Biomedical subjects

Y Zhao

Publications and source records attributed to Y Zhao.

At least 433 records · Page 24Linked to original sources

PubMed1999

S-adenosylmethionine attenuates the lipopolysaccharide-induced expression of the gene for tumour necrosis factor alpha.

Intracellular deficiency of S-adenosylmethionine (AdoMet) and elevated serum concentrations of tumour necrosis factor alpha (TNF) are hallmarks of toxin-induced liver injury. In these models, the administration of either exogenous AdoMet or antibody/soluble receptor for TNF attenuates the injury. We have demonstrated previously that the administration of exogenous AdoMet to AdoMet-deficient rats attenuated lipopolysaccharide (LPS)-induced liver injury and serum TNF concentrations. Here we report that AdoMet lowered the amount of TNF secreted by LPS-stimulated murine macrophage cells (RAW 264.7) in a dose-dependent manner. The inhibition of TNF release was correlated with changes in the steady-state TNF mRNA concentrations. Changes in TNF mRNA were not due to its altered stability and might have been due to an attenuation of the transcription rate of the TNF gene. The inhibition of TNF release in RAW cells was not mediated by GSH because treatment with AdoMet did not increase intracellular GSH. In addition, N-acetylcysteine, whereas it did increase GSH concentration, had no effect on LPS-stimulated TNF release in these cells. Exogenous AdoMet also attenuated LPS-induced serum TNF levels in normal rats sensitized with lead. Thus AdoMet administration might exert its hepatoprotective effects at least in part by its inhibitory effect on expression of the gene for TNF.

Acetylcysteine↗

PubMed1999

Engraftment of gene-marked hematopoietic progenitors in myeloma patients after transplant of autologous long-term marrow cultures.

We conducted a phase I hematopoietic stem cell (HSC) gene-marking trial in patients undergoing autologous blood or marrow stem cell transplant for the treatment of multiple myeloma. Between 500 and 1000 ml of bone marrow was harvested from each of 14 myeloma patients and 1 syngeneic donor. A mean of 3.3x10(9) cells per patient were plated in 20 to 50 long-term marrow culture (LTMC) flasks and maintained for 3 weeks. LTMCs were exposed on days 8 and 15 to clinical-grade neo(r)-containing retrovirus supernatant (G1Na). A mean of 8.23x10(8) day-21 LTMC cells containing 5.2x10(4) gene-marked granulocyte-macrophage progenitor cells (CFU-GM) were infused along with an unmanipulated peripheral blood stem cell graft into each patient after myeloablative therapy. Proviral DNA was detected in 71% of 68 tested blood and bone marrow samples and 150 of 2936 (5.1%) CFU-GM derived from patient bone marrow samples after transplant. The proportion of proviral DNA-positive CFU-GM declined from a mean of 9.8% at 3 months to a mean of 2.3% at 24 months postinfusion. Southern blots of 26 marrow and blood samples were negative. Semiquantitative PCR analysis indicated that gene transfer was achieved in 0.01-1% of total bone marrow and blood mononuclear cells (MNCs). Proviral DNA was also observed in EBV-transformed B lymphocytes, in CD34+ -enriched bone marrow cells, and in CFUs derived from the latter progenitors. Gene-modified cells were detected by PCR in peripheral blood and bone marrow for 24 months after infusion of LTMC cells. Sensitivity and specificity of the PCR assays were independently validated in four laboratories. Our data confirm that HSCs may be successfully transduced in stromal based culture systems. The major obstacle to therapeutic application of this approach remains the overall low level of genetically modified cells among the total hematopoietic cell pool in vivo.

Bone Marrow Cells↗

PubMed1999

Stat3 as an oncogene.

