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Biomedical subjects

Xing Zhang

Publications and source records attributed to Xing Zhang.

At least 55 records · Page 3Linked to original sources
PubMed2005

[Feasibility of chitosan as gene therapy vehicle].

BACKGROUND & OBJECTIVE: Recently, chitosan, as a nonviral vehicle for transferring DNA molecules into the cells, has attracted much attention because of its cationic properties. This study was to investigate characteristics and transfection activity of chitosan-pDNA microparticles, and confirm the feasibility of chitosan as gene therapy vehicle, which may be employed in further in vivo study. METHODS: Plasmid DNAs were amplified in Eschericha Coli JM109 with transfection of enhanced green fluorescence protein (EGFP) gene, and isolated according to the protocol of Qiagen plasmid midi kit. Chitosan-pDNA microparticles were prepared by the coacervation method, and observed under transmission electronic microscope. In vitro releasing experiment and ultraviolet spectrophotometry were used to measure DNA loading capacity and loading efficiency. The location of pDNA in the microparticles was analyzed by gel retardation assay. Transfection efficiency of chitosan-pDNA microparticles was evaluated by detecting gene expression of EGFP in HEK293 cells after transfection. RESULTS: Chitosan-pDNA microparticles were global, and the size ranged 100-200 nm, with the average of (138 +/- 43) nm. DNA loading capacity and loading efficiency of the microparticles were (46.8 +/- 9.0)% and 100%, respectively. Gel retardation assay confirmed that DNA was wholly encapsulated in the microparticles. In vitro study revealed that the release of DNA from chitosan-pDNA microparticles included two-step process, namely burst effect with a slow second phase. Most of DNA could be released at the time of 48 h. In vitro transfection results indicated that chitosan could efficiently deliver pEGFP into HEK293 cells and stably express green florescence protein. CONCLUSIONS: Chitosan can efficiently transfer target DNA molecules into mammalian cells, continuously release DNA, and stably express DNA. It can be potentially employed as a gene therapy vehicle.

Cell Line↗
PubMed2004

In vitro and in vivo analyses of a Phe/Tyr switch controlling product specificity of histone lysine methyltransferases.

The functional significance of mono-, di-, and tri-methylation of lysine residues within histone proteins is under investigation. Evidence from several model organisms suggests that different methylated states of H3 Lys(9) (H3K9) are generated by specific histone methyltransferases (MTases) to mark distinct types of silent chromatin. Sequence alignment of all histone lysine MTases with known product specificity suggested that a key residue in the active site determines how many methyl groups they add. We examined this possibility both in vitro and in vivo and found that a Phe at the position equivalent to Phe(281) of Neurospora crassa DIM-5 or Phe(1205) of human G9a allows the enzyme to perform di and tri-methylation, whereas a Tyr at this position is restrictive, inhibiting tri-methylation and thus yielding a mono- or di-MTase. Phe to Tyr mutants of both DIM-5 and G9a restrict product specificity in vitro and in vivo without compromising overall catalysis. These mutants were employed to probe the biological significance of mono-, di-, and tri-methylation of H3K9 in both mouse embryonic stem cells and N. crassa. G9a F1205Y, when expressed in G9a (-/-) embryonic stem cells, rescued only H3K9 mono-methylation, but not di-methylation, to wild-type levels yet silenced Mage-a gene expression. When expressed in dim-5 strains, DIM-5 F281Y generated significant levels of mono- and di-H3K9 methylation (which are not observed in wild type Neurospora) as well as tri-methyl H3K9. The altered DIM-5 rescued the growth defect characteristic of dim-5 N. crassa but did not fully rescue the gross DNA hypomethylation of dim-5 strains.

Amino Acid Sequence↗
PubMed2004

Sodium 4-phenylbutyrate induces apoptosis of human lung carcinoma cells through activating JNK pathway.

