PubMed Health⌕ Search

Biomedical subjects

D C Tang

Publications and source records attributed to D C Tang.

At least 19 recordsLinked to original sources
PubMed2001

Polymorphisms within the angiotensinogen gene (GT-repeat) and the risk of stroke in pediatric patients with sickle cell disease: a case-control study.

Stroke is one of the most devastating complications of patients with sickle cell disease (SCD). Currently, there are no known molecular or genetic markers that can be used to assess the risk of stroke in this population. We have previously shown that relative hypertension may be one risk factor for stroke in SCD. In a case-control study, we investigated the association between GT-repeat polymorphism within the angiotensinogen (AGT) gene and the risk of stroke in pediatric patients with SCD. After informed consent was obtained, 63 patients (21 stroke subjects and 42 nonstroke control subjects matched according to age and sex) with SCD followed at local pediatric hematology clinics were genotyped to test the association of specific GT-repeat alleles of the AGT gene and occurrence of stroke. There were statistical differences in the distribution of the genotypes among stroke and nonstroke SCD patients (chi(2) = 10.82, df = 11, P < 0.05). We also found GT-repeat alleles A3 and/or A4 of the AGT gene conferred a four-fold increase in the risk of stroke (odds ratio [OR] = 4, P < 0.05). The attributable odds ratio for allele A3 and A4 is 2.24 and 4.33, respectively (P < 0.005). Our results suggest that GT-repeat within the AGT gene may be associated with risk of stroke in pediatric SCD. The relative risk of stroke in the presence of alleles A3 and/or A4 is fourfold greater than in the absence of these alleles. If these data are substantiated in a larger cohort of patients, our results indicate that the determination of GT-repeat of AGT gene may be a useful genetic marker to assess the risk for stroke of patients with SCD. Am. J. Hematol. 68:164-169, 2001. Published 2001 Wiley-Liss, Inc.

Adolescent↗
PubMed2001

Rapid differentiation of five common alpha-thalassemia genotypes by polymerase chain reaction.

The alpha-thalassemias are common genetic disorders that arise from reduced synthesis of the alpha-globin chains. At present, large-scale carrier screening and clinically valuable antenatal detection programs have not been established for the congenital disorder alpha-thalassemia (alpha-thal). We have developed a simple nonradioactive polymerase chain reaction (PCR) approach that can detect and differentiate several common alpha-globin gene deletional alpha-thals regardless of the break points. When three primer sets were used--two gene-specific sets for the alpha1- and alpha2-globin genes and one set for the beta-actin gene (serving as an internal control)--PCR products from genomic DNA were simultaneously amplified and analyzed after coamplification and gel electrophoresis. The number of alpha-globin genes present in the subjects was determined by the intensity of alpha1 and alpha2 bands normalized with that of beta-actin when using densitometry. Our results demonstrate that five common genotypes of deletional alpha-thal are differentiated by the ratios of alpha1/beta-actin and alpha2/beta-actin. We also examined the feasibility of coupling this allele-specific amplification to a color-complementary assay. This easy and reproducible PCR assay is suitable for identifying alpha-thal carriers in screenings of large populations and improving genetic counseling.

Actins↗
PubMed2001

Protection against tetanus by needle-free inoculation of adenovirus-vectored nasal and epicutaneous vaccines.

The effectiveness of vaccination programs would be enhanced greatly through the availability of vaccines that can be administered simply and, preferably, painlessly without the need for timed booster injections. Tetanus is a prime example of a disease that is readily preventable by vaccination but remains a major threat to public health due to the problems associated with administration of the present vaccine. Here we show that a protective immune response against live Clostridium tetani infection in mice can be elicited by an adenovirus vector encoding the tetanus toxin C fragment when administered as a nasal or epicutaneous vaccine. The results suggest that these vaccination modalities would be effective needle-free alternatives. This is the first demonstration that absorption of a small number of vectored vaccines into the skin following topical application of a patch can provide protection against live bacteria in a disease setting.

Adenoviridae↗
PubMed2001

Myosin light chain kinase- and PKC-dependent contraction of LES and esophageal smooth muscle.

