Biomedical subjects

T H Chang

Publications and source records attributed to T H Chang.

At least 37 records · Page 2Linked to original sources

PubMed1998

A novel GFPneo vector designed for the isolation and analysis of enhancer elements in transfected mammalian cells.

We have designed a new approach to the direct cloning and rapid analysis of mammalian enhancer elements by fusing green fluorescent protein and neomycinphosphotransferase under the control of a thymidine kinase minimal promoter. DNA fragments containing known or potential enhancer elements can be inserted into a polylinker upstream of GFPneo and re-isolated from stably transfected cell lines by a direct transgene-specific polymerase chain reaction (PCR), for further analysis. C2C12 muscle cells were transfected with four vectors containing the GFPneo fusion gene regulated by the cytomegalovirus promoter, the myoD distal core enhancer and myoblast- and myotube-specific enhancers from the desmin gene. GFPneo shows robust epifluorescence by microscopy and flow cytometry and retains sufficient neo activity to permit selection of G418-resistant clones. The fluorescence signal pattern of GFPneo expressed under the control of the desmin enhancers mirrors their transcriptional profile during myogenic differentiation. This finding demonstrates the value of GFPneo as a tool to analyse differentiation stage-specific regulatory DNA elements in stably transfected mammalian cell lines. We were able to re-isolate the myoD enhancer mediating GFPneo expression from a stably transfected C2C12 clone by a transgene-specific PCR reaction, demonstrating the feasibility of using this new vector system for the isolation of regulatory sequences.

Animals↗

PubMed1998

Dbp5p, a cytosolic RNA helicase, is required for poly(A)+ RNA export.

The DBP5 gene encodes a putative RNA helicase of unknown function in the yeast Saccharomyces cerevisiae. It is shown here that Dbp5p is an ATP-dependent RNA helicase required for polyadenylated [poly(A)+] RNA export. Surprisingly, Dbp5p is present predominantly, if not exclusively, in the cytoplasm, and is highly enriched around the nuclear envelope. This observation raises the possibility that Dbp5p may play a role in unloading or remodeling messenger RNA particles (mRNPs) upon arrival in the cytoplasm and in coupling mRNP export and translation. The functions of Dbp5p are likely to be conserved, since its potential homologues can be found in a variety of eukaryotic cells.

Amino Acid Sequence↗

PubMed1998

Stimulation of endothelial cell production of vasoconstrictive substances by hypertensive sera.

The endothelial cell regulates vessel tone by elaborating a number of vasoactive substances such as thromboxane and endothelin, both of which are highly vaosconstrictive, and prostacyclin and nitric oxide, both of which are vasodilatory. The current study examines the postulate that one of the mechanisms responsible for the increased vessel tone found in hypertension is the presence of substances in the sera of patients with this disorder that stimulates selectively the endothelial cell production of thromboxane and endothelin. Sera from ten patients with mild hypertension and from 11 age-matched controls were incubated with human umbilical arterial endothelial cells and the concentrations of endothelin, thromboxane, prostacyclin, and nitric oxide produced by the cells was measured in the supernatant. The results of the assays showed that the amounts of thromboxane and endothelin produced by endothelial cells in response to stimulation by hypertensive sera were significantly higher than the amounts produced in response to control sera; in comparison, the amounts of prostacyclin and nitric oxide produced by the cells in response to either hypertensive sera or control sera were not significantly different. The findings suggest that a mechanism that may be responsible for the increased vascular tone found in hypertension is the presence of substances in hypertensive sera that stimulate endothelial cells selectively to produce increased amounts of the vasoconstrictive hormones, endothelin and thromboxane.

6-Ketoprostaglandin F1 alpha↗

PubMed1998

Effects of N-methyl berbamine on delayed outward potassium current in isolated rat hepatocytes.

AIM: To study the effects of N-methyl berbamine (NMB) on the delayed outward potassium currents (Ik) in isolated rat hepatocytes. METHODS: With patch-clamp techniques and whole-cell recording method, holding potential -50 mV, command potential +30 to +140 mV, duration 900 ms. RESULTS: NMB reduced Ik in a concentration-dependent manner. When the concentrations of NMB were 20, 50, 400 nmol.L-1 and 50 mumol.L-1, the amplitude values of Ik were decreased to 3.6 +/- 0.4 (P > 0.05), 2.1 +/- 1.6 (P > 0.05), 3.7 +/- 1.6 (P < 0.05), 2.3 +/- 1.3 nA (P < 0.01) from 4.4 +/- 1.0 (n = 4), 2.5 +/- 1.8 (n = 4), 5.8 +/- 2.1 (n = 5), 4.6 +/- 1.3 (n = 6) nA of control, respectively. The inhibitory rates were 10%, 15%, 37%, and 51%, respectively. CONCLUSION: NMB was a K+ channel inhibitor.