Sodium 4-phenylbutyrate (PB) has been used in the therapy of urea cycle defects for many years. Recently, it has been shown to cause cellular differentiation, growth arrest, and apoptosis in certain malignancies. We have analyzed the effects of PB on human lung carcinoma cells. PB has distinct patterns of effects on different lung carcinoma cells, inducing apoptosis in NCI-H460 and NCI-H1792 cells, causing G1 arrest in A549 and SK-LU-1 cells, but having no effect on a non-transformed bronchial epithelial cell line HBE4-E6/E7. We investigated the role of MAP kinase family members, extracellular signal-regulated kinase (ERK), JNK, and p38 mitogen-activated protein kinase (MAPK), as well as other important cell survival signaling molecules in PB-induced apoptosis. We observed activation of JNK and ERK by PB in the lung cancer cells. JNK was activated only in the two apoptotic cells, whereas ERK was activated in both the apoptotic and the growth-arrested cells, demonstrating a correlation between apoptosis and activation of JNK in response to PB. Both JNK inhibitor and JNK RNA interference (RNAi) inhibited PB-induced apoptosis, whereas MEK inhibitor did not, supporting that apoptosis induced by PB is through activation of JNK. De novo protein synthesis is required for the PB-induced JNK activation and induction of apoptosis. However, the production of known upstream activators of JNK, namely Fas/Fas ligand, tumor necrosis factor (TNF)-alpha, TNF-beta, and TRAIL, are not altered by PB treatment. Therefore, PB activates JNK through an unidentified and cell type-specific mechanism. Understanding of this mechanism is of therapeutic value in treating cancer patients with PB.

Adenocarcinoma↗
PubMed2004

Structure of the conserved core of the yeast Dot1p, a nucleosomal histone H3 lysine 79 methyltransferase.

Methylation of Lys79 on histone H3 by Dot1p is important for gene silencing. The elongated structure of the conserved core of yeast Dot1p contains an N-terminal helical domain and a seven-stranded catalytic domain that harbors the binding site for the methyl-donor and an active site pocket sided with conserved hydrophobic residues. The S-adenosyl-L-homocysteine exhibits an extended conformation distinct from the folded conformation observed in structures of SET domain histone lysine methyltransferases. A catalytic asparagine (Asn479), located at the bottom of the active site pocket, suggests a mechanism similar to that employed for amino methylation in DNA and protein glutamine methylation. The acidic, concave cleft between the two domains contains two basic residue binding pockets that could accommodate the outwardly protruding basic side chains around Lys79 of histone H3 on the disk-like nucleosome surface. Biochemical studies suggest that recombinant Dot1 proteins are active on recombinant nucleosomes, free of any modifications.

Amino Acid Sequence↗
PubMed2004

Multiple roles of T7 RNA polymerase and T7 lysozyme during bacteriophage T7 infection.

T7 RNA polymerase selectively transcribes T7 genes during infection but is also involved in DNA replication, maturation and packaging. T7 lysozyme is an amidase that cuts a bond in the peptidoglycan layer of the cell wall, but it also binds T7 RNA polymerase and inhibits transcription, and it stimulates replication and packaging of T7 DNA. To better understand the roles of these two proteins during T7 infection, mutants of each were constructed or selected and their biochemical and physiological behavior analyzed. The amidase activity of lysozyme is needed for abrupt lysis and release of phage particles but appears to have no role in replication and packaging. The interaction between polymerase and lysozyme stimulates both replication and packaging. Polymerase mutants that gain the ability to grow normally in the absence of an interaction with lysozyme still fail to shut down late transcription and, remarkably, have become hypersensitive to inhibition when lysozyme is able to bind. These lysozyme-hypersensitive polymerases behave without lysozyme similarly to wild-type polymerase with lysozyme: both remain longer at the promoter before establishing a lysozyme-resistant elongation complex and both increase the length of pausing when elongation complexes encounter an eight-base recognition sequence involved in DNA packaging. Replication origins contain T7 promoters, but the role of T7 RNA polymerase in initiating replication is not understood well enough to more than speculate how the lysozyme-polymerase interaction stimulates replication. Maturation and packaging is apparently initiated through interaction between prohead-terminase complexes and transcription elongation complexes paused at the sequence TATCTGT(T/A), well conserved at the right-end of the concatemer junction of T7-like phages. A model that is consistent with the structure of an elongation complex and a large body of mutational and biochemical data is proposed to explain sequence-specific pausing and potential termination at the consensus recognition sequence (C/T)ATCTGT(T/A).

