Biomedical subjects

Kewei Zhang

Publications and source records attributed to Kewei Zhang.

8 recordsLinked to original sources

PubMed2007

Event-specific quantitative detection of nine genetically modified maizes using one novel standard reference molecule.

With the development of genetically modified organism (GMO) detection techniques, the Polymerase Chain Reaction (PCR) technique has been the mainstay for GMO detection, and real-time PCR is the most effective and important method for GMO quantification. An event-specific detection strategy based on the unique and specific integration junction sequences between the host plant genome DNA and the integrated gene is being developed for its high specificity. This study establishes the event-specific detection methods for TC1507 and CBH351 maizes. In addition, the event-specific TaqMan real-time PCR detection methods for another seven GM maize events (Bt11, Bt176, GA21, MON810, MON863, NK603, and T25) were systematically optimized and developed. In these PCR assays, the fluorescent quencher, TAMRA, was dyed on the T-base of the probe at the internal position to improve the intensity of the fluorescent signal. To overcome the difficulties in obtaining the certified reference materials of these GM maizes, one novel standard reference molecule containing all nine specific integration junction sequences of these GM maizes and the maize endogenous reference gene, zSSIIb, was constructed and used for quantitative analysis. The limits of detection of these methods were 20 copies for these different GM maizes, the limits of quantitation were about 20 copies, and the dynamic ranges for quantification were from 0.05 to 100% in 100 ng of DNA template. Furthermore, nine groups of the mixed maize samples of these nine GM maize events were quantitatively analyzed to evaluate the accuracy and precision. The accuracy expressed as bias varied from 0.67 to 28.00% for the nine tested groups of GM maize samples, and the precision expressed as relative standard deviations was from 0.83 to 26.20%. All of these indicated that the established event-specific real-time PCR detection systems and the reference molecule in this study are suitable for the identification and quantification of these GM maizes.

Base Sequence↗

PubMed2006

GOLD HULL AND INTERNODE2 encodes a primarily multifunctional cinnamyl-alcohol dehydrogenase in rice.

Lignin content and composition are two important agronomic traits for the utilization of agricultural residues. Rice (Oryza sativa) gold hull and internode phenotype is a classical morphological marker trait that has long been applied to breeding and genetics study. In this study, we have cloned the GOLD HULL AND INTERNODE2 (GH2) gene in rice using a map-based cloning approach. The result shows that the gh2 mutant is a lignin-deficient mutant, and GH2 encodes a cinnamyl-alcohol dehydrogenase (CAD). Consistent with this finding, extracts from roots, internodes, hulls, and panicles of the gh2 plants exhibited drastically reduced CAD activity and undetectable sinapyl alcohol dehydrogenase activity. When expressed in Escherichia coli, purified recombinant GH2 was found to exhibit strong catalytic ability toward coniferaldehyde and sinapaldehyde, while the mutant protein gh2 completely lost the corresponding CAD and sinapyl alcohol dehydrogenase activities. Further phenotypic analysis of the gh2 mutant plants revealed that the p-hydroxyphenyl, guaiacyl, and sinapyl monomers were reduced in almost the same ratio compared to the wild type. Our results suggest GH2 acts as a primarily multifunctional CAD to synthesize coniferyl and sinapyl alcohol precursors in rice lignin biosynthesis.

Alcohol Oxidoreductases↗

PubMed2006

Clinical oral doses of dexamethasone decreases intrinsic clearance of quinidine, a cytochrome P450 3A substrate in dogs.

We investigated the effect of dexamethasone (DEX) at clinical doses on the pharmacokinetics of quinidine (QN) in dogs. Dogs (5 healthy 1-year-old male beagles) were orally administered DEX once daily for 5 days at 2.5 or 7.5 mg/day. QN (2 mg/kg) was intravenously injected 3 weeks before and one day after the DEX treatment. The plasma concentration of QN was determined by high-performance liquid chromatography with fluorometric detection. Plasma concentrations of albumin and alpha(1)-acid glycoprotein (AGP) were determined by a bromocresol green method and a single immunodiffusion method, respectively. In order to calculate unbound concentrations of QN in plasma, the binding kinetics of QN in plasma was examined by an ultrafiltration method using pooled plasma from the 5 dogs when they were drug-free. Total body clearance of QN was decreased dose-dependently By the DEX treatment, although the decrease was not statistically significant. Elimination half-lives significantly increased (more than twice at 7.5 mg), and intrinsic clearance significantly decreased (about 50%). The volume of distribution increased significantly (about two-fold). Plasma levels of AGP significantly decreased, and the unbound fraction of QN in plasma significantly increased. Our results demonstrate that clinical doses of DEX significantly affect the pharmacokinetics of QN, a CYP3A substrate in dogs, by decreasing CYP3A activity and plasma AGP levels. There is a possibility that adverse drug-drug interaction occurs during DEX therapy through its effects on CYP3A activity and plasma AGP levels.

