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D C Tang

Publications and source records attributed to D C Tang.

35 records · Page 2Linked to original sources
PubMed1994

Activation properties of myosin light chain kinase during contraction/relaxation cycles of tonic and phasic smooth muscles.

In intact smooth muscle, myosin light chain kinase (MLCK) is phosphorylated at its regulatory site by Ca2+/calmodulin-dependent protein kinase II resulting in an increase in the concentration of Ca2+/calmodulin required for half-maximal activation of the enzyme (KCaM). We investigated the physiological significance of MLCK phosphorylation during cycles of contraction and relaxation in tonic (tracheal) and phasic (uterine) smooth muscles. MLCK phosphorylation and dephosphorylation occurred at rates sufficient to modulate the Ca2+ sensitivity of light chain phosphorylation. In contractions of both smooth muscles (though using different sources of activating Ca2+), increases in [Ca2+]i preceded light chain phosphorylation; but, the rate of increase in light chain phosphorylation was significantly greater than the rate of increase in [Ca2+]i. The onset of MLCK phosphorylation with the resultant increase in KCaM coincided with the diminished rate of light chain phosphorylation. During spontaneous contractions of uterine smooth muscle, the Ca2+ transient was characterized by an initial rapid increase, a sustained plateau, and rapid decline. During the sustained phase of the Ca2+ transient, MLCK phosphorylation increased and coincided with dephosphorylation of light chain and relaxation. These results indicate that MLCK is sensitive to small increases in intracellular Ca2+ during the initiation of contraction and that the enzyme subsequently becomes desensitized to Ca2+/calmodulin, thereby limiting the extent of light chain phosphorylation.

Animals↗
PubMed1994

Gene gun transfection of animal cells and genetic immunization.

Gene gun technology at this point has the most utility in animal protein expression as a back-up technology. In other words, when other conventional systems fail, it will generally work. Most notable is its usefulness for hard-to-transfect cells or in some particular in situ applications. Improvements in the gun itself and in the microprojectiles present the potential for this technology to expand in utility. The one area in which it now appears to be the method of choice is genetic immunization.

Animals↗
PubMed1994

Butyrate-inducible and tumor-restricted gene expression by adenovirus vectors.

Efficient introduction of biologically active genes into tumor cells in vivo would facilitate cancer gene therapy. In this study, recombinant adenoviruses mediated high-level expression of reporter genes in both human and murine tumor cell lines and freshly resected human solid tumors in vitro. When adenovirus vectors were inoculated intratumorally in a living animal, reporter activity was detected only in the tumor nodule and not in other sites, in contrast to intracardiac injections in which expression was found in several organs. Adenovirus-mediated gene expression was further enhanced by butyrate treatment.

Adenoviruses, Human↗
PubMed1993

GTP gamma S-induced phosphorylation of myosin light chain kinase in smooth muscle.

Phosphorylation of myosin light chain kinase by a Ca(2+)-dependent protein kinase increases the concentration of Ca2+/calmodulin required for half-maximal activation. The Ca2+ concentrations required for myosin light chain kinase phosphorylation in permeable smooth muscle are similar to those required for myosin light chain phosphorylation. Both GTP gamma S and carbachol increase the Ca2+ sensitivity of myosin light chain kinase phosphorylation as well as light chain phosphorylation. It is proposed that a similar G-protein mediated mechanism regulates the Ca(2+)-dependent phosphorylation of these two contractile proteins in smooth muscle.

Animals↗
PubMed1993

Cloning and expression of mutant glucocorticoid receptors from glucocorticoid-sensitive and -resistant human leukemic cells.

