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Biomedical subjects

E Chan

Publications and source records attributed to E Chan.

At least 37 records · Page 2Linked to original sources
PubMed2001

Alcohol hypersensitivity, increased locomotion, and spontaneous myoclonus in mice lacking the potassium channels Kv3.1 and Kv3.3.

The Shaw-like potassium (K(+)) channels Kv3.1 and Kv3.3 are widely coexpressed in distinct neuronal populations in the CNS, possibly explaining the relatively "mild" phenotypes of the Kv3.1 and the Kv3.3 single mutant. Kv3.1-deficient mice show increased cortical gamma- and decreased delta-oscillations (Joho et al., 1997, 1999); otherwise, the Kv3.1-mutant phenotype is relatively subtle (Ho et al., 1997; Sánchez et al., 2000). Kv3.3-deficient mice display no overt phenotype (Chan, 1997). To investigate whether Kv3.1 and Kv3.3 K(+) channels are functionally redundant, we generated the Kv3.1/Kv3.3 double mutant. Kv3.1/Kv3.3-deficient mice were born at the expected Mendelian frequencies indicating that neither Kv3.1 nor Kv3.3 K(+) channels are essential for embryonic development. Although there are no obvious changes in gross brain anatomy, adult Kv3.1/Kv3.3-deficient mice display severe ataxia, tremulous movements, myoclonus, and hypersensitivity to ethanol. Mice appear unbalanced when moving, whereas at rest they exhibit whole-body jerks every few seconds. In spite of the severe motor impairment, Kv3.1/Kv3.3-deficient mice are hyperactive, show increased exploratory activity, and display no obvious learning or memory deficit. Myoclonus, tremor, and ethanol hypersensitivity are only seen in the double-homozygous Kv3.1/Kv3.3-deficient mice, whereas increased locomotor and exploratory activity are also present in double-heterozygous mice. The graded penetrance of mutant traits appears to depend on the number of null alleles, suggesting that some of the distinct phenotypic traits visible in the absence of Kv3.1 and Kv3.3 K(+) channels are unrelated and may be caused by localized dysfunction in different brain regions.

Animals↗
PubMed2001

Lack of complex I activity in human cells carrying a mutation in MtDNA-encoded ND4 subunit is corrected by the Saccharomyces cerevisiae NADH-quinone oxidoreductase (NDI1) gene.

The gene for the single subunit, rotenone-insensitive, and flavone-sensitive internal NADH-quinone oxidoreductase of Saccharomyces cerevisiae (NDI1) can completely restore the NADH dehydrogenase activity in mutant human cells that lack the essential mitochondrial DNA (mtDNA)-encoded subunit ND4. In particular, the NDI1 gene was introduced into the nuclear genome of the human 143B.TK(-) cell line derivative C4T, which carries a homoplasmic frameshift mutation in the ND4 gene. Two transformants with a low or high level of expression of the exogenous gene were chosen for a detailed analysis. In these cells the corresponding protein is localized in mitochondria, its NADH-binding site faces the matrix compartment as in yeast mitochondria, and in perfect correlation with its abundance restores partially or fully NADH-dependent respiration that is rotenone-insensitive, flavone-sensitive, and antimycin A-sensitive. Thus the yeast enzyme has become coupled to the downstream portion of the human respiratory chain. Furthermore, the P:O ratio with malate/glutamate-dependent respiration in the transformants is approximately two-thirds of that of the wild-type 143B.TK(-) cells, as expected from the lack of proton pumping activity in the yeast enzyme. Finally, whereas the original mutant cell line C4T fails to grow in medium containing galactose instead of glucose, the high NDI1-expressing transformant has a fully restored capacity to grow in galactose medium. The present observations substantially expand the potential of the yeast NDI1 gene for the therapy of mitochondrial diseases involving complex I deficiency.

Binding Sites↗
PubMed2001

A transcription function for the T cell-specific adapter (TSAd) protein in T cells: critical role of the TSAd Src homology 2 domain.