Animals↗

PubMed2006

Effect of oral administration of clinically relevant doses of dexamethasone on regulation of cytochrome P450 subfamilies in hepatic microsomes from dogs and rats.

OBJECTIVE: To evaluate the effect of oral administration of dexamethasone (DEX) at clinically relevant doses on metabolic activities of cytochrome P450 (CYP) isoenzymes in dogs and rats. ANIMALS: 15 healthy 1-year-old male Beagles and 20 healthy 10-week-old male Wistar rats. PROCEDURE: Hepatic microsomes were harvested from dogs treated orally with DEX at 2.5 and 7.5 mg for 5 days and from rats treated orally with DEX at 0.75, 6, and 48 mg/kg for 5 days. 7-ethoxyresorufin, tolbutamide, bufuralol, and midazolam were used as CYP1A, CYP2C, CYP2D, and CYP3A substrates, respectively. Concentrations of metabolites formed by CYPs were measured by use of high-performance liquid chromatography, except for the resorufin concentrations measured by use of a fluorometric method. Reaction velocity-substrate concentration data were analyzed to obtain maximum reaction velocity (Vmax) and Michaelis-Menten constant (Km). RESULTS: Values of Vmax for midazolam 4-hydroxylation were significantly decreased by treatment with DEX at 2.5 and 7.5 mg in dogs, although values of Km were not affected. Values of Vmax for bufuralol 1'-hydroxylation were also decreased by treatment with DEX. In rats, values of Vmax for midazolam 4- hydroxylation were significantly decreased by treatment with DEX at 0.75 and 6 mg/kg but significantly increased at 48 mg/kg. Other reactions were not affected by treatment with DEX. CONCLUSIONS AND CLINICAL RELEVANCE: Our results indicate that DEX downregulates the CYP3A subfamily when administered at clinically relevant doses to dogs. The effect of downregulation of CYP3A in dogs treated with DEX should be considered to avoid adverse effects from coadministration of drugs.

Administration, Oral↗

PubMed2005

Event specific qualitative and quantitative polymerase chain reaction detection of genetically modified MON863 maize based on the 5'-transgene integration sequence.

Because of the genetically modified organisms (GMOs) labeling policies issued in many countries and areas, polymerase chain reaction (PCR) methods were developed for the execution of GMO labeling policies, such as screening, gene specific, construct specific, and event specific PCR detection methods, which have become a mainstay of GMOs detection. The event specific PCR detection method is the primary trend in GMOs detection because of its high specificity based on the flanking sequence of the exogenous integrant. This genetically modified maize, MON863, contains a Cry3Bb1 coding sequence that produces a protein with enhanced insecticidal activity against the coleopteran pest, corn rootworm. In this study, the 5'-integration junction sequence between the host plant DNA and the integrated gene construct of the genetically modified maize MON863 was revealed by means of thermal asymmetric interlaced-PCR, and the specific PCR primers and TaqMan probe were designed based upon the revealed 5'-integration junction sequence; the conventional qualitative PCR and quantitative TaqMan real-time PCR detection methods employing these primers and probes were successfully developed. In conventional qualitative PCR assay, the limit of detection (LOD) was 0.1% for MON863 in 100 ng of maize genomic DNA for one reaction. In the quantitative TaqMan real-time PCR assay, the LOD and the limit of quantification were eight and 80 haploid genome copies, respectively. In addition, three mixed maize samples with known MON863 contents were detected using the established real-time PCR systems, and the ideal results indicated that the established event specific real-time PCR detection systems were reliable, sensitive, and accurate.

Animals↗

PubMed2005

Identification and quantification of three genetically modified insect resistant cotton lines using conventional and TaqMan real-time polymerase chain reaction methods.

