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Biomedical subjects

C Zhu

Publications and source records attributed to C Zhu.

At least 127 records · Page 7Linked to original sources
PubMed1999

A modified Boyden chamber assay for tumor cell transendothelial migration in vitro.

We have developed a modified Boyden chamber system for assessing tumor cell transmigration across endothelial monolayer in vitro. This radiometric-based method allows us to take into account the cells which have traversed but detached from the filter, a significant fraction not included in the existing method of visual counting. The method was applied to examine six malignant and one non-malignant cell lines. The variable invasive potentials of these cells were found to correlate with their ability to disrupt the endothelial cell monolayers.

Biological Assay↗
PubMed1999

[Detection of ABO genotypes by simultaneous PCR-RFLP method].

OBJECTIVE: To study human ABO genotyping by means of multiplex PCR and restriction fragment length polymorphism. METHODS: Two specific fragments of ABO gene were simultaneously amplified in a single tube, and then the double restriction digestion with RE Kpn I and Alu I was performed in the same tube. The amplified products were analyzed by PAGE and silver staining. A total of 125 Han unrelated individuals living in Wuhan were genotyped. RESULTS: Six genotypes of ABO were detected and the distribution was in good agreement with Hardy-Weinberg equilibrium. CONCLUSION: The results demonstrate that PCR-RFLP based approach is convenient, reliable and should be of value in forensic application.

ABO Blood-Group System↗
PubMed1999

[Expression of glycoprotein hepatitis C virus in mammalian cell and application of purified protein for detection of antibody against E2 in hepatitis C patients].

OBJECTIVE: E2 glycoprotein of hepatitis C virus was expressed in mammalian cell and purified for detection of antibody against E2 in hepatitis C patients. METHODS: E2/NS1 gene derived from HCV was inserted into expression vector containing six His tag. The recombinant plasmid was transfected into mammalian cells to express E2 glycoprotein expression. E2 glycoprotein was purified by affinity chromophotography. The purified protein was used to establish EIA method for detection of antibodies against E2 in hepatitis C patients. RESULT: Expressed E2 glycoprotein was 7.0 x 10(4). Purification of the purified E2 protein was 90.2%. Twenty-nine patients were anti-E2 antibody positive(82.9%). CONCLUSION: It was the first time to establish EIA method for detection of anti-E2 antibody by purified E2 glycoprotein in China. E2 glycoprotein expressed in mammalian cells had good immunogenity and could increase the sensibility of anti-HCV detection. It suggests that E2 glycoprotein may be useful for development of new anti-HCV reagents.

Hepatitis C↗
PubMed1999

[Histology and physiological function of dogs after extirpation of bronchi].

OBJECTIVE: To investigate the effect of extirpation of bronchi on histology and physiological function. METHODS: 21 dogs were divided in to two groups: control (3) and experimental (18). After bronchi extirpation one week, two weeks, one month, three months, six months, and twelve months, we used specimens to test the pulmonary surfactant (PS) and to observe the morphological changes. RESULTS: After the operation, the residual lung nearby the operative area had traumatic changes. Around the aforementioned area, the structure of lung was normal. Specially after six months, the lung returned to good status. We found that two weeks after operation, the contents of PS increased remarkably (P < 0.05). After one month the parameter returned to normal (P > 0.05). CONCLUSIONS: The residual lung after bronchi extirpation had ventilation and air-exchange functions. The lung function was preserved directly.

Animals↗
PubMed1999

[Diagnostic value of ultrasonography in cervical lymph node metastasis].