T cell-specific adapter (TSAd) protein is an Src homology 2 (SH2) domain-containing adapter molecule implicated in T cell receptor for antigen (TCR)-mediated interleukin 2 (IL-2) secretion in T cells. Here, we demonstrate that a substantial fraction of TSAd is found in the T cell nucleus. Nuclear import of TSAd is an active process that depends on TSAd SH2 domain recognition of a phosphotyrosine-containing ligand. Importantly, we show that TSAd can act as a potent transcriptional activator in T cells. Furthermore, the TSAd SH2 domain appears to be essential for this transcription-activating function independent of its role in nuclear import. Biochemical analyses suggest that a single TSAd SH2 domain ligand of 95-100 kD may be involved in these processes. Consistent with a role as a transcription activator, cotransfection of TSAd with an IL-2 promoter-reporter gene construct results in a considerable upregulation of IL-2 promoter activity. Further, we show that this augmentation requires a functional TSAd SH2 domain. However, TSAd does not appear to modulate the activity of the major recognized IL-2 gene transcription factors, nuclear factor kappaB (NF-kappaB), nuclear factor of activated T cells (NFAT), or activator protein 1 (AP-1). These findings point to the function of TSAd as a novel transcription-regulatory protein in T cells and illustrate the importance of the TSAd SH2 domain in this role.

Active Transport, Cell Nucleus↗
PubMed2001

Down-regulation of matrix Gla protein messenger RNA in human colorectal adenocarcinomas.

Matrix Gla protein (MGP) is a vitamin K-dependent extracellular matrix protein commonly found in a variety of tissues. In this study, we describe the potential use of MGP gene expression as the tumor marker of colorectal cancer. A decrease in expression of the MGP gene was also discovered in colorectal cancer using differential screening of cDNA libraries. The MGP expression in 80 human colorectal adenocarcinomas was quantified by a Northern blot analysis to better define the expression pattern of MGP in colorectal cancer. The expression of MGP mRNA was reduced in 63 of 80 (79%) colorectal adenocarcinomas (P<0.001) as compared to the mRNA in adjacent normal tissue, implying that a decrease in MGP expression is associated with colorectal cancer development. The proportion of tumors with downregulated expression of MGP was lower in Duke's A/B than Duke's C/D (34 of 47 versus 26 of 33, respectively) tumors and was lower in moderate differentiation than poor differentiation (44 of 64 versus 16 of 16, respectively). However, chi(2) analysis does not reveal any correlation between a loss of MGP expression and tumor progression or differentiation state. In conclusion, the downregulation of MGP mRNA generally occurs in colorectal adenocarcinomas. Although the role of MGP in cancer development is unknown, the reduced expression of MGP may be used to distinguish the normal colorectal cells from malignant cells.

Adenocarcinoma↗
PubMed2001

Tolerance induction by acylated peptides: suppression of EAE in the mouse with palmitoylated PLP peptides.

Treatment of SJL mice either before or after challenge with palmitoylated PLP139-151 (PAL139-151) completely suppressed or considerably reduced both acute and relapsing stages of EAE induced with PLP139-151. In the presence of Pertussis toxin, treatment with PAL139-151 was less effective, but treatment with a mixture of PAL139-151 and PAL178-191, the palmitoylated PLP epitope to which T cell recognition spreads, resulted in almost complete protection. Proliferation of lymphocytes from treated mice were sharply reduced, and adoptive transfer of lymph node lymphocytes from treated mice to naive recipients resulted in the reduction of the acute phase of EAE and in delayed relapses following challenge. The results suggest that treatment with PAL139-151 leads to both anergy and the generation of regulatory cells.

Adoptive Transfer↗
PubMed2001

Near-field scanner for moving molecules.

We have fabricated using electron beam nanolithography a fixed slit near-field optical scanning device which uses near-field fluorimetry to achieve 200 nm spatial resolution of objects moving over the slits. We explore the basic physics of operating narrow slits in the waveguide cutoff mode and present data from the passage of extended double-stranded DNA molecules passing over the slits as a first example of how this device can be used to do ultrahigh spatial resolution mapping of long polymers.

Algorithms↗
PubMed2001

p15(PAF), a novel PCNA associated factor with increased expression in tumor tissues.

Proliferating cell nuclear antigen (PCNA) is an essential protein in both DNA replication and DNA damage repair. A novel 15 kD protein, p15(PAF), was identified as a PCNA-associated factor in a yeast two-hybrid screen using PCNA as the bait. p15(PAF) is localized primarily in the nucleus. p15(PAF) shares the conserved PCNA binding motif with several other PCNA binding proteins including CDK inhibitor p21. Overexpression of p15(PAF) competes with p21-PCNA binding. Mutation of this motif in p15(PAF) abolished its PCNA-binding activity. Notably, p15(PAF) expression in several types of tumor tissues was significantly increased, especially in esophageal tumors. Like PCNA, p15(PAF) may possess prognostic significance in a broad array of human cancers.

