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Biomedical subjects

C K Tan

Publications and source records attributed to C K Tan.

At least 37 records · Page 2Linked to original sources
PubMed1999

Architecture of the active DNA polymerase delta.proliferating cell nuclear antigen.template-primer complex.

The relative positions of components of the DNA-dependent DNA polymerase delta (pol delta).proliferating cell nuclear antigen (PCNA).DNA complex were studied. We have shown that pol delta incorporates nucleotides close to a template biotin-streptavidin complex located 5' (downstream) to the replicating complex in the presence or absence of PCNA. PCNA-dependent synthesis catalyzed by pol delta was nearly totally (95%) inhibited by a biotin. streptavidin complex located at the 3'-end of a template with a 15-mer primer (upstream of the replicating complex), but was only partially inhibited with a 19-mer primer. With either primer, PCNA-independent synthesis was not affected by the biotin. streptavidin complex. Quantification of results with primers of varying length suggested that pol delta interacts with between 8 and 10 nucleotides of duplex DNA immediately proximal to the 3'-OH primer terminus. Using UV photocross-linking, we determined that the 125-kDa subunit of pol delta, but not the 50-kDa subunit, interacted with a photosensitive residue of a substrate oligonucleotide. Interaction apparently takes place through the C terminus of p125. Based on these results, we conclude that PCNA is located "behind" pol delta in the polymerization complex during DNA synthesis and that only the large subunit of pol delta (two-subunit form) interacts directly with DNA. A detailed model of the enzymatically active complex is proposed.

Animals↗
PubMed1999

A two-dimensional support for selective binding of polyhistidine-tagged proteins: identification of a proliferating cell nuclear antigen point mutant with altered function in vitro.

Whatman 3MM paper was chemically modified to generate nickel-charged iminodiacetic acid paper (Ni2+-IDA paper). Bacteria were transformed with Escherichia coli expression plasmids coding for either unmodified proliferating cell nuclear antigen (PCNA) or PCNA containing a genetically engineered polyhistidine tract (his-tag) located at its NH2 terminus. They were then grown, induced, and lysed, and macromolecules were transferred to Ni2+-IDA paper. After exhaustive washing, his-tagged PCNA but not unmodified PCNA remained bound to the paper. Moreover, bound his-tagged PCNA was biochemically active in an in situ DNA synthesis assay with exogenous template-primer and purified calf thymus DNA polymerase delta (pol delta). Ni2+-IDA paper was used to identify a PCNA- point mutant that, relative to wild-type PCNA, promotes increased DNA synthesis by pol delta beyond a model abasic template site. In addition, metal-charged IDA paper promises to be generally useful for functional screening of cells expressing cloned proteins.

Animals↗
PubMed1999

Association between serum uric acid and cardiovascular risk factors among elderly people in Taiwan.

In order to understand the distribution of serum uric acid and the relationship between serum uric acid and the cardiovascular risk factor among elderly people, a cross-sectional study was conducted in Chung-Shing-Shin-Tseun community in Taiwan in May 1998. All individuals aged 65 and over were collected. A total of 1123 persons, out of 1774 registered residents, were contacted by face-to-face interview. The response rate was 63.3%. However, only 586 respondents had blood tests and completed questionnaires. Analysis in this study was based on these 586 subjects. The mean uric acid values were 7.4 +/- 1.8 mg/dl in men and 6.3 +/- 1.6 mg/dl in women, respectively (p < 0.001). Multivariate linear regression showed that serum uric acid was significantly correlated with sex and body mass index. Simple correlation showed that serum uric acid was significantly correlated with diastolic pressure, total cholesterol, triglyceride and creatinine. Age, systolic pressure and fasting glucose were not related to serum uric acid. In our conclusion, the uric acid values are high among elderly people. The serum uric acid levels are significantly associated with the cardiovascular risk factors among elderly people.

Age Factors↗
PubMed1998

Translesional synthesis on DNA templates containing an estrogen quinone-derived adduct: N2-(2-hydroxyestron-6-yl)-2'-deoxyguanosine and N6-(2-hydroxyestron-6-yl)-2'-deoxyadenosine.

