra show the signatures of the substrate bound in the active site of the asparagine 145 form of the enzyme but these signatures are accompanied by those for the ionized product. After several minutes these signatures disappear to be replaced with those closely resembling the un-ionized product in the active site of wild-type dehalogenase. Similarly, for the product complex, the absorption and Raman spectra initially show evidence for ionized product in the active site of D145N, but these are rapidly replaced by signatures closely resembling the un-ionized product bound to wild-type enzyme. It is proposed that product bound to the active site of asparagine 145 dehalogenase catalyzes the deamidation of the asparagine side chain to produce the wild-type aspartate 145. For the complexes involving substrate, the asparagine 145 enzyme population contains a small amount of the WT enzyme, formed by spontaneous deamidation, that produces product. In turn, these product molecules catalyze the deamidation of Asn 145 in the major enzyme population. Thus, conversions of substrate to product and of D145N to D145D dehalogenase go on simultaneously. The spontaneous deamidation of asparagine 145 has been characterized by allowing the enzyme to stand at RT in Hepes buffer at pH 7.5. Under these conditions deamidation occurs with a rate constant of 0.0024 h-1. The rate of product-catalyzed deamidation in Hepes buffer at 22 degrees C was measured by stopped-flow kinetics to be 0.024 s-1, 36000 times faster than the spontaneous process. A feature near 1570 cm-1 could be observed in the early Raman spectra of both substrate and product-enzyme complexes. This band is not associated with either substrate or product and is tentatively assigned to an ester-like species formed by the attack of the product's 4-O- group on the carbonyl of asparagine's side chain and the subsequent release of ammonia. A reaction scheme is proposed, incorporating these observations.

Amides↗
PubMed1999

Mechanisms of hypoxia-induced endothelial cell death. Role of p53 in apoptosis.

Endothelial cell death may contribute to tissue injury from ischemia. Little is known, however, about the characteristics of endothelial cell death in response to hypoxia. Using an in vitro model, we found that human umbilical vein endothelial cells were resistant to hypoxia-induced cell death with only a 2% reduction in viability at 24 h and 45% reduction in viability at 48 h. Overexpression of a mutant, IkappaBalpha, via adenoviral vector did not potentiate cell death in hypoxia, indicating that nuclear factor-kappaB activation was not involved in cytoprotection. Cell death in hypoxia was determined to be apoptotic by 3' labeling of DNA using terminal deoxynucleotidyl transferase staining and reversibility of cell death with a caspase inhibitor. Exposure of endothelial cells to hypoxia did not alter levels of proapoptotic and antiapoptotic Bcl-2 family members Bax and Bcl-XL by immunoblot analysis. In contrast, changes in p53 protein levels correlated with the induction of apoptosis in hypoxic endothelial cells. Inhibition of the proteasome increased p53 protein levels and accelerated cell death in hypoxia. Overexpression of p53 by adenoviral transduction was sufficient to initiate apoptosis of normoxic endothelial cells. These data provide a framework for the study of factors regulating endothelial cell survival and death in hypoxia.

Adenoviridae↗
PubMed1999

Back pain and agricultural work among farmers: an analysis of the Colorado Farm Family Health and Hazard Surveillance Survey.

