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Biomedical subjects

Jing Cheng

Publications and source records attributed to Jing Cheng.

54 records · Page 3Linked to original sources
PubMed2004

Structural insights into molecular function of the metastasis-associated phosphatase PRL-3.

Phosphatases and kinases are the cellular signal transduction enzymes that control protein phosphorylation. PRL phosphatases constitute a novel class of small (20 kDa), prenylated phosphatases with oncogenic activity. In particular, PRL-3 is consistently overexpressed in liver metastasis in colorectal cancer cells and represents a new therapeutic target. Here, we present the solution structure of PRL-3, the first structure of a PRL phosphatase. The structure places PRL phosphatases in the class of dual specificity phosphatases with closest structural homology to the VHR phosphatase. The structure, coupled with kinetic studies of site-directed mutants, identifies functionally important residues and reveals unique features, differentiating PRLs from other phosphatases. These differences include an unusually hydrophobic active site without the catalytically important serine/threonine found in most other phosphatases. The position of the general acid loop indicates the presence of conformational change upon catalysis. The studies also identify a potential regulatory role of Cys(49) that forms an intramolecular disulfide bond with the catalytic Cys(104) even under mildly reducing conditions. Molecular modeling of the highly homologous PRL-1 and PRL-2 phosphatases revealed unique surface elements that are potentially important for specificity.

Amino Acid Sequence↗
PubMed2004

One-tube nested RT-PCR enabled by using a plastic film and its application for the rapid detection of SARS-virus.

A general strategy based on the use of a plastic film that allows reverse transcription and nested-PCR in a single closed-tube has been developed. The reaction mixture for the second PCR amplification is quarantined in the cap of the reaction tube during the first round amplification by a piece of plastic film, and later introduced into the PCR amplicons from the first round reaction by centrifugation without opening the reaction tube. The main advantages of our method are its high sensitivity, specificity, simplicity, cost effectiveness, low risk of contamination and the ease in establishment of conditions for nested-PCR. The method has been successfully applied to the detection of the genomic RNA from SARS-CoV, the detection limit of this method is comparable to that of the conventional two-tube nested PCR system. This method could be easily adapted to the detection of other targets.

Biocompatible Materials↗
PubMed2004

Negative dielectrophoretic force assisted construction of ordered neuronal networks on cell positioning bioelectronic chips.

Developing new methods and technologies in order to pattern neurons into regular networks is of utmost scientific interest in the field of neurological research. An efficient method here is developed for trapping neurons and constructing ordered neuronal networks on bioelectronic chips by using arrayed negative dielectrophoretic (DEP) forces. A special bioelectronic chip with well defined positioning electrode arrays was designed and fabricated on silicon substrate. When a high frequency AC signal was applied, the cell positioning bioelectronic chip (CPBC) is able to provide a well-defined non-uniform electric field, and thus generate negative DEP forces. The parameters, such as size of positioning electrode, conductivity of working solution, amplitude and frequency of power signal and cell concentration, were investigated to optimize the performance of the CPBC. When the neuron suspension was added onto the energized bioelectronic chip, the neurons were immediately trapped and quickly formed the predetermined pattern. Neurons may adhere and then be cultured directly on the CPBC, and show good neuron viability and neurite development. The formation of the ordered neuronal networks after two-week culture demonstrates that negative dielectrophoretic force assisted construction of ordered neuronal networks is effective, and it could be used to assist in monitoring functional activities of neuronal networks.

Animals↗
PubMed2004

Monitoring of gene expression profiles and isolation of candidate genes involved in pollination and fertilization in rice ( Oryza sativa L.) with a 10K cDNA microarray.

To monitor gene expression profiles during pollination and fertilization in rice at a genome scale, we generated 73,424 high-quality expressed sequence tags (ESTs) derived from the green/etiolated shoot and pistil (0-5 h after pollination, 5hP) of rice, which were subsequently used to construct a cDNA microarray containing ca. 10 000 unique rice genes. This microarray was used to analyze gene expression in pistil unpollinated (UP), 5hP and 5DAP(5 days after pollination), anther, shoot, root, 10-day-old embryo (10EM) and 10-day-old endosperm (10EN). Clustering analysis revealed that the anther has a gene-expression profile more similar to root than to pistil and most pistil-preferentially expressed genes respond to pollination and/or fertilization. There are 253 ESTs exhibiting differential expression (e +/- 2-fold changes) during pollination and fertilization, and about 70% of them can be assigned a putative function. We also recovered 20 genes similar to pollination-related and/or fertility-related genes previously identified as well as genes that were not implicated previously. Microarray and real-time PCR analyses showed that the array sensitivity was estimated at 1-5 copies of mRNA per cell, and the differentially expressed genes showed a high correlation between the two methods. Our results indicated that this cDNA microarray constructed here is reliable and can be used for monitoring gene expression profiles in rice. In addition, the genes that differentially expressed during pollination represent candidate genes for dissecting molecular mechanism of this important biological process in rice.

