Biomedical subjects

C C Wang

Publications and source records attributed to C C Wang.

At least 217 records · Page 12Linked to original sources

PubMed1998

Assay for enzyme activity by following the absorbance change of pH-indicators.

Based on the absorbance change of indicators with the concentration of hydrogen ion released from an enzyme-catalyzed reaction, a convenient colorimetric method was established for the assay of acidic phospholipase A2 and glycogen phosphorylase b. Brilliant yellow and bromothymol blue were chosen as indicators for assays of acidic phospholipase A2 and glycogen phosphorylase b by following the absorbance changes at 495 and 615 nm, respectively. The method is simple, sample-saving, sensitive and valid for a wide range of enzyme concentrations. It can be extended for assaying other enzymes catalyzing reactions with hydrogen ion concentration changes.

Animals↗

PubMed1998

The role of proteolysis during differentiation of Trypanosoma brucei from the bloodstream to the procyclic form.

The in vitro differentiation of Trypanosoma brucei from bloodstream to procyclic (insect) forms is accompanied by diminishing variant surface glycoprotein (VSG) and increasing levels of procyclin and phosphoenolpyruvate carboxykinase (PEPCK). In this study, we examined the fate of several glycolytic enzymes of T. brucei during differentiation. We observed a down-regulation of glycosomal phosphoglycerate kinase (gPGK) during differentiation. In contrast, intracellular levels of glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH), aldolase (ALD), and phosphoglucoisomerase (PGI) remained unchanged during differentiation and apparently continued to be synthesized in the procyclic form. To determine the potential role of proteasomes and other proteases during the differentiation process, we tested the effect of lactacystin, a specific inhibitor of proteasome activity, and morpholinourea-Phe-homoPhe-benz-alpha-pyrone (P27), a selective inhibitor of cysteine proteases, on the in vitro differentiation of T. brucei. Cells differentiated normally in the presence of 1 microM lactacystin, which confirmed our previous observation that this differentiation does not require crossing any phase boundaries in the cell cycle (Mutomba and Wang, Mol Biochem Parasitol 1996;80:89-102). But the cells thus differentiated did not increase in number and retained gPGK. Cells differentiated under 2 microM P27 also proceeded at a normal rate but failed to multiply and retained gPGK. However, most of the differentiated cells under 2 microM P27 also retained VSG on the cell membrane surface and expressed higher levels of procyclin suggesting that a cysteine protease(s) may be involved in releasing VSG and partially reducing procyclin during differentiation. This cysteine protease(s) has been tentatively identified in the procyclic cells as a 48 kDa protein through labeling of cysteine protease(s) with a biotinylated P27 homolog K02 (morpholinourea-Phe-homoPhe-vinylsulfone).

Acetylcysteine↗

PubMed1998

Rational design of novel antimicrobials: blocking purine salvage in a parasitic protozoan.

All parasitic protozoa obtain purine nucleotides solely by salvaging purine bases and/or nucleosides from their host. This observation suggests that inhibiting purine salvage may be a good way of killing these organisms. To explore this idea, we attempted to block the purine salvage pathway of the parasitic protozoan Tritrichomonas foetus. T. foetus is a good organism to study because its purine salvage depends primarily on a single enzyme, hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase), and could provide a good model for rational drug design through specific enzyme inhibition. Guided by the crystal structure of T. foetus HGXPRTase, we used structure-based drug design to identify several non-purine compounds that inhibited this enzyme without any detectable effect on human HGPRTase. One of these compounds, 4-[N-(3, 4-dichlorophenyl)carbamoyl]phthalic anhydride (referred to as TF1), was selected for further characterization. TF1 was shown to be a competitive inhibitor of T. foetus HGXPRTase with respect to both guanine (in the forward reaction; Ki = 13 microM) and GMP (in the reverse reaction; Ki = 10 microM), but showed no effect on the homologous human enzyme at concentrations of up to 1 mM. TF1 inhibited the in vitro growth of T. foetus with an EC50 of approximately 40 microM. This inhibitory effect was associated with a decrease in the incorporation of exogenous guanine into nucleic acids, and could be reversed by supplementing the growth medium with excess exogenous hypoxanthine or guanine. Thus, rationally targeting an essential enzyme in a parasitic organism has yielded specific enzyme inhibitors capable of suppressing that parasite's growth.

Animals↗

PubMed1998

Binding of a burst-phase intermediate formed in the folding of denatured D-glyceraldehyde-3-phosphate dehydrogenase by chaperonin 60 and 8-anilino-1-naphthalenesulphonic acid.