STATs are latent transcription factors that mediate cytokine- and growth factor-directed transcription. In many human cancers and transformed cell lines, Stat3 is persistently activated, and in cell culture, active Stat3 is either required for transformation, enhances transformation, or blocks apoptosis. We report that substitution of two cysteine residues within the C-terminal loop of the SH2 domain of Stat3 produces a molecule that dimerizes spontaneously, binds to DNA, and activates transcription. The Stat3-C molecule in immortalized fibroblasts causes cellular transformation scored by colony formation in soft agar and tumor formation in nude mice. Thus, the activated Stat3 molecule by itself can mediate cellular transformation and the experiments focus attention on the importance of constitutive Stat3 activation in human tumors.

3T3 Cells↗

PubMed1999

Molecular cloning of a cDNA encoding 14-3-3 protein from the protozoan Tetrahymena pyriformis and its mRNA expression during synchronous division.

The 14-3-3 proteins have been identified in a wide variety of eukaryotic cells and diverse biochemical properties have been ascribed to them. Here we have cloned a cDNA encoding 14-3-3 protein from a cDNA library of Tetrahymena pyriformis. This cDNA (Tp14-3-3) encoded an open reading frame consisting of 244 amino acids with predicated molecular mass as 28.1kDa. The predicted protein shares 59.2%, 56.5% and 59.2% identity to Entamoeba histolytica 14-3-3-1, Schizosaccharomyces pombe Rad 24 and human 14-3-3 zeta/delta respectively. On the basis of comparison with other 14-3-3 proteins, two of the putative functional domains (dimerization domain and annexin-similarity domain) were found in Tp14-3-3. In order to know the role of Tp14-3-3 in Tetrahymena, its mRNA levels during synchronous division were examined by Northern blot analysis. There was a marked increase in Tp14-3-3 mRNA level at 45min after the end of heat treatment, followed by a gradual decrease. These results suggest that the Tp14-3-3 mRNA level might vary during the cell cycle. The accumulation of Tp14-3-3 mRNA before cell division was assumed to be a prerequisite for the initiation of synchronous cell division.

14-3-3 Proteins↗

PubMed1999

Abnormalities of tumor suppressor genes P16 and P15 in primary maxillofacial squamous cell carcinomas.

As members of the same gene family, tumor suppressor genes P16/CDKN2/INK4A and P15/INK4B have a high degree of structural and functional homology with both P16 and P15 proteins involved directly in the regulation of cell cycles. However, the status of P16 and P15 genes in primary maxillofacial squamous cell carcinomas (MSCC) has not been reported. Studies on abnormalities of these genes including homozygous deletion, methylation of the 5'CpG islands, and mutations were carried out in 65 primary MSCC with polymerase chain reaction (PCR), methylation-specific PCR (MSP), PCR-SSCP (single-strand conformation polymorphism), and DNA sequencing techniques. Of the 65 tumors, 22 (34%) were methylated; 7 (11%) displayed point mutations. The total frequency of alteration of the P16 gene was 43% (28/65). The methylation rate of P15 was 12% (8/65). No homozygous deletion was found in either the P16 gene or P15 gene. In all MSCC samples, almost half (49%) harbored an alteration of the P16 or P15 gene. The P16 gene was altered more frequently than P15, and therefore is inactivated by methylation or mutation in a significant proportion of MSCC. The P15 gene appeared to play a lesser role in tumorigenesis of these tumors.

Adult↗

PubMed1999

Chemoenzymatic synthesis of the Salmonella group E1 core trisaccharide using a recombinant beta-(1-->4)-mannosyltransferase.

The chemical synthesis of the bacterial O-antigen from Salmonella serogroup E1, 3-O-(4-O-beta-D-mannopyranosyl-alpha-L-rhamnopyranosyl)-alpha-D-galactos e, presents a particular challenge because it contains a beta-(1-->4) mannosidic linkage to L-rhamnose. We report a chemoenzymatic synthesis of this crucial antigenic material which culminates in the enzymatic formation of the critical beta-mannosyl connection catalyzed by Salmonella GDP-alpha-D-Man:alpha Rha1-->3 alpha Gal-PP-Und beta-(1-->4)-mannosyltransferase (ManT beta 4). In comparison with previous synthetic routes, this method is advantageous since it utilizes intermediates, available in significant yield, which can be readily derivatized from the reducing end to present flexibility for analog construction, while the enzymatic construction of the Man1-->4Rha glycosidic bond is both rapid and occurs in high yield. Furthermore, the reported spectroscopic and enzymatic structural characterization of the trisaccharide product furnishes the first indisputable functional link between wbaO and ManT beta 4 and clearly sets the stage for the future mechanistic study and exploitation of this fascinating glycocatalyst.

