Biomedical subjects

Y H Tan

Publications and source records attributed to Y H Tan.

At least 37 records · Page 2Linked to original sources

PubMed2002

Percutaneous nephrolithotomy in the management of complex upper urinary tract calculi: the Singapore General Hospital experience.

INTRODUCTION AND OBJECTIVES: Presently, percutaneous nephrolithotomy (PCNL) is a well-established and effective method of treating upper urinary tract stones at our institution. The aim of this paper was to evaluate a single surgeon's four-year experience of PCNL in the Singapore General Hospital. MATERIALS AND METHODS: Between January 1996 and December 2000, 300 PCNLs were performed on 280 renal units. The mean age was 53.7 years. PCNL was performed on 57 complete staghorn calculi (20.4%), 83 partial staghorn calculi (29.6%), 66 large pelvic calculi (23.6%), 60 impacted uretero-pelvic junction (UPJ) and upper ureteric stones (21.4%) that failed extracorporeal shock wave lithotripsy (ESWL) and 14 symptomatic lower pole calculi (5%). RESULTS: The stone-free rate on discharge was 88.2% (n = 247). At 3 months and 1-year post PCNL, the stone-free rate was 91.1% (n = 255) and 95.7% (n = 268), respectively. The average postoperative stay was 4.5 days. Complications included 1 urosepsis post-PCNL (0.4%) and 2 arterio-venous fistulae (0.7%). Only 1 patient (0.4%) required blood transfusion. Thirty-four patients (12.1%) required ESWL and 4 needed ureteroscopy (1.3%). CONCLUSION: In experienced hands, the use of PCNL for upper urinary tract calculi is safe and effective.

Adult↗

PubMed2001

The hepatitis C virus core protein interacts with NS5A and activates its caspase-mediated proteolytic cleavage.

Viral proteins interact with one another during viral replication, assembly, and maturation. Systematic interaction assays of the hepatitis C virus (HCV) proteins using the yeast two-hybrid method have uncovered a novel interaction between core and NS5A. This interaction was confirmed by in vitro binding assays, and coimmunoprecipitation in mammalian cells. Core and NS5A are also colocalized in COS-7 cells. Interestingly, NS5A is cleaved to give specific-size fragments, when core is coexpressed in mammalian cells. Overexpression of core produced many dying and rounded cells and effects such as DNA laddering and the truncation of poly(ADP-ribose) polymerase 1 (PARP1), both indicators of apoptosis. These observations led us to investigate the link between the induction of apoptosis by core and the cleavage of NS5A. The proteolysis of NS5A and these apoptotic events can be inhibited by caspase inhibitor, Z-VAD, indicating that core induces apoptosis and the cleavage of NS5A by caspases. In cells infected by the HCV, core may provide the intrinsic apoptotic signal, which produces truncated forms of NS5A. The biological function of core-NS5A interaction and the downstream effect of NS5A cleavage are discussed.

Animals↗

PubMed2001

Conserved synteny between the Fugu and human PTEN locus and the evolutionary conservation of vertebrate PTEN function.

Mutations of PTEN, which encodes a protein-tyrosine and lipid phosphatase, are prevalent in a variety of human cancers. The human genome 'draft' sequence still lacks organization and much of the PTEN and adjacent loci remain undefined. The pufferfish, Fugu rubripes, by virtue of having a compact genome represents an excellent template for rapid vertebrate gene discovery. Sequencing of 56 kb from the Fugu pten (fpten) locus identified four complete genes and one partial gene homologous to human genes. Genes neighboring fpten include a PAPS synthase (fpapss2) differentially expressed between non-metastatic/metastatic human carcinoma cell lines, an inositol phosphatase (fminpp1) and an omega class glutathione-S-transferase (fgsto). We have determined the order of human BAC clones at the hPTEN locus and that the locus contains hPAPSS2 and hMINPP1 genes oriented as are their Fugu orthologs. Although the human genes span 500 kb, the Fugu genes lie within only 22 kb due to the compressed intronic and intergenic regions that typify this genome. Interestingly, and providing striking evidence of regulatory element conservation between widely divergent vertebrate species, the compact 2.1 kb fpten promoter is active in human cells. Also, like hPTEN, fpten has a growth and tumor suppressor activity in human glioblastoma cells, demonstrating conservation of protein function.

