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Biomedical subjects

T L Chang

Publications and source records attributed to T L Chang.

At least 19 recordsLinked to original sources
PubMed2004

Ankyrin is a target of spectrin's E2/E3 ubiquitin-conjugating/ligating activity.

Ubiquitin is a small protein of 8.6 kDa molecular weight. When polyubiquitin is attached to target proteins, they are tagged for destruction by cytoplasmic organelles called proteasomes. We now know that ubiquitination of target proteins also regulates functions as diverse as the sorting of proteins to different intracellular destinations, cell signaling, cell division, gene transcription, and protein-protein interactions. The ubiquitination of target proteins requires a cascade of enzymes: E1 ubiquitin activating enzyme, E2 ubiquitin conjugating enzyme and E3 ubiquitin ligating enzyme. Recently we have demonstrated that the red blood cell (RBC) membrane skeletal protein, spectrin, has E2/E3 enzymatic activities in its alpha-subunit, that can transfer ubiquitin to itself. We have now created a cell free assay using biotinylated ubiquitin that allows detection of target proteins by streptavidin peroxidase. This approach coupled with immunoprecipitation, purification and micro liquid chromatography coupled to tandem mass spectrometry has identified ankyrin as a target of spectrin's E2/E3 activity. Western blotting, with ubiquitin antibody, of purified ankyrin and its well characterized functional domains, has demonstrated that both the spectrin and band 3 binding domains are ubiquitinated in vivo.

Ankyrins↗
PubMed2004

Band 3 is a target protein of spectrin's E2/E3 activity: implication for sickle cell disease and normal red blood cell aging.

We have demonstrated that a 125 kDa red blood cell (RBC) membrane protein, being a target of spectrin's E2/E3 activity, is ubiquitinated band 3. This demonstration was based on copurification of this biotinylated-ubiquitinated protein with band 3, immunoprecipitation with band 3 antibody and analysis of proteins associated with strepavidin sepharose by micro liquid chromatography coupled to tandem mass spectrometry (microLC/MS/MS). Further, we demonstrated the presence of ubiquitinated band 3 in vivo by Western blotting of purified band 3 with a monoclonal antibody (FK2) against ubiquitin. The implications of these results for sickle cell disease and RBC aging are discussed.

Anemia, Sickle Cell↗
PubMed2002

Watch out for the K-wire: painful experiences in two cases.

According to reviews in the literature, only a few case reports have mentioned the unusual migration of K-wires during orthopaedic surgery since 1991. This report emphasises the potential migration of the K-wire during plastic surgery in order to avoid the possible catastrophic complications.

Abdomen↗
PubMed2000

Role of individual monomers of a dimeric initiator protein in the initiation and termination of plasmid rolling circle replication.

Plasmids of the pT181 family encode initiator proteins that act as dimers during plasmid rolling circle (RC) replication. These initiator proteins bind to the origin of replication through a sequence-specific interaction and generate a nick at the origin that acts as the primer for RC replication. Previous studies have demonstrated that the initiator proteins contain separate DNA binding and nicking-closing domains, both of which are required for plasmid replication. The tyrosine residue at position 191 of the initiator RepC protein of pT181 is known to be involved in nicking at the origin. We have generated heterodimers of RepC that consist of different combinations of wild type, DNA binding, and nicking mutant monomers to identify the role of each of the two monomers in RC replication. One monomer with DNA binding activity was sufficient for the targeting of the initiator to the origin, and the presence of Tyr-191 in one monomer was sufficient for the initiation of replication. On the other hand, a dimer consisting of one monomer defective in DNA binding and the other defective in origin nicking failed to initiate replication. Our results demonstrate that the monomer that promotes sequence-specific binding to the origin must also nick the DNA to initiate replication. Interestingly, whereas Tyr-191 of the initiator was required for nicking at the origin to initiate replication, it was dispensable for termination, suggesting that alternate amino acids in the initiator may promote termination but not initiation.

Bacterial Proteins↗
PubMed2000

Interleukin-4 mediates cell growth inhibition through activation of Stat1.

