Biomedical subjects

T Chen

Publications and source records attributed to T Chen.

At least 163 records · Page 9Linked to original sources

PubMed1999

Characterization of Sam68-like mammalian proteins SLM-1 and SLM-2: SLM-1 is a Src substrate during mitosis.

Sam68, the 68-kDa Src substrate associated during mitosis, is an RNA-binding protein with signaling properties that contains a GSG (GRP33, Sam68, GLD-1) domain. Here we report the cloning of two Sam68-like-mammalian proteins, SLM-1 and SLM-2. These proteins have an approximately 70% sequence identity with Sam68 in their GSG domain. SLM-1 and SLM-2 have the characteristic Sam68 SH2 and SH3 domain binding sites. SLM-1 is an RNA-binding protein that is tyrosine phosphorylated by Src during mitosis. SLM-1 bound the SH2 and SH3 domains of p59(fyn), Grb-2, phospholipase Cgamma-1 (PLCgamma-1), and/or p120(rasGAP), suggesting it may function as a multifunctional adapter protein for Src during mitosis. SLM-2 is an RNA-binding protein that is not tyrosine phosphorylated by Src or p59(fyn). Moreover, SLM-2 did not associate with the SH3 domains of p59(fyn), Grb-2, PLCgamma-1, or p120(rasGAP), suggesting that SLM-2 may not function as an adapter protein for these proteins. The identification of SLM-1 and SLM-2 demonstrates the presence of a Sam68/SLM family whose members have the potential to link signaling pathways with RNA metabolism.

Adaptor Proteins, Signal Transducing↗

PubMed1999

A2B adenosine and P2Y2 receptors stimulate mitogen-activated protein kinase in human embryonic kidney-293 cells. cross-talk between cyclic AMP and protein kinase c pathways.

Mitogen-activated protein kinase (MAPK) cascades underlie long-term mitogenic, morphogenic, and secretory activities of purinergic receptors. In HEK-293 cells, N-ethylcarboxamidoadenosine (NECA) activates endogenous A2BARs that signal through Gs and Gq/11. UTP activates P2Y2 receptors and signals only through Gq/11. The MAPK isoforms, extracellular-signal regulated kinase 1/2 (ERK), are activated by NECA and UTP. H-89 blocks ERK activation by forskolin, but weakly affects the response to NECA or UTP. ERK activation by NECA or UTP is unaffected by a tyrosine kinase inhibitor (genistein), attenuated by a phospholipase C inhibitor (U73122), and is abolished by a MEK inhibitor (PD098059) or dominant negative Ras. Inhibition of protein kinase C (PKC) by GF 109203X failed to block ERK activation by NECA or UTP, however, another PKC inhibitor, Ro 31-8220, which unlike GF 109203X, can block the zeta-isoform, and prevents UTP- but not NECA-induced ERK activation. In the presence of forskolin, Ro 31-8220 loses its ability to block UTP-stimulated ERK activation. PKA has opposing effects on B-Raf and c-Raf-1, both of which are found in HEK-293 cells. The data are explained by a model in which ERK activity is modulated by differential effects of PKC zeta and PKA on Raf isoforms.

Animals↗

PubMed1999

Parathyroid hormone-related protein interacts with RNA.

Parathyroid hormone-related protein (PTHrP) is a secreted protein that acts as an autocrine and paracrine mediator of cell proliferation and differentiation. In addition to its biological activity that is mediated through signal transduction cascades, there is evidence for an intracellular role for PTHrP in cell cycle progression and apoptosis. These effects are mediated through a mid-region nuclear targeting sequence (NTS) that localizes PTHrP to the region of the nucleolus where ribonucleoprotein complexes form in vivo. In this work, we show that endogenous, transfected, and in vitro translated PTHrP proteins bind homopolymeric and total cellular RNAs at salt concentrations up to 1 M. A peptide representing the PTHrP NTS was effective in competing with the wild-type protein for RNA binding, whereas a similar peptide representing the nucleolin NTS was not. Site-directed mutagenesis revealed that the binding of PTHrP to RNA was direct and was dependent on preservation of a core GXKKXXK motif, embedded in the PTHrP NTS, which is shared with other RNA-binding proteins. The current observations are the first to document RNA binding by a secreted cellular protein and predict a role for PTHrP in regulating RNA metabolism that may be related to its localization in the nucleolus of cells in vivo.