Cluster Analysis↗
PubMed2004

[A transgenic mouse that targets the expression of Cre recombinase in hepatocyte].

OBJECTIVE: To construct a mouse that specifically expresses Cre recombinase in hepatocyte. METHODS: A hepatocyte specific transgenic construct containing mouse albumin promoter, the Cre recombinase gene and the poly (A) of human growth factor gene was generated. The linearized constructs were introduced into the fertilized eggs by microinjection to obtain the transgenic mice. The transcriptional specificity of Cre recombinase was detected by reverse transcription polymerase chain reaction (RT-PCR). The expression and function of Cre recombinase were detected by PCR and Southern Blot after crossing the Alb-Cre transgenic mice with the Smad4 conditional knockout mice. RESULTS: The linearized constructs were microinjected into 837 fertilized eggs, and then the 797 effective eggs of microinjected eggs were implanted into the oviducts of 27 pseudo pregnant mice. In the 53 offspring, there were 6 mice carrying the transgene identified by polymerase chain reaction (PCR) and Southern Blot. Cre recombinase transcripts were detected in the livers and testis of the Alb-Cre transgenic mice using RT-PCR. The Cre recombinase was expressed in the livers of the double heterozygous for Alb-Cre and Smad4 floxed allele, and the exon 8 floxed by loxP site was deleted. CONCLUSION: A hepatocyte-specific Cre transgenic mouse was generated successfully. The Cre recombinase expressed specifically in liver and could mediate the recombination between loxP sites in vivo.

Animals↗
PubMed2003

The inhibitory effects of manganese on steroidogenesis in rat primary Leydig cells by disrupting steroidogenic acute regulatory (StAR) protein expression.

Manganese is known to impede the male reproductive function, however, the mechanisms through which the adverse effects are mediated are not clearly elucidated. In order to get insight into those mechanisms, the effects of manganese on the biosynthesis of testosterone by primary rat Leydig cells were examined. Primary Leydig cells were exposed to various concentrations of manganese chloride for different periods of time. Dose and time-dependent reductions of human chorionic gonadotropin (hCG)-stimulated testosterone level were observed in the culture medium. The expression of Steroidogenic Acute Regulatory (StAR) protein and the activities of P450 side-chain cleavage (P450scc) and 3beta-hydroxysteroid dehydrogenase (3beta-HSD) enzymes were also detected. The expression of StAR protein stimulated by hCG was suppressed by manganese chloride at all concentrations (0.01, 0.1, 1.0 mM) and time points (2, 4, 24, 48 h) tested. Progesterone productions treated with 22R-hydroxycholesterol or pregnenolone were reduced after treated by manganese chloride for 24 or 48 h, respectively. The manganese exposure effect on cell viability was significant at 1.0 and 1.5 mM at 24 h, while at 48 h it was significant at every concentration tested. The decreasing effect of manganese on mitochondrial membrane potential was significant at every concentration measured and every time point tested. These data suggest that manganese exposure for 2 and 4 h inhibited rat primary Leydig cell steroidogenesis by decreasing StAR protein expression while 24 and 48 h exposure of manganese chloride caused adverse effects on both StAR protein and P450scc and 3beta-HSD enzyme activity to reduce steroidogenesis. Manganese may also disrupt StAR expression and/or function secondary to mitochondrial dysfunction.

3-Hydroxysteroid Dehydrogenases↗
PubMed2003

Preliminary studies on Alzheimer's disease using cDNA microarrays.

Alzheimer's disease (AD) is characterized by a progressive impairment of memory and intellectual functioning. The disease is a complex multi-factorial disease with the involvement of several possible genes. To study AD, a high-density cDNA array was developed to characterize the mRNA expression profile of 14160 genes. The preliminary results support the amyloid cascade hypothesis as the mechanism of the disease.

