Biomedical subjects

Hui Zhou

Publications and source records attributed to Hui Zhou.

125 records · Page 7Linked to original sources

PubMed2003

[Airway beta-defensin-2 gene transfer enhances the bacterial clearing of rat lung].

beta-defensins possess a broad spectrum of antimicrobial activity. In this study its in vivo antibacterial effect was evaluated by using gene transfer. Rat beta-defensin-2 (rBD2) recombinant pBK-CMV-rBD2 and pCD-NA-3, 1-Myc-His(+)-rBD2 were constructed. Then, by use of liposome agent, the recombinants were delivered into rat airway via tracheal injection. The rBD-2 mRNA expression was detected in the trachea by RT-PCR and its protein expression was determined in the lungs by the tag His immunostaining, 24 hours after inoculation via trachea, the count of P. areuginosa in the lung of rat transfected with pBK-CMV-rBD2 markedly decreased, compared with the control (n = 8, P = 0.003). The data presented in this study provide evidence that airway beta-defensin-2-gene transfer can protect the rat against bacterial infection in vivo, suggesting the beta-defensins as part of the innate host defense system can be of potential applicability.

Animals↗

PubMed2002

Quantitative determination of trimebutine maleate and its three metabolites in human plasma by liquid chromatography-tandem mass spectrometry.

A sensitive and selective HPLC-MS-MS method was developed for the determination of trimebutine maleate (TM) and its major metabolites N-monodemethyltrimebutine (TM-MPB), N-didemethyltrimebutine (APB) and 3,4,5-trimethoxybenzoic acid (TMBA) in human plasma. The analytes were extracted from plasma samples by liquid-liquid extraction and chromatographed on a YMC J'sphere C(18) column. The mobile phase consisted of 2 mM ammonium acetate buffer (pH 6.5)-methanol (20:80, v/v), and at a flow-rate of 0.2 ml/min. Detection was carried out on a triple quadrupole tandem mass spectrometer in multiple reactions monitoring (MRM) mode using positive-negative switching electrospray ionization (ESI). The method was validated over the concentration range of 1-100 ng/ml for trimebutine maleate and APB, 1-500 ng/ml for MPB, and 50-10,000 ng/ml for TMBA. Inter- and intra-day precision (RSD%) for trimebutine maleate and its three metabolites were all within +/-15% and the accuracy was within 85-115%. The limit of quantitation was 1 ng/ml for trimebutine maleate, TM-MPB and APB, and 50 ng/ml for TMBA. The extraction recovery was on average 58.2% for trimebutine maleate, 69.6% for MPB, 51.2% for APB and 62.5% for TMBA. The method was applied to the pharmacokinetic study of trimebutine maleate and its metabolites in healthy Chinese volunteers.

Chromatography, High Pressure Liquid↗

PubMed2002

Diversity and structure of the archaeal community in the leachate of a full-scale recirculating landfill as examined by direct 16S rRNA gene sequence retrieval.

The diversity and structure of the archaeal community in the effluent leachate from a full-scale recirculating landfill was characterized by direct 16S rRNA gene (16S rDNA) retrieval. Total-community DNA was extracted from the microbial assemblages in the landfill leachate, and archaeal 16S rDNAs were amplified with a universally conserved primer and an Archaea-specific primer. The amplification product was then used to construct a 16S rDNA clone library, and 70 randomly selected archaeal clones in the library were grouped by restriction fragment length polymorphism (RFLP) analysis. Sequencing and phylogenetic analysis of representatives from each unique RFLP type showed that the archaeal library was dominated by methanogen-like rDNAs. Represented in the kingdom of Euryarchaeota were phylotypes highly similar to the methanogenic genera Methanoculleus, Methanosarcina, Methanocorpusculum, Methanospirillum and Methanogenium, where the clone distribution was 48, 11, 3, 1 and 1, respectively. No sequences related to known Methanosaeta spp. were retrieved. Four rDNA clones were not affiliated with the known methanogenic Archaea, but instead, they were clustered with the uncultured archaeal sequences recently recovered from anaerobic habitats. Two chimeric sequences were identified among the clones analyzed.

Archaea↗

PubMed2002

DNA-binding and cleavage studies of macrocyclic copper(II) complexes.