OBJECTIVE: To research the accuracy of ultrasonography in the diagnosis of cervical lymph node metastasis and the value of that of cervical lymph node occult metastasis. METHOD: Fifty-five patients(61 sides) operated by neck dissection were double-blindedly studied by preoperation palpation and ultrasonography comparing with postoperating pathological examination. RESULT: Fifty-two sides were confirmed to be pathology lymph node metastasis from 61 sides. 40 and 48 sides were checked out by palpation and ultrasonography, respectively. The sensitivity, specificity and accuracy of palpation and ultrasonography were 76.9% and 92.3% (P < 0.05), 77.8% and 88.9%(P > 0.05) and 77.0% and 91.8%(P < 0.05), respectively. Six sides were checked out by ultrasonography (occult metastasis) in 12 sides of palpation negative. CONCLUSION: The accuracy of ultrasonography was prior to that of palpation in examining cervical lymph node metastsis. Ultrasonography could check out about half occult metastasis and should be regarded as a routine examining items in diagnosing cervical lymph node metastasis.

Adult↗
PubMed1999

[Transposition of Tn5096 in a agricultural antibiotic 120 roducer Streptomyces hygrospinocus var. beijingensis RF220].

No transformant was obtained when pCZA168(bla, tsr, Tn5096, ColEI rep. Strep repts) was used to transform S. hygropinocus RF220. pIJ702 isolated from S. hygroscopicus N103 was transformed into RF220 at a low frequency. pIJ702 plasmid was cured in RF220 transformant and it was re-transformed into its cured FR220 strain, but the transformation frequency was not increased significantly, suggesting that restriction-modification system in FR220 was existent and complicated. Four transformants containing pCZA168 were obtained, when the RF220 strain was grown in medium with ampicillin, glycine and the protoplast was stored at -70 degrees C. Restriction analysis of plasmid from transformants indicated that the DNA fragment from E. coli in pCZA168 was deleted. With transposition of Tn5096, two mutants blocked in antibiotic biosynthesis of 120 and some mutants with variation in antibiotic level were obtained, this showed that the Tn5096 transposed in different positions of chromosomal DNA in RF220 and resulted in the production of 120 in different level.

Anti-Bacterial Agents↗
PubMed1999

[Identification study on pig's hoofnail].

The identification characteristics of pig's hoofnail on properties and tissue slice was initially probed from the viewpoint of identification in this paper in order to make basis for the systematic research on replacing Squama manitis.

Animals↗
PubMed1999

[Analysis of genome character and amino acid residues 2,209 to 2,248 of NS5 A region of hepatitis C virus in relation to the response to interferon therapy].

OBJECTIVE: To observe nucleotide sequence variations and amino acid residues 2,209 to 2,248 of the NS5 A region of hepatitis C virus in relation to interferon therapy in Chinese patients infected with HCV genotype 1b. METHODS: We collected sera from 22 patients infected with HCV genotype 1b, performed nested RT-PCR using extracted serum RNA from sera, amplified NS5 A region 2,209 to 2,248 and sequenced the PCR product directly. The deduced amino acid sequences were compared with the corresponding region of the HCV-J prototype strain. RESULTS: Only 4 isolates were intermediate type, and no mutant type was found. In 18 patients treated with IFN-alpha, only 4 were HCV RNA negative at present, No correlation was found between amino acid mutation and IFN response. CONCLUSION: Variations in the NS5 A region between amino acid residues 2,209 to 2,248 failed to predict IFN response in Chinese patients infected with HCV genotype 1b. IFN may accelerate nucleotide variation but has little influence on amino acid variations.

Antiviral Agents↗
PubMed1998

A critical role for DNA end-joining proteins in both lymphogenesis and neurogenesis.

XRCC4 was identified via a complementation cloning method that employed an ionizing radiation (IR)-sensitive hamster cell line. By gene-targeted mutation, we show that XRCC4 deficiency in primary murine cells causes growth defects, premature senescence, IR sensitivity, and inability to support V(D)J recombination. In mice, XRCC4 deficiency causes late embryonic lethality accompanied by defective lymphogenesis and defective neurogenesis manifested by extensive apoptotic death of newly generated postmitotic neuronal cells. We find similar neuronal developmental defects in embryos that lack DNA ligase IV, an XRCC4-associated protein. Our findings demonstrate that differentiating lymphocytes and neurons strictly require the XRCC4 and DNA ligase IV end-joining proteins and point to the general stage of neuronal development in which these proteins are necessary.

