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Biomedical subjects

Q Ren

Publications and source records attributed to Q Ren.

At least 19 recordsLinked to original sources
PubMed2006

Molecular characterization and expression of the antimicrobial peptide defensin from the housefly (Musca domestica).

A 430-bp cDNA encoding the insect antimicrobial peptide defensin was cloned from the housefly, and designated Musca domestica defensin (Mdde). The open reading frame of the cDNA encoded a 92-amino acid peptide with an N-terminal signal sequence followed by a propeptide that is processed by cleavage to a 40-amino acid mature peptide. Northern analysis and in situ hybridization identified the corresponding mRNA in the fat body of bacterially challenged houseflies and in the epidermis of the body wall of naive and challenged houseflies. The Gram-negative bacterium (Escherichia coli) is a strong inducer of the gene. By RT-PCR, Mdde mRNA was also detected in naive and challenged insects. These findings suggest that the defensin gene is constitutively expressed in the epidermis of the housefly body wall. The predicted mature form of Mdde was expressed as a recombinant peptide in E. coli and Pichia pastoris. The recombinant Mdde expressed in Pichia was active against Gram-positive and some Gram-negative bacteria.

Amino Acid Sequence↗
PubMed2005

The ergosterol biosynthesis pathway, transporter genes, and azole resistance in Aspergillus fumigatus.

The continuous use of triazoles can result in the development of drug resistance. Azole-resistant clinical isolates, spontaneous and induced mutants of Aspergillus fumigatus have been documented. The azoles block the ergosterol biosynthesis pathway by inhibiting the enzyme 14-alpha-demethylase, product of the CYP51. Fungal azole resistance involves both amino acid changes in the target site that alter drug-target interactions and those that decrease net azole accumulation. The reduced intracellular accumulation has also been correlated with overexpression of multidrug resistance (MDR) efflux transporter genes of the ATP-binding cassette (ABC) and the major facilitator superfamily (MFS) classes. About 20 genes are involved in the A. fumigatus ergosterol biosynthesis pathway. There are several duplicated genes in this pathway. Interestingly, erg3 and erg11 showed two copies in A. fumigatus. In general, Aspergillus spp. have proportionally more MFS transporter encoding genes than Saccharomyces cerevisiae, S. pombe, and Neurospora crassa. The drug H+ (12 and 14 spanners) sub-families are also proportionally greater than in the other species. Although the numbers of ABC transporter encoding genes are comparable, again the Aspergillus spp. have more ABC transporters related to multidrug permease than the other fungal species.

ATP-Binding Cassette Transporters↗
PubMed2003

A Controllable and Observable Protein Cystallization Facility.

A Controllable and Observable Protein Crystallization Facility (ACOP) was developed in 1999 for space experiment in the Get Away Special canister of Space Shuttle. To regulate the vapor diffusion rate, in a crystallization chamber, five cells each containing precipitant solution of different concentrations, exposed to one protein cell in turn. Five layers of chambers were overlapped coaxially. The crystallization process was observed and recorded by digital cameras. Structure of facility and result of image sequence were given.

Crystallization↗
PubMed2002

Evaluation of apolipoprotein A-II as a positional candidate gene for familial Type II diabetes, altered lipid concentrations, and insulin resistance.