Three hexaaza macrocyclic copper (II) complexes with different functional groups have been synthesized and characterized by elemental analysis and infrared spectra. Absorption and fluorescence spectral, cyclic voltammetric and viscometric studies have been carried out on the interaction of [CuL(1)]Cl(2) (L(1)[double bond]3,10-bis(2-methylpyridine)-1,3,5,8,10,12-hexaazacyclotetradecane), [CuL(2)]Cl(2) (L(2)[double bond]3,10-bis(2-propionitrile)-1,3,5,8,10,12-hexaazacyclotetradecane) and [CuL(3)]Cl(2) (L(3)=3,10-bis(2-hydroxyethyl)-1,3,5,8,10,12-hexaazacyclotetradecane) with calf thymus DNA. The results suggest that three complexes can bind to DNA by different binding modes. The spectroscopic studies together with viscosity experiments and cyclic voltammetry suggest that [CuL(1)](2+) could bind to DNA by partial intercalation via pyridine ring into the base pairs of DNA. [CuL(2)](2+) may bind to DNA by hydrogen bonding and hydrophobic interaction while [CuL(3)](2+) may be by weaker hydrogen bonding. The functional groups on the side chain of macrocycle play a key role in deciding the mode and extent of binding of complexes to DNA. Noticeably, the three complexes have been found to cleave double-strand pUC18 DNA in the presence of 2-mercaptoethanol and H(2)O(2).

Animals↗

PubMed2002

A novel gene organization: intronic snoRNA gene clusters from Oryza sativa.

Based on the analysis of structural features and conserved elements, 27 novel snoRNA genes have been identified from rice. All of them belong to the C/D box-containing snoRNA family except for one that belongs to the H/ACA box type. The newly found genes fall into six clusters that comprise at least three snoRNA genes, and in one case as many as nine genes. Interestingly, four of the six clusters are located within the largest intron of a protein coding gene. The majority of intronic snoRNA gene clusters are simply formed by multiple copies of the same species of snoRNA gene that possess the identical functional elements. This implies a possible mechanism of duplication for the origin of repeating snoRNA coding regions in one intron. However, a few intronic snoRNA gene clusters consisting of different snoRNAs species were also observed. Polycistronic precursors from two independently transcribed clusters were demonstrated by RT-PCR and individual snoRNAs processed from the polycistronic precursors were positively determined by reverse transcription assay. Analyses of the intergenic spacers in the clusters showed that, in addition to a very high AT content, the processing signals in rice snoRNA polycistronic transcripts might be different from those of yeast. Our results demonstrate that, in both plants and mammals, numerous snoRNAs can be produced simultaneously from an mRNA precursor of a host gene despite the different arrangements. The intronic snoRNA gene cluster is a novel gene organization, which is so far unique to plants. The conservation of intronic snoRNA gene clusters in plants was further demonstrated by the study of a similar snoRNA gene organization in the first intron of a Hsp70 gene from wild rice and Zizania caduciflora.

Base Sequence↗

PubMed2002

Characterization of a brain-enriched chaperone, MRJ, that inhibits Huntingtin aggregation and toxicity independently.

Molecular chaperones are involved in a wide range of cellular events, such as protein folding and oligomeric protein complex assembly. DnaK- and DnaJ-like proteins are the two major classes of molecular chaperones in mammals. Recent studies have shown that DnaJ-like family proteins can inhibit polyglutamine aggregation, a hallmark of many neurodegenerative diseases, including Huntington's disease (HD). Although most DnaJ-like proteins studied are ubiquitously expressed, some have restricted expression, so it is possible that some specific chaperones may affect polyglutamine aggregation in specific neurons. In this report, we describe the isolation of a DnaJ-like protein MRJ and the characterization of its chaperone activity. Tissue distribution studies showed that MRJ is highly enriched in the central nervous system. In an in vitro cell model of HD, overexpressed MRJ effectively suppressed polyglutamine-dependent protein aggregation, caspase activity, and cellular toxicity. Collectively, these results suggest that MRJ has a relevant functional role in neurons.

Adenosine Triphosphatases↗

PubMed2002

The Schizosaccharomyces pombe mgU6-47 gene is required for 2'-O-methylation of U6 snRNA at A41.

Through a computer search of DNA databases, we have identified the homologs of the mgU6-47 snoRNA gene from the yeast Schizosaccharomyces pombe, the fly Drosophila melanogaster and human. The three box C/D-containing snoRNA genes showed no significant similarity in their sequences except for an 11 nt long complementarity to U6 snRNA, suggesting that the mechanism of snoRNA guided snRNA methylation is conserved from mammals to yeast. The corresponding snoRNAs have been positively detected by reverse transcription and northern blotting. Taking advantage of the fission yeast system, we have disrupted the yeast mgU6-47 gene and demonstrated that it is absolutely required for site-specific 2'-O-methylation of U6 at position A41. No growth differences between mgU6-47 gene-disrupted and wild-type cells were observed, suggesting that the mgU6-47 gene, as for most rRNA methylation guides, is dispensable in yeast. Nevertheless, it was revealed by temperature shift assay that abolition of A41 methylation in yeast U6 snRNA might cause a small decrease in mRNA splicing efficiency. The timing of S.pombe U6 pre-RNA transport in the nucleus for splicing and methylation was also analyzed and is described.