Amino Acid Sequence↗

PubMed1999

Elevation of cyclic adenosine 3',5'-monophosphate potentiates activation of mitogen-activated protein kinase by growth factors in LNCaP prostate cancer cells.

Prostate cells are simultaneously exposed to a variety of peptide growth factors and neuropeptides that elevate cAMP. Both the growth factors and cAMP have large effects on the growth, differentiation, and movement of many cell types. Because mitogen-activated protein kinase (MAPK) is central to these effects, we analyzed the ways in which these agonists interact in regulating MAPK in prostate cancer cells. We show that, in LNCaP prostate cancer cells, elevation of intracellular cAMP can potentiate the ability of epidermal growth factor (EGF), interleukin 6, and serum to activate MAPK and that this potentiation depends on protein kinase A and Rap1. The response to cAMP is different in the androgen-independent prostate cancer cell line PC-3, where elevation of cAMP slightly inhibits MAPK activation by EGF. We also show that treatment of LNCaP with the calcium ionophore A23187 or the phorbol ester phorbol 12-myristate 13-acetate activates MAPK, but the activation of MAPK by these agonists is inhibited rather than potentiated by increasing cAMP. Finally, we show that phorbol 12-myristate 13-acetate and interleukin 6 can potentiate the signaling activity of EGF. We conclude that neuroendocrine factors that elevate cAMP sensitize LNCaP prostate cancer cells to signaling by peptide growth factors and that low levels of mixtures of growth factors can activate intracellular signaling to a greater degree than would be predicted from the activity of the individual agonists.

Blood Proteins↗

PubMed1999

ICAM-1 co-stimulation has differential effects on the activation of CD4+ and CD8+ T cells.

T cells play a central role in the initiation, maintenance and regulation of the immune response. Effector responses of T cells are controlled by complex combinations of lymphokines and adhesion/co-stimulatory molecule signals. To isolate the effects of the adhesion/co-stimulatory molecule ICAM-1, we have stimulated purified murine CD4+ and CD8+ T cells with plate-bound anti-CD3 in the presence or absence of plate-bound soluble ICAM-1. In this report, we demonstrate that the co-immobilization of soluble ICAM-1 and anti-CD3 leads to a much greater increase in IL-2 production by CD8+ T cells than CD4+ T cells. The ICAM-1-induced enhancement we observed has differential sensitivity to LFA-1 blockade, depending on the T cell subsets and cytokine evaluated. These effects may play an important role in the generation and modulation of immune responses.

Animals↗

PubMed1999

Evaluation of polymers based on a silicone backbone as pseudostationary phases for electrokinetic chromatography.

The feasibility of polymeric phases based on a silicone polymer backbone as pseudostationary phases for electrokinetic chromatography has been investigated. Silicone phases were studied because of the range of chemistries that could be developed based on these backbones, and because successful development of silicone phases would make it possible to employ much of the stationary phase chemistry developed in the past thirty years. Three silicone polymer structures have been investigated, but only one had sufficient aqueous solubility to permit application in electrokinetic chromatography. This phase was characterized by a variety of methods and was shown to be a mixture of partially hydrolyzed poly(bis-(3-cyanopropyl) siloxanes. When employed as a pseudostationary phase, this material provided selective and efficient separations. The electrophoretic mobility of the silicone polymer is greater than that of sodium dodecyl sulfate (SDS) micelles and poly(sodium 10-undecenylsulfate), providing an extended migration time range. A striking characteristic of the polymer is that the electrophoretic mobility is greater than typical electroosmotic mobilities. The chemical selectivity of the phase is significantly different from that of SDS micelles or poly(sodium 10-undecenylsulfate). The silicone phase is a more cohesive, basic and polar phase than SDS micelles. In buffers modified with a high concentration of organic solvents, the chromatographic properties of the silicone polymer are inferior to those of the poly(sodium 10-undecenylsulfate). The greatest limitation of silicone polymers for this application appears to be limited aqueous solubility, which will make it difficult to realize a family of such polymers with different chemical selectivities.

