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Biomedical subjects

D Yu

Publications and source records attributed to D Yu.

At least 37 records · Page 2Linked to original sources
PubMed2001

The repressive activity of hepatitis C virus core protein on the transcription of p21(waf1) is regulated by protein kinase A-mediated phosphorylation.

Hepatitis C virus (HCV) core protein is known to repress the transcription of p21(waf1) directly in a p53-independent manner. In this study, the region of HCV core protein responsible for the transcriptional repression of p21 promoter was determined. N-terminal half of core protein almost completely lost the ability to repress p21 promoter, indicating that the domain required for the majority of p21 repression is located between amino acid positions 84 and 191. The trans-repression activity of HCV core mutant S99L on p21 gene expression was similar to that of wild type core protein whereas mutation of the 116th amino acid Ser into either Ile or Ala completely abolished the repressive ability of HCV core protein. In addition, the trans-repression activity of HCV core mutant S116D was similar to that of wild type core protein, suggesting that an acidic aspartate residue can mimic the effect of phosphorylation. When treated with a protein kinase A (PKA) inhibitor, H-89, the inhibitory activity of wild-type HCV core protein was dose-dependently decreased and was completely lost at the concentration of 5 microM. On the contrary, the repression activity of HCV core protein was increased by treatment with a PKA activator, dibutyryl-cAMP, indicating that the p21 repressive activity of HCV core protein is regulated by phosphorylation at S-116 by protein PKA

3T3 Cells↗
PubMed2001

Wild type p53 sensitizes soft tissue sarcoma cells to doxorubicin by down-regulating multidrug resistance-1 expression.

BACKGROUND: p53 mutations occur in almost half of all soft tissue sarcomas (STS) and may contribute to multidrug resistance (MDR) in patients with STS. Doxorubicin (Dox) is one of the most active single agents in STS but is less effective in STS with p53 mutations. The effect of reintroducing wild type (wt) p53 into STS cells harboring p53 mutations on the cytotoxicity of DOX in vitro and in vivo was studied. METHODS: The following cell lines were used in this study: SKLMS-1 STS cells, which do not express wt p53; two wt p53 stable transfectant cells derived from SKLMS-1 cells; and SKLMS-1 transfectant cells from a p53 temperature-sensitive mutant that expresses wt p53 at 32 degrees C and mutant p53 at 38 degrees C. The cytotoxicity of Dox was examined by [3-(4,5-dimethylthiazzol-2-yl)-2,5-diphenltetrazolium] (MTT) and clonogenetic assay, and the effect of reintroducing wt p53 on tumor suppression by Dox was evaluated with a tumorigenicity assay. DNA fragmentation was used to detect apoptosis. MDR-1 P-glycoprotein (P-gp) expression was detected by Western blot and immunohistochemical analyses of protein levels and by Northern blot analysis of mRNA levels, respectively. The intracellular accumulation of Dox was detected by flow cytometric analysis. RESULTS: The 50% inhibitory concentration (IC(50)) of Dox for the SKLMS-1 wt p53 transfectants decreased 16-fold compared with SKLMS-1 parental cells expressing mutant p53. Colony formation of SKLMS-1 cells after Dox treatment also was inhibited by wt p53 reintroduction. The tumorigenicity of SKLMS-1 cells was inhibited by wt p53 reintroduction alone or by Dox treatment alone and was inhibited further when p53 introduction was combined with Dox treatment in severe combined immunodeficient mice. Although no difference in DNA fragmentation, Bax expression, or Bcl-2 expression was detected among wt p53 transfectants and parental SKLMS-1 cells after Dox treatment, MDR-1 P-gp expression was decreased in wt p53 transfectants compared with parental SKLMS-1 cells. Furthermore, higher intracellular accumulations of Dox were found in wt p53 transfectants than that in SKLMS-1 cells. CONCLUSIONS: Reintroduction of wt p53 into STS cells harboring p53 mutations can enhance their chemosensitivity to Dox through the inhibition of MDR-1 P-gp expression. Thus, the combination of p53 gene therapy and chemotherapy may increase the therapeutic efficacy in the treatment of patients with STS.