Amino Acid Sequence↗
PubMed2001

Dominant effector genetics in mammalian cells.

We have expressed libraries of peptides in mammalian cells to select for trans-dominant effects on intracellular signaling systems. As an example-and to reveal pharmacologically relevant points in pathways that lead to Taxol resistance-we selected for peptide motifs that confer resistance to Taxol-induced cell death. Of several peptides selected, one, termed RGP8.5, was linked to upregulation of expression of the gene ABCB1 (also known as MDR1, for multiple drug resistance) in HeLa cells. Our data indicate that trans-dominant effector peptides can point to potential mechanisms by which signaling systems operate. Such tools may be useful in functional genomic analysis of signaling pathways in mammalian disease processes.

Amino Acid Motifs↗
PubMed2001

Population pharmacokinetics of carbamazepine in Singapore epileptic patients.

AIMS: To document the population pharmacokinetics of carbamazepine in patients with epilepsy living in Singapore, the majority of whom are of Chinese origin and others of minority races. METHODS: Steady-state plasma carbamazepine concentration data were gathered during routine care from various hospitals in Singapore for patients with epilepsy. Age, body weight, gender, race, formulation and concurrent medication (for other illnesses) were the fixed effects (covariates) tested simultaneously for their influence on the population mean of carbamazepine clearance, using the nonlinear mixed-effects model, in the NONMEM program. RESULTS: No age, gender, race, or formulation-related effect was found. Body weight (W), age (A) and concurrent medication with phenobarbitone (PB) emerged as the determinants of carbamazepine clearance (CL). The final regression model for carbamazepine clearance found best to describe the data was CL = 40.7 x A(0.494) x W(-1.17) x 1.44PB where CL is in l day(-1) kg(-1), A is in years, W is in kg and PB = 0 for a patient on carbamazepine only and PB = 1 for a patient on concomitant PB. The corresponding interindividual variability (CV%) in CL, described by using an exponential model, was 21.4%, and the residual error, described by using an exponential error model, was 18.2%. Predictive performance of this population covariate model was evaluated by Bayesian forecasting in a similar, but independent cohort of patients. There was no statistically significant bias between predicted and measured plasma carbamazepine concentrations. The population mean value of carbamazepine clearance obtained was similar to that previously reported for patients with a very different ethnic (Caucasians and Blacks) or geographical background (South Africa, Europe and USA). CONCLUSIONS: The derived covariate regression model reasonably predicted concentrations in the separate validation Singapore patient data set. The correlation between carbamazepine clearance and patient-specific characteristics may thus allow dosage adjustment to be made to achieve target steady-state plasma concentrations.

Adolescent↗
PubMed2001

Effect of ubidecarenone on warfarin anticoagulation and pharmacokinetics of warfarin enantiomers in rats.

Interaction between the antioxidant ubidecarenone and the oral anticoagulant warfarin enantiomers was investigated in rats. The decreased hypoprothrombinemic response, assessed by means of percent changes of prothrombin complex activity and clotting factor VII activity, to warfarin, was observed following oral administration of 1.5 mg/kg racemic warfarin to rats during an 8-day oral regimen (10 mg/kg daily) of ubidecarenone. The antioxidant had no apparent effect on the in vitro rat serum protein binding of warfarin enantiomers. Treatment with ubidecarenone did not affect the absorption and distribution of the S- and R-enantiomers of warfarin, but produced a significant increase in the total serum clearance values of both R- and S-warfarin in rats. This effect was more pronounced with R-warfarin than with S-warfarin. The increased clearance values are attributable to acceleration of certain metabolic pathways and renal excretion of the warfarin enantiomers.

Animals↗
PubMed2001

Laforin is a cell membrane and endoplasmic reticulum-associated protein tyrosine phosphatase.

Lafora disease (LD) is the only progressive myoclonus epilepsy with polyglucosan bodies. Among conditions with polyglucosan bodies, LD is unique for the subcellular location of its polyglucosans in neuronal perikarya and dendrites and not in axons. Here we report that the protein encoded by the EPM2A gene, which is mutated in LD, localizes at the plasma membrane and the endoplasmic reticulum and that it is a functional protein tyrosine phosphatase. The significance of these findings in the epilepsy of LD and in the origin and characteristic subcellular location of Lafora bodies is discussed.

Amyotrophic Lateral Sclerosis↗
PubMed2001

Compound heterozygous familial hypercholesterolemia and familial defective apolipoprotein B-100 produce exaggerated hypercholesterolemia.