Acetates↗

PubMed1999

Flow cytometric detection of HLA antibodies using a spectrum of microbeads.

We describe here the use of HLA antigen coated beads for specificity and class determination of HLA antibodies by flow cytometry. The HLA specificity of antibodies was determined by use of beads containing eight levels of fluorescence. HLA antigens isolated from eight cultured cells were coated onto these beads so that each bead was the equivalent of one cell. By using four sets of eight beads, an equivalent of 32 cells could be examined in four test tubes. A total of 76 class I and 25 class II specificities could be determined by the 32 class I bead-panel and 32 class II bead-panel used, respectively. We noted no cross-reactivity of reactions between class I and II. The sensitivity of the test was shown to be higher than that of the standard cytotoxicity by dilution experiments and detection of additional cross-reacting antigens. By use of these coated beads, we achieved improved standardized detection of HLA antibodies. Antigen-coated beads have several advantages over the use of spleens or lymphocytes. (a) A highly selected panel of antigens can be routinely used. (b) Class I and class II antibodies can be readily distinguished from each other, even when they are present as mixtures in one serum. (c) Non-HLA antibodies are not detected because the beads do not have any other antigens than HLA on them. (d) The quantity of antigens coated on beads is more uniform than that found in cells from different individuals. (e) Beads are more convenient for storage and daily use.

Antibodies, Monoclonal↗

PubMed1999

Charged microbeads are not transported across the human stratum corneum in vitro by short high-voltage pulses.

There have been several reports of particle transport due to high-voltage pulsing of human skin. Here, several different short, high-voltage pulsing protocols were used in vitro to study the possible transport of highly charged, fluorescent polystyrene particles (14 nm to 2.1 microns in diameter; surface charges of -4.05 x 10(3) e to -2.77 x 10(7) e) across the skin. Two different methods were used to trap and measure particles on the other side of the skin. The first used a polycarbonate membrane to trap the particles, determining the amount of transport by enumeration under a fluorescence microscope. The second used spectrofluorimetry to measure the amount of particles transported. After pulsing, particles were found in randomly distributed clusters on the surface of the skin. No detectable transport across the stratum corneum for any size particle was observed.

Administration, Cutaneous↗

PubMed1999

The effects of secondary structure and O2 on the formation of direct strand breaks upon UV irradiation of 5-bromodeoxyuridine-containing oligonucleotides.

BACKGROUND: 5-Bromodeoxyuridine is a radiosensitizing agent that is currently being evaluated in clinical trials as an adjuvant in the treatment of a variety of cancers. gamma-Radiolysis and UV irradiation of oligonucleotides containing 5-bromodeoxyuridine result in the formation of direct strand breaks at the 5'-adjacent nucleotide by oxidation of the respective deoxyribose. We investigated the effects of DNA secondary structure and O2 on the induction of direct strand breaks in 5-bromodeoxyuridine-containing oligonucleotides. RESULTS: The efficiency of direct strand break formation in duplex DNA is dependent upon O2 and results in fragments containing 3'-phosphate and the labile 3'-ketodeoxyadenosine termini. The ratio of the 3'-termini is also dependent upon O2 and structure. Deuterium product isotope effects and tritium-transfer studies indicate that hydrogen-atom abstraction from the C1'- and C2'-positions occurs in an O2- and structure-dependent manner. CONCLUSIONS: The reaction mechanisms by which DNA containing 5-bromodeoxyuridine is sensitized to damage by UV irradiation are dependent upon whether the substrate is hybridized and upon the presence or absence of O2. Oxygen reduces the efficiency of direct strand break formation in duplex DNA, but does not affect the overall strand damage. It is proposed that the sigma radical abstracts hydrogen atoms from the C1'- and C2'-positions of the 5'-adjacent deoxyribose moiety, whereas the nucleobase peroxyl radical selectively abstracts the C1'-hydrogen atom from this site. This is the second example of DNA damage amplification by a nucleobase peroxyl radical, and might be indicative of a general reaction pattern for this family of reactive intermediates.