Amino Acid Sequence↗

PubMed2001

Identification and molecular characterisation of the complete genome of a Singapore isolate of hepatitis C virus: sequence comparison with other strains and phylogenetic analysis.

The complete genomic sequence of a Singapore isolate of the hepatitis C virus (HCV) was obtained from serum of an individual chronically infected with HCV. Nine overlapping cDNA clones covering the entire viral genome was amplified by reverse-transcription-polymerase chain reaction (RT-PCR), This isolate (HCV-S1) comprised 9,609 nucleotides (nt), including 341 nt of the complete 5' untranslated region (5' UTR), a single open reading frame of 3,011 amino acids (aa) and 235 nt of the complete 3' UTR. Its genotype was identified as type lb from analyses of its sequences in the 5' UTR, NS3 and NS5B regions. When compared against nine reported HCV isolates, the overall aa homology of HCV-SI was closest with an Australian strain, HCV-A (94%) and a Japanese strain, HCV-JT (93.9%). Phylogenetic analysis revealed that it was most closely related to the Taiwan strain, HCV-TW and another Japanese strain, HCV-K1-R1.

5' Untranslated Regions↗

PubMed2001

Mutations that affect dimer formation and helicase activity of the hepatitis C virus helicase.

Interaction between viral proteins is necessary for viral replication and viral particle assembly. We used the yeast two-hybrid assay to identify interactions among all the mature proteins of the hepatitis C virus. The interaction between NS3 and NS3 was one of the strongest viral protein-protein interactions detected. The minimal region required for this interaction was mapped to a specific subdomain of 174 amino acids in the N terminus of the helicase region. Random mutations in the minimal region were generated by PCR, and mutants that failed to interact with a wild-type minimal fragment were isolated using the yeast two-hybrid assay as a screen. Three of these mutations resulted in a reduction or a loss of interaction between helicases. Analytical gel filtration showed that in the presence of an oligonucleotide, wild-type helicases form dimers whereas the mutants remain mostly monomeric. All three mutants were partially or almost inactive when assayed for helicase activity in vitro. Mixing a mutant helicase (Y267S) with wild-type helicase did not dramatically affect helicase activity. These data indicate that dimerization of the helicase is important for helicase activity. The mutations that reduce self-association of the helicase may define the key residues involved in NS3-NS3 dimerization.

Amino Acid Sequence↗

PubMed2000

Isolation of a novel protein tyrosine phosphatase inhibitor, 2-methyl-fervenulone, and its precursors from Streptomyces.

High-throughput screening identified an extract from Streptomyces sp. IM 2096 with inhibitory activity toward several protein tyrosine phosphatases (PTPs). Four 1,2,4-triazine compounds 2096A-D (1-4) were isolated from this extract and their structures elucidated by interpretation of spectroscopic data and confirmed by degradation and synthesis. The novel glycocyamidine derivatives 1 and 2 are diastereomers and may interconvert. Both are inactive in the PTP inhibition assay. Compounds 1 and 2 are unstable and partially decompose to 3 and glycocyamidine (5) at room temperature. Compound 3, known as MSD-92 or 2-methyl-fervenulone, is a broad-specificity PTP inhibitor with comparable potency to vanadate. The imidazo[4, 5-e]-1,2,4-triazine (4), inactive in the PTP-inhibition assay, may be a degradation product of 3.

Enzyme Inhibitors↗

PubMed1998

A single plasmid vector (pSTAR) mediating efficient tetracycline-induced gene expression.