Upon dilution, D-glyceraldehyde-3-phosphate dehydrogenase (GADPH) that has been fully inactivated, but only partially unfolded, in dilute guanidine hydrochloride (GuHCl) recovers activity completely. The fully unfolded enzyme, however, is re-activated only to a limited extent after dilution, and refolds rapidly in a burst phase to a partially folded intermediate characterized by increases in both the emission intensity of intrinsic fluorescence and binding to 8-anilino-1-naphthalenesulphonic acid (ANS). This intermediate aggregates with a time lag of a few minutes, and the aggregation can be suppressed completely by chaperonin 60 (GroEL). Stoichiometric analysis of the suppression of GAPDH re-activation by GroEL suggests that the tetradecameric GroEL binds to a dimeric GAPDH folding intermediate. This intermediate can be re-activated by ATP or ATP/chaperonin 10 (GroES) to an extent considerably greater than that obtained on spontaneous re-activation of the fully denatured enzyme upon dilution. Probing with a fluorescent derivative of NAD+ shows that this folding intermediate does not have a native conformation at the active site. The similar profiles of the effects of GroEL and ANS on the re-activation of GAPDH denatured by different concentrations of GuHCl suggest that GroEL and ANS recognize and bind to the same folding intermediate, which is similar to the relatively stable, partially unfolded, state of the enzyme denatured in 0.5-1.0 MGuHCl. However, the complexes of the intermediate with GroEL or ANS appear to be different, in that GroEL, but not ANS, suppresses aggregation and assists folding in the presence of ATP.

Adenosine Triphosphate↗

PubMed1998

Role of substance P and the neurokinin 1 receptor in acute pancreatitis and pancreatitis-associated lung injury.

Substance P, acting via the neurokinin 1 receptor (NK1R), plays an important role in mediating a variety of inflammatory processes. However, its role in acute pancreatitis has not been previously described. We have found that, in normal mice, substance P levels in the pancreas and pancreatic acinar cell expression of NK1R are both increased during secretagogue-induced experimental pancreatitis. To evaluate the role of substance P, pancreatitis was induced in mice that genetically lack NK1R by administration of 12 hourly injections of a supramaximally stimulating dose of the secretagogue caerulein. During pancreatitis, the magnitude of hyperamylasemia, hyperlipasemia, neutrophil sequestration in the pancreas, and pancreatic acinar cell necrosis were significantly reduced in NK1R-/- mice when compared with wild-type NK1R+/+ animals. Similarly, pancreatitis-associated lung injury, as characterized by intrapulmonary sequestration of neutrophils and increased pulmonary microvascular permeability, was reduced in NK1R-/- animals. These effects of NK1R deletion indicate that substance P, acting via NK1R, plays an important proinflammatory role in regulating the severity of acute pancreatitis and pancreatitis-associated lung injury.

Acute Disease↗

PubMed1998

Study on the interactions between protein disulfide isomerase and target proteins, using immobilization on solid support.

Interaction between protein disulfide isomerase, possessing not only isomerase but also chaperone-like activity, and olygomeric enzyme, GAPDH, has been studied using technique of immobilization on insoluble support. PDI dimers bound to CNBr-activated Sepharose were shown to possess high TPOR activity as well as the ability to reactivate lysozyme. Immobilized PDI was not found to interact neither with soluble tetrameric GAPDH, nor with soluble denatured GAPDH. However, soluble PDI binds effectively to immobilized GAPDH monomers; Kd was found to be 3.7 x 10(-6) M, stoichiometry 0.824 mole PDI monomers per mole GAPDH monomers. Immobilized GAPDH tetramers do not interact with PDI. These observations are also confirmed by the data on electrophoresis of proteins bound to immobilized GAPDH monomers and tetramers. The ability of PDI to interact with denatured protein form, but not with the native one, is considered to be evidence of chaperone-like activity of the enzyme.

Disulfides↗

PubMed1998

Enzymes as chaperones and chaperones as enzymes.

Chaperones and foldases are two groups of accessory proteins which assist maturation of nascent peptides into functional proteins in cells. Protein disulfide isomerase, a foldase, and ATP-dependent proteases, responsible for degradation of misfolded proteins in cells, both have intrinsic chaperone activities. Trigger factor and DnaJ, well known Escherichia coli chaperones, show peptidyl prolyl isomerase and protein disulfide isomerase activities respectively. It is suggested that the combination of chaperone and enzyme activities in one molecule is the result of evolution to increase molecular efficiency.