In smooth muscle cells enzymatically isolated from circular muscle of the esophagus (ESO) and lower esophageal sphincter (LES), ACh-induced contraction and myosin light chain (MLC) phosphorylation were similar. Contraction and phosphorylation induced by purified MLC kinase (MLCK) were significantly greater in LES than ESO. ACh-induced contraction and MLC phosphorylation were inhibited by calmodulin and MLCK inhibitors in LES and by protein kinase C (PKC) inhibitors in ESO. Contraction of LES and ESO induced by the PKC agonist 1,2-dioctanoylglycerol (DG) was unaffected by MLCK inhibitors. Caldesmon and calponin concentration-dependently inhibited ACh-induced contraction of ESO and not LES. In ESO, caldesmon antagonist GS17C reversed caldesmon- but not calponin-induced ACh inhibition. GS17C caused contraction of permeabilized ESO but had much less effect on LES. GS17C-induced contraction was not affected by MLCK inhibitors, suggesting that MLCK may not regulate caldesmon-mediated contraction. DG-induced contraction of ESO and LES was inhibited by caldesmon and calponinin, suggesting that these proteins may regulate PKC-dependent contraction. We conclude that calmodulin and MLCK play a role in ACh-induced LES contraction, whereas the classical MLCK may not be the major kinase responsible for contraction and phosphorylation of MLC in ESO. ESO contraction is PKC dependent. Caldesmon and/or calponin may play a role in PKC-dependent contraction.

Acetylcholine↗
PubMed2000

The role of trans-acting factors and DNA-bending in the silencing of human beta-globin gene expression.

The molecular mechanisms which govern the develop-mental specificity of human beta-globin gene transcription have been studied in K562 cells, a human eyrthroleukemia line that expresses minimal beta-globin. Protein-binding analysis reveals that the 5' region contains three elements bound by trans-acting factors, beta-protein 1 (BP1) and beta-protein 2 (BP2). In vitro mutagenesis of each individual element in a beta-globin vector containing chloramphenicol acetyl-transferase (pCAT) followed by transient transfection into K562 cells increased levels of CAT activity 5. 5-fold higher than wild-type (wt) betaCAT, consistent with their silencing role. Mutagenesis of all three elements, however, resulted in activity significantly lower than wt betaCAT. BP1 and BP2 motifs have overlapping binding sites for high mobility group proteins (HMG1+2), DNA-bending factors, shown here to extrinsically bend the beta-globin promoter. Theoretically, mutations in all beta-protein binding sites could affect the binding of HMG1+2 sufficiently to impede DNA-protein and/or protein-protein interactions needed to facilitate constitutive gene expression. Placing two turns of DNA between BP1 and BP2 motifs also increased expression 3-fold, indicative of spatial constraints required for optimal silencing. However, insertion of the HMG1+2 DNA-bending motif (also equivalent to two turns) facilitates beta-silencing by re-establishment of BP1-BP2 proximity. Thus a combination of general DNA-bending and specific transcriptional factors appear to be involved in beta-globin silencing in the embryonic/fetal erythroid stage.

Adult↗
PubMed1999

DNA-based non-invasive vaccination onto the skin.

Non-invasive vaccination onto the skin (NIVS) could improve vaccination programs because the procedure requires no specially trained personnel and may eliminate many problems associated with needle injections. There is also evidence that the efficacy of a skin-targeted vaccine may be optimal when the antigen is expressed within the outer layer that is in constant contact with potential pathogens. We report here that non-invasive gene delivery by pipetting adenovirus- or liposome-complexed plasmid DNA onto the outer layer of skin could achieve localized transgene expression within a restricted subset of skin in mice and the elicitation of an immune response against the protein encoded by the DNA. These results provide a proof of principle that NIVS may appear as a novel method for the administration of DNA-based vaccines.

Adenoviridae↗
PubMed1999

Thrombopoietin has a differentiative effect on late-stage human erythropoiesis.