Apoptosis↗
PubMed2001

Angiogenin activates Erk1/2 in human umbilical vein endothelial cells.

Angiogenin is a potent angiogenic factor that binds to endothelial cells and is endocytosed and rapidly translocated to the nucleus where it is concentrated in the nucleolus and binds to DNA. Angiogenin also activates cell-associated proteases, induces cell invasion and migration, stimulates cell proliferation, and organizes cultured cells to form tubular structures. The intracellular signaling pathways that mediate these various cellular responses are not well understood. Here we report that angiogenin induces transient phosphorylation of extracellular signal-related kinase1/2 (Erk1/2) in cultured human umbilical vein endothelial cells. Angiogenin does not affect the phosphorylation status of stress-associated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) and p38 mitogen-activated protein (MAP) kinases. PD98059--a specific inhibitor of MAP or Erk kinase 1 (MEK 1), the upstream kinase that phosphorylates Erk1/2--abolishes angiogenin-induced Erk phosphorylation and cell proliferation without affecting nuclear translocation of angiogenin. In contrast, neomycin, a known inhibitor of nuclear translocation and cell proliferation, does not interfere with angiogenin-induced Erk1/2 phosphorylation. These data indicate that both intracellular signaling pathways and direct nuclear functions of angiogenin are required for angiogenin-induced cell proliferation and angiogenesis.

Active Transport, Cell Nucleus↗
PubMed2001

[Analysis of inflammation related gene expression spectrum in ankylosing spondylitis patients using cDNA microarray].

OBJECTIVE: To test the difference of gene expression patterns in peripheral blood mononuclear cells (PBMCs) among patients with ankylosing spondylitis (AS) or rheumatoid arthritis (RA) and healthy volunteers and to find potential discriminating genes. METHODS: The PBMC gene expression spectra of 7 patients with active AS, 6 patients with active RA, and 7 healthy volunteers were tested by microarray assay with 588 target gene filters. Semi-quantitative RT-PCR was performed to test the expression rates of thirteen inflammation related genes which had been with high expression rates in at least 4 arthritis patients by microarray assay, among 22 AS patients, 8 RA patients, and 7 healthy volunteers. RESULTS: The expression rates of inflammation related genes by cDNA microarray assay in healthy volunteer group, AS group, and RA group were 8.4%, 32.5% and 44.8% respectively (P < 0.001). The positive rate in RA patients was much higher than that in AS patients (P < 0.012). A significant increase of positive rate of these genes was also noticed in 2 active pulmonary tuberculosis patients. The expression rates of the 13 genes which were positive in at least 4 arthritis patients tested by semiquantitative RT-PCR in 22 AS patients, 8 RA patients and 7 healthy volunteers were very similar to those expression rates tested by cDNA microarray. CONCLUSION: The gene expression profiles of PBMC among AS patients are significantly abnormal and are significantly different from those among RA patients. The high expression of the 13 cytokines is not specific to AS.

Adolescent↗
PubMed2001

Modulation of immunostimulatory activity of CpG oligonucleotides by site-specific deletion of nucleobases.

The effect of nucleobase deletion in the 3'- or the 5'-flanking sequence to a CpG-motif on immunostimulatory activity of CpG-containing oligonucleotides was examined by cell proliferation, secretion of IL-12 and IL-6 in mouse spleen cell cultures, and by spleen enlargement in mice. Deletion of one or two nucleobases in the 3'-flanking sequence to a CpG-motif at certain positions did not affect immunostimulatory activity, while similar deletions in the 5'-flanking sequence increased immunostimulatory activity compared with the parent oligo.

3' Flanking Region↗
PubMed2001

Cell toxicity caused by products of the p(L) operon of bacteriophage lambda.