Active Transport, Cell Nucleus↗

PubMed2002

G(1)-phase specific apoptosis in liver carcinoma cell line induced by copper-1,10-phenanthroline.

Reactive oxygen species play an important role in the mediation of cell killing. But the mechanistic links between reactive oxygen species (ROS) and cell death remains unclear. There was a speculation that ROS, especially hydroxyl radicals can induce necrosis but not apoptosis in cells treated with copper-1,10-phenanthroline, IICu(OP)(2). In this paper, liver carcinoma cell line (Bel-7402) was treated with IICu(OP)(2) and its effect was examined by several means. Cells were found to undergo changes characteristic of apoptosis. Hoechst staining showed apoptotic body appeared in the cells induced by IICu(OP)(2). When DNA extracted from the cells treated with IICu(OP)(2) was analyzed by agarose gel electrophoresis it generated 'ladder' pattern of discontinuous DNA fragments. Sub-G(1) peak was detected in treated cells. Furthermore, two different flow cytometric methods were used, each allowing us to relate the apoptotic cells to the position the cell-cycle position. Apoptosis induced by IICu(OP)(2) was limited to G(1)-phase cells. Using cyclin analysis, the expression of cyclin E in G(1) was blocked. Thus, it was concluded that IICu(OP)(2) can induce G(1)-phase specific apoptosis in Bel-7402.

Annexin A5↗

PubMed2002

Thermodynamics and kinetics of the cleavage of DNA catalyzed by bleomycin A5.

Microcalorimetry and UV-vis spectroscopy were used to conduct thermodynamic and kinetic investigations of the scission of calf thymus DNA catalyzed by bleomycin A5 (BLM-A5) in the presence of ferrous ion and oxygen. The molar reaction enthalpy for the cleavage, the Michaelis-Menten constant for calf thymus DNA and the turnover number of BLM-A5 were calculated by a novel thermokinetic method for an enzyme-catalyzed reaction to be -577 +/- 19 kJ.mol-1, 20.4 +/- 3.8 microm and 2.28 +/- 0.49 x 10-2 s-1, respectively, at 37.0 degrees C. This DNA cleavage was a largely exothermic reaction. The catalytic efficiency of BLM-A5 is of the same order of magnitude as that of lysozyme but several orders of magnitude lower than those of TaqI restriction endonuclease, NaeI endonuclease and BamHI endonuclease. By comparing the molar enthalpy change for the cleavage of calf thymus DNA induced by BLM-A5 with those for the scission of calf thymus DNA mediated by adriamycin and by (1,10-phenanthroline)-copper, it was found that BLM-A5 possessed the highest DNA cleavage efficiency among these DNA-damaging agents. These results suggest that BLM-A5 is not as efficient as a DNA-cleaving enzyme although the cleavage of DNA by BLM-A5 follows Michaelis-Menten kinetics. Binding of BLM-A5 to calf thymus DNA is driven by a favorable entropy increase with a less favorable enthalpy decrease, in line with a partial intercalation mode involved in BLM-catalyzed breakage of DNA.

Animals↗

PubMed2002

Two norditerpenoid ester alkaloids from Aconitum bulleyanum.

Two new norditerpenoid alkaloids, 8-acetyl-14-p-methoxybenzoate of talatisamine (1) and 14-p-methoxybenzoate of talatisamine (2), were isolated from Aconitum bulleyanum Diels. The structures were elucidated on the basis of spectroscopic and chemical studies.

Aconitine↗

PubMed2002

Primitive neuroectodermal tumor (PNET) in the differential diagnosis of malignant kidney tumors.

Primitive neuroectodermal tumors (PNETs) of the kidney, a rare neoplastic disease of high malignancy with a tendency towards early metastasis, affect young adults (26-30 years) irrespective of the gender. Differential diagnosis from other renal tumors is very important for an effective therapy. Herein, we report on a 24-year-old male patient with a renal tumor consisting of small, round cells, and summarize the diagnostic procedures that establish the diagnosis of PNET. Light microscopy revealed not only areas containing small, round cells forming rosettes and pseudorosettes, but also areas containing spindle cells. Expression of CD 99 in combination with neural markers, such as NSE, was detected by immunohistochemistry, and further evidence of neural differentiation was provided by electron microscopy. Image cytometry revealed a peridiploid DNA-stemline. A reciprocal translocation of the chromosomes 11 and 22 [t(11;22)(q24;q12)] with expression of a EWS/FLI-1 fusion transcript was demonstrated by molecular pathology. Using these methods, the diagnosis of PNET was firmly established, and the tumor was treated by surgical resection and subsequent adjuvant chemotherapy. Eighteen months after therapy, the patient is in excellent health condition without any evidence of tumor recurrence.

