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Biomedical subjects

Rong Shi

Publications and source records attributed to Rong Shi.

At least 37 records · Page 2Linked to original sources
PubMed2004

Proteomic analysis on structural proteins of Severe Acute Respiratory Syndrome coronavirus.

Recently, a new coronavirus was isolated from the lung tissue of autopsy sample and nasal/throat swabs of the patients with Severe Acute Respiratory Syndrome (SARS) and the causative association with SARS was determined. To reveal further the characteristics of the virus and to provide insight about the molecular mechanism of SARS etiology, a proteomic strategy was utilized to identify the structural proteins of SARS coronavirus (SARS-CoV) isolated from Vero E6 cells infected with the BJ-01 strain of the virus. At first, Western blotting with the convalescent sera from SARS patients demonstrated that there were various structural proteins of SARS-CoV in the cultured supernatant of virus infected-Vero E6 cells and that nucleocaspid (N) protein had a prominent immunogenicity to the convalescent sera from the patients with SARS, while the immune response of spike (S) protein probably binding with membrane (M) glycoprotein was much weaker. Then, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the complex protein constituents, and the strategy of continuous slicing from loading well to the bottom of the gels was utilized to search thoroughly the structural proteins of the virus. The proteins in sliced slots were trypsinized in-gel and identified by mass spectrometry. Three structural proteins named S, N and M proteins of SARS-CoV were uncovered with the sequence coverage of 38.9, 93.1 and 28.1% respectively. Glycosylation modification in S protein was also analyzed and four glycosylation sites were discovered by comparing the mass spectra before and after deglycosylation of the peptides with PNGase F digestion. Matrix-assisted laser desorption/ionization-mass spectrometry determination showed that relative molecular weight of intact N protein is 45 929 Da, which is very close to its theoretically calculated molecular weight 45 935 Da based on the amino acid sequence deduced from the genome with the first amino acid methionine at the N-terminus depleted and second, serine, acetylated, indicating that phosphorylation does not happen at all in the predicted phosphorylation sites within infected cells nor in virus particles. Intriguingly, a series of shorter isoforms of N protein was observed by SDS-PAGE and identified by mass spectrometry characterization. For further confirmation of this phenomenon and its related mechanism, recombinant N protein of SARS-CoV was cleaved in vitro by caspase-3 and -6 respectively. The results demonstrated that these shorter isoforms could be the products from cleavage of caspase-3 rather than that of caspase-6. Further, the relationship between the caspase cleavage and the viral infection to the host cell is discussed.

Amino Acid Sequence↗
PubMed2004

[Preparation of the microarray for detecting hepatitis D virus].

OBJECTIVE: To prepare the microarray for detection of hepatitis D virus (HDV). METHOD: Several pairs of specific PCR primers were designed according to the conserved region of HDV genome. The DNA microarray were prepared by blotting the PCR products onto the surface of glass slides with the use of robotics, and restriction display PCR (RD-PCR) was employed to label the samples. RESULT: Sequences analysis showed that the products of PCR amplification were the specific gene fragments of HDV. Hybridization signals on the gene chip demonstrated good specificity and sensitivity of the microarray for HDV detection. CONCLUSION: Microarray-based clinical HDV detection can be sensitive and effective.

Base Sequence↗
PubMed2004

[Application of microarrays in screening the tumor-specific genes in the genome of K562 cells].

OBJECTIVE: To screen tumor-specific genes of K562 cells using DNA microarray technique. METHODS: The genomic DNA of normal white blood cells and cultured K562 cells were respectively purified and digested with Sau3A I, and the digested DNA fragments of K562 cells were cloned into TA cloning vector to construct the corresponding genomic DNA library. The insert genomic DNA fragments were amplified from the library to prepare the microarray using Cartesian 5500 Microarrayer. The digested genomic DNA fragments of normal white blood cells were labeled with fluorescent Cy3 by restriction display PCR (RD-PCR), followed by hybridization with the microarray, after which the slide was washed and scanned with ScanArray. RESULTS: Among the 426 target genes, 42 differential genes were identified in the genomic DNA of K562 cells in comparison with the normal white blood cells. One of the genes was identified as the breakpoint cluster gene (BCR) after sequence analysis. CONCLUSIONS: The DNA microarrays we constructed may effectively identify the tumor-specific genes in K562 cells, and DNA microarray technique can be helpful in elucidating the molecular mechanisms of tumorigenesis at the genomic level.