AIMS/HYPOTHESIS: We hypothesized that apolipoprotein A-II sequence variation was responsible for the observed linkage of Type II (non-insulin-dependent) diabetes mellitus to the apolipoprotein A-II region in Northern European families ascertained for multiple diabetic siblings, and might also influence insulin sensitivity and secretion, non-esterified fatty acids, and lipids. METHODS: We recruited 698 members of 63 families for pedigree studies and additional unrelated people providing 117 diabetic and 130 control subjects. We screened the apolipoprotein A-II gene by single strand conformation polymorphism analysis and fluorescent sequence analysis. Variants were typed by oligonucleotide ligation assay, restriction digest of amplification products, or radioactive fragment analysis for the microsatellite polymorphism. Association of each variant with Type II diabetes was tested in the case-control population by chi-square analysis, or using transmission disequilibrium test in families. Haplotypes were established in families using SIMWALK and tested for association with diabetes and quantitative traits. RESULTS: No detected variant altered the coding sequence of the gene. Three single nucleotide polymorphisms showed modest evidence for an association, but no variant or haplotype was associated with diabetes in families. Similarly, we found no association with non-esterified fatty acid concentrations, HDL concentrations, or fasting insulin. In contrast, we found evidence for an association of some haplotypes and individual variants with 2-h post-challenge glucose and measures of insulin secretion. CONCLUSION/INTERPRETATION: Apolipoprotein A-II is not likely to explain the observed linkage of Type II diabetes, but variation in this gene could alter insulin secretion and post-challenge glucose.

Apolipoprotein A-II↗
PubMed2001

Physical interaction of CcmC with heme and the heme chaperone CcmE during cytochrome c maturation.

Biogenesis of c-type cytochromes requires the covalent attachment of heme to the apoprotein. In Escherichia coli, this process involves eight membrane proteins encoded by the ccmABCDEFGH operon. CcmE binds heme covalently and transfers it to apocytochromes c in the presence of other Ccm proteins. CcmC is necessary and sufficient to incorporate heme into CcmE. Here, we report that the CcmC protein directly interacts with heme. We further show that CcmC co-immunoprecipitates with CcmE. CcmC contains two conserved histidines and a signature sequence, the so-called tryptophan-rich motif, which is the only element common to cytochrome c maturation proteins of bacteria, archae, plant mitochondria, and chloroplasts. We report that mutational changes of these motifs affecting the function of CcmC in cytochrome c maturation do not influence heme binding of CcmC. However, the mutants are defective in the CcmC-CcmE interaction, suggesting that these motifs are involved in the formation of a CcmC-CcmE complex. We propose that CcmC, CcmE, and heme interact directly with each other, establishing a periplasmic heme delivery pathway for cytochrome c maturation.

Amino Acid Sequence↗
PubMed2001

Complete genome sequence of an M1 strain of Streptococcus pyogenes.

The 1,852,442-bp sequence of an M1 strain of Streptococcus pyogenes, a Gram-positive pathogen, has been determined and contains 1,752 predicted protein-encoding genes. Approximately one-third of these genes have no identifiable function, with the remainder falling into previously characterized categories of known microbial function. Consistent with the observation that S. pyogenes is responsible for a wider variety of human disease than any other bacterial species, more than 40 putative virulence-associated genes have been identified. Additional genes have been identified that encode proteins likely associated with microbial "molecular mimicry" of host characteristics and involved in rheumatic fever or acute glomerulonephritis. The complete or partial sequence of four different bacteriophage genomes is also present, with each containing genes for one or more previously undiscovered superantigen-like proteins. These prophage-associated genes encode at least six potential virulence factors, emphasizing the importance of bacteriophages in horizontal gene transfer and a possible mechanism for generating new strains with increased pathogenic potential.

Bacteriophages↗
PubMed2001

Histone H2A.Z acetylation modulates an essential charge patch.

Histone H2A.Z is structurally and functionally distinct from the major H2As. To understand the function of H2A.Z acetylation, we performed a mutagenic analysis of the six acetylated lysines in the N-terminal tail of Tetrahymena H2A.Z. Tetrahymena cannot survive with arginines at all six sites. Retention of one acetylatable lysine is sufficient to provide the essential function of H2A.Z acetylation. This essential function can be mimicked by deleting the region encompassing all six sites, or by mutations that reduce the positive charge of the N terminus at the acetylation sites themselves, or at other sites in the tail. These properties argue that the essential function of H2A.Z acetylation is to modify a "charge patch" by reducing the charge of the tail.

Acetylation↗
PubMed2000

Recovery of active medium-chain-length-poly-3-hydroxyalkanoate polymerase from inactive inclusion bodies using ion-exchange resin.