Bromodeoxyuridine↗

PubMed1999

Analysis of volition latency on antisaccadic eye movements.

The antisaccadic paradigm can be applied to test the suppression of reflexive saccades and the activation of volitional saccades simultaneously. The impaired frontal cortex has been shown to have difficulty in suppressing reflexive saccade (prosaccade) to make a successful antisaccade. Degraded antisaccade performance can also be observed in patients with Parkinson's disease (PD). The studies of PD based on the prosaccadic and antisaccadic paradigms have shown controversial findings; the latency between patients and age-matched controls could be either with or without significant difference. Even with this inconsistency, our previous study and recent analysis have supported that the latency of both prosaccade and antisaccade increases significantly for patients with PD. The objective of this study is to investigate whether prolonged antisaccade latency is caused by the affected volitional decision process (volition latency) or simply by the delayed initiation of saccade with direction opposite to the cue, by measuring prosaccade and antisaccade latency from the intermingled paradigms. Eleven mildly affected patients with idiopathic PD and eight age-matched normal subjects were tested in this study. As compared to the age-matched control, the results showed that prosaccade, antisaccadic, and volition latency of the patients was significantly elevated (P<0.01). We conclude that antisaccade performance for the PD patients was degraded for both the volition decision process and the initiation of saccade with direction opposite to the cue. Also, volition latency analysis is a more objective method than prosaccade and antisaccade latency analysis, which can be compared among results obtained from different analysis methodologies.

Aged↗

PubMed1999

Large-scale synthesis of hematoregulatory nonapeptide SK&F 107647 by fragment coupling.

Linear and convergent routes for the large-scale preparation of the hematoregulatory nonapeptide (Glp-Glu-Asp)2-DAS-(Lys)2 (2, SK&F 107647) were investigated. A convergent approach ('3 + 2'-route employing Boc-and benzyl ester protecting groups) was selected for the preparation of multihundred-gram quantities of 2. Key steps were the preparation and the coupling of tripeptide hydrochloride (HCl.H)2-DAS-(Lys(Z)-OBn)2 (6, DAS-2,7-L,L-diaminosuberic acid) and tripeptide Glp-Glu(OBn)-Asp(OBn)-OH (26). Several coupling reagents were investigated in order to reduce the amount of epimerization of this fragment coupling. TDBTU [O-(3,4-dihydro-4-oxo-1,2,3-benzotriazin-3-yl-1,1,3,3-tetrameth yluronium tetrafluoroborate] was identified as the condensation reagent of choice. Using this synthetic route > 97% pure final product in an overall yield of 35% calculated on di-Boc protected 2,7-L,L-diaminosuberic acid was prepared.

Indicators and Reagents↗

PubMed1999

Human cytokine responses induced by gram-positive cell walls of normal intestinal microbiota.

The normal microbiota plays an important role in the health of the host, but little is known of how the human immune system recognizes and responds to Gram-positive indigenous bacteria. We have investigated cytokine responses of peripheral blood mononuclear cells (PBMC) to Gram-positive cell walls (CW) derived from four common intestinal indigenous bacteria, Eubacterium aerofaciens (Eu.a. ), Eubacterium limosum (Eu.l.), Lactobacillus casei (L.c.), and Lactobacillus fermentum (L.f.). Our results indicate that Gram-positive CW of the normal intestinal microbiota can induce cytokine responses of the human PBMC. The profile, level and kinetics of these responses are similar to those induced by lipopolysaccharide (LPS) or CW derived from a pathogen, Streptococcus pyogenes (S.p.). Bacterial CW are capable of inducing production of a proinflammatory cytokine, tumour necrosis factor-alpha (TNF-alpha), and an anti-inflammatory cytokine, IL-10, but not that of IL-4 or interferon-gamma (IFN-gamma). Monocytes are the main cell population in PBMC to produce TNF-alpha and IL-10. Induction of cytokine secretion is serum-dependent; both CD14-dependent and -independent pathways are involved. These findings suggest that the human cytokine responses induced by Gram-positive CW of the normal intestinal microbiota are similar to those induced by LPS or Gram-positive CW of the pathogens.