Amino Acid Sequence↗
PubMed2004

[Isolation of the target gene from cDNA restriction fragments using 70-mer oligo microarray].

OBJECTIVE: To study the method for using a 70-mer oligo microarray as the probe to isolate target genes from the cDNA restriction fragments. METHOD: Samples of Saccharomyces cerevisiae mRNA was extracted after heat shock culture and reversely transcribed into the double-stranded cDNAs, which were prepared into restriction cDNA fragments using restriction display (RD) method. The microarray was printed using a single 70-mer specific oligo designed to according to the SSA1 gene of yeast. The cDNA restriction fragments were labeled by PCR method with the Cy5 universal primer before hybridization with the microarray. The microarray was stripped after washing and scanning, and the strip solution was collected for another round of PCR amplification using the universal primer without fluorescence. The PCR product was then cloned into PUC18 T vector and transformed into to E.coli JM109 cells for amplification, and the plasmids were extracted and sequenced for identification. RESULTS: BLAST results showed that the target gene was cloned successfully. CONCLUSION: The target gene can be isolated directly using the 70-mer oligo microarray as the probe from the cDNA fragments prepared by RD method, without the necessity of building a cDNA library. This method can also be used in further research to acquire the differentially expressed genes after the oligo microarray hybridization.

Base Sequence↗
PubMed2004

Detection of hepatitis D virus by cDNA microarray method.

BACKGROUND: Viral hepatitis is considered a major public health problem in most areas of the world. In acute and chronic infections, hepatitis D virus (HDV) infection often leads to a more severe disease. This study was designed to prepare microarrays for HDV detection. METHODS: The specific primers of PCR were designed according to the conserved region of HDV. The cDNA microarrays were prepared by spotting PCR products onto the surface of glass slides by robotics. Restriction display PCR (RD-PCR) was used to label the samples. RESULTS: Sequences were aligned, and the results showed that the products of PCR amplification were the specific gene fragments of HDV. Hybridizing signals on gene chip showed the specificity and sensitivity in detecting HDV were satisfactory. CONCLUSION: Using PCR amplified products to construct gene chips for clinical diagnosis of HDV is a quick, simple and effective method.

Base Sequence↗
PubMed2004

[Oligonucleotide microarray preparation using enhanced poly-L-lysine glass slides].

OBJECTIVE: To modify conventional poly-L-lysine coating for oligonucleotide microarray preparation so as to enhance the sensitivity of the microarray. METHOD: The proposed chemical approaches included silanizing the slides with 3-glycid-oxypropyltrimethoxysilane (GOPS) after cleaning, followed by slide coating with polymers (poly-L-lysine) that was covalently bound to the modified glass. Subsequent attachment of the oligonucleotide to the modified slide surface was achieved after 1,4-phenylene diisothiocyanate (PDITC) activation of the surface. Various experiments were carried out, such as the immobilization efficiency and hybridization assays to test the modified slides, which were then used tentatively in the preparation of microarrays for SARS coronavirus detection. RESULTS: The improved surface had high immobilization efficiency, good uniformity and satisfactory hybridization efficiency, better than those slides with conventional poly-L-lysine coating. In addition, such modified slides rendered the microarrays more resistant to consecutive probing/stripping cycles. CONCLUSION: The modified slide surface is satisfactory to immobilize unmodified oligonucleotide by covalent binding, which enhances not only the sensitivity of the prepared oligonucleotide microarray but also the binding of the oligonucleotide to the slide surface.

DNA, Viral↗
PubMed2004

[Parental education to reduce blood lead levels in children with mild and moderate lead poisoning: a randomized controlled study].