Miscoding properties induced by estrogen quinone-derived DNA adducts were analyzed using an in vitro experimental system to quantify base substitutions and deletions. Site-specifically modified oligodeoxynucleotides containing a single N2-(2-hydroxyestron-6-yl)-2'-deoxyguanosine (2-OHE1-N2-dG) or N6-(2-hydroxyestron-6-yl)-2'-deoxyadenosine (2-OHE1-N6-dA) were prepared postsynthetically and used as templates in primer extension reactions catalyzed by mammalian DNA polymerases (pol) alpha, beta, and delta. The 2-OHE1-N2-dG adduct blocked primer extension reactions more strongly than 2-OHE1-N6-dA. Using pol alpha and delta, 2-OHE1-N2-dG promoted incorporation of dCMP (6.3 and 3.1%, respectively), the correct base, opposite the lesion: when pol delta was used, misincorporation of dTMP (0.52%) was detected. 2-OHE1-N6-dA also promoted incorporation of dTMP, the correct base, opposite the lesion, accompanied by misincorporation of dCTP (0.54% for pol alpha and 3.2% for pol delta) and one-base deletion (0.3-0.5%). Using pol beta, no miscoding was detected. The miscoding occurred only when replicative DNA polymerases were used. Kinetic data were consistent with those obtained from the analysis of fully extended products formed by pol alpha or pol beta. These results indicate that endogenous estrogen quinone-derived DNA adducts have miscoding potential: G --> A and A --> G transitions and deletions are predicted in mammalian cells.

Animals↗
PubMed1997

Proliferating cell nuclear antigen promotes DNA synthesis past template lesions by mammalian DNA polymerase delta.

Consistent with previous observations, proliferating cell nuclear antigen (PCNA) promotes DNA synthesis by calf thymus DNA polymerase delta (pol delta) past several chemically defined template lesions including model abasic sites, 8-oxo-deoxyguanosine (dG) and aminofluorene-dG (but not acetylaminofluorene-dG). This synthesis is potentially mutagenic. The model abasic site was studied most extensively. When all deoxyribonucleoside triphosphates and a template bearing a model abasic site were present, DNA synthesis by pol delta beyond this site was stimulated 53-fold by addition of homologous PCNA. On an unmodified template (lacking any lesions), PCNA stimulated pol delta by 1.3-fold. Product analysis demonstrated that as expected from the "A-rule," fully and near-fully extended primers incorporated predominantly dAMP opposite the template lesion. Moreover, corollary primer extension studies demonstrated that in the presence (but not the absence) of PCNA, pol delta preferentially elongated primers containing dAMP opposite the model abasic template site. p21, a specific inhibitor of PCNA-dependent DNA replication, inhibits PCNA-stimulated synthesis past model abasic template sites. We propose that DNA synthesis past template lesions by pol delta promoted by PCNA results from the fundamental mechanism by which PCNA stimulates pol delta, i.e., stabilization of the pol delta. template-primer complex.

2-Acetylaminofluorene↗
PubMed1997

The small subunit is required for functional interaction of DNA polymerase delta with the proliferating cell nuclear antigen.

DNA polymerase delta is usually isolated as a heterodimer composed of a 125 kDa catalytic subunit and a 50 kDa small subunit of unknown function. The enzyme is distributive by itself and requires an accessory protein, the proliferating cell nuclear antigen (PCNA), for highly processive DNA synthesis. We have recently demonstrated that the catalytic subunit of human DNA polymerase delta (p125) expressed in baculovirus-infected insect cells, in contrast to the native heterodimeric calf thymus DNA polymerase delta, is not responsive to stimulation by PCNA. To determine whether the lack of response to PCNA of the recombinant catalytic subunit is due to the absence of the small subunit or to differences in post-translational modification in insect cells versus mammalian cells, we have co-expressed the two subunits of human DNA polymerase delta in insect cells. We have demonstrated that co-expression of the catalytic and small subunits of human DNA polymerase delta results in formation of a stable, fully functional heterodimer, that the recombinant heterodimer, similar to native heterodimer, is markedly stimulated (40- to 50-fold) by PCNA and that the increase in activity seen in the presence of PCNA is the result of an increase in processivity. These data establish that the 50 kDa subunit is essential for functional interaction of DNA polymerase delta with PCNA and for highly processive DNA synthesis.

Animals↗
PubMed1997

Expression of Fos-like immunoreactivity in the brain and spinal cord of rats following middle cerebral artery occlusion.

This study examined c-fos protein expression in the brain and spinal cord of rats following permanent occlusion of the middle cerebral artery (MCA) above the rhinal fissure. At 1 h after right-sided MCA occlusion, Fos-like immunoreactivity (Fos-LI) was detected in neurons not only in the ipsilateral cerebral cortex but also in the spinal cord. In the latter, Fos-LI was localized in the nucleus and perikarya of neurons in the grey matter, notably the large motor neurons in the ventral horn. Fos-LI was most intense at 2-4 h, but became undetectable after 48 h in the cerebral cortex and 72 h in the spinal cord. In sham-operated animals, Fos-LI was almost undetectable or virtually absent. It was also not detected in the core territory supplied by the MCA at any time points after arterial occlusion. When the ischaemia-induced neuronal damage in both the cerebral cortex and spinal cord was evaluated by Nissl staining, some neurons appeared atrophic. We conclude that the induction of Fos-LI in neurons of the cerebral cortex and spinal cord is linked respectively to early onset-short stimulation and persistent excitatory or disinhibition phenomenon as a result of focal ischaemic brain injury.