BACKGROUND: Familial hypercholesterolemia (FH) and familial defective apolipoprotein B-100 (FDB) represent ligand-receptor disorders that are complementary. Individuals with both FH and FDB are unusual. We report a family with both disorders and the impact of the mutations on the phenotypes of the family members. METHODS: We used single strand conformation polymorphism (SSCP) and denaturing gradient gel electrophoresis (DGGE) for genetic analysis of all 18 exons and the promoter region of the LDL receptor and DGGE for genetic analysis of the apolipoprotein B-100 (apo B-100) gene. The functional significance of the apo B-100 mutation was studied using a U937 cell proliferation assay. Fasting serum lipid profiles were determined for the index case and seven first-degree relatives. RESULTS: One of the patient's sisters had a missense mutation (Asp(407)-->Lys) in exon 9 of the LDL receptor and a serum LDL-cholesterol concentration of 4.07 mmol/L. Four other first-degree relatives had hyperlipidemia but no LDL-receptor mutation. However, these subjects had a mutation of the apo B-100 gene (Arg(3500)-->Trp). The cell proliferation rate of U937 cells fed with LDL from other subjects with the same mutation was fourfold less than that of controls. The index case had both FH- and FDB-related mutations. Her serum LDL-cholesterol (9.47 mmol/L) was higher than all other relatives tested. CONCLUSIONS: Existence of both FH and FDB should be considered in families with LDL-receptor mutations in some but not all individuals with hypercholesterolemia or when some individuals in families with FH exhibit exaggerated hypercholesterolemia.

Adolescent↗
PubMed2001

High levels of HER-2 expression alter the ability of epidermal growth factor receptor (EGFR) family tyrosine kinase inhibitors to inhibit EGFR phosphorylation in vivo.

The epidermal growth factor receptor (EGFR) and HER-2 tyrosine kinases have been implicated in the development, progression, and severity of several human cancers and are attractive targets for therapeutic intervention. SU11925 was developed as a small molecule inhibitor of the tyrosine kinase activity of both EGFR and HER-2. In cellular assays, SU11925 exhibited similar potency against EGFR and HER-2, inhibiting EGF-stimulated EGFR autophosphorylation in A431 (human epidermoid carcinoma) cells with an IC(50) of 30 nM and HER-2 phosphorylation in SK-OV-3TP5 (human ovarian carcinoma) cells with an IC(50) of 38 nM. In contrast to its similar activity against the two targets in cellular assays, approximately 10-fold higher plasma concentrations of SU11925 were required to inhibit HER-2 phosphorylation in HER-2-overexpressing tumors compared with EGFR phosphorylation in EGFR-overexpressing tumors in vivo. Consistent with the proposed mechanism of action of this inhibitor, SU11925 inhibited the s.c. growth of EGFR- and HER-2-dependent tumors in athymic mice at doses that produced substantial inhibition of target receptor phosphorylation in vivo. An unexpected finding from these studies was that higher plasma concentrations of SU11925 were required to inhibit EGFR phosphorylation in vivo in tumors that also express high levels of HER-2 than in tumors that express EGFR alone. This observation, which suggests that it is more difficult to inhibit EGFR phosphorylation in vivo in cells that express high levels of HER-2, was confirmed with ZD1839 (Iressa), a selective EGFR inhibitor that also targets the tyrosine kinase catalytic site. The potential clinical implications of this observation are discussed.

Animals↗
PubMed2000

MEK kinase 1 gene disruption alters cell migration and c-Jun NH2-terminal kinase regulation but does not cause a measurable defect in NF-kappa B activation.

MEK kinase 1 (MEKK1) is a 196-kDa mitogen-activated protein kinase (MAPK) kinase kinase that, in addition to regulating the c-Jun NH(2)-terminal kinase (JNK) pathway, is involved in the control of cell motility. MEKK1(-/-) mice are defective in eyelid closure, a TGFalpha-directed process involving the migration of epithelial cells. MEKK1 expression in epithelial cells stimulates lamellipodia formation, a process required for cell movement. In addition, mouse embryo fibroblasts derived from MEKK1(-/-) mice are inhibited in their migration relative to MEKK1(+/+) fibroblasts. MEKK1 is required for JNK but not NF-kappaB activation in response to virus infection, microtubule disruption, and stimulation of embryonic stem cells with lysophosphatidic acid. MEKK1 is not required for TNFalpha or IL-1 regulation of JNK or NF-kappaB activation in macrophages or fibroblasts. Thus, MEKK1 senses microtubule integrity, contributes to the regulation of fibroblast and epithelial cell migration, and is required for activation of JNK but not NF-kappaB in response to selected stress stimuli.