Interleukin-4 (IL-4) activates Stat6 (signal transducer and activator of transcription 6) and plays multiple roles in regulation of the immune system. IL-4 also triggers phosphorylation of insulin receptor substrate (IRS), leading to stimulation of cell growth. Moreover, IL-4 inhibits proliferation of a variety of cells, but the molecular mechanism of its growth inhibitory effect is not understood. In this study, we demonstrated that IL-4 inhibited cell growth of colon carcinoma cell lines (HT29 and WiDr) but promoted cell growth of Burkitt's lymphoma cell lines (BL30 and BL41) in a dose-dependent manner. The growth inhibition was not dependent on Stat6 activation, because Stat6 was activated at similar levels in all cell lines in response to IL-4. Strikingly, IL-4 activated Stat1 in colon carcinoma cell lines but not in Burkitt's lymphoma cell lines. Therefore, these results suggest that IL-4 induced Stat1 activation, resulting in growth inhibition of colon carcinoma cell lines. Importantly, we present evidence that Stat1 is necessary for IL-4-mediated growth inhibition using Stat1-deficient and Stat1-reconstituted cells. The growth inhibitory effect of IL-4 was diminished in Stat1-deficient cells, whereas it was restored in Stat1-reconstituted cells. In addition, the expression of dominant-negative Stat1 in HT29 cells led to the loss of growth inhibition in response to IL-4. Taken together, our data suggest that IL-4 activates Stat1, leading to cell growth inhibition in colon cancer cells. Thus, this study demonstrates, for the first time, a molecular mechanism by which IL-4 inhibits cell growth.

Animals↗
PubMed1999

Determination of benzodiazepines in clinical serum samples: comparative evaluation of REMEDi system, aca analyzer, and conventional HPLC performance.

Emergency toxicology or drug screening in clinical settings requires rapid qualitative and quantitative analysis with acceptable levels of sensitivity and specificity. The aim of this study was to comparatively evaluate the performance of the multi-column HPLC-based REMEDi drug profiling system (Bio-Rad), the aca analyzer (Du Pont), and the bench standard conventional HPLC method in the identification of 12 clinically important benzodiazepines. In this study, the presence of benzodiazepines in 133 patients' serum samples were qualitatively and comparatively analyzed by these three procedures. Among these methods, 120 of 133 samples were identified as benzodiazepine-positive by conventional HPLC (90%); 127 by aca analyzer (95%); and 84 by REMEDi (63%). Detection sensitivity of REMEDi for most of the benzodiazepines was found satisfactory when concentrations were greater than 1.0 microg/mL. When benzodiazepine concentrations were in the ranges of 0.3-1.0 microg/mL, detection sensitivity became varied among the benzodiazepine family of drugs by REMEDi method. REMEDi procedure should not be considered as the method of choice for detection of benzodiazepines if expected concentration levels are below 0.3 microg/mL, with the exception of bromazepam. Conventional HPLC displayed the highest sensitivity and specificity for the detection of benzodiazepines. In our studies, 36 REMEDi-negative samples were positive by HPLC, although in 16 of the 36 REMEDi negative samples (13.3%), the presence of benzodiazepines was detected but only listed as candidates without positive identification of the individual compounds by REMEDi. In our series, however, there were no false positives by the REMEDi method whereas aca procedure showed 13 false positive results (9%) and 6 cases of false negative (4%). Our data indicate that the REMEDi procedure can be used on serum samples for rapid qualitative screening of clinically important high levels of benzodiazepines with high specificity. However, due to the relatively low sensitivity of REMEDi in samples with low benzodiazepine levels and relatively low specificity by aca, all samples should be further confirmed by conventional HPLC procedure.

Autoanalysis↗
PubMed1999

Restoration of partially edentulous patients using customized implant abutments.

The ideal placement of implants is not always possible in partially edentulous patients. The diverse and unique implant positions that occur in clinical practice may be difficult or impossible to restore through the use of conventional abutments. Customized abutments permit the fabrication of aesthetic restorations that correct deficiencies in implant angulation, alignment, and position. These abutments also enhance the soft tissue emergence profile of the restorations and allow the prosthetic margins to be properly positioned in all dimensions. Additional benefits include ease of treatment delivery and comparative expense.