A plasmid vector (pSTAR) has been constructed which confers neomycin resistance for selecting stably transfected cells, possesses a cloning cassette for placing a gene of interest under the control of the tetO DNA motif, and expresses rtTAnls which, upon association with tetracycline, binds to and drives gene expression from the tetO DNA motif. The plasmid pSTAR/LacZ, which has the gene for beta-galactosidase inserted into the cloning cassette, was transfected into Chinese hamster ovary (CHO) cells and selected for stably transfected cells. In pooled transfectants of CHO, tetracycline induced the expression of beta-galactosidase in 10-30% of cells. Using clonal transfectants, beta-galactosidase expression was induced by tetracycline in essentially every cell. Furthermore, induction of beta-galactosidase expression by tetracycline was both dose- and time-dependent. Similar tetracycline-induced beta-galactosidase expression is also observed in other cell types. The pSTAR vector is thus suited to facilitate the application of tetracycline-induced gene expression in diverse research areas.

Animals↗

PubMed1998

Mouse PRL-2 and PRL-3, two potentially prenylated protein tyrosine phosphatases homologous to PRL-1.

Protein tyrosine phosphatases (PTPs) play a fundamental role in regulating diverse cellular processes. PRL-1 is a unique nuclear PTP that is induced in mitogen-stimulated cells and regenerating liver. Database searches using the PRL-1 sequence led to the identification of mouse PRL-2 and PRL-3 which exhibit 87% and 76% identity to mouse PRL-1 in their amino acid sequences. All three mouse PRL proteins contain a C-terminal consensus sequence for prenylation. All PRL proteins bear significant sequence homology to Cdc14p and the recently identified tumor suppressor PTEN/MMAC1, in regions other than the conserved PTP signature motif. The nucleotide sequences of the coding regions of mouse PRL-2 and PRL-3 are, respectively, 71% and 62%, identical to mouse PRL-1, while the 5' un-translated regions of mouse PRL-1, PRL-2, and PRL-3 are much more divergent. Northern blot analysis revealed that PRL-2 is preferentially expressed in skeletal muscle, while PRL-3 is preferentially expressed in both skeletal muscle and heart, although both PRL-2 and PRL-3 are expressed at lower levels in other tissues.

Amino Acid Sequence↗

PubMed1998

Biologic activity of oligonucleotides with polarity and anomeric center reversal.

Human papillomavirus (HPV) type 16 E6 and E7 inactivate the tumor suppressors p53 and pRB, respectively. Both viral oncoproteins play important roles in maintaining the transformed phenotype of cells. In this study, we examine the effects of antisense oligodeoxynucleotides with polarity and anomeric center reversal (alpha/beta-ODNs). ODNs of the general structure 5'alphaN3'3'NNN5'5'alphaN3'3'NNNN5'5'alphaN3+ ++'3'N5' were synthesized using phosphoramidite DNA chemistry. These alpha/beta-ODNs were complementary in sequence to regions flanking the start codons of HPV type 16 E6 and E7 genes. The anti-HPV type 16 alpha/beta-ODNs were able to form stable duplexes with their complementary RNA, which then serve as substrates for RNase H hydrolysis. Anti-HPV type 16 alpha/beta-ODNs also specifically inhibited the growth of two cervical carcinoma cell lines, CaSki and SiHa, both of which harbor HPV type 16 DNA. A decrease in E7 protein expression was also observed. Injection of nude mice with SiHa cells induces tumors. Treatment of these tumor-bearing mice with anti-HPV type 16 alpha/beta-ODNs led to substantially smaller tumors. These results show that alpha/beta-ODNs can exert antisense activities both in vitro and in vivo on the E6 and E7 genes of HPV type 16.

Animals↗

PubMed1997

Effects of cycloprotobuxine-A on atrial fibrillation.