To further explore the mechanism of the effect of thrombopoietin (TPO) on erythropoiesis, we used a two-phase culture system to investigate the effect of TPO on late-stage human erythroid lineage differentiation. In serum-free suspension and semisolid cultures of human peripheral blood derived erythroid progenitors, TPO alone did not produce benzidine-positive cells. However, in serum-containing culture, TPO alone stimulated erythroid cell proliferation and differentiation, demonstrated by erythroid colony formation, production of benzidine-positive cells and haemoglobin (Hb) synthesis. Monoclonal anti-human erythropoietin antibody and anti-human erythropoietin receptor antibody completely abrogated the erythroid differentiative ability of TPO in the serum-containing systems. This implied that binding of EPO and EPO-R was essential for erythropoiesis and the resultant signal transduction may be augmented by the signals emanating from TPO-c-Mpl interaction. Experiment of withdrawal of TPO further demonstrated the involvement of TPO in late-stage erythropoiesis. RT-PCR results showed that there was EPO-R but not c-Mpl expression on developing erythroblasts induced by TPO in serum-containing system. Our results establish that TPO affects not only the proliferation of erythroid progenitors but also the differentiation of erythroid progenitors to mature erythroid cells.

Cell Differentiation↗
PubMed1998

Identification of upstream regulatory elements that repress expression of adult beta-like globin genes in a primitive erythroid environment.

Our investigations have focused on localizing cis-elements responsible for the down regulation of the adult beta-like globin genes (delta and beta) in immature, or primitive erythroid tissues. We studied their activity after transfection into K562 cells, an erythroleukemia cell line with an embryonic-fetal phenotype. Analyzed DNA sequences included delta and beta 5' flanking regions extending from approximately -500 to +50bp (promoter regions), truncated delta and beta 5' flanking regions extending from approximately -250 to +50 bp, and chimeric promoter constructions, which consisted of a distal delta or beta fragment fused to a proximal beta or delta sequence. In CAT reporter constructions no appreciable level of CAT activity was supported by the beta globin promoter, and only low level activity by the delta promoter. Truncation of the beta globin promoter led to a 2-3 fold increase in promoter activity. In contrast, deletion of the upstream portion of the delta promoter led to a 10 fold decrease in expression. Coupling of the upstream beta globin sequence from approximately -500 to -250 bp to the truncated delta promoter fragment led to complete extinction of transcription activity, consistent with a negative regulatory effect of the beta globin gene upstream element(s). Fusion of the upstream portion of the delta promoter to the truncated beta globin promoter yielded a modest increase in promoter strength relative to the truncated beta gene promoter, indicating the presence of a positive transcriptional element(s) in the upstream delta globin regulatory region. Site-directed mutagenesis of binding sites for the repressor proteins BP1 and BP2 in the upstream portion of the beta globin gene flanking region led to a 4-6 fold increase in promoter activity. DNase I footprinting of the upstream delta-globin region revealed protected sequences corresponding to consensus binding sites for GATA-1 and BP2. These results confirm that sequences in the upstream promoter region of the adult beta globin gene contribute to its factor-mediated suppression early in development and then may modulate its expression at a later stage.

Binding Sites↗
PubMed1998

Activation of the human delta-globin gene promoter in primary adult erythroid cells.

Restoration of the CCAAT box or insertion of an erythroid Krüppel-like factor (EKLF) binding site in the delta promoter activates its expression in several erythroid cell lines. We extended these studies using a novel primary human adult erythroid cell (hAEC) system to investigate these effects at the late erythroblast stage. Restoration of the CCAAT box at -70 bp, or insertion of an EKLF binding site at -85 bp or -95 bp in the promoter significantly increased delta globin gene expression in hAEC. Our results demonstrate that the altered CCAAT box (CCAAC) and the lack of an EKLF binding site in delta-globin contribute to its low level of expression in the hAEC model as well.

Adult↗
PubMed1997

Overexpression of adenovirus-encoded transgenes from the cytomegalovirus immediate early promoter in irradiated tumor cells.