BACKGROUND: Back pain is the most prevalent occupational health problem experienced by much of the world's workforce. However, agricultural work-related back pain occurring among US farmers working on small operations or family farms is usually not included in surveillance. With data collected by Colorado Farm Family Health and Hazard Surveillance Survey, this study reports characteristics of and risk factors for back pain among adult farmers living in eight Colorado counties. METHODS: A stratified probability sample of 500 farms was selected in proportion to the number of farms in study areas. During the 4-year period from 1993 through 1996, 458 farms were enrolled in the study and 759 farmers were interviewed using a questionnaire. Information on self-reported back pain and potential risk factors among 742 white farmers was evaluated and reported here. RESULTS: A total of 194 farmers (26.2%) reported to have had at least one episode of back pain lasting for 1 week or more. Males had a slightly higher prevalence of back pain than females (28.6% vs. 22.5%) and the lower back was the predominantly affected part of body among both males and females. In 45.4% of males and 43.9% of females back pain was brought on by repeated activities. Males' activities at work were more likely to cause back pain while females' activities at home were more likely to cause back pain. Three factors were found to be significantly associated with back pain: being depressed (odds ratio (OR) = 3.68, 95% confidence interval (CI) = 2.23-6.09), farming/ranching as main activities (OR = 1.66, CI = 1.17-2.36), and worked in agriculture for 10 to 29 years (OR = 1.62, CI = 1.14-2.30). CONCLUSIONS: Our analyses indicate that back pain is an occupational health problem among farmers on small operations or family farms and that back pain affected males and females differently. The finding of significant positive associations between depression, farming activities, and back pain warrants further attention.

Adult↗
PubMed1999

Cigarette smoking among medical college students in Wuhan, People's Republic of China.

BACKGROUND: This study examined cigarette smoking among a sample of medical college students in Wuhan, People's Republic of China. Specific cultural factors associated with cigarette smoking in China were discussed and issues which need to be studied in the future are presented. METHODS: A self-administrated questionnaire was administered to a sample of 1,611 medical students in 1995. Smoking status was based on self-reported use of cigarettes within the preceding 180 days. Information about demographic characteristics, home residence, annual family income, and smoking status of family members was also obtained. RESULTS: A total of 1,540 (1,201 males and 339 females) completed and returned the questionnaires (response rate 95.6%). None of the 339 female students surveyed reported cigarette smoking. A total of 37.7% (95% confidence interval (CI) = 35.0-40.5%) of the male medical students were current smokers, and 5. 3% (95% CI = 4.0-6.6%) were former smokers. The mean age of beginning to smoke was 16.5 years (95% CI = 8.7-24.3 years). The major reasons for first smoking were stress (42.8%, 95% CI = 38.2-47. 4%), curiosity (34.4%, 95% CI = 29.6-38.4%), and loneliness (33.7%, 95% CI = 28.7-37.4%). Multivariate analyses suggested that age, college year, and having a family member who smoked were significantly associated with cigarette smoking. CONCLUSIONS: The results indicated a need to provide comprehensive tobacco use prevention programs among medical students.

Adolescent↗
PubMed1999

Generation of enhancer trap lines in Arabidopsis and characterization of expression patterns in the inflorescence.

Eleven thousand, three hundred and seventy enhancer/promoter trap lines in Arabidopsis were generated via T-DNA transformation utilizing the binary vector pD991 that contains a minimal promoter fused to the uidA reporter gene. Overall 31% of the lines generated exhibit a staining pattern in the inflorescence. Flanking DNA has been cloned from 15 lines exhibiting inflorescence staining patterns by either thermal asymmetric interlaced PCR (TAIL-PCR), inverse PCR (IPCR), or partial library construction. Seeds from these lines are available from the ABRC and NASC Arabidopsis stock centers and DNA pools are available from the ABRC.

Arabidopsis↗
PubMed1999

Nonfatal agricultural injuries among Colorado older male farmers.

OBJECTIVES: This study described characteristics and potential risk factors for nonfatal agricultural injuries among older male farmers age 60 years and older in Colorado. METHODS: A telephone survey was conducted among 113 older male farmers between 1993 and 1995. Agricultural injuries and potential risk factors were evaluated. RESULTS: A total of 27 injuries were sustained. The leading external cause of injuries was livestock, and most of the injuries affected lower and upper extremities of the body. Injured farmers were more likely to be persons who had gone deeply into debt or who were using prescription medications. DISCUSSION: Older farmers are at risk of injuries. The study results support the need to consider physical limitations of aging in developing prevention programs in agriculture.

Accidents, Occupational↗