Adult↗

PubMed2002

Selection of peptide ligands binding to fibroblast growth factor receptor 1.

Inappropriate expression of fibroblast growth factors (FGFs) or activation of FGF receptors (FGFRs) could contribute to several human angiogenic pathologies. In an attempt to design antagonists of FGF, we developed a screening procedure for identifying peptide ligands binding to FGFR1. To retain the natural conformation of FGFR1 during screening, we expressed recombinant FGFR1 on the surface of Sf9 insect cells. A 6-mer phage display peptide library was then screened on the cell surface and a group of hydrophobic peptide sequences were identified. Further experiments demonstrated that the phages displaying these sequences can specifically bind to FGFR1. The docking analysis suggests that the peptide ValTyrMetSerProPhe can specifically bind to the hydrophobic surface of FGFR1. The synthetic peptide Ac-ValTyrMetSerProPhe-NH2 can inhibit mitogenic activity of aFGF and has the potential to become a therapeutic agent as an aFGF antagonist.

3T3 Cells↗

PubMed2002

Interaction of Huntington disease protein with transcriptional activator Sp1.

Polyglutamine expansion causes Huntington disease (HD) and at least seven other neurodegenerative diseases. In HD, N-terminal fragments of huntingtin with an expanded glutamine tract are able to aggregate and accumulate in the nucleus. Although intranuclear huntingtin affects the expression of numerous genes, the mechanism of this nuclear effect is unknown. Here we report that huntingtin interacts with Sp1, a transcription factor that binds to GC-rich elements in certain promoters and activates transcription of the corresponding genes. In vitro binding and immunoprecipitation assays show that polyglutamine expansion enhances the interaction of N-terminal huntingtin with Sp1. In HD transgenic mice (R6/2) that express N-terminal-mutant huntingtin, Sp1 binds to the soluble form of mutant huntingtin but not to aggregated huntingtin. Mutant huntingtin inhibits the binding of nuclear Sp1 to the promoter of nerve growth factor receptor and suppresses its transcriptional activity in cultured cells. Overexpression of Sp1 reduces the cellular toxicity and neuritic extension defects caused by intranuclear mutant huntingtin. These findings suggest that the soluble form of mutant huntingtin in the nucleus may cause cellular dysfunction by binding to Sp1 and thus reducing the expression of Sp1-regulated genes.

Animals↗

PubMed2002

[Effect of anti-HPV16 E6-ribozyme on the sensitivity of cervical carcinoma cell line to chemotherapy].

OBJECTIVE: To study the role of anti-HPV16E6-ribozyme in modulating the sensitivity of cervical carcinoma cell line to chemotherapy. METHODS: By way of lipofectin transfection, anti-HPV16E6-ribozyme antibody and empty eukaryotic expression plasmids were respectively transfected into CaSKi cells that were designated as CaSKi-R and CaSKi-P cells accordingly. Ribozyme expression in the transfected cells was subsequently observed with RNA dot blot, and the amount of E6 mRNA expression detected by Northern blotting. Cell count was performed for determining the growth rate of the non-transfected and transfected CaSKi cells and colony formation test was employed to examine the sensitivity of the cells to chemotherapy. PI/Annexin V staining was conducted to determine the apoptosis rates of each cell line in response to chemotherapy. RESULTS: As shown by dot blot analysis, stable expression of anti-HPV16E6-ribozyme was achieved in CaSKi-R cells, in which lower E6 mRNA expression level was observed by means of Northern blotting in comparison with those in CaSKi-P and CaSKi cells. Significant slow-down of the growth rate occurred in CaSKi-R cells as compared with the growth rate of the other 2 cell lines, but no differences in relative cloning efficiency and apoptosis rates of the 3 cell lines were observed in response to taxol treatment (P>0.05), while cis-platinum treatment produced comparatively deceased relative cloning efficiency and increased apoptosis rate in CaSKi-R (P<0.01). CONCLUSION: Transfection by anti-HPVE6-ribozyme may inhibit the growth of CaSKi cells and increase their sensitivity to cis-platinum but not to taxol.