Induction of a lambda prophage causes the death of the host cell even in the absence of phage replication and lytic functions due to expression of functions from the lambda p(L) operon. We genetically modified the lambda prophage to determine which lambda p(L) operon functions were involved in cell killing. Viability assays and flow cytometry were used to monitor cell death and filamentation. The kil gene was shown to cause cell death and filamentation as described previously. Another killing activity was mapped within the p(L) operon to the gam gene. Inspection of the DNA sequence showed that there are two possible translation start points for both kil and gam. In both cases, the shorter of the two possible products could cause cell killing. The shorter products were also sufficient for the known filamentation and recombination activities of the respective Kil and Gam functions. The expression level of the p(L) operon is down-regulated by Cro repressor. In the absence of Cro, higher p(L) expression levels allow either Kil or Gam to be lethal or growth inhibitory, whereas at lowered expression in Cro-repressed conditions, only Kil is lethal. The filamentation function of Kil and recombination activity of Gam are unaffected at Cro-repressed levels of expression.

Bacterial Proteins↗
PubMed2001

High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides.

Homologous DNA recombination is a fundamental, regenerative process within living organisms. However, in most organisms, homologous recombination is a rare event, requiring a complex set of reactions and extensive homology. We demonstrate in this paper that Beta protein of phage lambda generates recombinants in chromosomal DNA by using synthetic single-stranded DNAs (ssDNA) as short as 30 bases long. This ssDNA recombination can be used to mutagenize or repair the chromosome with efficiencies that generate up to 6% recombinants among treated cells. Mechanistically, it appears that Beta protein, a Rad52-like protein, binds and anneals the ssDNA donor to a complementary single-strand near the DNA replication fork to generate the recombinant. This type of homologous recombination with ssDNA provides new avenues for studying and modifying genomes ranging from bacterial pathogens to eukaryotes. Beta protein and ssDNA may prove generally applicable for repairing DNA in many organisms.

Amino Acid Sequence↗
PubMed2001

Cutting edge: systemic inhibition of angiogenesis underlies resistance to tumors during acute toxoplasmosis.

The ability of various infections to suppress neoplastic growth has been well documented. This phenomenon has been traditionally attributed to infection-induced concomitant, cell-mediated antitumor immunity. We found that infection with Toxoplasma gondii effectively blocked neoplastic growth of a nonimmunogenic B16.F10 melanoma. Moreover, this effect was independent of cytotoxic T or NK cells, production of NO by macrophages, or the function of the cytokines IL-12 and TNF-alpha. These findings suggested that antitumor cytotoxicity was not the primary mechanism of resistance. However, infection was accompanied by strong, systemic suppression of angiogenesis, both in a model system and inside the nascent tumor. This suppression resulted in severe hypoxia and avascular necrosis that are incompatible with progressive neoplastic growth. Our results identify the suppression of tumor neovascularization as a novel mechanism critical for infection-induced resistance to tumors.

Acute Disease↗
PubMed2001

Transcriptional upregulation and activation of p55Cdc via p34(cdc2) in Taxol-induced apoptosis.

Paclitaxel (Taxol) is a potent and highly effective antineoplastic agent for the treatment of advanced, drug-refractory, metastatic breast cancers. Taxol not only induces tubulin polymerization, stabilizes microtubules, blocks cell cycle progression, and induces apoptosis, but it also alters gene expression. Here, we have identified that Taxol can upregulate expression of the gene encoding the cell cycle protein p55Cdc by using cDNA array technique. Taxol induced p55Cdc mRNA expression through activation of the p55Cdc promoter, which led to increase p55Cdc protein expression. Taxol also activated p55Cdc-associated kinase. In addition, overexpression of the p55Cdc gene resulted in cell death in both HeLa cells and NIH3T3 cells in a dose-dependent manner. A dominant-negative mutant of p34(cdc2) blocked Taxol-induced p55Cdc activation and inhibited p55Cdc-induced and Taxol-induced cell death. Our data suggest that transcriptional upregulation of p55Cdc and activation of p55Cdc by Taxol-mediated p34(cdc2) activation play a critical role in Taxol-induced cell death.

