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C C Tseng

Publications and source records attributed to C C Tseng.

At least 37 records · Page 2Linked to original sources
PubMed2000

Real-time PCR for quantitative detection of Toxoplasma gondii.

The protozoan Toxoplasma gondii is one of the most common infectious pathogenic parasites and can cause severe medical complications in infants and immunocompromised individuals. We report here the development of a real-time PCR-based assay for the detection of T. gondii. Oligonucleotide primers and a fluorescence-labeled TaqMan probe were designed to amplify the T. gondii B1 gene. After 40 PCR cycles, the cycle threshold values (C(T)) indicative of the quantity of the target gene were determined. Typically, a C(T) of 25.09 was obtained with DNA from 500 tachyzoites of the T. gondii RH strain. The intra-assay coefficients of variation (CV) were 0.4, 0.16, 0.24, and 0.79% for the four sets of quadruplicate assays, with a mean interassay CV of 0.4%. These values indicate the reproducibility of this assay. Upon optimization of assay conditions, we were able to obtain a standard curve with a linear range (correlation coefficient = 0.9988) across at least 6 logs of DNA concentration. Hence, we were able to quantitatively detect as little as 0.05 T. gondii tachyzoite in an assay. When tested with 30 paraffin-embedded fetal tissue sections, 10 sections (33%) showed a C(T) of <40 and were scored as positive for this test. These results were consistent with those obtained through our nested-PCR control experiments. We have developed a rapid, sensitive, and quantitative real-time PCR for detection of T. gondii. The advantages of this technique for the diagnosis of toxoplasmosis in a clinical laboratory are discussed.

Animals↗
PubMed2000

Regulation of glucose-dependent insulinotropic polypeptide release by protein in the rat.

Glucose-dependent insulinotropic polypeptide (GIP) release has been demonstrated predominantly after ingestion of carbohydrate and fat. These studies were conducted to determine the effects of protein on GIP expression in the rat. Whereas no significant changes in duodenal mucosal GIP mRNA levels were detected in response to peptone, the duodenal GIP concentration increased from 8.4+/-1.5 to 19.8+/-3.2 ng GIP/mg protein at 120 min (P<0.01). Plasma GIP levels also increased from 95+/-5.2 pg/ml to a peak of 289+/-56.1 pg/ml at 120 min (P<0.01). To determine whether the effects of protein on GIP were due to stimulation of acid secretion, rats were pretreated with 10 mg/kg omeprazole, after which mucosal and plasma GIP concentrations were partially attenuated. To further examine the effects of luminal acid, rats were administered intraduodenal 0.1 M HCl for 120 min, which significantly enhanced GIP expression. These studies indicate that nutrient protein provides a potent stimulus for GIP expression in the rat, an effect that occurs at the posttranslational level and may be mediated in part through the acid-stimulatory properties of protein. The effects of acid on GIP are consistent with a role for GIP as an enterogastrone in the rat.

Animals↗
PubMed2000

Role of gut-enriched Krüppel-like factor in colonic cell growth and differentiation.

Cancer cells differ from normal cells in many aspects, including hyperproliferation and loss of differentiation. Recent research has focused on the role of transcription factors in regulating abnormal cell growth. Gut-enriched Krüppel-like factor (GKLF) is a newly identified eukaryotic zinc finger protein expressed extensively in the gastrointestinal tract. In the current study, we demonstrated that GKLF mRNA levels were significantly decreased in the dysplastic epithelium of the colon, including adenomatous polyp and cancer. GKLF immunostains in the normal colon were higher at the surface epithelium and gradually decreased toward the crypt, but this gradient was not present in the adenomatous and cancerous mucosa. Constitutive overexpression of GKLF DNA in a human colonic adenocarcinoma cell line (HT-29) decreased [(3)H]thymidine incorporation, whereas suppression of GKLF gene increased DNA synthesis, indicating that downregulation of the GKLF gene might contribute to cellular hyperproliferation. Cyclin D1 (CD1) protein level and CD1-associated kinase activity were decreased in HT-29 cell overexpressed GKLF cDNA, and CD1 promoter activity was profoundly suppressed by GKLF. When HT-29 cells were cultured in the presence of sodium butyrate, GKLF mRNA levels increased as cells acquired more differentiated phenotypes. These results suggest that GKLF plays an important role in regulating cell growth and differentiation in the colonic epithelium and that downregulation of GKLF expression may cause colonic cells to become hyperproliferative. Furthermore, GKLF appears to be a transcriptional repressor of the CD1 gene.

Adenomatous Polyposis Coli↗
PubMed2000

Role of G protein-coupled receptor kinases in glucose-dependent insulinotropic polypeptide receptor signaling.