Carbohydrate Conformation↗

PubMed1999

A fetal fatty-acid oxidation disorder as a cause of liver disease in pregnant women.

BACKGROUND: Acute fatty liver of pregnancy and the HELLP syndrome (hemolysis, elevated liver-enzyme levels, and a low platelet count) are serious hepatic disorders that may occur during pregnancy in women whose fetuses are later found to have a deficiency of long-chain 3-hydroxyacyl-coenzyme A (CoA) dehydrogenase. This enzyme resides in the mitochondrial trifunctional protein, which also contains the active site of long-chain 2,3-enoyl-CoA hydratase and long-chain 3-ketoacyl-CoA thiolase. We undertook this study to determine the relation between mutations in the trifunctional protein in infants with defects in fatty-acid oxidation and acute liver disease during pregnancy in their mothers. METHODS: In 24 children with 3-hydroxyacyl-CoA dehydrogenase deficiency, we used DNA amplification and nucleotide-sequence analyses to identify mutations in the alpha subunit of the trifunctional protein. We then correlated the results with the presence of liver disease during pregnancy in the mothers. RESULTS: Nineteen children had a deficiency only of long-chain 3-hydroxyacyl-CoA dehydrogenase and presented with hypoketotic hypoglycemia and fatty liver. In eight children, we identified a homozygous mutation in which glutamic acid at residue 474 was changed to glutamine. Eleven other children were compound heterozygotes, with this mutation in one allele of the alpha-subunit gene and a different mutation in the other allele. While carrying fetuses with the Glu474Gln mutation, 79 percent of the heterozygous mothers had fatty liver of pregnancy or the HELLP syndrome. Five other children, who presented with neonatal dilated cardiomyopathy or progressive neuromyopathy, had complete deficiency of the trifunctional protein (loss of activity of all three enzymes). None had the Glu474Gln mutation, and none of their mothers had liver disease during pregnancy. CONCLUSIONS: Women with acute liver disease during pregnancy may have a Glu474Gln mutation in long-chain hydroxyacyl-CoA dehydrogenase. Their infants are at risk for hypoketotic hypoglycemia and fatty liver.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1999

Cellular applications of a sensitive and selective fiber-optic nitric oxide biosensor based on a dye-labeled heme domain of soluble guanylate cyclase.

Nitric oxide-selective sensors have been prepared with the heme domain of soluble guanylate cyclase (sGC), the only known receptor for signal transduction involving nitric oxide. Expressed in and purified from E. coli, the heme domain contains a stoichiometric amount of heme that has electronic and resonance Raman spectra almost identical to those of heterodimeric (native) sGC purified from bovine lung. The small size of the heme domain, its inability to bind oxygen, and its high affinity for nitric oxide make it well-suited for sensor applications. The heme domain has been labeled with a fluorescent reporter dye and changes in this dye's intensity are observed based on the sGC heme domain's characteristic binding of nitric oxide. The current sensors are prepared with 100-microns optical fiber but could also be prepared using submicrometer fiber tips. These sensors have fast, linear, and reversible responses to nitric oxide and are unaffected by numerous common interferents, such as oxygen, nitrite and nitrate. The sensor limit of detection is 1 microM nitric oxide. Glutathione has been shown to decrease the sensitivity of the sensor; however, the sensor response remains linear and can be calibrated on the basis of the glutathione concentration present in the biological environment of interest. The sensors have been used to measure extracellular nitric oxide production by BALB/c mouse macrophages. Minimal nitric oxide was produced by untreated cells, while high levels of nitric oxide were released from activated cells, e.g., 111 +/- 2 microM in a given cell culture.