3T3 Cells↗
PubMed2001

A novel chordin-like protein inhibitor for bone morphogenetic proteins expressed preferentially in mesenchymal cell lineages.

Chordin is a bone morphogenetic protein (BMP) inhibitor that has been identified as a factor dorsalizing the Xenopus embryo. A novel secreted protein, CHL (for chordin-like), with significant homology to chordin, was isolated from mouse bone marrow stromal cells. Injection of CHL RNA into Xenopus embryos induced a secondary axis. Recombinant CHL protein inhibited the BMP4-dependent differentiation of embryonic stem cells in vitro and interacted directly with BMPs, similar to chordin. However, CHL also weakly bound to TGFbetas. In situ hybridization revealed that the mouse CHL gene, located on the X chromosome, was expressed predominantly in mesenchyme-derived cell types: (1) the dermatome and limb bud mesenchyme and, later, the subdermal mesenchyme and the chondrocytes of the developing skeleton during embryogenesis and (2) a layer of fibroblasts/connective tissue cells in the gastrointestinal tract, the thick straight segments of kidney tubules, and the marrow stromal cells in adults. An exception was expression in the neural cells of the olfactory bulb and cerebellum. Interestingly, the spatiotemporal expression patterns of CHL were distinct from those of chordin in many areas examined. Thus, CHL may serve as an important BMP regulator for differentiating mesenchymal cells, especially during skeletogenesis, and for developing specific neurons.

Alternative Splicing↗
PubMed2001

A highly efficient Escherichia coli-based chromosome engineering system adapted for recombinogenic targeting and subcloning of BAC DNA.

Recently, a highly efficient recombination system for chromosome engineering in Escherichia coli was described that uses a defective lambda prophage to supply functions that protect and recombine a linear DNA targeting cassette with its substrate sequence (Yu et al., 2000, Proc. Natl. Acad. Sci. USA 97, 5978-5983). Importantly, the recombination is proficient with DNA homologies as short as 30-50 bp, making it possible to use PCR-amplified fragments as the targeting cassette. Here, we adapt this prophage system for use in bacterial artificial chromosome (BAC) engineering by transferring it to DH10B cells, a BAC host strain. In addition, arabinose inducible cre and flpe genes are introduced into these cells to facilitate BAC modification using loxP and FRT sites. Next, we demonstrate the utility of this recombination system by using it to target cre to the 3' end of the mouse neuron-specific enolase (Eno2) gene carried on a 250-kb BAC, which made it possible to generate BAC transgenic mice that specifically express Cre in all mature neurons. In addition, we show that fragments as large as 80 kb can be subcloned from BACs by gap repair using this recombination system, obviating the need for restriction enzymes or DNA ligases. Finally, we show that BACs can be modified with this recombination system in the absence of drug selection. The ability to modify or subclone large fragments of genomic DNA with precision should facilitate many kinds of genomic experiments that were difficult or impossible to perform previously and aid in studies of gene function in the postgenomic era.

Animals↗
PubMed2001

Up-regulation of vascular endothelial growth factor in breast cancer cells by the heregulin-beta1-activated p38 signaling pathway enhances endothelial cell migration.