ATP-Dependent Proteases↗

PubMed1998

Steady-state kinetics of the hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus: the role of threonine-47.

Tritrichomonas foetus, an anaerobic flagellated protozoan, causes urogenital trichomoniasis in cattle. Hypoxanthine-guanine-xanthine phosphoribosyl transferase (HGXPRTase), an essential enzyme in T. foetus required for salvaging exogenous purine bases, has been regarded as a promising target for anti-tritrichomonial chemotherapy. The steady-state kinetic analyses of synthesis and pyrophosphorolysis of IMP, GMP, and XMP and product inhibition studies have been used to elucidate the reaction mechanisms. Double-reciprocal plots of initial velocities versus the varying concentrations of one substrate at a fixed concentration of the other show intersecting lines indicating a sequential mechanism for both the forward and the reverse reactions. In terms of the kcat/Km ratios, hypoxanthine is the most effective substrate whereas guanine and xanthine are converted equally well into their corresponding nucleotides. The minimum kinetic model from the data in product inhibition studies is an ordered bi-bi mechanism, where the substrates bind to the enzyme (first PRPP followed by the purine bases), and the products released (first PPi followed by purine nucleotide) in a defined order. The Kms for PPi in the T. foetus HGXPRTase-catalyzed reactions are unusually high, close to the millimolar range. Since the crystal structure of this enzyme [Somoza et al. (1996) Biochemistry 35, 7032-7040] suggests potential binding between the threonine-47 in a conserved cis-peptide loop and PPi whereas human HGPRTase has lysine-68 [Eads et al. (1994) Cell 78, 325-334] at the corresponding position, we prepared a T47K enzyme mutant and found in the T47K-catalyzed reaction a 4-10-fold decrease of Km for PPi. The lack of ionic interactions between Thr-47 and PPi and an increased distance between the loop and the active site as compared to the human HGPRTase are thus proposed to be responsible for the high Km for PPi in the T. foetus HGXPRTase-catalyzed reaction.

Amino Acid Substitution↗

PubMed1998

Murine cerberus homologue mCer-1: a candidate anterior patterning molecule.

Xenopus cerberus (Xcer) is a cytokine expressed in anterior mesendoderm overlapping and surrounding Spemann's gastrula organiser. When misexpressed in blastomeres, Xcer can induce ectopic heads with well-defined brain, cement gland, olfactory placodes, cyclopic eye, and occasionally liver and heart. We report here the identification of mCer-1, a murine gene related to cerberus. Both mCer-1 and Xcer appear to belong to the cystine knot superfamily, which includes TGF beta s and BMPs. In Xenopus animal cap assays, mCer-1 and Xcer induced cement glands and markers of anterior neural tissue and endoderm, characteristic of BMP inhibition. Furthermore, both antagonised the ventrolateral mesoderm-inducing activity of coexpressed BMP4. In mouse embryos, mCer-1 was expressed at early gastrulation in a stripe of primitive endoderm along the future anterior side of the egg cylinder, a region essential for anterior patterning. A second phase of expression was detected in anterior embryonic mesendoderm, and by late-streak stages most of the anterior half of the embryo was positive, except for the node and cardiac progenitors. Expression was later seen in the cranial portion of the two most-recently formed somites and in two stripes within presomitic mesoderm. In embryos lacking Otx2, a homeogene with a demonstrated role in anterior patterning, mCer-1 was still expressed in an anterior zone, although often abnormally. The data suggest that mCer-1 shares structural, functional, and expression characteristics with Xcer and may participate in patterning the anterior of the embryo and nascent somite region, in part, through a BMP-inhibitory mechanism.

Amino Acid Sequence↗

PubMed1998

Determinants of tachycardia recurrences after radiofrequency ablation of idiopathic ventricular tachycardia.

Clinical and electrophysiologic parameters were analyzed to define the factors potentially related to tachycardia recurrences in 79 patients undergoing successful radiofrequency ablation of idiopathic right or left ventricular tachycardia. It was found that the endocardial activation time at the successful ablation site was the only independent predictor of tachycardia recurrences.

Adolescent↗

PubMed1998

Results of accelerated radiotherapy for supraglottic carcinoma: a Massachusetts General Hospital and Massachusetts Eye and Ear Infirmary experience.