The glucose-dependent insulinotropic polypeptide receptor (GIPR) is a member of class II G protein-coupled receptors. Recent studies have suggested that desensitization of the GIPR might contribute to impaired insulin secretion in type II diabetic patients, but the molecular mechanisms of GIPR signal termination are unknown. Using HEK L293 cells stably transfected with GIPR complementary DNA (L293-GIPR), the mechanisms of GIPR desensitization were investigated. GIP dose dependently increased intracellular cAMP levels in L293-GIPR cells, but this response was abolished (65%) by cotransfection with G protein-coupled receptor kinase 2 (GRK2), but not with GRK5 or GRK6. Beta-arrestin-1 transfection also induced a significantly decrease in GIP-stimulated cAMP production, and this effect was greater with cotransfection of both GRK2 and beta-arrestin-1 than with either alone. In betaTC3 cells, expression of GRK2 or beta-arrestin-1 attenuated GIP-induced insulin release and cAMP production, whereas glucose-stimulated insulin secretion was not affected. GRK2 and beta-arrestin-1 messenger RNAs were identified by Northern blot analysis to be expressed endogenously in betaTC3 and L293 cells. Overexpression of GRK2 enhanced agonist-induced GIPR phosphorylation, but receptor endocytosis was not affected by cotransfection with GRKs or beta-arrestin-1. These results suggest a potential role for GRK2/beta-arrestin-1 system in modulating GIP-mediated insulin secretion in pancreatic islet cells. Furthermore, GRK-mediated receptor phosphorylation is not required for endocytosis of the GIPR.

Arrestins↗
PubMed1999

A requirement for phosphatidylinositol 3-kinase in pseudopod extension.

Phagocytosis requires actin assembly and pseudopod extension, two cellular events that coincide spatially and temporally. The signal transduction events underlying both processes may be distinct. We tested whether phagocytic signaling resembles that of growth factor receptors, which induce actin polymerization via activation of phosphatidylinositol 3-kinase (PI 3-kinase). Fcgamma receptor-mediated phagocytosis was accompanied by a rapid increase in the accumulation of phosphatidylinositol 3,4,5-trisphosphate in vivo, and addition of wortmannin (WM) or LY294002, two inhibitors of PI 3-kinase(s), inhibited phagocytosis but not Fcgamma receptor-directed actin polymerization. However, both compounds prevented maximal pseudopod extension, suggesting that PI 3-kinase inhibition produced a limitation in membrane required for pseudopod extension. Availability of plasma membrane was not limiting for phagocytosis, because blockade of ingestion in the presence of WM was not overcome by reducing the number of particles adhering to macrophages. However, decreasing bead size, and hence the magnitude of pseudopod extension required for particle engulfment, relieved the inhibition of phagocytosis in the presence of WM or LY294002 by up to 80%. The block in phagocytosis of large particles occurred before phagosomal closure, because both compounds inhibited spreading of macrophages on substrate-bound IgG. Macrophage spreading on IgG was accompanied by exocytic insertion of membrane from an intracellular source, as measured by the dye FM1-43. These results indicate that one or more isoforms of PI 3 kinase are required for maximal pseudopod extension but not phagocytosis per se. We suggest that PI 3-kinase is required for coordinating exocytic membrane insertion and pseudopod extension.

Androstadienes↗
PubMed1999

The roles of histidine residues at the starch-binding site in streptococcal-binding activities of human salivary amylase.

Human salivary alpha-amylase participates in the initial digestion of starch and may be involved in the colonization of viridans streptococci in the mouth. To elucidate the role of histidine residues located near the starch-binding site on the streptococcal-binding activity, the wild type and three histidine mutants, H52A, H299A and H305A were constructed and expressed in a baculovirus system. While His52 is located near the non-reducing end of the starch-binding pocket (subsite S3/S4), the residues His299 and His305 are located near the subsites S1/S1'. For the wild type, the cDNA encoding the leader and secreted sequences of human salivary amylase was amplified by polymerase chain reaction from a human submandibular salivary-gland cDNA library, and subcloned into the baculovirus shuttle vector pVL1392 downstream of the polyhedrin promoter. Oligonucleotide-based, site-directed mutagenesis was used to generate the mutants expressed in the baculovirus system. Replacing His52 or His299 or His305 to Ala residue did not alter the bacterial-binding activity significantly, but these mutants did show differences in their catalytic activities. The mutant H52A showed negligible reduction in enzymatic activity compared to that of wild type for the hydrolysis of starch and oligosaccharides. In contrast, the H299A and H305A mutants showed a 12 to 13-fold reduction (90-92%) in starch-hydrolysing activity. In addition, the k(cat) for the hydrolysis of oligosaccharides by H299A decreased by as much as 11-fold for maltoheptaoside. This reduction was even higher (40-fold) for the hydrolysis of p-nitrophenyl maltoside, with a significant change in K(M). The mutant H305A, however, exhibited a reduction in k(cat) only, with no changes in the K(M) for the hydrolysis of oligosaccharides. The reduction in the k(cat) for the H305A mutant was almost 93% for maltoheptaoside hydrolysis. The pH activity profile for the H305A mutant was also significantly different from that of the wild type and the other two mutants. These results suggest that, although histidines at the starch-binding site of salivary amylase are involved in starch binding and catalysis, they may not participate in Streptococcus gordonii G9B binding.