Amino Acid Sequence↗
PubMed1997

Distribution of NADPH-diaphorase and nitric oxide synthase-containing neurons in the intramural ganglia of guinea pig urinary bladder.

The cell population and distribution of NADPH-diaphorase positive and NOS immunoreactive intramural ganglion cells were examined on stretched whole-mount preparations of the guinea pig urinary bladder which was divided into 3 regions: base, body and dome. The results showed that the highest frequency both of NADPH-d and NOS positive neurons was observed in the bladder base. Cell counts in the whole bladder showed that the number of NADPH-d positive neurons was much more than that of NOS immunoreactive neurons. Using neuron specific enolase (NSE) positive neurons as a reference (100%), NADPH-d positive neurons accounted for 84% while NOS immunoreactive neurons only made up 45% of the total neuronal population. These results, along with previous studies on the function of nitric oxide, suggest that nitric oxide may be involved in the relaxation activity in the bladder base during micturition. The significant difference in the number of NADPH-d positive and NOS immunoreactive neurons suggests that the localisation of one enzyme does not necessarily reflect the presence of the other.

Animals↗
PubMed1997

Acute viral hepatitis E: clinical and serologic studies in Singapore.

Seroprevalence of hepatitis E is now documented in many countries around the world, but studies of its clinical manifestations and serologic course have been confined to endemic areas. We have prospectively evaluated the occurrence, evolution, and outcome of acute hepatitis E in our patients. Fifteen patients (11 men, 4 women; median age: 41 years) were diagnosed to have acute, sporadic hepatitis E between July 1993 and January 1995; 10 of the 15 were followed up. Sera anti-hepatitis E virus (HEV) immunoglobulin (Ig)G and IgM antibodies and HEV ribonucleic acid in the blood and stool were tested at weeks 1 and 2; serial tests for hepatitis E antibodies and liver function were carried out at months 1, 3, 6, 9, 12, and 18. Coinfection with hepatitis A and superinfection on chronic hepatitis B were found in 3 and 2 patients, respectively. One patient had transient passage of virus in the stool, but none was viremic. Eighty-seven percent of patients lost their IgM antibodies within 3 months, but anti-HEV IgG, once present, persisted throughout follow-up. All patients but one had complete recovery. A higher than reported level of alanine transaminase (mean: 28.5 times normal) and the lack of viremia during acute infection in our patients may be due to increased immune-mediated viral clearance.

Acute Disease↗
PubMed1997

Electron microscopic study of sprouting dendrites in the ciliary ganglia of cat and monkey (Macaca fascicularis) following pre- and post-ganglionic axotomy.

The present paper reports the ultrastructure of dendritic sprouting and formation of associated synapses in the ciliary ganglion of cat and monkey induced by pre- and post-ganglionic axotomy. In both series of experiments, sprouting dendrites were observed mostly at 3-5 days postoperatively; such profiles were identified by their dense packing of mitochondria and glycogen-like granules. In longitudinal section, such profiles appeared as expanded extensions from the normal-looking dendritic trunks. None were observed to arise directly from the neuronal soma. After preganglionic nerve section, the cross-sectional diameters of such profiles measured 2.2 +/- 1.0 microm (range: 0.9-6.2 microm) in cat and 2.4 +/- 0.7 microm (range: 0.9-5.5 microm) in monkey. After postganglionic nerve section, the cross-sectional diameters of such profiles measured 2.1 +/- 0.7 microm (range: 0.8-4.5 microm) in cat and 2.8 +/- 1.4 microm (range: 1.1-7.0 microm) in monkey. After preganglionic axotomy, in both cat and monkey, the axon terminals began to degenerate at 3 days postoperatively and disappeared by 5 days postoperatively. However, at later postoperative survival periods, the axon terminals reappeared and were observed to make synaptic contacts with the sprouting dendrites. Some of the sprouting dendrites were observed to degenerate, some as early as 3 days postoperatively; such profiles did not appear to have any synapse on them. After postganglionic axotomy, such sprouting dendritic profiles were also observed to make synaptic contacts with axon terminals; some were only closely associated with profiles filled with synaptic vesicles. The results thus suggest that through the formation of new synapses, sprouting of dendrites may have a role to play in neuronal survival after axotomy.