Heregulin (HRG) belongs to a family of polypeptide growth factors that bind to receptor tyrosine kinases ErbB3 and ErbB4. HRG binding induces ErbB3 and ErbB4 heterodimerization with ErbB2, activating downstream signal transduction. Vascular endothelial growth factor (VEGF) is a primary regulator of physiological angiogenesis and is a major mediator of pathological angiogenesis, such as tumor-associated neovascularization. In this study, we demonstrate that HRG-beta1 increased secretion of VEGF from breast cancer cells in a time- and dosage-dependent manner and that this increase resulted from up-regulation of VEGF mRNA expression via transcriptional activation of the VEGF promoter. Deletion and mutational analysis revealed that a CA-rich upstream HRG response element located between nucleotide-2249 and -2242 in the VEGF promoter mediated HRG-induced transcriptional up-regulation of VEGF. While investigating the downstream signaling pathways involved in HRG-mediated up-regulation of VEGF, we found that HRG activated extracellular signal-regulated protein kinases, Akt kinase, and p38 mitogen-activated protein kinase (MAPK). However, only the specific inhibitor of p38 MAPK (SB203580), not extracellular signal-regulated kinase inhibitor PD98059 nor the inhibitor of phosphatidylinositol 3-kinase-Akt pathway (Wortmannin), blocked the up-regulation of VEGF by HRG. The HRG-stimulated secretion of VEGF from breast cancer cells resulted in increased migration of murine lung endothelial cells, an activity that was inhibited by either VEGF-neutralizing antibody or SB203580. These results show that HRG can activate p38 MAPK to enhance VEGF transcription via an upstream HRG response element, leading to increased VEGF secretion and angiogenic response in breast cancer cells.

Breast Neoplasms↗
PubMed2001

Effect of 2'-O-methyl antisense ORNs on expression of thymidylate synthase in human colon cancer RKO cells.

Translation of thymidylate synthase (TS) mRNA is controlled by its own protein end-product TS in a negative autoregulatory manner. Disruption of this regulation results in increased synthesis of TS and may lead to the development of cellular drug resistance to TS-directed anticancer agents. As a strategy to inhibit TS expression, antisense 2'-O-methyl RNA oligoribonucleotides (ORNs) were designed to directly target the 5' upstream cis-acting regulatory element (nucleotides 80-109) of TS mRNA. A 30 nt ORN, HYB0432, inhibited TS expression in human colon cancer RKO cells in a dose-dependent manner but had no effect on the expression of beta-actin, alpha-tubulin or topoisomerase I. TS expression was unaffected by treatment with control sense or mismatched ORNs. HYB0504, an 18 nt ORN targeting the same core sequence, also repressed expression of TS protein. However, further reduction in oligo size resulted in loss of antisense activity. Following HYB0432 treatment, TS protein levels were reduced by 60% within 6 h and were maximally reduced by 24 h. Expression of p53 protein was inversely related to that of TS, suggesting that p53 expression may be directly linked to intracellular levels of TS. Northern blot analysis demonstrated that TS mRNA was unaffected by HYB0432 treatment. The half-life of TS protein was unchanged after antisense treatment suggesting that the mechanism of action of antisense ORNs is mediated through a process of translational arrest. These findings demonstrate that an antisense ORN targeted at a critical cis-acting element on TS mRNA can specifically inhibit expression of TS protein in RKO cells.

Colonic Neoplasms↗
PubMed2001

Modulation of the nuclear factor kappa B pathway by Shp-2 tyrosine phosphatase in mediating the induction of interleukin (IL)-6 by IL-1 or tumor necrosis factor.

Shp-2, a src homology (SH)2-containing phosphotyrosine phosphatase, appears to be involved in cytoplasmic signaling downstream of a variety of cell surface receptors, although the mechanism is unclear. Here, we have determined a role of Shp-2 in the cytokine circuit for inflammatory and immune responses. Production of interleukin (IL)-6 in response to IL-1 alpha or tumor necrosis factor (TNF)-alpha was nearly abolished in homozygous mutant (Shp-2(-/)-) fibroblast cells. The targeted Shp-2 mutation has no significant effect on the activation of the three types of mitogen-activated protein (MAP) kinases, extracellular signal-regulated kinase (Erk), c-Jun NH(2)-terminal kinase (Jnk), and p38, by IL-1/TNF, indicating that Shp-2 does not work through MAP kinase pathways in mediating IL-1/TNF-induced IL-6 synthesis. In contrast, IL-1/TNF-stimulated nuclear factor (NF)-kappa B DNA binding activity and inhibitor of kappa B (I kappa B) phosphorylation was dramatically decreased in Shp-2(-/)- cells, while the expression and activity of NF-kappa B-inducing kinase (NIK), Akt, and I kappa B kinase (IKK) were not changed. Reintroduction of a wild-type Shp-2 protein into Shp-2(-/)- cells rescued NF-kappa B activation and IL-6 production in response to IL-1/TNF stimulation. Furthermore, Shp-2 tyrosine phosphatase was detected in complexes with IKK as well as with IL-1 receptor. Thus, this SH2-containing enzyme is an important cytoplasmic factor required for efficient NF-kappa B activation. These results elucidate a novel mechanism of Shp-2 in cytokine signaling by specifically modulating the NF-kappa B pathway in a MAP kinase-independent fashion.