AIM: To study the effects of cycloprotobuxine-A (Cyc-A) on atrial fibrillation. METHODS: Atrial fibrillations in vivo and in vitro were induced by arrhythmogenic drugs. Action potentials were measured by the standard microelectrode technique. RESULTS: Cyc-A, similar to or slightly stronger than amiodarone (Ami), decreased incidences of atrial fibrillation elicited by CaCl2-acetylcholine in mice and increased doses of aconitine, ouabain, or adrenaline to elicit atrial fibrillation in isolated guinea pig atria. Cyc-A 0.3-100 mumol.L-1 decreased the normal automaticity and 0.3-30 mumol.L-1 attenuated or almost abolished the isoprenaline-induced abnormal increase in automaticity in sinus nodal cells. In isolated left atria, Cyc-A 0.3-30 mumol.L-1 inhibited the abnormal rhythmic activity elicited by adrenaline, prolonged action potential duration (APD) and effective refractory period, and reduced excitability. At 3-30 mumol.L-1, Cyc-A also decreased the maximal velocity of depolarization (Vmax). Cyc-A antagonized the acetylcholine-induced shortening of APD. These electrophysiologic effects were similar to those of amiodarone, but Ami did not affect the Vmax. CONCLUSION: Cyc-A produces a protective effect against experimental atrial fibrillation via a prolongation of repolarization, a decease of automaticity, and an inhibition of excitability.

Acetylcholine↗

PubMed1996

Suc1-associated neurotrophic factor target (SNT) protein is a major FGF-stimulated tyrosine phosphorylated 90-kDa protein which binds to the SH2 domain of GRB2.

In Swiss 3T3 fibroblasts, basic fibroblast growth factor (bFGF) stimulates the unique tyrosine phosphorylation of a protein complex around 90 kDa, as ascertained by high resolution 2-D PAGE and anti-phosphotyrosine blotting. The majority of this complex consists of the protein(s) designated previously as SNT (suc1-associated neurotrophic factor target). Tyrosine phosphorylated SNT binds to both p13suc1 protein and the SH2 domain of Grb2. Binding of SNT to Grb2 is likely to be mediated through the consensus binding motif, pYXN, on SNT. The binding of SNT to p13suc1 is independent of the pYXN motif. Tyrosine phosphorylated SNT is localised in the plasma membrane where it could form a complex with Grb2 and Sos, enabling the initiation of a novel FGF-specific signalling pathway.

3T3 Cells↗

PubMed1996

Casein kinase II associates with Egr-1 and acts as a negative modulator of its DNA binding and transcription activities in NIH 3T3 cells.

Although the activation domains within early growth response gene protein 1 (Egr-1) have been mapped, little is known of the kinases which phosphorylate Egr-1 and how phosphorylation correlates with the transcriptional activity of Egr-1. In this study we report that casein kinase II (CKII) co-immunoprecipitates with Egr-1 from NIH 3T3 cell lysates. The association of Egr-1 and CKII requires the C terminus of Egr-1 and CKII phosphorylates Egr-1 in vitro. The in vitro phosphorylation of Egr-1 by CKII and that induced by serum in vivo was compared by examining the CNBr-digested fragments of the phosphorylated Egr-1. CKII strongly phosphorylates fragments 7 and 10 which cover part of the activation/nuclear localization and DNA binding domains of Egr-1. CKII also phosphorylates, albeit weakly, fragments 5 and 8 which cover part of activation domain and the entire repression domain of Egr-1, respectively. Strong phosphorylation on fragment 10 as well as fragment 5 was also observed in Egr-1 immunoprecipitated from serum-induced, 32P-labeled cells. CKII phosphorylation of Egr-1 resulted in a decrease of its DNA binding as well as its transcriptional activities.

3T3 Cells↗

PubMed1996

Identification of p90, a prominent tyrosine-phosphorylated protein in fibroblast growth factor-stimulated cells, as 80K-H.