Amidohydrolases↗
PubMed2004

A Structural-informatics approach for tracing beta-sheets: building pseudo-C(alpha) traces for beta-strands in intermediate-resolution density maps.

We report the development of two computational methods to assist density map interpretation at intermediate resolutions: sheettracer for building pseudo-C(alpha) models of beta-sheets, and a deconvolution method for enhancing features attributed to major secondary structural elements. Sheettracer is tightly coupled with sheetminer, which was developed to locate sheet densities in intermediate-resolution density maps. The results from sheetminer are used as inputs to sheettracer, which employs a multi-step ad hoc morphological analysis of sheet densities to trace individual strands of beta-sheets. The methods were tested on simulated density maps from 12 protein crystal structures that represent a reasonably complete sampling of sheet morphology. The sheet-tracing results were quantitatively assessed in terms of sensitivity, specificity and rms deviations. Furthermore, sheettracer and the deconvolution method were rigorously tested on experimental maps of the lambda2 protein of reovirus at resolutions of 7.6A and 11.8A. Our results clearly demonstrate the capability of sheettracer in building pseudo-C(alpha) models of beta-sheets in intermediate-resolution density maps and the power of the deconvolution method in enhancing the performance of sheettracer. These computational methods, along with other related ones, should facilitate recognition and analysis of folding motifs from experimental data at intermediate resolutions.

Algorithms↗
PubMed2004

Dimethylation of histone H3 lysine 9 is a critical mark for DNA methylation and gene silencing in Arabidopsis thaliana.

The Arabidopsis KRYPTONITE gene encodes a member of the Su(var)3-9 family of histone methyltransferases. Mutations of kryptonite cause a reduction of methylated histone H3 lysine 9, a loss of DNA methylation, and reduced gene silencing. Lysine residues of histones can be either monomethylated, dimethylated or trimethylated and recent evidence suggests that different methylation states are found in different chromatin domains. Here we show that bulk Arabidopsis histones contain high levels of monomethylated and dimethylated, but not trimethylated histone H3 lysine 9. Using both immunostaining of nuclei and chromatin immunoprecipitation assays, we show that monomethyl and dimethyl histone H3 lysine 9 are concentrated in heterochromatin. In kryptonite mutants, dimethyl histone H3 lysine 9 is nearly completely lost, but monomethyl histone H3 lysine 9 levels are only slightly reduced. Recombinant KRYPTONITE can add one or two, but not three, methyl groups to the lysine 9 position of histone H3. Further, we identify a KRYPTONITE-related protein, SUVH6, which displays histone H3 lysine 9 methylation activity with a spectrum similar to that of KRYPTONITE. Our results suggest that multiple Su(var)3-9 family members are active in Arabidopsis and that dimethylation of histone H3 lysine 9 is the critical mark for gene silencing and DNA methylation.

Arabidopsis↗
PubMed2004

Cleavage of p130Cas in anoikis.

p130Cas is a multifunctional signaling adaptor protein. It integrates and relays signals generated from a variety of extracellular stimuli and regulates a number of cellular activities including cell death. In this study, we analyzed the regulation and function of p130Cas in anoikis, a type of apoptosis caused by disruption of cell-matrix interactions. We found that p130Cas was specifically cleaved during anoikis in anoikis-sensitive epithelial cells, but not in anoikis-resistant tumor cells. There is a close correlation between p130Cas cleavage and anoikis. Furthermore, we found that the cleavage of p130Cas, as well as another focal adhesion component FAK, is different from that of caspase substrate PARP and spectrin. Although caspases and calpain were found to be involved in the cleavage of p130Cas, there appear to be other unidentified proteases that are mainly responsible for the cleavage of p130Cas, particularly at the early stage of anoikis. Overexpression of the p130Cas cleavage product induced apoptosis. Taken together, these data suggest that there are novel proteases involved in the cleavage of p130Cas during anoikis, which may be functionally involved in the onset of anoikis. p130Cas may have a dual role in the regulation of anoikis. On one hand, it mediates a survival signal from cell-matrix interactions when cells are attached to the extracellular matrix. On the other hand, it participates in executing cell death when cell-matrix interactions are disrupted. These observations provide new insights into the understanding of the function of p130Cas and the molecular mechanism of anoikis.