Animals↗

PubMed1999

Control of hippocampal morphogenesis and neuronal differentiation by the LIM homeobox gene Lhx5.

The mammalian hippocampus contains the neural circuitry that is crucial for cognitive functions such as learning and memory. The development of such circuitry is dependent on the generation and correct placement of the appropriate number and types of neurons. Mice lacking function of the LIM homeobox gene Lhx5 showed a defect in hippocampus development. Hippocampal neural precursor cells were specified and proliferated, but many of them failed to either exit the cell cycle or to differentiate and migrate properly. Lhx5 is therefore essential for the regulation of precursor cell proliferation and the control of neuronal differentiation and migration during hippocampal development.

Animals↗

PubMed1999

Transforming growth factor-beta (TGF-beta) type I and type II receptors are both required for TGF-beta-mediated extracellular matrix production in lung fibroblasts.

Transforming growth factor-beta (TGF-beta) regulates a variety of cellular activities including cell growth, differentiation and extracellular matrix production. The TGF-beta type I and type II serine/threonine kinase receptors (TbetaRI and TbetaRII) have been identified as signal-transducing TGF-beta receptors. This study was undertaken to examine the role of the type I and type II receptors in TGF-beta-induced extracellular matrix production of lung fibroblasts. We constructed expression plasmids containing truncated derivatives of TbetaRI and TbetaRII that lacked the cytoplasmic serine/threonine kinase domain (TbetaRI deltaK and TbetaRII deltaK), and transfected them into lung fibroblasts. TbetaRII deltaK expressed by lung fibroblasts was able to bind 125I-TGF-beta1, whereas TbetaRI deltaK was unable to bind ligand when expressed alone. Co-expression with TbetaRII was required for binding and cross-linking of TGF-beta1 to TbetaRI deltaK. Lung fibroblasts upregulate tenascin and fibronectin production when treated with TGF-beta1. The kinase-defective deletions of both TbetaRI and TbetaRII were dominant-acting inhibitors of TGF-beta signal transduction. Expression of either TbetaRI deltaK or TbetaRII deltaK alone was sufficient to block TGF-beta-induced tenascin and fibronectin production of lung fibroblasts. The results indicate that both TbetaRI and TbetaRII were required for TGF-beta signaling in regulation of extracellular matrix production by lung fibroblasts.

Animals↗

PubMed1999

Expression, characterization, and mutagenesis of the Yersinia pestis murine toxin, a phospholipase D superfamily member.

A phospholipase D (PLD) superfamily was recently identified that contains proteins of highly diverse functions with the conserved motif HXKX4DX6G(G/S). The superfamily includes a bacterial nuclease, human and plant PLD enzymes, cardiolipin synthases, phosphatidylserine synthases, and the murine toxin from Yersinia pestis (Ymt). Ymt is particularly effective as a prototype for family members containing two conserved motifs, because it is smaller than many other two-domain superfamily enzymes, and it can be overexpressed. Large quantities of pure recombinant Ymt allowed the formation of diffraction-quality crystals for x-ray structure determination. Dimeric Ymt was shown to have PLD-like activity as demonstrated by the hydrolysis of phosphatidylcholine. Ymt also used bis(para-nitrophenol) phosphate as a substrate. Using these substrates, the amino acids essential for Ymt function were determined. Specifically, substitution of histidine or lysine in the conserved motifs reduced the turnover rate of bis(para-nitrophenol) phosphate by a factor of 10(4) and phospholipid turnover to an undetectable level. The role of the conserved residues in catalysis was further defined by the isolation of a radiolabeled phosphoenzyme intermediate, which identified a conserved histidine residue as the nucleophile in the catalytic reaction. Based on these data, a unifying two-step catalytic mechanism is proposed for this diverse family of enzymes.