Alkaloids↗

PubMed1997

The human papillomavirus-16 E6 oncoprotein decreases the vigilance of mitotic checkpoints.

The E6 and E7 proteins of the high risk human papillomaviruses (HPVs) are consistently expressed in HPV-positive cervical carcinomas. We investigated the ability of HPV-16 E6 and E7 to disrupt mitotic checkpoints in normal diploid human cells. Acute expression of HPV-16 E6, but not HPV-16 E7, decreased the fidelity of multiple checkpoints controlling entry into and exit from mitosis. After irradiation, nearly 50% of cells containing HPV-16 E6 readily entered mitosis as opposed to less than 10% of control cells. Consistent with this, asynchronous populations of cells expressing HPV-16 E6 had increased cdc2-associated histone H1 kinase activity relative to control populations. In addition, HPV-16 E6 increased sensitivity to chemically-induced S-phase premature mitosis and decreased mitotic spindle assembly checkpoint function relative to control populations. HPV-16 E6 mutants with a reduced ability to target p53 for degradation were unable to abrogate mitotic checkpoints, suggesting a possible mechanism by which HPV-16 E6 disrupts mitotic checkpoints. Expression of a mutant p53 gene yielded an intermediate phenotype relative to HPV-16 E6, generating moderate increases in sensitivity to chemically-induced S-phase PCC and mitotic spindle disruption and a heightened propensity to enter mitosis after irradiation.

CDC2 Protein Kinase↗

PubMed1997

Genetic interactions of conserved regions in the DEAD-box protein Prp28p.

The yeast PRP28 g ene has been implicated in nuclear precursor messenger RNA (pre-mRNA) splicing, a two-step reaction involved in a multitude of RNA structural alterations. Prp28p, the gene product of PRP28 , is a member of the evolutionarily conserved DEAD-box proteins (DBPs). Members of DBPs are involved in a variety of RNA-related biochemical processes, presumably by their putative RNA helicase activities. Prp28p has been speculated to play a role in melting the duplex between U4 and U6 small nuclear RNAs (snRNAs), leading to the formation of an active spliceosome. To study the function of Prp28p and its interactions with other components of the splicing machinery, we have isolated and characterized a large number of prp28 conditional mutants. Strikingly, many of these prp28 mutations are localized in the highly conserved motifs found in all the DBPs. Intragenic reversion analysis suggests that regions of motifs II, III and V, as well as of motifs I and IV, in Prp28p are likely to be in close proximity to each other. Our results thus provide the first hint of the local structural arrangement for Prp28p, and perhaps for other DBPs as well.

DEAD-box RNA Helicases↗

PubMed1997

Disregulation of mitotic checkpoints and regulatory proteins following acute expression of SV40 large T antigen in diploid human cells.

SV40 large T antigen (T) inactivates the tumor suppressor proteins p53 and pRb, and can induce cells to enter DNA replication at inappropriate times. We show here that T also compromises three cell cycle checkpoints that regulate the entry into and exit from mitosis. Human diploid fibroblasts infected with a retrovirus expressing T displayed an attenuated radiation-induced mitotic delay, were more susceptible to chemical-induced uncoupling of mitosis from the completion of DNA replication, and were more likely to exit mitosis and rereplicate their DNA when mitotic spindle assembly was inhibited. Consistent with altered mitotic checkpoint control, cells expressing T displayed elevated protein levels and/or associated activities of the mitotic regulatory proteins cyclin A, cyclin B, Cdc25C and p34(cdc2). These changes in mitotic control were evident within 5-10 population doublings after retroviral infection, indicating a direct effect of T expression. Cells acutely infected with the T-expressing retrovirus suffered numerical and structural chromosome aberrations, including increases in aneuploidy, dicentric chromosomes, chromatid exchanges and chromosome breaks and gaps. These findings indicate that T rapidly disrupts mitotic checkpoints that help maintain genomic stability, and suggest mechanisms by which T induces chromosome aberrations and promotes the immortalization and neoplastic transformation of human cells.