Alanine↗
PubMed1999

Double cancers of the kidney and ureter complicated with emphysematous pyelonephritis within the parenchyma of the renal tumour.

Emphysematous pyelonephritis and double cancers of the kidney and urinary tract are rare. We describe here a case of a diabetic man who had simultaneous renal cell carcinoma of the right kidney and transitional cell carcinoma of the right ureter complicated with emphysematous pyelonephritis within the tumour parenchyma of renal cell carcinoma. Imaging and pathology are demonstrated.

Aged↗
PubMed1999

Effects of a nitric oxide scavenger, carboxy-PTIO, on isoflurane MAC and cerebellar nitric oxide synthase activity in rats.

Recent studies have indicated that nitric oxide may play a role in inhalation anaesthesia. Inhibition of nitric oxide synthase reduces the minimum alveolar concentration (MAC) for inhalation anaesthetics and decreases cerebellar nitric oxide synthase (NOS) activity in rats. In this study, we have explored further the role of nitric oxide in isoflurane anaesthesia by examining the effects of a nitric oxide scavenger, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO 0.075-0.6 mg kg-1) on MAC values and cerebellar NOS activity in rats. Bolus injection of carboxy-PTIO at doses greater than 0.15 mg kg-1 reduced the MAC value of isoflurane (mean 1.36 (SEM 0.07)% at 0.15 mg kg-1, 1.39 (0.14)% at 0.3 mg kg-1 and 1.31 (0.06)% at 0.6 mg kg-1 vs control value of 1.61 (0.19)%. Administration of carboxy-PTIO 0.125 and 0.15 mg kg-1 resulted in increased cerebellar NOS activity during isoflurane anaesthesia (P < 0.05). These findings suggest that the level of nitric oxide may set a baseline from which isoflurane then acts.

Anesthetics, Inhalation↗
PubMed1999

Effect of GIP and GLP-1 antagonists on insulin release in the rat.

Glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are potent insulinotropic peptides released from the small intestine. To examine their relative contribution to postprandial insulin release, a specific GIP antagonist (ANTGIP) and a GLP-1 antagonist, exendin-(9-39)-NH2, were infused into rats after an intragastric glucose meal. In control rats, plasma glucose and insulin levels rose gradually during the first 20 min and then decreased. Exendin-(9-39)-NH2 administration inhibited postprandial insulin secretion by 32% at 20 min and concomitantly increased plasma glucose concentrations. In contrast, ANTGIP treatment not only induced a 54% decrease in insulin secretion but also a 15% reduction in plasma glucose levels 20 min after the glucose meal. In vivo studies in rats demonstrated that glucose uptake in the upper small intestine was significantly inhibited by the ANTGIP, an effect that might account for the decrease in plasma glucose levels observed in ANTGIP-treated rats. When the two antagonists were administered to rats concomitantly, no potentiating effect on either insulin release or plasma glucose concentration was detected. Glucose meal-stimulated GLP-1 release was not affected by ANTGIP administration, whereas postprandial glucagon levels were diminished in rats receiving exendin-(9-39)-NH2. The results of these studies suggest that GIP and GLP-1 may share a common mechanism in stimulating pancreatic insulin release. Furthermore, the GIP receptor appears to play a role in facilitating glucose uptake in the small intestine.

Absorption↗
PubMed1998

A requirement for ARF6 in Fcgamma receptor-mediated phagocytosis in macrophages.

Phagocytosis requires extension of F-actin-rich pseudopods and is accompanied by membrane fusion events. Members of the ARF family of GTPases are essential for many aspects of membrane trafficking. To test a role for this family of proteins in Fcgamma receptor-mediated phagocytosis, we utilized the fungal metabolite brefeldin A (BFA). The addition of 100 microM BFA to a subclone of RAW 264.7 macrophages disrupted the appearance and function of the Golgi apparatus as indicated by altered immunofluorescent distribution of beta-COP and reduced efflux of BODIPY C5-ceramide, a phospholipid that normally accumulates in the Golgi apparatus. In contrast, BFA had no effect on phagocytosis of IgG-coated erythrocytes. These results suggested that activation of BFA-sensitive ARFs is not required for phagocytosis. ARF6 is unique among members of the ARF family in that its membrane association is unaffected by BFA. Expression of ARF6 mutants defective in either GTP hydrolysis (Q67L) or binding (T27N) inhibited phagocytosis of IgG-coated erythrocytes and attenuated the focal accumulation of F-actin beneath the test particles. These results indicate a requirement for ARF6 in Fcgamma receptor-mediated phagocytosis and suggest that ARF6 is an important mediator of cytoskeletal alterations after Fcgamma receptor activation.