Animals↗
PubMed2004

Insecticidal activity of deoxypodophyllotoxin, isolated from Juniperus sabina L, and related lignans against larvae of Pieris rapae L.

In the course of screening for naturally occurring insecticides from plants from the northwestern part of China, a petroleum ether extract of Juniperus sabina L was found to show insecticidal activity against fifth-instar larvae of Pieris rapae L. From the extract, an insecticidal compound was isolated by bioassay-guided fractionation. The compound was identified as deoxypodophyllotoxin (1) by comparison of its spectroscopic characteristics with literature data. In bioassays, 1 showed antifeedant activity against fifth-instar larvae of P rapae at 0.05-1.00 g litre(-1) and its AFC50 (concentration for 50% antifeedant activity) values at 12 and 48h were 0.170 and 0.060 g litre(-1), respectively. In that concentration range, all treated insects died within 48 h after treatment and compound 1 showed delayed insecticidal activity. At 0.015-0.100 g litre(-1), 1 showed insecticidal activity, with an LC50 of 0.020 g litre(-1). The related compound deoxypicropodophyllotoxin (2), however, showed lower antifeedant and insecticidal activities than 1 in bioassay. This indicated that the trans-lactone ring is an important moiety for enhancing activity in these compounds. Comparison of the insecticidal activities of 1 and another related compound, podophyllotoxin (3), suggested that varying the substituent at C-4 is an exciting possibility for synthesizing more potent analogues.

Animals↗
PubMed2004

Putative autocleavage of outer capsid protein micro1, allowing release of myristoylated peptide micro1N during particle uncoating, is critical for cell entry by reovirus.

Several nonenveloped animal viruses possess an autolytic capsid protein that is cleaved as a maturation step during assembly to yield infectious virions. The 76-kDa major outer capsid protein micro1 of mammalian orthoreoviruses (reoviruses) is also thought to be autocatalytically cleaved, yielding the virion-associated fragments micro1N (4 kDa; myristoylated) and micro1C (72 kDa). In this study, we found that micro1 cleavage to yield micro1N and micro1C was not required for outer capsid assembly but contributed greatly to the infectivity of the assembled particles. Recoated particles containing mutant, cleavage-defective micro1 (asparagine --> alanine substitution at amino acid 42) were competent for attachment; processing by exogenous proteases; structural changes in the outer capsid, including micro1 conformational change and sigma1 release; and transcriptase activation but failed to mediate membrane permeabilization either in vitro (no hemolysis) or in vivo (no coentry of the ribonucleotoxin alpha-sarcin). In addition, after these particles were allowed to enter cells, the delta region of micro1 continued to colocalize with viral core proteins in punctate structures, indicating that both elements remained bound together in particles and/or trapped within the same subcellular compartments, consistent with a defect in membrane penetration. If membrane penetration activity was supplied in trans by a coinfecting genome-deficient particle, the recoated particles with cleavage-defective micro1 displayed much higher levels of infectivity. These findings led us to propose a new uncoating intermediate, at which particles are trapped in the absence of micro1N/micro1C cleavage. We additionally showed that this cleavage allowed the myristoylated, N-terminal micro1N fragment to be released from reovirus particles during entry-related uncoating, analogous to the myristoylated, N-terminal VP4 fragment of picornavirus capsid proteins. The results thus suggest that hydrophobic peptide release following capsid protein autocleavage is part of a general mechanism of membrane penetration shared by several diverse nonenveloped animal viruses.

Animals↗
PubMed2004

Structure of the Q237W mutant of HhaI DNA methyltransferase: an insight into protein-protein interactions.