Animals↗
PubMed1998

Phosphorylation of anticancer nucleoside analogs by human mitochondrial deoxyguanosine kinase.

The kinetic properties of recombinant human mitochondrial deoxyguanosine kinase (dGK, EC 2.7.1.113) for 2'-deoxyguanosine and the clinically important nucleoside analogs 2-chloro-2'-deoxyadenosine (CdA), 9-beta-D-arabinofuranosylguanine (araG) and 2',2',-difluorodeoxyguanosine (dFdG) were determined. The Michaelis-Menten kinetic parameters, comparing ATP and UTP as phosphate donors, demonstrated a marked increase in phosphorylation efficiency (VmaxKm) with UTP in comparison with ATP for both CdA and araG. The difluoro analog dFdG was an efficient substrate for recombinant dGK with an apparent Km of 16 microM with ATP as phosphate donor. We compared the kinetic properties of dGK with those of the related enzyme deoxycytidine kinase (dCK, EC 2.7.1.74). Although the purines 2'-deoxyguanosine (dGuo) and 2'-deoxyadenosine are substrates for both dGK and dCK, only CdA among the purine nucleoside analogs tested was an efficient substrate for both dCK and dGK. In competition with dGuo, the most efficient analog for phosphorylation by dGK was araG, as indicated by a lower Ki value than for CdA and dFdG. Of the purine analogs tested as substrates for dCK, only CdA could compete with 2'-deoxycytidine (dCyd). No inhibition of dCK-mediated dCyd phosphorylation was found by either araG or dFdG. In crude cell extract of HeLa and Capan 2 cells, the major CdA phosphorylation was contributed by dCK, while most araG phosphorylation was a result of dGK activity. Our study with pure recombinant enzymes confirms that dGK is mainly responsible for araG and dFdG phosphorylation, whereas dCK is the most important enzyme for activation of CdA and 2',2'-difluorodeoxycytidine (dFdC).

Antineoplastic Agents↗
PubMed1998

A convenient method of aligning large DNA molecules on bare mica surfaces for atomic force microscopy.

Large DNA molecules remain difficult to be imaged by atomic force microscopy (AFM) because of the tendency of aggregation. A method is described to align long DNA fibers in a single direction on unmodified mica to facilitate AFM studies. The clear background, minimal overstretching, high reproducibility and convenience of this aligning procedure make it useful for physical mapping of genome regions and the studies of DNA-protein complexes.

Aluminum Silicates↗
PubMed1998

Enhanced cytotoxicity of nucleoside analogs by overexpression of mitochondrial deoxyguanosine kinase in cancer cell lines.

The cytotoxic anti-cancer purine nucleoside analogs 2-chloro-2'-deoxyadenosine (CdA), 9-beta-D-arabinofuranosylguanine (araG), and 2',2'-difluorodeoxyguanosine (dFdG) are phosphorylated by human mitochondrial deoxyguanosine kinase (dGK) in vitro. We overexpressed dGK as a fusion protein to the green fluorescent protein in the human pancreatic cancer cell lines PanC-1 and MIA PaCa-2 to determine the importance of dGK-mediated nucleoside analog phosphorylation. The transfected cells showed mitochondrial fluorescence patterns, and the mitochondrial locations of endogenous and overexpressed dGK were verified by Western blot analysis of cell extracts with polyclonal anti-dGK antibodies. The increase of dGK activity in the overexpressing cells was approximately 4-fold. These cell lines exhibited increased sensitivity to CdA, araG, and dFdG as compared with the untransfected parent cell lines. This is, to our knowledge, the first demonstration of a correlation between the activity of a mitochondrial deoxyribonucleoside kinase and the cytotoxicity of nucleoside analogs. Our data imply that the dGK activity is rate-limiting for the efficacy of nucleoside analogs in the cell lines investigated.