OBJECTIVE: To evaluate the effectiveness of parental education on reducing lead exposure of children by examining the changes in blood lead levels of children whose parents receiving or not receiving educational intervention. METHODS: Two hundred children with confirmed blood lead levels beyond 100 microg/L were selected. They were randomized into two groups, 107 children in study group and 93 in control. At the beginning of the study, parents of both study and control groups were called for interview to complete KABP questionnaire and lead study questionnaire. The study group was provided with interventional measures while control group was not contacted until the end of study. Intervention of parental education was undertaken by means of a TV program, a set of slides and a brochure, and focused on the questions regarding harmful effects of lead poisoning, the sources of environmental lead and prevention of this preventable disease. Tests for blood lead level were repeated for both study and control groups 3 months after the determination of the initial blood lead level. RESULTS: All the relevant knowledge of health effect, lead sources and prevention of childhood lead poisoning of participating parents of study group were improved significantly (chi(2) = 14.06, 13.07, 10.08, 28.26, P < 0.01) after educational intervention while parents control group also were significantly improved in the sub-catalogs of concept and prevention (chi(2) = 7.69, 8.64, P < 0.01), but not the health effect and sources of childhood lead poisoning. Children and parents' behavior in study group was improved accordingly and significantly. Less children ate popcorn (chi(2) = 4.08, P < 0.05), less children drank tap-water in the morning (chi(2) = 23.04, P < 0.01), more kids washed their hands before eating (chi(2) = 5.82, P < 0.05), less kids played on road side (chi(2) = 9.60, P < 0.01), and more parents changed their coat or took shower or washed hands before going home after work (chi(2) = 4.00, P < 0.05). But in the control group only the number of kids playing on road side was decreased significantly (chi(2) = 9.60, P < 0.01). A general decline in blood lead levels was detected in both groups with statistical significance at P < 0.01. However, the decrease in blood lead levels was more remarkable in the study group. There was average reduction of 55 microg/L (35%) in blood lead levels for study group (t = 4.979, P < 0.01) and an almost 33 microg/L (20%) for control (t = 3.398, P < 0.01). The reduction in blood lead level was 22 microg/L greater in study group (t = 3.531, P < 0.01). The study also showed that the effectiveness of the educational interventions depended upon various aspects. Fourteen variables were included in the stepwise multiple regression equation of blood lead level changes. Such as parents' occupational exposure to lead, the improvement of knowledge about prevention of childhood lead poisoning, the chang of habit of snacks intake, parents' education levels, the change of attitude of parents for the childhood lead poisoning, etc. CONCLUSION: Educating parents is proved to be an effective approach for children with mild and moderate lead poisoning.

Child↗
PubMed2003

Identifying androsterone (ADT) as a cognate substrate for human dehydroepiandrosterone sulfotransferase (DHEA-ST) important for steroid homeostasis: structure of the enzyme-ADT complex.

In steroid biosynthesis, human dehydroepiandrosterone sulfotransferase (DHEA-ST) in the adrenals has been reported to catalyze the transfer of the sulfonate group from 3'-phosphoadenosine-5'-phosphosulfate to dehydroepiandrosterone (DHEA). DHEA and its sulfate play roles as steroid precursors; however, the role of the enzyme in the catabolism of androgens is poorly understood. Androsterone sulfate is clinically recognized as one of the major androgen metabolites found in urine. Here it is demonstrated that this enzyme recognizes androsterone (ADT) as a cognate substrate with similar kinetics but a 2-fold specificity and stronger substrate inhibition than DHEA. The structure of human DHEA-ST in complex with ADT has been solved at 2.7 A resolution, confirming ADT recognition. Structural analysis has revealed the binding mode of ADT differs from that of DHEA, despite the similarity of the overall structure between the ADT and the DHEA binary complexes. Our results identify that this human enzyme is an ADT sulfotransferase as well as a DHEA sulfotransferase, implying an important role in steroid homeostasis for the adrenals and liver.

Adrenal Glands↗
PubMed2003

Possibility of using DNA chip technology for diagnosis of human papillomavirus.