Dental Abutments↗
PubMed1997

Burkholderia pseudomallei infection in the Singapore Armed Forces from 1987 to 1994--an epidemiological review.

Between 1987 and 1994, twenty-three cases of Burkholderia pseudomallei infection (melioidosis) were diagnosed in persons serving in the Singapore Armed Forces. There were four deaths resulting from complications of the infection. Unlike the situation in the general population, where the affected are mainly the elderly with underlying illness, the majority of cases in the Singapore Armed Forces were otherwise fit and healthy young servicemen. Serological surveys have shown the prevalence of the infection in Singapore to be 0.2% in the military as well as civilian population. As physical contact with soil is an unavoidable part of military training, military personnel continue to be at risk of exposure to this soil-related disease.

Adolescent↗
PubMed1996

Characterization of primary lesions caused by the plastome mutator of Oenothera.

Oenothera plants homozygous for a recessive allele at the plastome mutator (pm) locus show non-Mendelian mutation frequencies that are 1000-fold higher than spontaneous levels. Characterization of RFLP sites in a collection of mutants indicates that insertion-deletion hot spots in the pm lines are defined by tandem direct repeats, implicating replication slippage or misalignment during recombination. Several sites known to contain very short direct repeats were examined, and all were found to have been targeted in one or more plants of the mutant collection. To determine if replication slippage was occurring, two oligo-A stretches in non-coding DNA were examined, and 3 of 12 plants were found to have an additional adenine in a 13-base track. To search for other mutations that would not be visible as restriction fragment length polymorphisms, PCR-amplification products of the psbB gene were digested with a restriction endonuclease, denatured, and examined for single-strand conformational polymorphisms. Among 21 mutants, one 4-bp insertion and one point mutation were identified in psbB. The discovery that the plastome mutator can cause base substitutions as well as repeat-mediated insertions and deletions points to a likely defect in a component of the cpDNA replication machinery.

Base Sequence↗
PubMed1994

Inhibition of vesicular stomatitis virus RNA synthesis by protein hyperphosphorylation.

Vesicular stomatitis virus (VSV) RNA synthesis requires the template nucleocapsid, the polymerase (L) protein, and the cofactor phosphorylated (P/NS) protein. To determine whether the degree of phosphorylation regulated VSV RNA synthesis, infected Chinese hamster ovary cells were treated with okadaic acid (OKA), a serine/threonine phosphatase inhibitor. OKA reduced viral penetration and uncoating but had little or no effect on primary transcription or viral protein synthesis. However, approximately 80% of total viral RNA synthesis was inhibited when 2 microM or more OKA was added to infected cells after viral uncoating had taken place. Analysis of proteins and RNA species in infected cells labeled with 32P showed that OKA led to hyperphosphorylation of two viral phosphoproteins, the P/NS protein and matrix protein (M), resulting in inhibition of full-length RNA synthesis and subsequent secondary transcription. Pulse-chase experiments demonstrated that the hyperphosphorylated P/NS species was converted rapidly from the less phosphorylated form. Hyperphosphorylated P/NS as well as the less phosphorylated form, but not M, were found to be associated with nucleocapsids isolated from cytoplasmic extracts. These results suggest that phosphorylation played an important role in the regulation between viral transcription and viral RNA replication as well as the turning off of RNA replication. Thus, phosphatase inhibitors promise to be a valuable tool for dissecting the regulatory mechanisms involving phosphorylated viral proteins.

Animals↗
PubMed1994

Detection of specific antibody to mycobacterial antigen 60 in tuberculous pleural effusion.