Antibodies↗

PubMed2002

[Radiation sensitizing effect of anti HPV16E6-ribozyme on cervical carcinoma cell line].

BACKGROUND & OBJECTIVE: Human papillomavirus (HPV) are the most important etiologic factor for cervical carcinoma. E6 virus gene is one of the most important oncogene and expression levels of E6 transforming oncoproteins of high risk HPV genotypes, such as HPV16, appear to be necessary for maintaining the malignant phenotype. Radiation treatment represents a standardized and effective modality for contemporary cervical carcinoma therapy. The goal of this study was to investigate the radiation sensitizing effect of anti-HPV16 E6-ribozyme on cervical carcinoma cell line. METHODS: With the method of lipofectin transfection, the anti-HPV16 E6 ribozyme and empty eucaryotic expressing plasmids were transfected into CaSKi cell line, which named as CaSKi-R and CaSKi-P, respectively. The expression of ribozyme in transfected cells was observed by RNA dot blot. The amounts of E6 mRNA in the three kinds of cells were detected by Northern blot. The growth rates of the CaSKi and transfected cells were examined by cell count and their sensitivity to radiotherapy were examined by colony formation test. The apoptosis rates of each cell was determined by PI/Annexin V stained methods. Expressions of p53, bcl-2, and bax were determined by Flow cytometry analysis. RESULTS: Anti-HPV16 E6-ribozyme can be expressed stably in transfected CaSKi-R cells. Northern blot showed that E6 mRNA was less in CaSKi-R than in CaSKi and CaSKi-P. The growth rate of CaSKi-R was much slower than that of CaSKi and CaSKi-P. The sensitivity of CaSKi-R cells to radiotherapy increased more than that of CaSKi and CaSKi-P cells. The ability of colony formation decreased (P < 0.05), while the apoptosis rates of CaSKi-R cells increased more than that of CaSKi and CaSKi-P cells(P < 0.01). Anti-HPV16 E6-ribozyme did significantly upregulate expression of p53, bax protein, and downregulate the expression of bcl-2 protein before and after radiotherapy (P < 0.01). CONCLUSION: CaSKi-R cells transfected with Anti-HPVE 6-rivozyme showed growth inhibition and increased sensitivity to radiotherapy.

Apoptosis↗

PubMedDate not supplied

Rapid identification of vinca alkaloids by direct-injection electrospray ionisation tandem mass spectrometry and confirmation by high-performance liquid chromatography-mass spectrometry.

A simple and rapid method for the identification of Vinca alkaloids from a crude extract of Catharanthus roseus G. Don (Apocynaceae) by direct-injection electrospray ionisation (ESI) and tandem mass spectrometry (MS/MS) has been developed. The alkaloids vindoline, vindolidine, vincristine and vinblastine were evaluated in a commercial extract of C. roseus using this method. Catharanthine and its isomers 19S-vindolinine and vindolinine were detected in the commercial product by direct injection ESI/MS/MS and confirmed by preparation and by HPLC-ESI/MS. For the characterisation of different fragment fingerprints, ESI/MS/MS is a sensitive, rapid and convenient technique by which to identify some constituents in complex and mixed plant extracts.

Catharanthus↗

PubMedDate not supplied

Cytokinetic analysis of cell cycle and sub-phases in MOLT-4 cells by cyclin E + A/DNA multiparameter flow cytometry.

Cell cycle analysis has become increasingly important in verifying the effect of anti-tumor drugs and cytokinetic research. In the early methods of cell cycle analysis, the flow cytometry relied on DNA content, and therefore, the cell cycle could be only broken into three stages: G(0)/G(1), S, and G(2)/M phase. It could not distinguish the G(0), G(1), G(2), and M phase cells, let alone the sub-phases in G(1) phase. In cell cycle, expression of cyclin E living up to the maximal level in the cells undergoing transition from G(1) to S phase, and G(2) + M cells are cyclin E negative. Expression of cyclin A is progressively increasing during S phase and is maximal in G(2) phase cells. Therefore, in the current study we established a cyclin E + A/DNA multiparameter flow cytometric technique by using a mixture of cyclin E and cyclin A antibodies, which can identify six stages in the whole cell cycle: G(0), early G(1), late G(1), S, G(2), and M phase. Furthermore, we found that cyclin E + A/DNA multiparameter flow cytometry could also be used for stathmokinetic analysis of lymphocyte leukemia MOLT-4 cells after addition of the stathmokinetic agent vinblastine to cultures of exponentially growing MOLT-4 cells. We believe that this new technique will provide a much better tool for molecular cell biology research and especially for cell proliferation kinetics investigations.

Antineoplastic Agents, Phytogenic↗