A novel process for the purification of active medium-chain-length-polyhydroxyalkanoate (mcl-PHA) polymerase was developed. This process is based on solubilization and activation of inactive polymerase inclusion bodies by incubation with ion-exchange resin. The mcl-PHA polymerase 1 from Pseudomonas oleovorans was overproduced from the Palk promoter. Most of the polymerase produced was sequestered in the cytoplasm as an inactive form in insoluble aggregates. By incubating the protein aggregates with S-Sepharose ion-exchange resin in the presence of dithiothreitol and glycerol, the mcl-PHA polymerase could be extracted in an active and soluble form with a final yield of about 5.2 mg/g of cell dry weight. The solubilized polymerase was able to catalyse the in vitro synthesis of mcl-PHA without any additional cell components, suggesting its potential application for production of biopolymer. The procedure used here may be of general value in solubilizing and activating purified inactive labile enzymes.

Acyltransferases↗
PubMed2000

Development of an improved in vitro activity assay for medium chain length PHA polymerases based on CoenzymeA release measurements.

An improved activity assay for polyhydroxyalkanoate (PHA) polymerases from Pseudomonas oleovorans GPo1 was developed. The activity assay is based on the detection of released Coenzyme A (CoA) using 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB), a compound which specifically reacts with thiol groups. The formed adduct was measured spectrophotometrically with high sensitivity and accuracy. The assay was used to study the effect of several additives on the activity of granule-associated PHA polymerase. Mild non-ionic detergents such as Tween-20, Triton X-100, CHAPS and Hecameg all appeared to be strongly inhibitory. In contrast, bovine serum albumin (BSA) had a strong stimulatory effect on the activity and stability of the PHA polymerases. Using optimized conditions, activities up to 5.8 U/mg granule-bound polymerase have been measured.

Acyltransferases↗
PubMed2000

Properties of engineered poly-3-hydroxyalkanoates produced in recombinant Escherichia coli strains.

To prepare medium-chain-length poly-3-hydroxyalkanoates (PHAs) with altered physical properties, we generated recombinant Escherichia coli strains that synthesized PHAs with altered monomer compositions. Experiments with different substrates (fatty acids with different chain lengths) or different E. coli hosts failed to produce PHAs with altered physical properties. Therefore, we engineered a new potential PHA synthetic pathway, in which ketoacyl-coenzyme A (CoA) intermediates derived from the beta-oxidation cycle are accumulated and led to the PHA polymerase precursor R-3-hydroxyalkanoates in E. coli hosts. By introducing the poly-3-hydroxybutyrate acetoacetyl-CoA reductase (PhbB) from Ralstonia eutropha and blocking the ketoacyl-CoA degradation step of the beta-oxidation, the ketoacyl-CoA intermediate was accumulated and reduced to the PHA precursor. Introduction of the phbB gene not only caused significant changes in the monomer composition but also caused changes of the physical properties of the PHA, such as increase of polymer size and loss of the melting point. The present study demonstrates that pathway engineering can be a useful approach for producing PHAs with engineered physical properties.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed2000

FabG, an NADPH-dependent 3-ketoacyl reductase of Pseudomonas aeruginosa, provides precursors for medium-chain-length poly-3-hydroxyalkanoate biosynthesis in Escherichia coli.

Escherichia coli hosts expressing fabG of Pseudomonas aeruginosa showed 3-ketoacyl coenzyme A (CoA) reductase activity toward R-3-hydroxyoctanoyl-CoA. Furthermore, E. coli recombinants carrying the poly-3-hydroxyalkanoate (PHA) polymerase-encoding gene phaC in addition to fabG accumulated medium-chain-length PHAs (mcl-PHAs) from alkanoates. When E. coli fadB or fadA mutants, which are deficient in steps downstream or upstream of the 3-ketoacyl-CoA formation step during beta-oxidation, respectively, were transformed with fabG, higher levels of PHA were synthesized in E. coli fadA, whereas similar levels of PHA were found in E. coli fadB, compared with those of the corresponding mutants carrying phaC alone. These results strongly suggest that FabG of P. aeruginosa is able to reduce mcl-3-ketoacyl-CoAs generated by the beta-oxidation to 3-hydroxyacyl-CoAs to provide precursors for the PHA polymerase.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed2000