Alcohol Oxidoreductases↗
PubMed2003

The polymorphisms of intron1 sequences of HLA-A and -B.

Human leucocyte antigen (HLA) class I antigens are highly polymorphic membrane glycoproteins present on most nucleated cells. The polymorphism region is mainly located in exons 2 and 3 of HLA class I genes, which is flanked by introns 1 and 3. Thus, the sequence information of introns 1 and 3 is important for the genotyping of HLA. However, the information about them has not been extensively reported by now. In this work, the intron 1 sequences of HLA-A and -B of 51 standard genomic DNAs provided by the 13th International Histocompatibility Work Group (IHWG) were cloned and sequenced. The polymorphism of the intron sequences were also analyzed by the software Clustal W 1.82. Some sequences were chosen to compare with the standard sequences published in National Center for Biotechnology Information (NCBI). The comparison showed that all the sequences matched exactly with the standard sequences. The knowledge of intron 1 sequences could be very important not only for developing DNA-based typing strategies for the HLA-A and -B alleles but also for establishing an understanding of the evolutionary mechanisms involved in the polymorphism generation of HLA class I alleles.

HLA-A Antigens↗
PubMed2003

Blocking oligo--a novel approach for improving chip-based DNA hybridization efficiency.

For most of the commonly used DNA chips, the probes are usually single-stranded oligonucleotides and the targets are double-stranded DNAs (dsDNAs). Only one strand of the DNA serves as the target while the other competes with the probes immobilized on the chip for the target and therefore is regarded as the interfering strand. In this report, a novel technique was developed for improving the hybridization efficiency on DNA chips by using blocking oligos, which is complimentary to the target interfering strand to reduce the influence of the interfering strand. The hybridization efficiency of dsDNA was much lower than that of single-stranded DNA (ssDNA) when synthesized DNA targets were tested on the DNA chip. Blocking oligos can improve the hybridization efficiency of dsDNA to about 2/3 that of ssDNA. Blocking oligos have also been applied to PCR products of different lengths for hybridization. The hybridization efficiency with blocking oligos is about three times higher than that without blocking oligos. We have tested PCR products of 1054 and 435 bp using our blocking procedure, and the results are consistent.

DNA↗
PubMed2003

Detection of known mutations in hypertrophic cardiomyopathy using oligonucleotide microarrays assisted by improved base stacking hybridization.

With the assistance of improved base stacking hybridization, a low-density microarray, containing 12 capture probes, was used to identify 7 known hypertrophic cardiomyopathy-related mutations in the gene of MYH7 (beta-myosia heavy chain). The hybridization targets, amplified from 11 plasmids containing wild type or mutation sequences of MYH7 and healthy genomic DNA, were prepared by single-step fluorescence labeled asymmetric PCR. Six single base substitutions and a trinucleotide deletion were identified unambiguously, and the average discrimination ratio (Qmut) for artificial heterozygous samples was as high as 16.2.

Base Sequence↗
PubMed2002

Effects of the near-dipole-dipole interaction on gap solitons in resonantly absorbing gratings.

Gap solitons can exist in a periodic refractive-index grating modified by periodic layers of near-resonant two-level systems. In this work, we include the effect of the density-dependent near-dipole-dipole (NDD) interaction in order to generalize this model. For certain values of the grating parameters, we find that the NDD interaction significantly modifies the frequency bands in which gap solitons can exist. The difference between the model with and without the NDD interaction is discussed. The stability of gap solitons is studied numerically.

Journal Article↗
PubMed2002

Expression profiling of the response of Saccharomyces cerevisiae to 5-fluorocytosine using a DNA microarray.

5-Fluorocytosine is a commonly used antifungal agent. It acts by inhibiting the synthesis of fungal DNA and protein. In order to understand the global response of Saccharomyces cerevisiae to changes in DNA and protein synthesis caused by 5-fluorocytosine, genome-wide transcript profiling following 5-fluorocytosine exposure was obtained. A total of 96 genes were identified as responsive to 25 microg/ml fluorocytosine treatment for 90 min, which caused approximately 17% specific growth inhibition. The transcript levels of 57 genes were increased more than 2-fold, while it was found that the transcript levels of the other 39 genes decreased to a similar extent. Genes involved in DNA repair, synthesis and replication represented the highest proportion of induced genes identified, which may account for the easily acquired resistance to 5-fluorocytosine. Two enzyme encoding genes CTS1 and EGT2, which function in the separation of daughter cells from their mother cells, were down-regulated by a factor of 3.7 and 10.2, respectively, indicating that 5-fluorocytosine may also inhibit the separation of fungal cells.