Antigens, Viral, Tumor↗

PubMed1997

A mitosis-specific phosphorylation of the gap junction protein connexin43 in human vascular cells: biochemical characterization and localization.

Western blotting studies revealed that connexin43 (Cx43), one of the major gap junction proteins in human vascular endothelial cells, is posttranslationally modified during mitosis. This mitosis-specific modification results in a Cx43 species that migrates as a single protein band and was designated Cx43(m). Cx43(m) was shown to be the result of additional Ser/Thr phosphorylation as indicated by: (a) the increased gel mobility induced by both alkaline phosphatase and the Ser/ Thr-specific protein phosphatase-2A (PP2A) and (b) the removal of virtually all (32)P(i) from Cx43(m) by PP2A. Immunofluorescent confocal microscopy of mitotic cells revealed that Cx43 is intracellularly located, while in nonmitotic cells Cx43 is located at regions of cell-cell contact. Dye coupling studies revealed that mitotic endothelial cells were uncoupled from each other and from nonmitotic cells. After cytokinesis, sister cells resumed cell coupling independent of de novo protein synthesis. The mitosis-specific phosphorylation of Cx43 correlates with the transient loss of gap junction intercellular communication and redistribution of Cx43, suggesting that a protein kinase that regulates gap junctions is active in M-phase.

Animals↗

PubMed1997

Requirement of the DEAD-Box protein ded1p for messenger RNA translation.

The DED1 gene, which encodes a putative RNA helicase, has been implicated in nuclear pre-messenger RNA splicing in the yeast Saccharomyces cerevisiae. It is shown here by genetic and biochemical analysis that translation, rather than splicing, is severely impaired in two newly isolated ded1 conditional mutants. Preliminary evidence suggests that the protein Ded1p may be required for the initiation step of translation, as is the distinct DEAD-box protein, eukaryotic initiation factor 4A (eIF4A). The DED1 gene could be functionally replaced by a mouse homolog, PL10, which suggests that the function of Ded1p in translation is evolutionarily conserved.

Animals↗

PubMed1997

Dbp3p, a putative RNA helicase in Saccharomyces cerevisiae, is required for efficient pre-rRNA processing predominantly at site A3.

In Saccharomyces cerevisiae, ribosomal biogenesis takes place primarily in the nucleolus, in which a single 35S precursor rRNA (pre-rRNA) is first transcribed and sequentially processed into 25S, 5.8S, and 18S mature rRNAs, leading to the formation of the 40S and 60S ribosomal subunits. Although many components involved in this process have been identified, our understanding of this important cellular process remains limited. Here we report that one of the evolutionarily conserved DEAD-box protein genes in yeast, DBP3, is required for optimal ribosomal biogenesis. DBP3 encodes a putative RNA helicase, Dbp3p, of 523 amino acids in length, which bears a highly charged amino terminus consisting of 10 tandem lysine-lysine-X repeats ([KKX] repeats). Disruption of DBP3 is not lethal but yields a slow-growth phenotype. This genetic depletion of Dbp3p results in a deficiency of 60S ribosomal subunits and a delayed synthesis of the mature 25S rRNA, which is caused by a prominent kinetic delay in pre-rRNA processing at site A3 and to a lesser extent at sites A2 and A0. These data suggest that Dbp3p may directly or indirectly facilitate RNase MRP cleavage at site A3. The direct involvement of Dbp3p in ribosomal biogenesis is supported by the finding that Dbp3p is localized predominantly in the nucleolus. In addition, we show that the [KKX] repeats are dispensable for Dbp3p's function in ribosomal biogenesis but are required for its proper localization. The [KKX] repeats thus represent a novel signaling motif for nuclear localization and/or retention.

Amino Acid Sequence↗

PubMed1997

Purification and characterization of a 94 KD high molecular weight allergen from house dust mite, Dermatophagoides pteronyssinus.