BACKGROUND: With the development of the enzyme-linked immunosorbent assay (ELISA) method, serodiagnosis of tuberculosis has been studied by many investigators. Only a few studies have been performed in pleural fluid. This study was designed to evaluate the IgG antibody levels to mycobacterial antigen 60 (Ag60) in pleural fluid, and evaluate its role in the diagnosis of tuberculous pleurisy. METHODS: Eighteen patients with tuberculous pleural effusions and 18 patients with malignant pleural effusions were studied. The levels of IgG antibodies to Ag60 in pleural fluids were measured by ELISA method. RESULTS: The mean titers of IgG against Ag60 in pleural fluids of tuberculous patients (508.3 +/- 382.7 EU) were significantly higher than those of the mean value of the malignant group (131.2 +/- 83.2 EU). In the TB pleurisy group, patients with positive M. tuberculosis cultures from pleural fluids had significantly higher titers than those with negative cultures (796.5 +/- 394.7 vs 277.8 +/- 150.2 EU); patients with impaired immune function had significantly lower titers than those without (138.4 +/- 28.9 vs 650.6 +/- 358.1 EU). Using 250 EU as a cutoff value for a positive test, the sensitivity was 72.2% and the specificity, 94.4%. CONCLUSIONS: ELISA method using Ag60 is a rapid test with an acceptable sensitivity and excellent specificity for differentiation between tuberculous and malignant pleural effusion.

Adolescent↗
PubMed1991

Anergy in immature B lymphocytes. Differential responses to receptor-mediated stimulation and T helper cells.

We have compared the responses of purified neonatal and adult B lymphocytes to stimulation by anti-Ig antibodies, which are functional analogues of Ag, and by Th cells. Neonatal B cells are markedly deficient in proliferative responses to anti-Ig antibodies + IL-4 or to anti-Ig conjugated to dextran, both of which induce strong proliferation of adult B cells in the absence of T lymphocytes. Anti-Ig antibodies actually inhibit the functional responses of neonatal B cells, even to polyclonal stimuli such as LPS. However, Th cells induce both proliferation and Ig secretion by neonatal B cells in the presence of Ag that bind to B cell Ig and are subsequently presented by the B cells. Thus, in neonatal B lymphocytes, cross-linking of membrane Ig in the absence of Th cells has a net inhibitory effect, and this inhibition is overcome by T cell help. These results also suggest that unresponsiveness or tolerance to thymus-independent Ag is induced in the B cells themselves, but tolerance to thymus-dependent proteins resides primarily in the T cell compartment.

Aging↗
PubMed1991

Activation of functionally distinct subsets of CD4+ T lymphocytes.

The studies summarized in this review have established that cloned lines of CD4+ T cells that produce distinct cytokines differ markedly in their responses to different forms of antigenic stimulation. Furthermore, we are beginning to develop experimental systems for better defining the signals that stimulate the differentiation of resting T cells into functionally distinct subsets. From these studies it is possible to construct the following hypothetical model for the differentiation of mature CD4+ T cells. Resting cells produce IL2 as the principal growth factor, IFN gamma, and little or no IL4. Antigenic stimulation in the presence of IL4 (which may be produced by non-T cells) leads to the preferential expansion of IL4-producing cells. These cells secrete their cytokines maximally when stimulated with antigens presented by B cells, which are also the principal targets of these cytokines. Continued expansion of IL4-producing T cells may require antigen exposures that also stimulate the production of IL1 by macrophages. In the absence of IL4 and IL1 (and in the presence of costimulators that are not yet defined) the T cells that are preferentially expanded belong to the IL2-producing subset. In addition, each subset may produce cytokines that stimulate the expansion of that subset and inhibit the other (Fiorentino et al., 1989). It is apparent that a number of assays and reagents need to be developed if these results are to be extended to physiologic immune responses. First, it is important to identify surface molecules that may serve as phenotypic markers for functionally distinct subsets of CD4+ cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Heterogeneity of helper/inducer T lymphocytes. III. Responses of IL-2- and IL-4-producing (Th1 and Th2) clones to antigens presented by different accessory cells.

Murine CD4+ T cell clones have been classified into at least two subsets, Th1 and Th2, on the basis of their distinct lymphokine secretion profiles and functions. In the present study, we compared the functional responses of Th1 and Th2 clones to Ag presentation by splenic B cells and peritoneal macrophages. Th2 clones secreted IL-4 in response to Ag presented by resting B cells, but their optimal proliferation required the addition of IL-1 or a source of IL-1. The degree of IL-1 dependence varied among the four Th2 clones examined. In contrast, Th1 clones secreted IL-2 and proliferated in response to Ag presented by both B cells and macrophages, without any requirement for exogenous IL-1. Furthermore, the proliferation of Th2 clones in response to Ag presented by splenocytes or macrophages was inhibited by an IL-1R antagonist. These results indicate that IL-1 is an important costimulator for the expansion of the Th2 subset of CD4+ T cells. The different requirements for the proliferation of Th1 and Th2 cells may be responsible for the preferential expansion of one or the other subset under different conditions of immunization.