Antifungal Agents↗
PubMed2002

Response of gene expression in Saccharomyces cerevisiae to amphotericin B and nystatin measured by microarrays.

The response of the yeast Saccharomyces cerevisiae to two polyene antibiotics, amphotericin B and nystatin, was studied by genomic expression profiling. The two agents produced highly similar expression pattern changes, which was consistent with their known identical mechanisms of action on cell membranes. Detailed analysis was focused on the amphotericin B-treated sample in this study. Our data showed that genes involved in mitochondrial ribosomal protein synthesis were more severely repressed than those in the cytoplasm, which might contribute to the cytotoxicity of amphotericin B. To counteract the leakage of intracellular nutrients and ions from the pores in the cell membrane caused by amphotericin B, c. 17 genes involved in transport facilitation were induced, presumably to allow more efficient uptake of nutrients and ions. The expression level of five genes involved in ergosterol synthesis dropped and three genes related to cell wall biogenesis were induced, indicating that the cell membrane and cell wall were also affected by the presence of polyene antibiotics. It was observed that the pleiotropic drug resistance network in yeast cells was activated after exposure to amphotericin B, possibly contributing to the acquisition of amphotericin B resistance. Part of the gene expression alteration measured by microarray was confirmed by quantitative RT-PCR.

Amphotericin B↗
PubMed2002

[Preliminary clinical study on the correlation between allergic rhinitis and food factors].

OBJECTIVE: To study the correlation between the onset of allergic rhinitis(AR) and food factors. METHOD: All participants involving 101 patients with AR(10 cases accompanied with asthma) and 112 patients with nonallergic rhinitis(NAR) were studied in the form of prevalence survey. The components of food such as fat, salt and sugar intake were estimated through a food frequency questionnaire completed by the patients and controls. Scores were obtained depended on the food components. Histamine, dispensed in different concentrations, was dropped in the inferior turbinate to detect the nasal hyperreactivity(NH). RESULT: A significant difference (P < 0.05) was found between the food scores in AR and NAR groups. There was a greater significant difference (P < 0.01) between AR and NAR groups in NH. There was a negative correlation between food scores and histamine dosage both in AR group and AR and NAR group (P < 0.05). CONCLUSION: There is a correlation between the onset of AR and the food factors. The more the fat, salt and sugar are contained in the food, the more obvious the NH is.

Adult↗
PubMed2002

[Murine beta-casein gene sequences direct a human tissue plasminogen activator variant expressed in the milk of transgenic mice].

To investigate the ability of our cloned murine beta-casein locus to direct the exogenous gene expression in the milk of transgenic mice, the human t-PA variant mammary gland expression vector under the control of murine beta-casein gene regulatory elements was constructed, in which the human t-PA variant signal-pro peptide sequence was replaced with murine beta-casein signal peptide sequence and the human t-PA variant mature peptide cDNA was inserted into the second exon of beta-casein gene. The fusion gene was microinjected in the fertilized mice eggs. A total of 285 embryos were microinjected and transferred into 13 surrogate mother mice. Twelve positive transgenic mice were identified through PCR and Southern blot analysis among 42 new born mice. Human t-PA variant was expressed in the milk of 7 transgenic mice, the highest expression level attained to 3.6593 micrograms/ml. The results demonstrated that the murine beta-casein gene regulatory elements can direct the human t-PA variant gene successfully express in the milk of transgenic mice. It lays great foundation for the research on the beta-casein knock-in mice mammary gland bioreactor model construction.

Animals↗
PubMedDate not supplied

Parallel profiling of active transcription factors using an oligonucleotide array-based transcription factor assay (OATFA).

Cell/tissue-specific gene expression are tightly regulated by various combinations of multiple transcription factors (TFs). Here, we present an oligonucleotide array-based transcription factor assay (OATFA), which allows the simultaneous assay of multiple active TFs. In this proof-of-principle work, both purified TFs and cell extracts were analyzed using OATFA and further antibody-based validation confirmed the chip data. This method could simplify the assay of multiple TFs and may facilitate high-throughput profiling of large numbers of TFs.

Biological Assay↗