The molecular basis for the receptorless (r-) and activation-labile (act1) phenotypes of glucocorticoid-resistant mutants isolated from glucocorticoid-sensitive human leukemic CEM-C7 cells was determined. Clones isolated from a complementary DNA library prepared from r- ICR27TK.3 cells, in which one glucocorticoid receptor (GR) gene has been deleted, contained a single adenosine to thymidine transversion in the third position of codon 753, resulting in the substitution of phenylalanine for leucine. This mutant gene (GR753F) had only 13% of the trans-activating activity of the normal gene and produced a M(r) 92,000 receptor protein with the same r- phenotype seen in ICR27TK.3 cells. Analysis of complementary DNA clones isolated from a library prepared from parental glucocorticoid-sensitive 6TG1.1 cells showed that these cells express both a normal GR gene (GR+) and the GR753F gene. Thus, their genotype is GR+/GR753F. Analysis of clones isolated from a complementary DNA library prepared from glucocorticoid-resistant activation-labile 3R7. 6TG.4 cells revealed the presence of the GR753F gene and a second mutant gene (GR421Y) containing a guanosine to adenosine transition in the second position of codon 421, resulting in the replacement of the first cysteine of the proximal zinc finger of the DNA-binding domain by tyrosine. This mutant had no trans-activating activity but normal ligand-binding characteristics. Thus, the genotype of act1 3R7.6TG.4 cells is GR421Y/GR753F. Consequently, the sequence-specific DNA-binding activity of receptors in act1 cells is attributable to the GR753F gene, while the ligand-binding activity seen in intact cells is attributable to the GR421Y gene. These results provide a direct explanation for the r- and act1 phenotypes of glucocorticoid-resistant cells and demonstrate that glucocorticoid-sensitive cells derived from CEM-C7 cells contain a heterogeneous population of normal and mutant receptors.

Amino Acid Sequence↗
PubMed1993

The use of microparticle injection to introduce genes into animal cells in vitro and in vivo.

The biolistic technology has had a tremendous impact on plant and microbial research and development. The experience to date indicates that it should also have important uses with animal applications. The in-chamber system should prove increasingly valuable for transfecting non-dividing cells, primary cells, and other difficult-to-transfect targets. The hand-held version is currently useful for in situ transfection of cells in skin, but has limited effectiveness for internal organ transfection. This limitation may be lessened with further improvements in gun design and microprojectile coating. For gene therapy the most promising applications of this technology appear to be for genetic immunization and protocols which elicit an anti-tumor response.

Animals↗
PubMed1993

Phosphorylation of myosin light chain kinase: a cellular mechanism for Ca2+ desensitization.

Phosphorylation of the regulatory light chain of myosin by the Ca2+/calmodulin-dependent myosin light chain kinase plays an important role in smooth muscle contraction, nonmuscle cell shape changes, platelet contraction, secretion, and other cellular processes. Smooth muscle myosin light chain kinase is also phosphorylated, and recent results from experiments designed to satisfy the criteria of Krebs and Beavo for establishing the physiological significance of enzyme phosphorylation have provided insights into the cellular regulation and function of this phosphorylation in smooth muscle. The multifunctional Ca2+/calmodulin-dependent protein kinase II phosphorylates myosin light chain kinase at a regulatory site near the calmodulin-binding domain. This phosphorylation increases the concentration of Ca2+/calmodulin required for activation and hence increases the Ca2+ concentrations required for myosin light chain kinase activity in cells. However, the concentration of cytosolic Ca2+ required to effect myosin light chain kinase phosphorylation is greater than that required for myosin light chain phosphorylation. Phosphorylation of myosin light chain kinase is only one of a number of mechanisms used by the cell to down regulate the Ca2+ signal in smooth muscle. Since both smooth and nonmuscle cells express the same form of myosin light chain kinase, this phosphorylation may play a regulatory role in cellular processes that are dependent on myosin light chain phosphorylation.

Amino Acid Sequence↗
PubMed1992

Memory of butyrate induction by the Moloney murine sarcoma virus enhancer-promoter element.

We reported previously that a housekeeping gene could be converted to a butyrate-inducible gene by replacing its cognate promoter with the Moloney murine sarcoma virus enhancer-promoter element. In this study we report that the activated transcriptional state could be propagated from mother to daughter cells after the withdrawal of the inducer.