Animals↗
PubMed1996

Proliferating cell nuclear antigen promotes misincorporation catalyzed by calf thymus DNA polymerase delta.

A proliferating cell nuclear antigen (PCNA)-dependent complex, detectable after nondenaturing polyacrylamide gel electrophoresis, is formed between calf thymus DNA polymerase delta (pol delta) and synthetic oligonucleotide template-primers containing a mispaired nucleotide at the 3'-terminal position of the primer. This complex is indistinguishable in composition from that formed with a fully base paired template-primer. Extension of a mispaired primer terminus is a component of DNA polymerase fidelity. The fidelity of pol delta on synthetic oligonucleotide template-primers was compared with and without its specific processivity factor, PCNA. In the absence of PCNA, pol delta misincorporates less than one nucleotide for every 100,000 nucleotides incorporated correctly. Addition of PCNA to reactions reduces fidelity by at least 27-fold. PCNA also confers upon pol delta, the ability to incorporate (and/or not excise) the dTTP analog, 2'-deoxythymidine-5'-O-(alpha-phosphonomethyl)-beta, gamma-diphosphate. A model is proposed whereby the increased stability (decreased off-rate) of the pol delta.template-primer complex in the presence of PCNA facilitates unfavorable events catalyzed by pol delta. This model suggests an explicit mechanistic requirement for the intrinsic 3'-5'-exonuclease of pol delta.

Animals↗
PubMed1996

Purification and characterization of the catalytic subunit of human DNA polymerase delta expressed in baculovirus-infected insect cells.

The catalytic subunit of human DNA polymerase delta has been overexpressed in insect cells by a recombinant baculovirus. The recombinant protein has a Mr = approximately 125,000 and is recognized by polyclonal antisera against N-terminal and C-terminal peptides of the catalytic subunit of human DNA polymerase delta. The recombinant protein was purified to near homogeneity (approximately 1200-fold) from insect cells by chromatography on DEAE-cellulose, phosphocellulose, heparin-agarose, and single-stranded DNA-cellulose. The purified protein had both DNA polymerase and 3'-5' exonuclease activities. The properties of the recombinant catalytic subunit were compared with those of the native heterodimeric DNA polymerase delta isolated from fetal calf thymus, and the enzymes were found to differ in several respects. Although the native heterodimer is equally active with either Mn2+ or Mg2+ as divalent cation activator, the recombinant catalytic subunit is approximately 5-fold more active in Mn2+ than in Mg2+. The most striking difference between the two proteins is the response to the proliferating cell nuclear antigen (PCNA). The activity and processivity of native DNA polymerase delta are markedly stimulated by PCNA whereas it has no effect on the recombinant catalytic subunit. These results suggest that the small subunit of DNA polymerase delta is essential for functional interaction with PCNA.

Animals↗
PubMed1996

The mammalian DNA polymerase delta--proliferating cell nuclear antigen--template-primer complex: molecular characterization by direct binding.

Three direct assays, polyacrylamide gel electrophoresis-band mobility shift, agarose gel electrophoresis-band mobility shift, and nitrocellulose filter binding, were established to study complexes formed among mammalian DNA polymerase delta (pol delta), proliferating cell nuclear antigen (PCNA), and synthetic oligonucleotide template-primers. In all contexts, complex formation requires simultaneous presence of pol delta, PCNA, and template-primer. Moreover, we showed in one such assay that the complex formed contains each molecular component. Nuclease protection experiments demonstrate that complex formation protects template from degradation by DNase I. The mass determined for the pol delta.PCNA.template-primer complex was about 267 kDa, consistent with the participation of one molecule of pol delta, two or three molecules of PCNA and one molecule of template-primer. PCNA alone behaved as a trimer (mass determined to be about 87 kDa). Complex could be manipulated enzymologically. Measurement of off rates demonstrates directly that PCNA stabilizes the pol delta.template-primer complex.

Amino Acid Sequence↗
PubMed1996

Ultrastructural study of external cuneothalamic neurons and their synaptic relationships with primary afferents in the gerbil.