We have determined the structure of a mutant (Q237W) of HhaI DNA methyltransferase, complexed with the methyl-donor product AdoHcy. The Q237W mutant proteins were crystallized in the monoclinic space group C2 with two molecules in the crystallographic asymmetric unit. Protein-protein interface calculations in the crystal lattices suggest that the dimer interface has the specific characteristics for homodimer protein-protein interactions, while the two active sites are spatially independent on the outer surface of the dimer. The solution behavior suggests the formation of HhaI dimers as well. The same HhaI dimer interface is also observed in the previously characterized binary (M.HhaI-AdoMet) and ternary (M.HhaI-DNA-AdoHcy) complex structures, crystallized in different space groups. The dimer is characterized either by a non-crystallographic two-fold symmetry or a crystallographic symmetry. The dimer interface involves three segments: the amino-terminal residues 2-8, the carboxy-terminal residues 313-327, and the linker (amino acids 179-184) between the two functional domains--the catalytic methylation domain and the DNA target recognition domain. Both the amino- and carboxy-terminal segments are part of the methylation domain. We also examined protein-protein interactions of other structurally characterized DNA MTases, which are often found as a 2-fold related 'dimer' with the largest dimer interface area for the group-beta MTases. A possible evolutionary link between the Type I and Type II restriction-modification systems is discussed.

Crystallization↗
PubMed2004

[A meta-analysis of cohort studies on cancer mortality among workers exposure to chrysotile fiber alone].

OBJECTIVE: To determine there was excessive risk of malignant tumors or not among workers exposure to chrysotile fiber alone by applying a meta-analysis technique. METHODS: All data meeting the criteria of cohort studies on cancer mortality among workers exposed only to chrysotile would incorporate into the meta-analysis. The pooled standardized mortality ratios (SMR) and their corresponding 95% confidence intervals (95% CI) for main cancer sites were calculated using two approaches of unweighted ratio and random effects model. The heterogeneity and its sources of the results were examined with a Q-statistic and Z-score test. RESULTS: 26 chrysotile-exposed alone cohorts were summarized. The significantly elevated meta-SMRs for all deaths (1.28), all cancers (1.26), cancers of respiratory organs (2.24), cancer of lung (2.29) and cancer of stomach (1.27) were observed. The significantly elevated meta-SMRs for lung cancer within occupational strata were observed among textile workers (3.64), asbestos products manufacturers (3.07), miners and millers (2.24), cement products workers (1.22), and for stomach cancer among asbestos products manufacturers (1.48). Meta-SMRs for cancers at other sites were not significant. CONCLUSION: There were excessive risks of lung cancer and mesothelioma among workers exposure to chrysotile fiber alone, and likely no convincing indication of an etiological association between chrysotile exposure and cancers at other sites.

Asbestos↗
PubMed2004

[The expression and possible function of RhoA in human gastric cancer cell lines].

OBJECTIVE: To study the expression and possible function of RhoA in human gastric cancer cell lines. METHODS: The expression of RhoA in human gastrointestinal cancer cell lines was detected by Western blot. Antisense plasmid of RhoA was constructed by pGEFL and transferred into gastric cancer cell line AGS by lipofectamine. Cell survival was examined by MTT assays, and cell cycle was detected by flow cytometry. RESULTS: The expression of RhoA protein in 10 different kinds of human cancer cell lines was much higher than that in immortalized human intestinal epithelial cell line. After being transfected with antisense RhoA, with the decrease in RhoA protein expression, the growth rate of AGS was inhibited, and the number of cells in S phase was increased by 14%. CONCLUSION: RhoA is overexpressed in many human cancer cell lines. Some of the malignant characteristics of a gastric cancer cell line can be partially reversed by inhibiting RhoA expression.

Antisense Elements (Genetics)↗
PubMed2004

[Evaluation of telomerase activation in laryngeal mucosa adjacent to carcinoma].