Animals↗

PubMed1999

Molecular cloning and characterization of the human CLOCK gene: expression in the suprachiasmatic nuclei.

The Clock gene is an essential regulator of circadian rhythms. It encodes a member of the basic helix-loop-helix/PER-ARNT-SIM family of transcription factors known to play a central role in the control of diverse cellular events. Previously we described the functional identification and molecular isolation of the Clock gene in the mouse, its interaction with the BMAL1 protein, and the role of this complex as a transcriptional activator in the circadian pacemaker. Here, we report the cloning, exon organization, chromosomal location, and mRNA expression of the human CLOCK gene. The coding sequence of human CLOCK extends for 2538 bp and is 89% identical to its mouse ortholog; its deduced amino acid sequence is 846 residues long and is 96% identical to mouse CLOCK. Radiation hybrid mapping localized human CLOCK to the long arm of human chromosome 4 (4q12). Direct sequencing of a genomic CLOCK clone indicated that the coding sequence of human CLOCK extends over 20 exons and that its intron/exon organization is identical to that of the mouse ortholog. Northern blot analysis indicated widespread expression of two major transcripts of 8 and 10 kb, and in situ hybridization of human brain tissue revealed elevated expression of CLOCK mRNA in the suprachiasmatic nuclei, the locus of circadian control in mammals, and in the cerebellum. Comparison of cDNA clones revealed two single nucleotide polymorphisms in noncoding sequence flanking the CLOCK open reading frame. The central role of Clock in the organization of circadian rhythms suggests that it will be a useful candidate gene for genetic analyses of disorders associated with dysfunction of the circadian system.

Alleles↗

PubMed1999

[Rapid detection of non-deletional alpha-thalassemia mutations by PCR-LIS-SSCP]

OBJECTIVE: To establish a rapid and convenient single strand conformation polymorphism (SSCP) analysis method for detecting the point mutation of non-deletional alpha-thalassemia. METHODS: The 543bp DNA fragment spanning the hot spot region mainly responsible for non-deletional alpha-thalassemia was nested amplified using the selective amplification of alpha2 globin gene as a template and was denatured with low ionic strength(LIS) solution followed by SSCP analysis. RESULTS: LIS buffer was more efficient for ssDNA formation than formamide buffer was and the formation of ssDNA was very stable. In addition to a normal electrophoresis pattern, at least three SSCP profiles can be detected by the present method when the DNA samples bearing non-deletional genes of Hb H disease were screened. Confirmed by DNA sequencing analysis, the DNAs represented these profiles have turned out to be the different three mutants, i.e., the alphaCS mutation, the alpha QS mutation and the alphaWestmead mutation, respectively. Only 3 hours were needed to complete the electrophoresis procedure of this method. CONCLUSION: PCR-LIS-SSCP can be used as a tool in rapid screening for the alterations in human alpha-globin gene.

Journal Article↗

PubMed1999

Apoptosis of mouse liver nuclei induced in the cytosol of carrot cells.

We report here the apoptosis of mouse liver nuclei induced in the cytosol of carrot cells by cytochrome c. Several typical characteristics of apoptosis, such as chromatin condensation, margination and apoptotic bodies, were detected. The result of DNA gel electrophoresis showed that DNA was degraded into nucleosomal fragments. The terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labelling procedure was also performed to detect the breakage of 3'-OH ends of a DNA strand. Furthermore, we found that nuclear lamins were degraded from 88 kDa and 66 kDa to 37 kDa and 47 kDa fragments. The DNA fragmentation could be inhibited by AC-DEVD-CHO and AC-YVAD-CHO. The results indicate that the apoptosis in plant cells may share some similar pathways to apoptosis in animal cells.

Animals↗

PubMed1999

Identification of the block in targeted retroviral-mediated gene transfer.