Adenine Phosphoribosyltransferase↗
PubMed1992

Sodium butyrate selectively induces transcription of promoters adjacent to the MoMSV viral enhancer.

The long terminal repeat region of the Moloney murine sarcoma virus (MoMSV) was cloned upstream from the Chinese hamster ovary adenine phosphoribosyltransferase (APRT)-encoding gene (APRT) in order to enhance synthesis of the APRT protein. The replacement of the native promoter with the viral enhancer-promoter increased the enzymatic activity of APRT two- to threefold. Addition of sodium butyrate (NaBu) to the cell growth medium induced APRT activity ten- to 20-fold above wild-type levels in both transient and stable transfectants. The introduction of the APRT native promoter between the MoMSV enhancer-promoter and structural gene reduced the magnitude of the NaBu response. The bacterial cat gene was also stimulated by NaBu when linked to the viral enhancer-promoter. No NaBu response was found in constructs lacking the MoMSV enhancer region. Northern analysis and nuclear run-on experiments indicated that NaBu enhanced transcription of APRT mRNA in both transiently and stably transfected cells, but not in cells inhibited by cycloheximide. Thus, a butyrate-response element (BRE) is associated with the MoMSV enhancer and the action of the MoMSV BRE is promoter-dependent.

Adenine Phosphoribosyltransferase↗
PubMed1992

Regulation of the Ca2+ dependence of smooth muscle contraction.

Cellular mechanisms for the regulation of Ca(2+)-dependent myosin light chain phosphorylation were investigated in bovine tracheal smooth muscle. Increases in the free intracellular Ca2+ concentration ([Ca2+]i), light chain phosphorylation, and force were proportional to carbachol concentration. KCaM, the concentration of Ca2+/calmodulin required for half-maximal activation of myosin light chain kinase, also increased proportionally, presumably due to Ca(2+)-dependent phosphorylation of the kinase. Isoproterenol treatment inhibited agonist-induced contraction by decreasing [Ca2+]i and thereby light chain phosphorylation. Depolarization by increasing concentrations of KCl also resulted in proportional increases in [Ca2+]i, KCaM, light chain phosphorylation, and force. However, the [Ca2+]i required to obtain a given value of either light chain phosphorylation or KCaM was greater in KCl-depolarized tissues compared to carbachol-treated tissues. In muscles contracted with KCl, isoproterenol treatment resulted in diminished light chain phosphorylation and force without alterations in [Ca2+]i or KCaM. Thus, isoproterenol inhibition of KCl-induced contraction results from a cellular mechanism different from that found in agonist-induced contraction. In neither case does isoproterenol produce relaxation by altering the calmodulin activation properties of myosin light chain kinase.

Animals↗
PubMed1992

Genetic immunization is a simple method for eliciting an immune response.

To produce an immune reaction against a foreign protein usually requires purification of that protein, which is then injected into an animal. The isolation of enough pure protein is time-consuming and sometimes difficult. Here we report that such a response can also be elicited by introducing the gene encoding a protein directly into the skin of mice. This is achieved using a hand-held form of the biolistic system which can propel DNA-coated gold microprojectiles directly into cells in the living animal. Genetic immunization may be time- and labour-saving in producing antibodies and may offer a unique method for vaccination.

Animals↗
PubMed1991

Dynamic changes of myometrial activity, levels of PGF2 alpha and E2 in rabbits after insertion of four types of IUDs.