O-Glucuronidation of the lung carcinogen 4-(methylnitrosamino)-1- (3-pyridyl)-1-butanol (NNAL) by human UDP-glucuronosyltransferases 2B7 and 1A9.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and its major metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), are potent lung carcinogens in animals. UGT-mediated O-glucuronidation of NNAL is an important detoxification pathway for these carcinogens. To better characterize this pathway in humans, we screened a series of UGT-overexpressing cell lines and baculosome preparations for their ability to O-glucuronidate NNAL and examined multiple human liver and lung specimens for NNAL-glucuronidating activity and their levels of expression of NNAL-glucuronidating UGTs. Human liver microsomal fractions exhibited significant levels of NNAL-glucuronidating activity, with the NNAL-Gluc II diastereomer formed at a rate 3.4 times that observed for NNAL-Gluc I. As with liver microsomal fractions, NNAL-Gluc II was the major diastereomer formed by homogenates from UGT2B7-overexpressing HK293 cells or UGT2B7-overexpressing baculosomes; the major diastereomer formed by homogenates from UGT1A9-overexpressing V79 cells was NNAL-Gluc I. No significant O-glucuronidating activity of NNAL was detected in UGT1A1-, UGT1A4-, UGT1A6-, UGT2B4-, or UGT2B15-overexpressing HK293 or V79 cell homogenates, or in UGT1A1-, UGT1A3-, UGT1A7-, or UGT1A10-overexpressing baculosomes. Significant levels of UGT2B7 mRNA were detected by reverse transcriptase-polymerase chain reaction in human liver and at low levels in human lung specimens. UGT1A9 mRNA was detected in liver but not in lung. These results suggest that although both UGT2B7 and UGT1A9 play an important role in the overall glucuronidation of NNAL in humans, UGT2B7 potentially plays an important role in the detoxification of NNAL in the lung.

Animals↗
PubMed2000

[Relation of GFR and endothelin in the plasma in pilots and ground crew].

OBJECTIVE: To observe the specitic property of Glomeoular Filtration Rate (GFR) and its relation to endothelin of plasma in pilots. METHOD: GFR was assessed with single photon emisson computed tomography (SPECT), tracer for 99mTc-DTPA, enthlion of plasma were measured by radio immunossay in forty-six pilots, thirty ground crew and renal disease patients. RESULT: Endothelin were not correlation with GFR in pilots. GFR of pilots and ground crew had not a significant difference. Compared with ground crew and pilots, endothelin of renal disease patients had a significant increased, and GFR had a significant decreased. Age were a linear negative correlation with total GFR r = (0.84, P < 0.01), flying time, age had not correlation with endothelin. After thirty-one ground endothelin had a significant difference with ground crew. CONCLUSION: It suggested that the endothelin has no influence on GFR in pilots when it was increased with in the limits of a level, pilots and ground crew may use the same stand on GFR.

Adult↗
PubMed2000

[Effect of acute hypobaric hypoxia on renal function and structure in rats].

OBJECTIVE: To observe renal damage due to acute hypobaric hypoxia. METHOD: Thirty-six male wistar rats were randomly divided into three groups A, B and C (n = 12 for each). Group A served as control, while groups B and C were exposed to 5000 m altitude for 30 min. Sample of serum and renal tissues were taken from group B rats 20 min and from group C rats 24 h after the exposure respectively. RESULT: Serum ureanitrogen (BUN) and creatinine (Crea) increased significantly in group B and C rats after exposure to hypoxia (P< 0.01). Cl- and alkaline phosphatase (AKP) in serum also increased (P < 0.05), but renal endothelin (ET) decreased significantly (P < 0.01). All changes in group C rats tended to recover to normal level, except Cl- which continued to increase. Dilation of renal vessels was found under optic-microscope, and pathological changes were found in mitochondra and epithelial cells. CONCLUSION: Acute hypobaric hypoxia might be harmful to renal function and structure.

