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Biomedical subjects

C H Chen

Publications and source records attributed to C H Chen.

At least 19 recordsLinked to original sources
PubMed1998

Structural characteristics of brain glutamate decarboxylase in relation to its interaction and activation.

The conformation, stability, cofactor interactions, and activation of a recombinant 65-kDa form of rat brain glutamate decarboxylase (GAD65) were investigated by using UV/visible spectrophotometry, fluorescence spectroscopy, circular dichroism, and differential scanning microcalorimetry. The enzyme was prepared from Sf9 insect cells infected with a recombinant baculovirus containing the entire GAD65 coding region. The UV/visible absorption spectrum of purified holoenzyme (holoGAD) exhibits two peaks in the range of 300-450 nm, which are due to the formation of a Schiff base when pyridoxal phosphate (pyridoxal-P) binds to GAD. Fluorescence emission intensity (excited at 295 or 280 nm) was substantially enhanced when pyridoxal-P was removed from holoGAD and quenched when pyridoxal-P was added to the apoenzyme (apoGAD). These observations implied that a significant enzyme conformational change occurs during the formation of holoGAD. Circular dichroism provided additional evidence for a conformational change, as the ellipticity of both negative (202-242 nm) and positive (188-202 nm) bands decreased when pyridoxal-P was removed from holoGAD. Secondary structure determination estimated that holoGAD contains a higher content of alpha-helix (34% versus 24%) and a lower content of beta-sheet (18% versus 30%) than apo-GAD. Differential scanning microcalorimetry indicated that holoGAD exhibits a much larger enthalpy and a 3 degrees C higher temperature of thermal unfolding than apoGAD, suggesting that holoGAD has a much tighter conformation and greater stability than apoGAD. A model describing the interaction of pyridoxal-P with GAD is presented, which proposes that an intermediate complex involving ionic interaction between the phosphate group of pyridoxal-P and the positive, charged residues in the active site of GAD maintains the pyridoxal-P molecule in an appropriate position in the active center. Simultaneously, this complex formation is accompanied by a moderate enzyme conformational change, providing a favorable configuration that enables the epsilon-amino of the active-site lysine to react with the aldehyde group of pyridoxal-P. The formation of active holoGAD involves a large enzyme conformational change, which leads to increased stability.

Animals↗
PubMed1997

Mediation of Sonic hedgehog-induced expression of COUP-TFII by a protein phosphatase.

A Sonic hedgehog (Shh) response element was identified in the chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) promoter that binds to a factor distinct from Gli, a gene known to mediate Shh signaling. Although this binding activity is specifically stimulated by Shh-N (amino-terminal signaling domain), it can also be unmasked with protein phosphatase treatment in the mouse cell line P19, and induction by Shh-N can be blocked by phosphatase inhibitors. Thus, Shh-N signaling may result in dephosphorylation of a target factor that is required for activation of COUP-TFII-, Islet1-, and Gli response element-dependent gene expression. This finding identifies another step in the Shh-N signaling pathway.

Animals↗
PubMed1997

The coatomer protein beta'-COP, a selective binding protein (RACK) for protein kinase Cepsilon.

Distinct subcellular localization of activated protein kinase C (PKC) isozymes is mediated by their binding to isozyme-specific RACKs (receptors for activated C-kinase). Our laboratory has previously isolated one such protein, RACK1, and demonstrated that this protein displays specificity for PKCbeta. We have recently shown that at least part of the PKCepsilon RACK-binding site on PKCepsilon lies within the unique V1 region of this isozyme (Johnson, J. A., Gray, M. O., Chen, C.-H., and Mochly-Rosen, D. (1996) J. Biol. Chem. 271, 24962-24966). Here, we have used the PKCepsilon V1 region to clone a PKCepsilon-selective RACK, which was identified as the COPI coatomer protein, beta'-COP. Similar to RACK1, beta'-COP contains seven repeats of the WD40 motif and fulfills the criteria previously established for RACKs. Activated PKCepsilon colocalizes with beta'-COP in cardiac myocytes and binds to Golgi membranes in a beta'-COP-dependent manner. A role for PKC in control of secretion has been previously suggested, but this is the first report of direct protein/protein interaction of PKCepsilon with a protein involved in vesicular trafficking.