ADP-Ribosylation Factors↗
PubMed1998

The cysteine of the cytoplasmic tail of glucose-dependent insulinotropic peptide receptor mediates its chronic desensitization and down-regulation.

The glucose-dependent insulinotropic peptide receptor (GIP-R) is a member of G-protein-coupled, seven transmembrane-spanning receptors. Recent studies have shown that elevated serum GIP level in diabetic patients may induce chronic desensitization of the GIP-R, and that this mechanism could contribute to impaired insulin secretion. The cellular basis of down-regulation and chronic desensitization of GIP-R is unclear. To explore the role of the carboxyl terminus of the GIP-R in mediating these processes, five truncated GIP-Rs (T395, T399, T420, T431, T455) were created to delete consecutive serines from the carboxyl end. All mutants except T395 exhibit an identical ligand-binding affinity to the WT receptor. The T395 mutant, which had the entire carboxyl tail removed, does not bind to ligand. Down-regulation and desensitization was assessed by measuring the receptor number and the ability of agonist-induced cAMP or [Ca2+] generation after pre-exposure to 10(-7) M GIP for 24 h. The wild-type (WT) and T421, T431, T455 mutant GIP-Rs are maximally down-regulated by GIP preincubation, whereas T399 mutant does not, indicating that the sequence between amino acids 399 and 420 is critical for this process. Mutation analysis of this area by alanine scanning mutagenesis reveals two critical residues: serine 406 and cysteine 411. Replacement of serine 406 with arginine (S406R) or alanine (S406A) partly attenuates agonist-induced down-regulation and desensitization. In contrast, mutation of the cysteine 411 to glycine (C411G) or alanine (C411A) markedly attenuates both processes. Mutant SCRG, in which both serine 406 and cysteine 411 are mutated, behaves similar to C411G or C4111A. The data suggest that chronic desensitization and down-regulation of the GIP-R may be mediated by similar mechanisms, and that the cysteine in the carboxyl terminus plays an essential role in regulating both processes.

Amino Acid Sequence↗
PubMed1998

Dens invaginatus. Review of formation and morphology with 2 case reports.

Dens invaginatus manifests itself with an aberrant morphologic character because of altered patterns of tooth formation. Presented in this article are 2 cases of dens invaginatus in maxillary lateral incisors that were successfully treated nonsurgically. In the first case, a circular main canal was clearly observed surrounding the invaginated canal. The involved tooth in the second case responded to electric pulp testing, though a periapical radiolucency was evident; root canal treatment of the invaginated canal failed to resolve the pathosis. Debridement of both the main canal and the invaginated canal produced resolution. The complex morphologic nature of these root canal systems and the close relationship between the invaginated and main canals is demonstrated and discussed.

Adolescent↗
PubMed1998

Role of regulator of G protein signaling in desensitization of the glucose-dependent insulinotropic peptide receptor.

The glucose-dependent insulinotropic peptide receptor (GIP-R) is a member of the G protein-coupled receptors. Recent studies have indicated that elevated serum GIP concentrations in type II diabetic patients might induce desensitization of the GIP-R, and this mechanism could contribute to impaired insulin secretion. The cellular and molecular mechanisms governing GIP desensitization are unknown. Here, we report the results of studies on a new family of proteins known as regulators of G protein signaling (RGS) that have been shown to mediate the desensitization process of other receptors. GIP-R and RGS1, -2, -3, and -4 complementary DNAs were cotransfected into human embryonic kidney cells (L293). GIP-stimulated cAMP generation in control cells and in those coexpressing RGS1, -3, and -4 displayed a dose-dependent increase 10 min after GIP treatment. In contrast, RGS2 expression inhibited the GIP-induced cAMP response by 50%, a response similar to that of cells desensitized by preincubation with 10(-7) M GIP. In betaTC3 cells, preincubation of GIP attenuated GIP-induced insulin release by 45% at 15 min and by 55% at 30 min. Expression of RGS2 in the betaTC3 cells significantly decreased GIP-stimulated insulin secretion, whereas glucose-induced insulin release was not affected. RGS2 messenger RNA was identified by Northern blot analysis to be expressed endogenously in betaTC3 and L293 cells, and its level was significantly induced by GIP treatment in betaTC3 cells. Moreover, RGS2 bound Gs alpha protein in an in vitro system, suggesting that RGS2 attenuated the Gs-adenylate cyclase signaling pathway. These results suggest a potential role for RGS2 in modulating GIP-mediated insulin secretion in pancreatic islet cells.

Blotting, Northern↗