Alkaline Phosphatase↗
PubMed1999

Production of medium-chain-length poly(3-hydroxyalkanoates) from gluconate by recombinant Escherichia coli.

It was shown recently that recombinant Escherichia coli, defective in the beta-oxidation cycle and harboring a medium-chain-length (MCL) poly(3-hydroxyalkanoate) (PHA) polymerase-encoding gene of Pseudomonas, is able to produce MCL PHA from fatty acids but not from sugars or gluconate (S. Langenbach, B. H. A. Rehm, and A. Steinbüchel, FEMS Microbiol. Lett. 150:303-309, 1997; Q. Ren, Ph.D. thesis, ETH Zürich, Zürich, Switzerland, 1997). In this study, we report the formation of MCL PHA from gluconate by recombinant E. coli. By introduction of genes coding for an MCL PHA polymerase and the cytosolic thioesterase I ('thioesterase I) into E. coli JMU193, we were able to engineer a pathway for the synthesis of MCL PHA from gluconate. We used two expression systems, i.e., the bad promoter and alk promoter, for the 'thioesterase I- and PHA polymerase-encoding genes, respectively, which enabled us to modulate their expression independently over a range of inducer concentrations, which resulted in a maximum MCL PHA accumulation of 2.3% of cell dry weight from gluconate. We found that the amount of PHA and the 'thioesterase I activity are directly correlated. Moreover, the polymer accumulated in the recombinant E. coli consisted mainly of 3-hydroxyoctanoate monomers. On the basis of our data, we propose an MCL PHA biosynthesis pathway scheme for recombinant E. coli JMU193, harboring PHA polymerase and 'thioesterase I, when grown on gluconate, which involves both de novo fatty acid synthesis and beta-oxidation.

Acyltransferases↗
PubMed1999

Glucuronidation of the lung carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) by rat UDP-glucuronosyltransferase 2B1.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone and its major metabolite, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), are potent lung carcinogens in animals. UDP-glucuronosyltransferase (UGT)-mediated glucuronidation of NNAL is a potentially important detoxification pathway for these carcinogens. To identify the UGT isozyme(s) involved in this pathway, we examined the glucuronidation of NNAL in rat liver microsomes and homogenates from cell lines overexpressing specific UGT isozymes. NNAL glucuronidation was induced in liver microsomes from rats treated with family 2 UGT inducers including phenobarbitol and 3, 5-di-tert-butyl-4-hydroxytoluene, which exhibited 1.7- and 2.6-fold higher rates of glucuronidation than microsomes from control rats. The rates of NNAL glucuronidation in liver microsomes from GUNN (deficient in family 1 UGTs) and RHA parental control rats were similar. All rat liver microsomes used in the present study catalyzed the glucuronidation of (S)-NNAL at a rate between 3.5 and 5.5 times that of the glucuronidation of (R)-NNAL. Liver microsomes from Wistar rats exhibiting the low-androsterone glucuronidation phenotype characteristic of the UGT2B2-deficient genotype glucuronidated NNAL at a rate similar to microsomes from Wistar rats exhibiting the high-androsterone glucuronidation phenotype/UGT2B2 [+] genotype. Homogenates from UGT2B1-overexpressing cells catalyzed the glucuronidation of NNAL at a K(m) of 745 microM. As with rat liver microsomes, NNAL-Gluc I was the major diastereomer formed by UGT2B1. Glucuronidation of NNAL was not detected with homogenates from UGT2B12-overexpressing cells. These results suggest that UGT2B1 plays an important role in the glucuronidation of NNAL in the rat.

Animals↗