Animals↗
PubMed2000

Characterization of the human DNA methyltransferase splice variant Dnmt1b.

Tissue- and gene-specific patterns of cytosine-DNA methylation are characteristic features of vertebrate genomes. The generation and proper maintenance of DNA methylation patterns are essential for embryonic development, as demonstrated by the lethal phenotypes of mice with either a targeted disruption of Dnmt1, the gene responsible for the maintenance of DNA methylation, or targeted disruption of Dnmt3a or Dnmt3b, the genes involved in generation of newly formed methylation patterns. Recently, a novel mRNA, Dnmt1b, resulting from alternative splicing of Dnmt1 was identified (Hsu, D. W., Lin, M. J., Lee, T. L., Wen, S. C., Chen, X., and Shen, C. K., (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 9751-9756). The abundance of Dnmt1b mRNA was estimated by semiquantitative reverse transcription polymerase chain reaction and was suggested to encode a major C-5 DNA methyltransferase isoform. Here we report characterization of this novel DNA methyltransferase transcript, Dnmt1b, and its protein product in human cell lines and in freshly isolated human peripheral blood mononuclear cells. The abundance of Dnmt1b transcript, as determined by quantitative RNase protection analysis, was determined to range from 6% to 25% of Dnmt1 in human cells. Second generation antisense inhibitors targeted to the 5'- and 3'-ends of Dnmt1 inhibited the accumulation of both Dnmt1 and Dnmt1b in cells. Dnmt1b protein purified from a baculovirus expression system was demonstrated to be a functional DNA methyltransferase, and to have Michaelis constants for both DNA and S-adenosyl-L-methionine similar to baculovirus-expressed Dnmt1. However, antibodies raised against Dnmt1b epitopes demonstrated that Dnmt1b protein was present at approximately 2-5% of the level of Dnmt1 and therefore represents only a minor DNA methyltransferase isoform in human cells.

Amino Acid Sequence↗
PubMed2000

Magnetic resonance thermometry for predicting thermal damage: an application of interstitial laser coagulation in an in vivo canine prostate model.

Magnetic resonance image-guidance for interstitial thermal therapy has proven to be a valuable tool in its traditional role in device localization and, more recently, in monitoring heat deposition within tissue. However, a quantitative understanding of how temperature-time exposure relates to thermal damage is crucial if the predictive value of real-time MR thermal-monitoring is to be fully realized. Results are presented on interstitial laser coagulation of two canine prostate models which are shown to provide an opportunity to evaluate three models of thermal damage based on a threshold maximum temperature, an Arrhenius damage integral, and a temperature-time product. These models were compared to the resultant lesion margin as derived from post-treatment T(1)- and T(2)-weighted MR images, as well as from direct histological evaluation of the excised canine prostate. Histological evaluation shows that the thermal-injury boundary can be predicted from a threshold-maximum temperature of approximately 51 degrees C or an equivalent Arrhenius t(43) period of 200 minutes, but it is not reliably predicted using the temperature-time product. The methods described in this study are expected to have implications for the treatment of benign prostatic hyperplasia and prostate cancer with interstitial laser coagulation, which will be the focus of future human studies.

Animals↗
PubMed2000

Biaxial residual stress states of plasma-sprayed hydroxyapatite coatings on titanium alloy substrate.

Biaxial residual stress states of plasma-sprayed hydroxyapatite coatings (HACs) on titanium alloy substrate as a function of plasma power, powder feed rate and coating thickness were studied by X-ray 'sin2 psi' method. The Young's modulus of hydroxyapatite (HA), required for the stress analysis, was measured from the separated free coating by three-point bending test method. It was found that the directions of principal stresses were in proximity to and perpendicular to the spraying direction. The measured Young's moduli of HACs were much lower than the theoretical value reported. The denser, well-melted HAC exhibited a higher residual stress, as compared with the less dense, poor-melting HAC. The denser coatings could be effected by higher plasma power and lower powder feed rate. Significantly, the thicker 200 microm HAC exhibited higher residual stress than that of the thinner 50 microm HAC. The implications of residual stress in HAC for biomaterials are discussed in detail.

Alloys↗