Efficient expression of therapeutic genes in irradiated tumor cells would facilitate the conversion of a malignant tumor nodule into a cancer vaccine in situ. We reported previously that transgene expression from an adenoviral vector could be markedly enhanced by treating transduced tumor cells with butyrate. In this study, we demonstrated that a similar butyrate effect could be achieved in irradiated tumor cells. In addition, irradiating cells at doses of 2-40 Gy prior to transduction could also amplify recombinant adenoviral transgene products in a cell-type-specific manner. This suggests that adenovirus-mediated gene therapy, radiation therapy, and butyrate-mediated cancer therapy may potentially be formulated into one synergistic protocol for cancer treatment.

Adenoviridae↗
PubMed1997

Restoration of the CCAAT box or insertion of the CACCC motif activates [corrected] delta-globin gene expression.

Hemoglobin A2 (HbA2), which contains delta-globin as its non-alpha-globin, represents a minor fraction of the Hb found in normal adults. It has been shown recently that HbA2 is as potent as HbF in inhibiting intracellular deoxy-HbS polymerization, and its expression is therefore relevant to sickle cell disease treatment strategies. To elucidate the mechanisms responsible for the low-level expression of the delta-globin gene in adult erythroid cells, we first compared promoter sequences and found that the delta-globin gene differs from the beta-globin gene in the absence of an erythroid Krüppel-like factor (EKLF) binding site, the alteration of the CCAAT box to CCAAC, and the presence of a GATA-1 binding site. Second, serial deletions of the human delta-globin promoter sequence fused to a luciferase (LUC) reporter gene were transfected into K562 cells. We identified both positive and negative regulatory regions in the 5' flanking sequence. Furthermore, a plasmid containing a single base pair (bp) mutation in the CCAAC box of the delta promoter, restoring the CCAAT box, caused a 5.6-fold and 2.4-fold (P < .05) increase of LUC activity in transfected K562 cells and MEL cells, respectively, in comparison to the wild-type delta promoter. A set of substitutions that create an EKLF binding site centered at -85 bp increased the expression by 26.8-fold and 6.5-fold (P < .05) in K562 and MEL cells, respectively. These results clearly demonstrate that the restoration of either an EKLF binding site or the CCAAT box can increase delta-globin gene expression, with potential future clinical benefit.

Adult↗
PubMed1997

Amplification of recombinant adenoviral transgene products occurs by inhibition of histone deacetylase.

n-Butyrate (butyrate) has been shown to amplify transgene expression in cells infected with E1-defective adenoviruses. The present studies were undertaken in order to better define the actions of butyrate in the context of adenovirus gene expression, and to attempt to elucidate the mechanism by which butyrate mediates the transgene amplification. It was found that butyrate amplified viral transgene expression over a concentration range of 0.5-5 mM, and that the amplification required an exposure of 12-24 hr for maximal effect. Western blot analysis of representative viral proteins showed that butyrate treatment amplified DNA-binding protein, but not fiber protein. A transient adenoviral replication system suggested that butyrate had a modest inhibitory effect on replication of the E1-defective adenovirus. Use of a specific inhibitor of histone deacetylase, trichostatin A (TSA), reproduced the amplification of the viral transgene product achieved with the butyrate. In contrast, adenoviral transgene expression could not be amplified by TSA treatment in a cell line known to have a TSA-resistant histone deacetylase. Butyrate amplified steady-state gene expression of the viral transgene, but had no detectable effects on either DNA-binding protein or fiber steady-state gene expression. Nuclear run-off experiments showed that both butyrate and TSA caused an increase in the viral transgene transcription. It was concluded that inhibitors of histone deacetylase amplify adenoviral transgene expression at the transcriptional level.

Adenoviruses, Human↗
PubMed1996

Structure-function relations of smooth muscle calponin. The critical role of serine 175.