A chimeric retroviral vector (33E67) containing a CD33-specific single-chain antibody was generated in an attempt to target cells displaying the CD33 surface antigen. The chimeric envelope protein was translated, processed, and incorporated into viral particles as efficiently as wild-type envelope protein. The viral particles carrying the 33E67 envelope protein could bind efficiently to the CD33 receptor on target cells and were internalized, but no gene transfer occurred. A unique experimental approach was used to examine the basis for this postbinding block. Our data indicate that the chimeric envelope protein itself cannot participate in the fusion process, the most reasonable explanation being that this chimeric protein cannot undergo the appropriate conformational change that is thought to be triggered by receptor binding, a suggested prerequisite to subsequent fusion and core entry. These results indicate that the block to gene transfer in this system, and probably in most of the current chimeric retroviral vectors to date, is the inability of the chimeric envelope protein to undergo this obligatory conformational change.

3T3 Cells↗

PubMed1999

Detection of hepatitis B surface antigen mutants and their integration in human hepatocellular carcinoma.

Vaccine escape hepatitis B virus surface antigen (HBsAg) mutants are capable of independent replication and have been implicated in acute hepatitis. We now report the detection of these mutants with changes at various positions of the antigenic 'a' determinant in human hepatocellular carcinoma (HCC). Southern blot analysis indicated that the HBsAg mutant with the Glycine to Arginine change at position 145 was integrated in HCC, whereas those with a Threonine at position 133 instead of a Methionine were identified in the serum of aggressive HCC. Further studies on HBsAg mutants in HCC should provide new insights on their involvement in the hepatocarcinogenesis.

Arginine↗

PubMed1999

[Zinc deficiency inhibited the proliferation and differentiation of rat osteoblast in vitro].

The effect of zinc deficiency on proliferation and differentiation of rat osteoblast was studied in vitro. The osteoblasts were isolated from the rat calvaria, and were subcultured in DMEM medium. Zinc deficiency model in cell culture was made by chelating zinc from the medium with a special chelator TPEN. Incorporation of 3H-thymidine was measured to show the DNA synthesis at different time point, and cell cycle was analyzed by flow cytometry. A histochemistry method was used to detect changes of osteoblast cytoskeleton. Incorporation of 3H-proline was measured to show the synthesis of collagen. Alkaline phosphatase activity (AKP) and osteocalcin content were measured by enzyme dynamics method and radioimmunoassay respectively. The results showed that the incorporation of 3H-thymidine in the zinc deficient group was significantly less than that in the control group at different time points. The cells were blocked at G2/M phase of cell cycle, which is related to the impairment of cytoskeleton. Collagen synthesis, osteocalcin content and AKP activity in the zinc deficient group were reduced significantly as compared with those in the control group. But no differences in all the measurements were observed between the control group and the group of zinc deficiency supplemented with zinc. Therefore, it was concluded that zinc deficiency inhibited the proliferation and differentiation of rat osteoblast in vitro.

Alkaline Phosphatase↗

PubMed1999

[Application of gas chromatography-mass spectrometry for determination of the organochlorine pesticide residues in ginseng].

A gas chromatographic-electronionization-mass spectrometric method (GC-EIMS) was developed for the determination of organochlorine pesticide residues in Ginseng plants. The Ginseng samples were extracted with acetone, partioned using petroleum ester with sodium chloride saturated aqueous solution and clean-up with concentrated sulfuric acid. The organochlorine pesticide residues were analysed by GC-MS with monitoring selective mass ions. Ten organochlorine pesticides including alpha, beta, gamma and delta isomers of hexachlorocyclohexane (HCH), pp'-DDE, pp'-DDT, op'-DDT, pp'-DDT, pentachloronitrobenzene (Quintozene, PCNB), and hexachlorobenzene(HCB), were determined simultaneously. Targeted pesticide analytes were separated on DB-5 capillary column. The optimum chromatographic conditions and MS parameters were determined. The average recovery rates for those pesticide residues Ginseng were from 76.6% to 90.1% at 0.1 mg/kg of spiked level, except 58.3% for PCNB. The relative standard deviations were 9.3%-15.0%.

Drug Contamination↗