Myometrial activity, levels of PGF2 alpha and E2 in uterine flushings were measured in rabbits before and after insertion of four types of IUDs. The results showed that IUDs could increase the uterine contraction during the early stages of insertion. Thereafter, the uterine activity tended to be stable. The sequences of uterine contractility generally were: Cu-IUD greater than SS-IUD greater than LNG-Cu-IUD greater than LNG-IUD. The measurement of prostaglandins, determined by RIA, indicated that the concentrations of PGF2 alpha and E2 in Cu-IUD and SS-IUD groups were higher than those of controls for the early phase of insertion, which was not found after four months of use. Our results suggested that hyperactivity of the uterus in the early stage after insertion of IUDs might be relevant to side-effects like expulsion and pain. Copper released by Cu-IUD stimulates the uterine activity partially through increasing PGF2 alpha and E2 contents. In rabbits wearing LNG-IUD or LNG-Cu-IUD, both PGF2 alpha and E2 levels were low. The relatively low uterine mechanical and electrical activities were also observed in these two groups. Based on these data, it is concluded that development of LNG-Cu-IUD is feasible for reducing expulsion of IUDs.

Animals↗
PubMed1990

Transcriptional activation of the adenine phosphoribosyltransferase promoter by an upstream butyrate-induced Moloney murine sarcoma virus enhancer-promoter element.

It has been documented that the activity of a specific promoter can be occluded by the presence of another promoter element upstream. We present evidence for a phenomenon contradictory to that predicted by the promoter occlusion theory. Transcription from the hamster aprt (adenine phosphoribosyltransferase) promoter was augmented instead of repressed in transfected mouse L cells when an upstream Moloney murine sarcoma virus enhancer-promoter element was induced with butyrate. Without an adjacent Moloney murine sarcoma virus element, butyrate could not activate the aprt promoter.

Adenine Phosphoribosyltransferase↗
PubMed1989

Influence of naproxen on uterine PGF2 alpha and the antifertility effect of IUDs in rats.

An IUD was implanted into one horn of the uterus in each of our experimental rats. The animals were mated with males of proven fertility about 2 weeks later. At day 1 of gestation (positive mating smear), the animals were treated with oral naproxen at 2 mg/kg per day or 20 mg/kg per day for 10 consecutive days. Control animals received saline. At day 16 the animals were sacrificed and the implantation rates and fetal survival and pregnancy rates were determined. The IUD completely prevented fetal survival and largely reduced implantation rates. Naproxen did not modify these parameters in either the IUD or the non-IUD horn. In a separate set of experiments, the reduction in uterine levels of immunoreactive PGF2 alpha (iPGF) was measured during the implantation period. Naproxen treatment for 5 days with 2 mg/kg per day or 20 mg/kg per day reduced iPGF from 6.71 +/- 0.93 to 2.19 +/- 0.34 and 1.01 +/- 0.18 ng/100 mg tissue, respectively (P less than 0.01). These data show that the suppression of uterine PG synthesis by naproxen does not interfere with the antifertility effects of IUDs in the rat.

Animals↗
PubMedDate not supplied

Increased immunogenicity of tumors bearing mutant p53 and P1A epitopes after transduction of B7-1 via recombinant adenovirus.

The majority of human solid tumors are likely to express protein epitopes which can act as targets for cytotoxic T cells, but these are frequently not effectively recognized. We tested whether the introduction of the costimulatory molecule B7-1 using a recombinant adenovirus (Ad-B7) can result in effective induction of epitope-specific immunity in two tumor models that express defined endogenous protein epitopes: D459, a fibroblast-derived cell line transfected with a human missense mutant p53 (C to Y at position 135), and P815, a mastocytoma expressing the endogenous tumor epitope P1A. Under the conditions studied, both of these tumors grow and kill their hosts without evidence of significant immune rejection. However, after transduction with the adenovirus containing B7-1, both of these tumors lose tumorigenicity and elicit specific cellular immunity to the mutant p53 epitope in D459 and P1A in P815. In addition, animals exposed to B7-transduced tumor cells were protected from subsequent challenge with nontransduced tumor. Adenovirus has distinct advantages for this approach, as it has high infectivity not requiring in vitro culture, low lytic potential, and transient expression of sufficient duration for immunologic effectiveness but without significant concern over permanent genetic modification. We conclude that transduction of tumor cells with Ad-B7 can increase the immunogenicity of endogenous protein epitopes and may represent a practical therapeutic approach to system human cancers.

Adenoviridae↗