Animals↗
PubMed2001

Monitoring human serum transferrin by capillary zone electrophoresis with end-column amperometric detection.

Capillary zone electrophoresis was employed for the determination of human serum transferrin using end-column amperometric detection with a carbon fiber microelectrode at a constant potential of 1.9 V vs. saturated calomel electrode (SCE). The optimum conditions of separation and detection are 7.5 x 10(-4) mol/L Tris-3.44 x 10(-4) mol/L HCl for the buffer solution, 20 kV for the separation voltage, 5 kV and 10 s for the injection voltage and the injection time, respectively. The limit of detection is 6.7 x 10(-8) mol/L or 440 amol (S/N = 2). The relative standard deviations are 0.67% for the migration time and 1.5% for the electrophoretic peak current. The method was applied to the determination of transferrin in human serum. The recovery is between 93-104%.

Adult↗
PubMed2001

Rapid engineering of bacterial artificial chromosomes using oligonucleotides.

A rapid method obviating the use of selectable markers to genetically manipulate large DNA inserts cloned into bacterial artificial chromosomes is described. Mutations such as single-base changes, deletions, and insertions can be recombined into a BAC by using synthetic single-stranded oligonucleotides as targeting vectors. The oligonucleotides include the mutated sequence flanked by short homology arms of 35-70 bases on either side that recombine with the BAC. In the absence of any selectable marker, modified BACs are identified by specific PCR amplification of the mutated BAC from cultures of pooled bacterial cells. Each pool represents about 10 electroporated cells from the original recombination mixture. Subsequently, individual clones containing the desired alteration are identified from the positive pools. Using this BAC modification method, we have observed a frequency of one recombinant clone per 90-260 electroporated cells. The combination of high targeting frequency and the sensitive yet selective PCR-based screening method makes BAC manipulation using oligonucleotides both rapid and simple.

Bacterial Proteins↗
PubMed2001

Investigation on marking method for phenomenon on regrowth drug resistance in relapsed acute myelogenous leukemia.

A method for evaluating the regrowth drug resistance in relapsed acute myelogenous leukemia (AML) was developed. Drug sensitivity and proliferation of leukemic cells in vitro were determined using leukemic cell colony forming unit (CUF-L), MTT drug-sensitive test, percentage of S phase cells in cell cycle (S%), fluorescent index (FI) and drug resistant index (DRI) by detecting intracellular daunorubicin, expression of P-170 glycoprotein by APAAP assay, and abundance of Bcl-XL mRNA by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) methods. First, the correlation between scoring criteria and cell drug resistance and cell proliferation was investigated in newly untreated AML patients. Second, 20 patients with relapsed AML were marked. According to each tested result, its point(s) was scored. The results showed that among the 20 cases of relapsed AML, 9 were diagnosed as having regrowth drug resistance. It was concluded that the scoring method for regrowth drug resistance was first developed in AML. There was regrowth drug resistance in relapsed AML; clinically circumventing it would be of extreme significance for establishment of new approaches to the treatment in AML.

ATP Binding Cassette Transporter, Subfamily B↗