BACKGROUND: Carcinoma of the supraglottic larynx is a relatively common malignancy treated with either surgery, radiotherapy, or a combined approach. METHODS: The radiotherapy records of 190 patients with carcinoma of the supraglottic larynx treated at the Massachusetts General Hospital (MGH) and Massachusetts Eye and Ear Infirmary (MEEI) from 1981 to 1992 were reviewed. Of these patients, 164 were available for evaluation for local control and 169 for disease-specific survival. The patients were treated with accelerated hyperfractionated radiotherapy to 67.2-72 Gy/1.6 Gy per fraction twice a day in 6 weeks. The median follow-up was 56 months. Five-year actuarial local and regional control, relapse-free and overall survival, and voice-preservation rates were analyzed. RESULTS: For T1, T2, T3, and T4 tumors, local control rates were 96%, 86%, 76%, and 43%, respectively (p < .01), and the corresponding relapse-free survival rates were 78%, 82%, 64%, and 40% (p < .01). For the patients with N0, N1, and N2-3 disease, local control rates at the primary site were 86%, 74%, and 46%, respectively (p < .01), and the corresponding relapse-free survival rates were 79%, 53%, and 39% (p < .01). Including surgical salvage, the ultimate local control rates were 96%, 93%, 88%, and 51%, respectively (p < .01). Voice preservation rate for the entire group was 79% and for T1, T2, T3, and T4 tumors, rates were 96%, 80%, 72%, and 43%, respectively. CONCLUSIONS: Our experience with accelerated hyperfractionated radiotherapy for supraglottic carcinoma showed excellent locoregional control, relapse-free survival, and laryngeal preservation. The radiation toxicity was acceptable. The T and N stages were significant predictors of outcome, and the T4 tumors and node-positive neck disease portended a poor prognosis and therefore should be considered for protocols that include adjuvant therapies.

Carcinoma, Squamous Cell↗

PubMed1998

Trypanosoma brucei brucei: characterization of an ODC null bloodstream form mutant and the action of alpha-difluoromethylornithine.

Ornithine decarboxylase (ODC) is a key enzyme in the polyamine synthesis pathway in African trypanosomes. We report here the characterization of an ODC null bloodstream form Trypanosoma brucei brucei mutant, created by replacing the ODC gene with antibiotic resistant marker genes through transfection and homologous recombination. The null mutant expresses no ODC mRNA or protein and does not have ODC enzymatic activity. We tested the attenuation of the bloodstream form ODC- mutants in mice, and showed that these mutants cannot multiply and are quickly cleared from the blood. We also tested the effect of DFMO on this ODC null mutant.

Animals↗

PubMed1998

Hypoxanthine, guanine, xanthine phosphoribosyltransferase activity in Cryptosporidium parvum.

All parasitic protozoa examined to date are incapable of de novo synthesis of purine nucleotides and rely on salvage mechanisms for survival. We have identified hypoxanthine, guanine, xanthine phosphoribosyl-transferase activities in crude cell-free extracts of Cryptosporidium sporulated oocysts utilizing radiolabeled substrates. Guanine, hypoxanthine, and xanthine were converted to their corresponding mononucleotides with specific activities of 346, 280, and 108 nmol/min/mg protein, respectively. The conversion of the radiolabeled purines was examined in the presence of another, unlabeled, purine base. These competition assays showed that both hypoxanthine and guanine were capable of inhibiting conversion of hypoxanthine, guanine, and xanthine to the corresponding nucleotides. Xanthine had a much lower inhibitory effect on the conversion of guanine and hypoxanthine to the nucleotides, whereas adenine had no effect at all. Autoradiographic studies of Cryptosporidium-infected Madin-Darby canine kidney (MDCK) cells showed that radiolabeled hypoxanthine, guanine, and adenine were primarily incorporated by intracellular Cryptosporidium as well as by MDCK nuclei. No apparent incorporation of xanthine by either host cells or intracellular parasites occurred. Radiolabeled glycine and formate were incorporated only into the nuclei of MDCK cells, suggesting a lack of de novo synthesis of purine nucleotides in Cryptosporidium. Radiolabeled hypoxanthine and guanine were also incorporated by excysting Cryptosporidium sporozoites. Altogether, our results indicate the presence of HPRTase, GPRTase, and XPRTase activities. These activities may play an important role in purine salvage, and may localize to a single HGXPRTase enzyme, as in the case of Eimeria, Toxoplasma, and Plasmodium.

Adenine↗

PubMed1998