To explore the application of DNA chip technology for the detection and typing of Human Papillomavirus (HPV), the HPV6, 11, 16 and 18 gene fragments were isolated and printed onto aminosilane-coated glass slides by a PixSys 5500 microarrayer as probes to prepare the HPV gene chips. HPV samples, after being labeled with fluorescent dye by restriction display PCR (RD-PCR) technology, were hybridized with the microarray, which was followed by scanning and analysis. The experimental condition for preparing the HPV gene chips was investigated, and the possibility of HPV genotyping using gene chips was discussed. The technique that was established in this study for preparing HPV gene chips is practical. The results of the present study demonstrated the versatility and inspiring prospect of using this technology to detect and genotype HPV.

DNA Probes↗
PubMed2003

How estrogen-specific proteins discriminate estrogens from androgens: a common steroid binding site architecture.

Steroid hormones play an essential role in a wide range of physiological and pathological processes, such as growth, metabolism, aging, and hormone-sensitive cancers. Estrogens are no exception and influence growth, differentiation, and functioning of many target tissues, such as the mammary gland, uterus, hypothalamus, pituitary, bone, and liver. Although very similar in structure, each steroid class (i.e., estrogens, androgens, progestins, mineral corticoids, or glucocorticoids) is responsible for distinct physiological processes. To permit specific biological responses for a given steroid class, specific proteins are responsible for steroid bioactivation, action, and inactivation, yet they have low or no affinity to other classes. Estrogens make no exception and possess their own set of related proteins. To understand the molecular basis underlying estrogen recognition from other steroids, structural features of estrogen-specific proteins were analyzed along with their ability to discriminate between steroid hormones belonging to different classes. Hence, the study of all estrogen-specific proteins for which an atomic structure has been determined demonstrated that a common steroid-binding pocket architecture is shared by these proteins. This architecture is composed of the following elements: i) a glutamate residue acting as a proton acceptor coupled with a proton donor that interact with the steroid O3; ii) a proton donor (His or Ser) that interacts with O17; iii) a highly conserved sandwich-like structure providing steric hindrance and preventing C19 steroid from binding; and iv) several amino acid residues interacting with the C18. As these different estrogen-specific proteins are not related in overall sequence, the inference is that the steroid binding site in these proteins has originated by convergent evolution.

17-Hydroxysteroid Dehydrogenases↗
PubMed2003

A modified restriction display PCR method in sample-labelling of DNA microarray.

The restriction display PCR is a useful technique for studying the diversity of gene expression. This method involves ligating the digested genes with adapters and amplifying the gene fragments by PCR using universal and selective primers. In this study, we improved this restriction display PCR method by using Cy3-UP, a fluorescently labelled universal primer, in place of Cy3-dCTP in sample-labelling for DNA microarray. The results show that this new method increases significantly the sensitivity of the assay, and will have a wide application in the DNA microarray field.

Carbocyanines↗
PubMed2003

[Two restriction fluorescence labeling methods for enhancing the signal-to-noise ratio of cDNA microarray hybridization].

OBJECTIVE: To study the signal-to-noise ratio (SNR) of two restricted fluorescence labeling methods for examining gene expression profile by microarray hybridization. METHOD: Samples of Saccharomyces cerevisiae mRNA was labeled by traditional reverse transcription method and 2 restriction fluorescent labeling methods using respectively Cy-universal primer and extension incorporated Cy-dNTP. The labeled samples were examined by the microarray, followed by washing and scanning under the same conditions. RESULTS: The two restriction labeling methods showed superior results with lowered background and enhanced SNR and sensitivity, and Cy-universal primer labeling presented the best results. CONCLUSION: SNR can be enhanced by the restriction labeling methods, which improve the applicability of microarray technology.

Fluorescence↗
PubMed2003

Application of Agilent 2100 Bioanalyzer in detection of human papilloma virus.

OBJECTIVE: To explore the feasibility of using Agilent 2100 bioanalyzer in the detection and genotyping of human papilloma virus (HPV). METHODS: The consensus sequence of highly conserved region (L1) and genotype-specific gene fragments of all HPV genotypes (including HPV6, 11, 16 and 18) were amplified by PCR technique using general and type-specific primers respectively. Agilent 2100 Bioanalyzer and routine agarose gel electrophoresis were then employed respectively to examine the PCR products, and the accuracy, reproducibility and sensitivity of these 2 detection techniques were compared. RESULTS: Agilent 2100 Bioanalyzer showed better accuracy in determining the length of the gene fragments than agarose gel electrophoresis, the accuracy of the former reaching above 95% while the latter was only 85%. Bioanalyzer was also 100 times more sensitive than agarose electrophoresis. CONCLUSIONS: Agilent 2100 Bioanalyzer combined with PCR is specific, accurate and sensitive for HPV detection and genotyping.