Calponin has been implicated in the regulation of smooth muscle contraction through its interaction with F-actin and inhibition of the actin-activated MgATPase activity of phosphorylated myosin. Both properties are lost following phosphorylation (primarily at serine 175) by protein kinase C or calmodulin-dependent protein kinase II. To evaluate further the functional importance of serine 175, wild-type calponin and three site-specific mutants (S175A, S175D, and S175T) were expressed in Escherichia coli and compared with calponin purified from chicken gizzard smooth muscle in terms of actin binding, actomyosin MgATPase inhibition, and phosphorylation by protein kinase C and calmodulin-dependent protein kinase II. The affinities of skeletal muscle F-actin for wild-type and S175T calponins were similar to that for the tissue-purified protein (Kd = 0.8, 1.3, and 1.0 microM, respectively), whereas the affinities for S175A and S175D calponins were much lower (Kd = 26.8 and 44.2 microM, respectively). Tissue-purified, wild-type, and S175T calponins displayed comparable inhibition of the smooth muscle actin-activated myosin MgATPase, whereas S175A and S175D calponins were much less effective. Phosphorylation confirmed serine 175 as the principal site of phosphorylation by both kinases. These results indicate that the hydroxyl side chain at position 175 of calponin plays a critical role in the binding of calponin to actin and inhibition of the cross-bridge cycling rate.

Actins↗
PubMed1996

In vivo cytotoxicity assay for assessing immunity.

A rapid, quantitative, in vivo assay of cytotoxic responses would facilitate experimental evaluation of the potency of novel vaccine strategies. We have developed an in vivo cytotoxicity assay in which target cells expressing a luciferase reporter gene are implanted as monolayers on polystyrene disks onto the muscle tissue of mice. The luciferase activity retrievable from the adjacent tissue is used as an index of cytotoxicity. Implantation of B16 or NIH/3T3 cells expression the beta-galactosidase gene indicated that the target cells migrated to the muscle tissue from plastic within 4 h and then remained localized in the area of the disk. The amounts of luciferase retrievable from the adjacent tissue a few days post implantation readily detected the immune response induced by allo-immunization of fibroblasts or by production of interleukin-4 by tumor cells co-mixed with implanted reporter cells. Histologic analysis showed a correlation between the amount of luciferase retrieved and the number of viable target cells at the implantation site. Recruitment of immune effector cells which may be responsible for target cell death and luciferase elimination could be readily visualized. This simple cytotoxicity assay can be used as an in vivo assay of the net effect of cytotoxic immune responses.

3T3 Cells↗
PubMed1995

Adenovirus-based p53 gene therapy in ovarian cancer.

Mutations of the p53 tumor suppressor gene are the most common molecular genetic abnormality to be described in ovarian cancer. To determine the feasibility of mutant p53 as a molecular target for gene therapy in ovarian cancer, we constructed an adenovirus vector containing the wild-type p53 gene. The ability of this adenovirus construct (Ad-CMV-p53) to express p53 protein was examined by Western blot analysis in the H358 lung cancer cell line, which has a homozygous deletion of the p53 gene. The ability of the adenovirus vector system to infect ovarian cancer cells was tested using an adenovirus containing the beta-galactosidase reporter gene under the control of the CMV promoter (Ad-CMV-beta gal). The ovarian cancer cell line 2774, which contains an Arg273His p53 mutation, was infected with Ad-CMV-beta gal, and the infected cells were assayed for beta-galactosidase activity after 24 hr. To test the ability of wild-type p53 to inhibit cell growth, the 2774 cell line was infected with Ad-CMV-p53 or Ad-CMV-beta gal, and the effect of these agents on the growth of 2774 cells was determined using an in vitro growth inhibition assay. Western blot analysis of lysates from H358 cells infected with Ad-CMV-p53 showed expression of wild-type p53 protein. When 2774 cells were infected with Ad-CMV-beta gal at a multiplicity of infection (m.o.i.) of 10 PFU/cell, > 90% of cells showed beta-galactosidase activity, demonstrating that these cells are capable of efficient infection by the adenovirus vector. Growth of 2774 cells infected with Ad-CMV-p53 was inhibited by > 90% compared to noninfected cells. The ability of the adenovirus vector to mediate high-level expression of infected genes and the inhibitory effect of Ad-CMV-p53 on the 2774 cell line suggests that the Ad-CMV-p53 could be further developed into a therapeutic agent for ovarian cancer.

Adenoviridae↗