Antibodies, Monoclonal↗

PubMed1999

A new flavanone isolated from rhizoma smilacis glabrae and the structural requirements of its derivatives for preventing immunological hepatocyte damage.

From the rhizome of Smilax glabra Roxb., a new flavanone was isolated and named as smitilbin (1), together with 6 known compounds, engeletin (2), astilbin (3), dihydroquercetin (4), eurryphin (5), resveratrol (6), and 5-O-caffeoylshikimic acid (7). These compounds were applied to the assay of liver nonparenchymal cells (NPC) against hepatocytes (HC) isolated from mice with an immunological liver injury. Against the NPC-caused elevation of ALT (alanine transminase) in culture supernatant from HC, the pretreatment of NPC with flavanoids (1-3) dose-dependently blocked the ALT release while 4, the aglycone of 3, did not. The chromone 5 showed a much stronger inhibition. Compound 6 also showed the activity. However, 1-7 did not show any suppression of NPC or CCl4-induced ALT release when they were used to pretreat HC. These results suggest that compounds 1-3, 5, and 6 could protect the hepatocyte damage from NPC through selectively producing the dysfunction of NPC with an essential requirement of rhamnose, and the chromone part in their structures may be critical for exhibiting the activity rather than through protecting the hepatocyte membranes.

Animals↗

PubMed1999

Brachydactyly type B: clinical description, genetic mapping to chromosome 9q, and evidence for a shared ancestral mutation.

Autosomal dominant brachydactyly type B (BDB) is characterized by nail aplasia with rudimentary or absent distal and middle phalanges. We describe two unrelated families with BDB. One family is English; the other family is Canadian but of English ancestry. We assigned the BDB locus in the Canadian family to an 18-cM interval on 9q, using linkage analysis (LOD score 3.5 at recombination fraction [theta] 0, for marker D9S938). Markers across this interval also cosegregated with the BDB phenotype in the English family (LOD score 2.1 at straight theta=0, for marker D9S277). Within this defined interval is a smaller (7.5-cM) region that contains 10 contiguous markers whose disease-associated haplotype is shared by the two families. This latter result suggests a common founder among families of English descent that are affected with BDB.

Activin Receptors, Type I↗

PubMed1999

A role for the GSG domain in localizing Sam68 to novel nuclear structures in cancer cell lines.

The GSG (GRP33, Sam68, GLD-1) domain is a protein module found in an expanding family of RNA-binding proteins. The numerous missense mutations identified genetically in the GSG domain support its physiological role. Although the exact function of the GSG domain is not known, it has been shown to be required for RNA binding and oligomerization. Here it is shown that the Sam68 GSG domain plays a role in protein localization. We show that Sam68 concentrates into novel nuclear structures that are predominantly found in transformed cells. These Sam68 nuclear bodies (SNBs) are distinct from coiled bodies, gems, and promyelocytic nuclear bodies. Electron microscopic studies show that SNBs are distinct structures that are enriched in phosphorus and nitrogen, indicating the presence of nucleic acids. A GFP-Sam68 fusion protein had a similar localization as endogenous Sam68 in HeLa cells, diffusely nuclear with two to five SNBs. Two other GSG proteins, the Sam68-like mammalian proteins SLM-1 and SLM-2, colocalized with endogenous Sam68 in SNBs. Different GSG domain missense mutations were investigated for Sam68 protein localization. Six separate classes of cellular patterns were obtained, including exclusive SNB localization and association with microtubules. These findings demonstrate that the GSG domain is involved in protein localization and define a new compartment for Sam68, SLM-1, and SLM-2 in cancer cell lines.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Hprt mutant frequency and molecular analysis of Hprt mutations in Fischer 344 rats treated with thiotepa.