Tyrosine phosphorylation of cellular proteins occurs rapidly upon treatment of fibroblasts with acidic or basic fibroblast growth factors (aFGF, bFGF), suggesting a role for protein phosphorylation in the FGF signaling pathway. Stimulation of Swiss 3T3 cells and MRC-5 fibroblasts with bFGF results in the tyrosine phosphorylation of several proteins, of which the most prominent has been designated as p90. The phosphorylation of p90 is observed within 30 s of treating the cells with FGF but not with other growth factors. Microsequencing of p90 resolved on two-dimensional polyacrylamide gel electrophoresis indicated an N-terminal amino acid sequence which corresponded to a protein previously named as 80K-H. Polyclonal antibodies raised against the predicted C terminus of 80K-H recognized p90 on all Western blots. p90 was found to bind specifically to GRB-2-glutathione S-transferase fusion protein and to be immunoreactive with 80K-H antibody. In addition, anti-phosphotyrosine antibodies immunoprecipitated 80K-H from cell lysates of FGF-stimulated but not from control fibroblasts. The biological function of 80K-H is yet unknown. However, from this study and a previous observation of the obligatory dependence of p90 phosphorylation on FGF receptor occupation, it appears that 80K-H is involved in FGF signaling.

3T3 Cells↗

PubMed1996

Recombinant dengue type 1 virus NS5 protein expressed in Escherichia coli exhibits RNA-dependent RNA polymerase activity.

The complete nonstructural NS5 gene of dengue type 1 virus, Singapore strain S275/90 (D1-S275/90) was expressed in Escherichia coli as a glutathione S-transferase (GST) fusion protein (126 kDa). The GST-NS5 fusion protein was purified and the recombinant NS5 protein released from the fusion protein by thrombin cleavage. The recombinant NS5 had a predicted molecular weight of 100 kDa and reacted with antiserum against D1-S275/90 virus in Western blot analysis. The purified recombinant NS5 protein possessed RNA-dependent RNA polymerase activity which was inhibited (>99%) by antibodies against the recombinant NS5 protein. The polymerase product was shown to be a negative-stranded RNA molecule, of template size, which forms a double-stranded complex with the template RNA.

Animals↗

PubMed1996

Activation and association of Stat3 with Src in v-Src-transformed cell lines.

STAT proteins are a group of latent cytoplasmic transcription factors which function as signal transducers and activators of transcription. Stat1 and -2 were originally identified to function in interferon signaling, and Stat1 was also found to be activated by epidermal growth factor (EGF) and other cytokines. New members of the STAT gene family are identified. Among them, Stat3 has 52.5% amino acid sequence homology with Stat1 and is activated by platelet-derived growth factor (PDGF), colony-stimulating factor 1 (CSF-1), EGF, interleukin-6, and other cytokines. Treatment of cells with EGF activates Stat1 and Stat3, which become phosphorylated on tyrosine residues to form homo - or heterodimers and translocate into the nucleus, binding to the sis-inducible element (SIE) in the c-fos promoter. Somatic cell genetic analyses demonstrated that Jaks, a family of nontransmembrane protein tyrosine kinases, are required for the activation of Stat1 and Stat2 in interferon-treated cells. However, little is known about the activation of Stat3 by growth factors. Here we report that in all v-Src-transformed cell lines examined, Stat3 is constitutively activated to bind to DNA and the phosphorylation of tyrosine on Stat3 is enhanced by the induction of v-Src expression. We also report that Src is shown to be associated with Stat3 in vivo, as well as in vitro, and phosphorylates Stat3 in vitro. Stat3 is also activated by CSF-1, possibly through CSF-1 receptor-c Src association in NIH 3T3 cells overexpressing CSF-1 receptors. Together, the data suggest that Src is involved in activation of Stat3 in growth factor signal transduction.

3T3 Cells↗

PubMed1996

[Anti-atrial fibrillation effects of cyclovirobuxine-D and its electrophysiological mechanism studied on guinea pig atria].

Cyclovirobuxine-D (CVB-D) was shown to produce significant and dose-dependent protective effects against atrial fibrillation induced by CaCl2-Ach in mice. On atrial fibrillation induced by aconitine, ouabain or adrenaline in isolated guinea pig atria, the effects of CVB-D were similar to those of amiodarone. CVB-D 0.3-100 mumol.L-1 was shown to depress the automaticity of the isolated guinea pig right atria. In isolated left atria, CVB-D 0.3 mumol.L-1 was found to inhibit the abnormal automaticity elicited by adrenaline, to prolong the duration of action potential and effective refractory period and to reduce excitability. At high concentration (30 mumol.L-1), CVB-D was also found to decrease the maximal velocity of depolarization (Vmax) and to elongate the conduction time of initiation. Amiodarone 0.3-30 mumol.L-1 was shown to closely resemble CVB-D in electrophysiology without effect on Vmax.