As the genetically modified organisms (GMOs) labeling policies are issued in many countries, qualitative and quantitative polymerase chain reaction (PCR) techniques are increasingly used for the detection of genetically modified (GM) crops in foods. Qualitative PCR and TaqMan real-time quantitative PCR methods to detect and identify three varieties of insect resistant cotton, i.e., Mon531 cotton (Monsanto Co.) and GK19 and SGK321 cottons (Chinese Academy of Agricultural Sciences), which were approved for commercialization in China, were developed in this paper. Primer pairs specific to inserted DNAs, such as Cowpea trypsin inhibitor (CpTI) gene of SGK321 cotton and the specific junction DNA sequences containing partial Cry1A(c) gene and NOS terminator of Mon531, GK19, and SGK321 cotton varieties were designed to conduct the identified PCR assays. In conventional specific identified PCR assays, the limit of detection (LOD) was 0.05% for Mon531, GK19, or SGK321 in 100 ng of cotton genomic DNA for one reaction. Also, the multiplex PCR method for screening the three GM cottons was also established, which could save time and cost in practical detection. Furthermore, a real-time quantitative PCR assay based on TaqMan chemistry for detection of insect resistant gene, Cry1A(c), was developed. This assay also featured the use of a standard plasmid as a reference molecule, which contained both a specific region of the transgene Cry1A(c) and an endogenous stearoyl-acyl carrier protein desaturase (Sad1) gene of the cotton. In quantitative PCR assay, the quantification range was from 0.01 to 100% in 100 ng of the genome DNA template, and in the detection of 1.0, 3.0, and 5.0% levels of three insect resistant cotton lines, respectively, all of the relative standard deviations (RSDs) were less than 8.2% except for the GM cotton samples with 1.0% Mon531 or GK19, which meant that our real-time PCR assays involving the use of reference molecule were reliable and practical for GM insect resistant cottons quantification. All of these results indicated that our established conventional and TaqMan real-time PCR assays were applicable to detect the three insect resistant cottons qualitatively and quantitatively.

Animals↗

PubMed2005

Enhancement of resistance to aphids by introducing the snowdrop lectin gene gna into maize plants.

In order to enhance the resistance to pests, transgenic maize (Zea mays L.) plants from elite inbred lines containing the gene encoding snowdrop lectin (Galanthus nivalis L. agglutinin; GNA) under control of a phloem-specific promoter were generated through the Agrobacterium tumefaciens-mediated method. The toxicity of GNA-expressing plants to aphids has also been studied. The independently derived plants were subjected to molecular analyses. Polymerase chain reaction (PCR) and Southern blot analyses confirmed that the gna gene was integrated into maize genome and inherited to the following generations. The typical Mendelian patterns of inheritance occurred in most cases. The level of GNA expression at 0.13%-0.28% of total soluble protein was observed in different transgenic plants. The progeny of nine GNA-expressing independent transformants that were derived separately from the elite inbred lines DH4866, DH9942, and 8902, were selected for examination of resistance to aphids. These plants synthesized GNA at levels above 0.22% total soluble protein, and enhanced resistance to aphids was demonstrated by exposing the plants to corn leaf aphid (Rhopalosiphum maidis Fitch) under greenhouse conditions. The nymph production was significantly reduced by 46.9% on GNA-expressing plants. Field evaluation of the transgenic plants supported the results from the inoculation trial. After a series of artificial self-crosses, some homozygous transgenic maize lines expressing GNA were obtained. In the present study, we have obtained new insect-resistant maize material for further breeding work.

Agrobacterium tumefaciens↗

PubMed2005

Qualitative and quantitative PCR methods for event-specific detection of genetically modified cotton Mon1445 and Mon531.

Based on the DNA sequences of the junctions between recombinant and cotton genomic DNA of the two genetically modified (GM) cotton varieties, herbicide-tolerance Mon1445 and insect-resistant Mon531, event-specific primers and probes for qualitative and quantitative PCR detection for both GM cotton varieties were designed, and corresponding detection methods were developed. In qualitative PCR detection, the simplex and multiplex PCR detection systems were established and employed to identify Mon1445 and Mon531 from other GM cottons and crops. The limits of detection (LODs) of the simplex PCR were 0.05% for both Mon1445 and Mon531 using 100 ng DNA templates in one reaction, and the LOD of multiplex PCR analysis was 0.1%. For further quantitative detection using TaqMan real-time PCR systems for Mon1445 and Mon531, one plasmid pMD-ECS, used as reference molecule was constructed, which contained the quantitative amplified fragments of Mon1445, Mon531, and cotton endogenous reference gene. The limits of quantification (LOQs) of Mon1445 and Mon531 event-specific PCR systems using plasmid pMD-ECS as reference molecule were 10 copies, and the quantification range was from 0.03 to 100% in 100 ng of the DNA template for one reaction. Thereafter, five mixed cotton samples containing 0, 0.5, 0.9, 3 and 5% Mon1445 or Mon531 were quantified using established real-time PCR systems to evaluate the accuracy and precision of the developed real-time PCR detection systems. The accuracy expressed as bias varied from 1.33 to 8.89% for tested Mon1445 cotton samples, and from 2.67 to 6.80% for Mon531. The precision expressed as relative standard deviations (RSD) were different from 1.13 to 30.00% for Mon1445 cotton, and from 1.27 to 24.68% for Mon531. The range of RSD was similar to other laboratory results (25%). Concluded from above results, we believed that the established event-specific qualitative and quantitative PCR systems for Mon1445 and Mon531 in this study are acceptable and suitable for GM cotton identification and quantification.

Animals↗