Antineoplastic Agents↗
PubMed1998

Analysis of the DNA-binding site for Xenopus glucocorticoid receptor accessory factor. Critical nucleotides for binding specificity in vitro and for amplification of steroid-induced fibrinogen gene transcription.

In addition to the glucocorticoid receptor, DNA-binding proteins called accessory factors play a role in hormone activation of many glucocorticoid-responsive genes. Hormonal regulation of the gamma-fibrinogen subunit gene from the frog Xenopus laevis requires a novel DNA sequence that binds a liver nuclear protein called Xenopus glucocorticoid receptor accessory factor (XGRAF). Here we demonstrate that the recognition site for XGRAF encompasses GAGTTAA at positions -175 to -169 relative to the start site of transcription. This sequence is not closely related to the binding sites for known transcription factors. The two guanosines make close contact with XGRAF, as shown by the methylation interference assay. Single-point mutagenesis of every nucleotide in the 9-base pair region from positions -177 to -169 showed an excellent correlation between ability to bind XGRAF in vitro and ability to amplify hormone-induced transcription from DNA transfected into Xenopus primary hepatocytes. Conversely, XGRAF had little or no effect on basal transcription of the gamma-fibrinogen gene. Maximal hormonal induction also requires three half-glucocorticoid response elements (half-GREs) homologous to the downstream half of the consensus GRE. Interestingly, the XGRAF-binding site is immediately adjacent to the most important half-GRE. This close proximity suggests a new mechanism for activation of a gene lacking a conventional full GRE.

Animals↗
PubMed1998

Measuring two-dimensional receptor-ligand binding kinetics by micropipette.

We report a novel method for measuring forward and reverse kinetic rate constants, kf0 and kr0, for the binding of individual receptors and ligands anchored to apposing surfaces in cell adhesion. Not only does the method examine adhesion between a single pair of cells; it also probes predominantly a single receptor-ligand bond. The idea is to quantify the dependence of adhesion probability on contact duration and densities of the receptors and ligands. The experiment was an extension of existing micropipette protocols. The analysis was based on analytical solutions to the probabilistic formulation of kinetics for small systems. This method was applied to examine the interaction between Fc gamma receptor IIIA (CD16A) expressed on Chinese hamster ovary cell transfectants and immunoglobulin G (IgG) of either human or rabbit origin coated on human erythrocytes, which were found to follow a monovalent biomolecular binding mechanism. The measured rate constants are Ackf0 = (2.6 +/- 0.32) x 10(-7) micron 4 s-1 and kr0 = (0.37 +/- 0.055) s-1 for the CD16A-hIgG interaction and Ackf0 = (5.7 +/- 0.31) X 10(-7) micron 4 s-1 and kr0 = (0.20 +/- 0.042) s-1 for the CD16A-rIgG interaction, respectively, where Ac is the contact area, estimated to be a few percent of 3 micron 2.

Animals↗
PubMed1998

Determining force dependence of two-dimensional receptor-ligand binding affinity by centrifugation.

Analyses of receptor-ligand interactions are important to the understanding of cellular adhesion. Traditional methods of measuring the three-dimensional (3D) dissociation constant (Kd) require at least one of the molecular species in solution and hence cannot be directly applied to the case of cell adhesion. We describe a novel method of measuring 2D binding characteristics of receptors and ligands that are attached to surfaces and whose bonds are subjected to forces. The method utilizes a common centrifugation assay to quantify adhesion. A model for the experiment has been formulated, solved exactly, and tested carefully. The model is stochastically based and couples the bond force to the binding affinity. The method was applied to examine tumor cell adherence to recombinant E-selectin. Satisfactory agreement was found between predictions and data. The estimated zero-force 2D Kd for E-selectin/carbohydrate ligand binding was approximately 5 x 10(3) microm(-2), and the bond interaction range was subangstrom. Our results also suggest that the number of bonds mediating adhesion was small (<5).

Cell Adhesion↗