Aconitine↗

PubMed1996

Direct evidence for histamine H3 receptor-mediated inhibition of norepinephrine release from sympathetic terminals of guinea pig myocardium.

AIM: To study the histamine H3 receptors mediated inhibition of norepinephrine (NE) release from cardiac sympathetic terminals of guinea pig isolated atria. METHODS: Release of NE induced by electric field stimulation (50 mA, 5 ms) in the bath solution was measured by HPLC-ECD. RESULTS: The release of NE caused by field stimulation was attenuated by (R)-alpha-methyl-histamine (alpha-MeHA, 0.1 nmol.L-1(-10) mumol.L-1) in a concentration-dependent manner. Thioperamide concentration-dependently antagonized the inhibition of alpha-MeHA. Blockade of H1, H2, alpha 2, beta 2-receptors failed to prevent the inhibitory effect of alpha-MeHA. Thioperamide (1 nmol.L-1(-10) mumol.L-1), when used alone, concentration-dependently facilitated the release of NE evoked by field stimulation. CONCLUSION: The presynaptic histamine H3-receptors inhibited the NE release from cardiac sympathetic terminals.

Adrenergic Fibers↗

PubMed1995

Involvement of a putative protein-tyrosine phosphatase and I kappa B-alpha serine phosphorylation in nuclear factor kappa B activation by tumor necrosis factor.

Inhibitors of phosphotyrosyl protein phosphatases, pervanadate and phenylarsine oxide, abrogate tumor necrosis factor (TNF)-induced nuclear factor kappa B (NF-kappa B) nuclear translocation in transformed cell lines (U-937 and Jurkat) and primary fibroblasts (MRC-5 and REF). The inhibitors also abrogate NF-kappa B activation by the phosphoseryl/threonyl protein phosphatase inhibitor okadaic acid in U-937 cells. Inhibition of NF-kappa B activation is not due to a general inhibitory effect since neither pervanadate nor phenylarsine oxide treatment affected the constitutive DNA-binding activity of the transcription factors octamer-1 and cAMP response element-binding protein in U-937 cells, nor did these compounds inhibit the TNF-induced phosphorylation of proteins, viz. hsp-27, eukaryotic initiation factor 4e, and pp19, in MRC-5 fibroblasts. Overexpression of the protein-tyrosine phosphatase HPTP alpha resulted in a constitutive nuclear NF-kappa B-like DNA-binding activity in REF cells. Conversely, treatment of human protein-tyrosine phosphatase alpha-overexpressing cells with phenylarsine oxide led to a loss of the constitutive NF-kappa B activity. The presence of a tyrosine phosphorylation site on the inhibitor of NF-kappa B (I kappa B-alpha) suggested that it could be a target for TNF/okadaic acid-induced tyrosine dephosphorylation. However, no tyrosine phosphorylation was detected on I kappa B-alpha fron unstimulated cells, while TNF/okadaic acid-treated cells showed increased phosphorylation of I kappa B-alpha exclusively at serine residue(s). Treatment of cells with pervanadate inhibited TNF-induced I kappa B-alpha phosphorylation and degradation, whereas the serine protease inhibitors tosylphenylalanyl chloromethyl ketone and N alpha-p-tosyl-L-lysine chloromethyl ketone prevented TNF-induced I kappa B-alpha degradation and NF-kappa B nuclear translocation, but not the TNF-induced phosphorylation of I kappa B-alpha. The data suggest that TNF and okadaic acid induce the activation of a putative protein-tyrosine phosphatase(s), leading to I kappa B-alpha serine phosphorylation and degradation and NF-kappa B nuclear translocation.

Amino Acid Sequence↗