House dust mite allergens from Dermatophagoides pteronyssinus is an important cause of severe allergic asthma and rhinitis in many countries. Although several low to medium molecular weight allergens had been well characterized, limited studies on the high molecular weight IgE-binding components were reported. In this study, a 94 kD high molecular weight allergen from crude mite body extract of D. pteronyssinus was purified and characterized. Monoclonal antibody (mAb) affinity chromatography and high performance liquid chromatography were used to purify 94 kD allergen. Its antigenicity and allergenicity were confirmed by in vitro and in vivo studies. Two mAbs 2205-3.45 and 2220-7.25 specific to 94 kD high molecular weight component of D. pteronyssinus were generated. The epitopes recognized by these mAbs were species-specific. Enzyme-linked immunosorbent assay (ELISA) of IgE reactivity in the sera from 40 asthmatic children allergic to D. pteronyssinus showed that 37.5% of them had significantly higher optical density values (range 0.011 to 0.452) than normal (range 0.013 to 0.035). In in vivo skin test showed that 9 out of 20 (45%) asthmatic children were positive to 94 kD allergen. The results demonstrate that 94 kD high molecular weight component is an important allergen existing in house dust mite in Taiwan.

Allergens↗

PubMed1997

Serum responses to the combination of Epstein-Barr virus antigens from both latent and acute phases in nasopharyngeal carcinoma: complementary test of EBNA-1 with EA-D.

Elevated serum IgA to antigens of EBV is associated with nasopharyngeal carcinoma (NPC). We have tested 620 NPC sera by ELISA for the presence of antibodies to EBV-encoded DNA binding protein, EBV-specific DNA polymerase, early antigen-diffused (EA-D), EBV nuclear antigen 1 (EBNA-1), EBV-specific thymidine kinase, and BamHI Z fragment EBV replication antigen. Sensitivity of these proteins was in the range of 51.5-79.5% for IgA and 69.4-82.8% for IgG. The complementary use of EBNA-1 with EA-D, however, could increase the sensitivity significantly to 98.1%. Western blot analysis further showed that the combination of EBNA-1 and EA-D is most useful for the detection of NPC. This is the first report of using double biomarkers including EBV gene products from both latent and active infections. The results of this study suggest that EBV in NPC may not be latent alone and that the method may be valuable for the early detection, early treatment, and better survival rate of patients with NPC. Because the application of recombinant EBV protein in ELISA is cost-effective and feasible for mass screening, the method may be of worth for further clinical investigation.

Adult↗

PubMed1996

Purification and characterization of a naturally processed hepatitis B virus peptide recognized by CD8+ cytotoxic T lymphocytes.

In vitro studies in patients with hepatitis B virus (HBV) infection have suggested that hepatocytolysis induced by CD8+ cytotoxic T lymphocytes (CTLs) is the most important effector pathway in eliminating infected cells. The recognition is implicated in the endogenously processed HBV antigens in the context of HLA class I molecules presented on the liver cell membrane. However, the naturally occurring HBV peptide antigens have not yet been demonstrated. We report here that a naturally processed peptide antigen P2 was isolated from HLA class I molecules of HBV-infected liver cell membrane. The P2 peptide exhibited the activity of sensitizing target cells for lysis by CD8+ CTLs. The P2 sequence (YVNVNMGLK) purified from liver tissue was in concordance with that encoded by the viral genome for the HBV nucleocapsid antigen or HBcAg 88-96. P2 peptide could also be isolated from the EBV-transformed B cells that were transfected by HBcAg-expressing vector. The P2 epitope, sharing the HLA-A11 binding motifs, was recognized by HLA-A11-restricted CD8+ CTLs. The data provided direct evidence that, in hepatitis B patients, antigenic peptides of HBV were processed by hepatocytes, presented with the class I MHC molecules, and recognized by CD8+ CTLs.

Adult↗

PubMed1996

Induction of cyclin A gene expression by homocysteine in vascular smooth muscle cells.

Homocysteine is an important and independent risk factor for arteriosclerosis. We showed previously that homocysteine stimulates vascular smooth muscle cell proliferation, a hallmark of arteriosclerosis. We show here that homocysteine and serum increased DNA synthesis synergistically in both human and rat aortic smooth muscle cells (RASMCs). Treatment of quiescent RASMCs with 1 mM homocysteine or 2% calf serum for 36 h increased cyclin A mRNA levels by 8- and 14-fold, respectively, whereas homocysteine plus serum increased cyclin A mRNA levels by 40-fold, indicating a synergistic induction of cyclin A mRNA. Homocysteine did not increase the half-life of cyclin A mRNA (2.9 h), but it did increase the transcriptional rate of the cyclin A gene in nuclear run-on experiments. The positive effect of homocysteine on cyclin A gene transcription was confirmed by our finding that homocysteine increased cyclin A promoter activity and ATF-binding protein levels in RASMCs. Finally, 1 mM homocysteine increased cyclin A protein levels and cyclin A-associated kinase activity by threefold. This homocysteine-induced expression lesions by promoting proliferation of vascular smooth muscle cells.