Thiotepa is a bifunctional alkylating anticancer drug that is a rodent carcinogen and a suspected human carcinogen. In order to determine the sensitivity of mutant induction in the Hprt lymphocyte assay for detecting tumorigenic doses of thiotepa, Fischer 344 rats were treated for 4 weeks with thiotepa using a procedure adapted from a carcinogenesis protocol. At various times after beginning the treatment regimen, rats were killed and the lymphocyte Hprt assay was performed on splenic lymphocytes isolated from the animals. The 6-thioguanine-resistant T lymphocyte mutant frequency increased with time during the period of thiotepa exposure and declined slightly thereafter. Significant dose-dependent increases in mutant frequency were found using concentrations of thiotepa that eventually result in lymphoproliferative tumors. Hprt mRNA from mutant lymphocytes was reverse transcribed to cDNA, amplified by PCR and examined for mutations by DNA sequencing. This analysis indicated that the major type of point mutation was G:C-->T:A transversion and that 33% of the mutants contained simple or complex frameshifts. Also, a multiplex PCR performed on DNA from mutant clones that were expanded in vitro indicated that 34% of the clones had deletions in the Hprt gene. These results indicate that the induction of lymphocyte Hprt mutants is a sensitive biomarker for the carcinogenicity of thiotepa and that the types of mutations found in the lymphocyte Hprt gene reflect the kinds of DNA damage produced by thiotepa.

Adenine↗

PubMed1999

Age- and sex-related risk factors for adolescent suicide.

OBJECTIVE: To examine the impact of age and sex on adolescent suicide risk. METHOD: A standard psychological autopsy protocol was used to compare 140 suicide victims with 131 community controls. The risk factors for older (> or = 16 years) and younger, and for male and female suicide were compared. RESULTS: Mood disorders, parental psychopathology, lifetime history of abuse, availability of a gun, and past suicide attempt conveyed significant risk for suicide across all 4 demographic groups. Psychopathology, particularly substance abuse (alone and comorbid with mood disorder), was more common and conveyed a much higher risk for suicide in the older versus younger adolescents. Younger suicide victims showed lower suicidal intent. Males chose more irreversible methods, and conduct disorder was both more prevalent and a more significant risk factor in males. CONCLUSIONS: The increased rate of suicide in older versus younger adolescents is due in part to greater prevalence of psychopathology, namely substance abuse, and greater suicidal intent in the older population. The increased rate in males is less easily explained, but it may stem from method choice and the greater prevalence of and risk conveyed by conduct disorder in males.

Adolescent↗

PubMed1999

Loss of heterozygosity in (LewisxF344)F1 rat urinary bladder tumors induced with N-butyl-N-(4-hydroxybutyl)nitrosamine followed by dimethylarsinic acid or sodium L-ascorbate.

Dimethylarsinic acid (DMA), a main metabolite of arsenicals which are carcinogenic in man, exerts tumor-promoting activity on rat urinary bladder carcinogenesis initiated with N-butyl-N-(4-hydroxybutyl)nitrosamine (BBN). Sodium L-ascorbate (Na-AsA) is also a strong tumor promoter in this animal model. In this study, we used (LewisxF344)F, rats to compare molecular alterations in urinary bladder tumors caused by BBN followed by DMA or Na-AsA. Male, 6-week-old rats were given 0.05% BBN in their drinking water for 4 weeks, and then the rats in group 1 were maintained with no further treatment for 40 weeks. The animals of groups 2 and 3 were administered 0.01% DMA in their drinking water (group 2) or 5% Na-AsA in the powder diet (group 3) after the BBN treatment. Group 4 rats were given 0.05% BBN continuously for 36 weeks. At weeks 12, 20, 36 and 44, subgroups of rats were killed. Histopathological examination revealed promoting activity for DMA and, to a greater extent, Na-AsA on urinary bladder carcinogenesis. Loss of heterozygosity (LOH), detected with the polymerase chain reaction using 36 microsatellite markers, was found to be present in 2 of 9 (22%) urinary bladder tumors after treatment with DMA and 3 of 22 (14%) induced by continuous administration with BBN. No LOH was, however, detected in urinary bladder tumors after treatment with Na-AsA. The results thus suggest that the mechanisms of action of these two promoters, DMA and Na-AsA, may differ in rat urinary bladder carcinogenesis.

Animals↗