Animals↗
PubMed1988

Determination of the synthetic steroid norgestomet in bovine plasma by capillary column gas chromatography/negative chemical ionization mass spectrometry.

A sensitive and specific method is described for the determination of norgestomet in bovine plasma as low as 10 ppt with better than 83% average recovery and a relative standard deviation (RSD) of range 1-3%. Norgestomet is separated from the bulk of the endogenous substances in plasma by adsorption on a PrepSep C18 extraction column and elution with acetonitrile. The residue after evaporation of acetonitrile is reacted with O-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride to form syn and anti geometric isomers of the mono-oxime derivative. The derivative, after further clean-up and evaporation of solvent, is reconstituted with cyclohexane, and an aliquot is analyzed by capillary column gas chromatography/negative chemical ionization mass spectrometry using methane as the carrier gas. Selected ion monitoring was employed to detect the [M - HF]- fragment ion of the norgestomet mono-oxime derivative (m/z 547) and its dideuterated mono-oxime analog (m/z 549) which serves as the internal standard. Quantification is achieved by using the Quantitative Selected Ion Monitoring Processing System (QSIMPS) software to generate a standard curve of fragment ion area ratios v. concentration of norgestomet, and then calculate sample concentrations.

Animals↗
PubMed1984

In vitro processing of proopiocortin by membrane-associated and soluble converting enzyme activities from rat intermediate lobe secretory granules.

Secretory granules (SGs) from rat intermediate lobes (IL) were isolated in a highly purified form by differential centrifugation, followed by sucrose density gradient centrifugation. The purified IL-SGs were lysed by freezing and thawing. The granule lysate was then centrifuged to generate membrane and soluble fractions. Proopiocortin -converting enzyme (PCE) activity was assayed by incubation of [3H]arginine- or [3H] phenylalanine-labeled toad proopiocortin with the total granule lysate, the membrane, or the soluble fraction at pH 5.0. The processed products were identified by immunoprecipitation with ACTH and beta-endorphin antisera, followed by acid-urea-gel electrophoresis. The PCE activity in rat IL-SG lysate cleaved proopiocortin to 21,000 mol wt ACTH, 21,000 mol wt ACTH/lipotropin (LPH), 13,000 mol wt ACTH, beta LPH, beta-endorphin-like peptides, and alpha MSH-like peptides, similar to those synthesized by the toad intermediate lobe in situ. Treatment of the PCE cleavage products with carboxypeptidase B resulted in the liberation of free arginine. This observation together with the nature of the products formed suggest that the PCE activity cleaved at pairs of basic residues of proopiocortin , yielding one or more products that terminated with an arginine or an arginine-lysine. PCE activity was found in membrane and soluble granule fractions, and both activities were inhibited by leupeptin, p-chloromercuribenzoate, dithiodipyridine, and pepstatin A, but not by chloroquine or N-alpha-p-tosyl-L-lysine-chloromethylketone HCl. Diisopropyl fluorophosphate and other thiol protease reagents (p-chloromercuriphenyl sulfonic acid, iodoacetic acid, and HgCl2) had a small inhibitory effect. The products formed by PCE activities in the membrane and soluble fractions were similar to those cleaved by the total granule lysate. The membrane fraction primarily cleaved proopiocortin between ACTH and beta LPH to form 21,000 (21 K) mol wt ACTH and beta-LPH, similar to the first processing step in the IL in situ. The soluble fraction, however, showed a greater tendency to cleave proopiocortin between the 16 K N-terminal glycopeptide and ACTH, to yield twice as much 21 K ACTH/LPH product as the membrane fraction. The membrane-associated PCE activity was found to be easily solubilized by extraction with high salt (1 M NaCl), suggesting that it is not an integral granule membrane protein.

Adrenocorticotropic Hormone↗