The present study examined the synaptic organization of external cuneothalamic neurons and their relationships with primary afferents in the gerbil external cuneate nucleus (ECN) following an injection of horseradish peroxidase (HRP) into the anterodorsal cap of the ventrobasal thalamus in conjunction with a simultaneous injection of HRP into the contralateral brachial and cervical nerve plexuses. The thalamus-projecting neurons have been shown to be confined to the intermediate portion of the caudal half of the ECN at the light microscopic level (Lan et al., 1994c). In this study, HRP-labelled external cuneothalamic neurons were ultrastructurally characterized by their relatively small-sized soma bearing a variable number of somal spines. Their nucleus had a slightly indented contour with an eccentric nucleolus. The HRP-labelled somata were postsynaptic to many axon terminals, which were classified into round (Rs type; 53.0%), pleomorphic (Ps type; 32.7%), and flattened (Fs type; 14.3%) vesicle-containing boutons. The HRP-labelled dendritic elements were postsynaptic to a greater number of axon terminals, which were also classified into the round (Rd; 64.7%), pleomorphic (Pd; 25.2%), and flattened (Fd; 10.1%) type boutons. These presynaptic axonal boutons tended to synapse on distal and secondary dendrites of external cuneothalamic neurons. In the present simultaneous HRP labelling study, some of the primary afferent terminals made direct synaptic contacts with the dendrites of the external cuneothalamic neurons. In view of the multiple inputs onto the external cuneothalamic neurons, impinging particularly on their somata and secondary dendrites, it is suggested that the proprioceptive information reaching these neurons is intensively modulated and integrated before transmission ultimately to the cerebral sensorimotor cortex.

Animals↗
PubMed1996

Ultrastructural study of phenylethanolamine-N-methyltransferase, corticotropin-releasing factor and neurotensin immunoreactive neurons in the external cuneate nucleus of the gerbil.

The present study examined the existence of catecholamine-, corticotropin-releasing factor (CRF)- and neurotensin (NT)-containing neurons in the external cuneate nucleus (ECN) of the gerbil using single label pre-embedding immunocytochemistry in an attempt to shed light on the increasing evidence for autonomic involvement of the ECN. Peroxidase immunoreactivity of phenylethanolamine-N-methyl-transferase (PNMT), CRF or NT was identified in the heterogeneous population of the ECN neurons characterized by a deeply infolded nucleus. The label was localized in their somata, dendrites, myelinated axons and axon terminals. The immunolabelled dendrites were contacted by spherical (S) and flattened (F) types of presynaptic boutons containing spherical and flattened synaptic vesicles, respectively. The PNMT-labelled dendrites, however, were postsynaptic to an additional type of axon terminals containing pleomorphic (P) synaptic vesicles. Among the immunoreactive axon terminals, the PNMT-labelled boutons consisted of two types: S and F; in the CRF- and NT-labelled axon terminals, only the S type was observed. The catecholamine-containing ECN neurons differed from the CRF- and NT-immunoreactive neurons in their synaptic organization. The latter two were considered to be of the same cell population because of their similarities in ultrastructural features and synaptic relations. In view of a high frequency (48% for PNMT, 50% for CRF and 46% for NT) of the F-typed boutons associated with the three categories of immunolabelled neurons in the ECN, it is possible that they are under considerable inhibitory control. The presence of catecholamine, CRF and NT in the ECN suggests that the nucleus may be involved in the integration of proprioception-, exercise- or stress-evoked autonomic responses.

Animals↗
PubMed1996

An ultrastructural study of the ciliary ganglia of the cat and monkey (Macaca fascicularis) following preganglionic axotomy.

The present study describes ultrastructural changes in the ciliary ganglia of the cat and monkey following preganglionic axotomy. At 3, 5 and 7 days after operation, the nucleus of some neurons was irregular, with prominent indentations, and displaced to the periphery of the neuron. The surface of most neurons was irregular. Neurofilaments and glycogen-like granules were much increased in some neurons. At 21 and 28 days after operation, neurons again appeared normal. Dendritic profiles, packed with many mitochondria and glycogen-like granules, could often be observed from 3 days after operation. In longitudinal section such profiles represented expanded trunks of dendrites; dilated mitochondria and dense bodies were sometimes encountered within them. At later stages after operation, some of these profiles were synaptically contacted by, or closely associated with, axon terminals. In myelinated axons, mitochondria and glycogen-like granules were also increased in number and dilated profiles and dense bodies were found within the axoplasm. In unmyelinated axons, dilated profiles and myelin-like figures were present, as were vesiculo-tubular structures and dense bodies. Electron-dense and -lucent changes could both be observed in myelinated and unmyelinated axons. Almost all the axon terminals were affected 3 days after operation. Within such degenerating axon terminals, the synaptic vesicles had accumulated to form one or several clumps; sometimes the degenerating axon terminals had undergone filamentous hyperplasia. At 45 days after operation, hardly any axon terminals were encountered. Non-neuronal cells, including satellite cells, macrophages and Schwann cells, were actively involved in removing degenerating axons and other cell debris.

Animals↗