Electrophoresis, Agar Gel↗
PubMed2003

[Analysis of gene expression patterns of leukemia K562 cells after cytochalasin B treatment].

BACKGROUND & OBJECTIVE: The advanced technique of DNA microarray makes it possible to monitor the expression of thousands of genes simultaneously in one hybridization experiment. This technique accelerates demonstration of anti-tumor drug mechanisms and discovery of new drug targets. This study was designed to investigate the differential gene expression of K562 cells after cytochalasin B treatment using cDNA microarray. METHODS: Restriction display polymerase chain reaction (RD-PCR) products of 277 human genes were spotted on a glass slide in microarray. K562 cells grew in RPMI 1640 medium with 10 microg/ml cytochalasin B. After 24 hours, the total RNA was isolated from K562 cells, and mRNA was purified. Both mRNA from the treated K562 cells and the controlled K562 cells were reversely transcribed into cDNA and labeled with two different fluorescence dyes: Cy5 or Cy3, using a method of restriction digestion and PCR labeling (RD-PCR). The probes were hybridized to the cDNA microarrays. After high-stringent washing,the cDNA microarray was scanned for the fluorescent signals and showed difference between the two cells. RESULTS: Among the 277 target genes, 18 down-regulated genes were identified after cytochalasin B treatment. CONCLUSION: There is a consistent tendency toward lower-expressed genes in partial K562 cells after cytochalasin B treatment. Most down-regulated genes were correlated with cell proliferation, signal transduction, and transcription factor.

Cytochalasin B↗
PubMed2003

Gene expression study of Saccharomyces cerevisiae with the Agilent 2100 bioanalyser.

This study explores the restriction display-polymerase chain reaction (RD-PCR) application of a new chip-based nucleic acid analysis system (Agilent 2100 bioanalyser) in a gene differential expression study. Total RNAs is extracted from Saccharomyces cerevisiae, double-stranded complementary DNA (cDNA) is synthesised by reverse transcription from the purified messenger RNA (mRNA), RD-PCR conducted to obtain the cDNA fragments and bioanalyser and agarose gel electrophoresis compared for the analysis of RD-PCR products. The bioanalyser proved to be faster and more sensitive in separating and detecting gene fragments, and was also able to compare different gene fragments quantitatively. Using this technology, comparison of several differential gene fragments is performed.

DNA↗
PubMed2003

Rapid preparation of DNA microarray using PCR for hepatitis B and D virus detection.

OBJECTIVE: To prepare DNA microarray for detecting both hepatitis B and D virus as (HBV and HDV). METHODS: With the assistance of Oligo6.4 software, specific PCR primers targeting the conserved region of HBV and HDV were designed. The PCR products were purified and cloned into the pMD18-T vectors, followed by rapid identification. The recombinant plasmids were then extracted from positive clones and the target gene fragments underwent sequence analysis. RESULTS: The gene fragments of HBV and HDV were obtained by PCR, which were confirmed by sequence analysis to be specific gene fragments of HBV and HDV. CONCLUSION: Using PCR amplification products to prepare the DNA microarray is quick, simple and effective.

Hepatitis B virus↗
PubMed2003

[Study on development of DNA microarrays for human papillomavirus (HPV) diagnosis].

To study the technology for establishing DNA microarrays for the diagnosis of HPV. HPV6, 11, 16 and 18 gene fragments were isolated and printed onto aminosilane-coated glass slides by PixSys 5500 microarray printer as probes to prepare the HPV. HPV samples, after labeled with Cy3 or Cy5, were hybridaized with the microarray followed by scanning for analysis. The experimental condition for preparing the HPV gene chips was investigated and the possibility of HPV genotying using DNA microarrays was discussed. The technique established in this study for preparing HPV DNA microarrays is applicable and has potential clinical application significance.

Alphapapillomavirus↗