Activating Transcription Factors↗

PubMed1996

SV40 T antigen increases the expression and activities of p34cdc2, cyclin A, and cyclin B prior to immortalization of human diploid fibroblasts.

SV40 T antigen induces karyotype instability soon after it is expressed in human diploid fibroblasts and ultimately promotes cell immortalization and tumorigenesis. Protein levels and activities of mitotic cell cycle proteins have been shown to be elevated in several immortal cell lines relative to their normal parental cells, suggesting a possible role for the aberrant regulation of mitosis in karyotype instability. We show here that IMR-90 human diploid lung fibroblasts expressing the SV40 tumor antigens display increased protein levels and associated enzymatic activities of cyclin A, cyclin B, and p34cdc2 long before crisis and immortalization. These elevations cannot be explained by faster cell growth or altered cell cycle distributions. Increased protein levels were not totally accounted for by elevated levels of the corresponding mRNA, indicating that T antigen modulates expression at least partially by posttranscriptional mechanisms. These results indicate that perturbation of mitotic regulatory proteins precedes crisis, and imply that altered mitotic control is a direct consequence of T antigen expression rather than an outcome of secondary events associated with immortalization.

Amino Acid Sequence↗

PubMed1996

Mobile cecum syndrome: a report of two cases.

Mobile cecum syndrome is characterized by chronic right lower quadrant pain with the evidence of neither appendicitis nor other pathological findings at operation. Two cases of mobile cecum syndrome are reported; both had intermittent right lower quadrant cramping pain for months. One had received appendectomy because of chronic right lower quadrant pain at another hospital about nine months before admission here. The symptoms did not improve postoperatively, and the patient underwent laparotomy under impression of partial intestinal obstruction. The other patient underwent laparotomy under impression of chronic appendicitis. At surgery, both were found to have cecum and ascending colon were not attached to the posterior parietal wall, and cecopexy was performed. They have now been symptom free for one year. Cecopexy appears therefore to be an effective method of treatment of mobile cecum syndrome.

Abdominal Pain↗

PubMed1995

Expression of the Epstein-Barr virus DNA polymerase in Escherichia coli for use as antigen for the diagnosis of nasopharyngeal carcinoma.

Epstein-Barr virus (EBV) encoded DNA polymerase (POL) was cloned and over-expressed in Escherichia coli. Western blot analysis confirmed the presence of antibody to this POL protein in sera from nasopharyngeal carcinoma (NPC) patients. By Western blot analysis, moderate to high concentration of IgG POL-specific antibodies were present in 43 of 48 NPC sera and only 4 of 48 healthy, seropositive controls. The POL-specific IgG antibodies appear as early as stage I of NPC, suggesting that the recombinant POL protein can be a useful diagnostic marker for early diagnosis of the disease. It was also found that human sera containing high titer of cytomegalovirus (CMV) antibodies or herpes simplex virus type 1 (HSV-1) antibodies did not cross-react with the recombinant EBV POL, despite the homology shared by DNA polymerase proteins of these viruses.

Amino Acid Sequence↗

PubMed1995

A method of stimulating an immune response with a hapten immobilized on a nitrocellulose membrane.

Hapten refers to a chemical compound of small molar mass (typically less than 1000 daltons) that can bind with an antibody, but cannot initiate an immune response by itself unless it is conjugated to a protein carrier of larger molar mass. A novel method to prepare a hapten to generate anti-hapten immunity without covalent conjugation to a carrier was developed. Coating both water-soluble and -insoluble haptens onto a nitrocellulose membrane effectively presented haptens to the system and caused the generation of specific anti-hapten B lymphocytes and antibodies by immunization both in vitro and in vivo. This method has a potential to substitute for conventional hapten carrier conjugation to generate anti-hapten immunity.

Adsorption↗