OBJECTIVE: To investigate telomerase activity in laryngeal nonmalignant mucosa adjacent to carcinoma and discuss its effect on extension and recurrence of laryngeal carcinoma. METHOD: Telomerase repeat amplification protocol was used and combined with histological examination. RESULT: The positive percentage of telomerase activation was even higher in laryngeal nonmalignant mucosa distant toward surgically negative margin of carcinoma than in laryngeal carcinomas themselves (P<0.05). CONCLUSION: Telomerase activation occurred probably before the morphologic alteration, which may imply expansion of the cells with potential of malignant transformation.

Adult↗
PubMed2004

[Investigation on extraction conditions of tebufenozide residue from cabbage using supercritical fluid extraction].

An extraction method of tebufenozide using supercritical fluid extraction (SFE) and high performance liquid chromatography (HPLC) was developed. The selected conditions were 48.3 MPa (7000 psi), 60 degrees C, 20 min of static time, dynamic extraction with 10 mL of CO2, 0.04 mL/g of methanol as static modifier, and 5 mL of acetone as collecting solvent. Under these conditions, the recovery of tebufenozide extracted by SFE was 100.75%. The extract was analyzed directly by HPLC with a photodiode array detector. The chromatographic conditions were: UV detection wavelength, 245 nm; column, a C18 column; mobile phase, acetonitrile-water (55:45, v/v); flow rate, 1.0 mL/min; injection volume, 5 microL.

Brassica↗
PubMed2004

Cohort studies on cancer mortality among workers exposed only to chrysotile asbestos: a meta-analysis.

OBJECTIVE: To determine whether there was excessive risk of cancer among workers exposed to chrysotile fiber alone by applying a meta-analysis technique. METHODS: All data meeting the criteria of cohort studies on cancer mortality among workers exposed only to chrysotile were incorporated into meta-analysis. Pooled standardized mortality ratios (SMRs) and their corresponding 95% confidence intervals (CIs) for main cancer sites were calculated using two approaches of unweighted ratio and random effect model. The heterogeneity and its sources of the results were examined with a Q-statistic and Z-score test. The dose-response effect as reflected in the percentage of all deaths due to mesothelioma served as a proxy measure of chrysotile exposure. RESULTS: A cohort of twenty six workers exposed to chrysotile alone was summarized. The significantly elevated meta-SMRs for all deaths (1.27), all cancers (1.28), cancers of respiratory organs (2.51), cancers of lung (2.35) and cancers of stomach (1.24) were observed. The significantly elevated meta-SMRs for lung cancer within occupational strata were observed among textile workers (3.55), asbestos product manufacturers (3.30), miners and millers (2.24), cement product workers (1.22), and for stomach cancer among asbestos product manufacturers (1.49). Meta-SMRs for cancers at other sites were not significant. Meta-SMR for lung cancer showed an increasing trend with an elevated percentage of all deaths from mesothelioma, but no such trend for stomach cancer. CONCLUSION: There are excessive risks of lung cancer and mesothelioma among workers exposed to chrysotile fiber alone, and likely no convincing indication of an etiological association between chrysotile exposure and cancers at other sites.

Asbestos, Serpentine↗
PubMed2003

Reovirus polymerase lambda 3 localized by cryo-electron microscopy of virions at a resolution of 7.6 A.

Reovirus is an icosahedral, double-stranded (ds) RNA virus that uses viral polymerases packaged within the viral core to transcribe its ten distinct plus-strand RNAs. To localize these polymerases, the structure of the reovirion was refined to a resolution of 7.6 A by cryo-electron microscopy (cryo-EM) and three-dimensional (3D) image reconstruction. X-ray crystal models of reovirus proteins, including polymerase lambda 3, were then fitted into the density map. Each copy of lambda 3 was found anchored to the inner surface of the icosahedral core shell, making major contacts with three molecules of shell protein lambda 1 and overlapping, but not centering on, a five-fold axis. The overlap explains why only one copy of lambda 3 is bound per vertex. lambda 3 is furthermore oriented with its transcript exit channel facing a small channel through the lambda 1 shell, suggesting how the nascent RNA is passed into the large external cavity of the pentameric capping enzyme complex formed by protein lambda 2.

Cryoelectron Microscopy↗