Amino Acid Sequence↗
PubMed1997

Ontogeny of the immune system: gamma/delta and alpha/beta T cells migrate from thymus to the periphery in alternating waves.

The embryonic thymus is colonized by the influx of hemopoietic progenitors in waves. To characterize the T cell progeny of the initial colonization waves, we used intravenous adoptive transfer of bone marrow progenitors into congenic embryos. The experiments were performed in birds because intravenous cell infusions can be performed more efficiently in avian than in mammalian embryos. Progenitor cells, which entered the vascularized thymus via interlobular venules in the capsular region and capillaries located at the corticomedullary junction, homed to the outer cortex to begin thymocyte differentiation. The kinetics of differentiation and emigration of the T cell progeny were analyzed for the first three waves of progenitors. Each progenitor wave gave rise to gamma/delta T cells 3 d earlier than alpha/beta T cells. Although the flow of T cell migration from the thymus was uninterrupted, distinct colonization and differentiation kinetics defined three successive waves of gamma/delta and alpha/beta T cells that depart sequentially the thymus en route to the periphery. Each wave of precursors rearranged all three TCR Vgamma gene families, but displayed a variable repertoire. The data indicate a complex pattern of repertoire diversification by the progeny of founder thymocyte progenitors.

Adoptive Transfer↗
PubMed1997

B cells in the bursa of Fabricius express a novel C-type lectin gene.

The avian bursa of Fabricius provides an essential microenvironment for B cell development and Ab repertoire expansion by a gene conversion mechanism. To explore regulatory interactions between B lineage cells and the bursal microenvironment, we sought to identify genes encoding cell surface molecules selectively expressed on bursal B cells. We report in this work the identification of the chB1 gene that encodes a C-type lectin molecule that is a distant relative of the mammalian B cell differentiation Ag, CD72. The chB1 gene is expressed by intrabursal B cells and a B cell line, DT40, that also diversifies its Ig V region genes by gene conversion. Two forms of this type II membrane protein, differing in their cytoplasmic domains, are generated by the differential usage of two translational initiation sites. The longer chB1 isoform, which is the most abundant, contains a consensus immunoreceptor tyrosine-based inhibitory motif in its cytoplasmic domain. Cross-linkage of the chB1 molecules inhibits proliferation of bursal B cells and the DT40 cell line. The chB1 lectin-like molecule may thus modulate intrabursal B cell development.

Amino Acid Sequence↗
PubMed1997

Comparison of continuous left ventricular volumes by transthoracic two-dimensional digital echo quantification with simultaneous conductance catheter measurements in patients with cardiac diseases.

Automated border detection enables real-time tracking of left ventricular (LV) volume by 2-dimensional transthoracic echocardiography. This technique has not been previously compared with simultaneously measured continuous LV volumes at rest or during transients in humans. We performed 18 studies in 16 patients (age 50 +/- 15 years, range 22 to 70; ejection fraction 63 +/- 20%, range 15% to 85%) in which continuous LV volumes acquired by digital echo quantification (DEQ) were compared with simultaneous conductance catheter volume obtained by cardiac catheterization. Both volume signals were calibrated by thermodilution-derived cardiac output and ventriculogram-derived ejection fraction. Volume traces acquired at rest were averaged to generate a comparison cycle. The averaged volume waveforms acquired by DEQ and by conductance catheter were similar during all phases of the cardiac cycle and significantly correlated (conductance catheter = slope. DEQ + intercept, slope = 0.94 +/- 0.09, intercept = 5 +/- 8 ml, r2 = 0.86 +/- 0.12, all p <0.0001). Steady-state hemodynamic parameters calculated using either averaged volume signal were significantly correlated. Transient obstruction of the inferior vena cava yielded a 45 +/- 13% decrease in end-diastolic volume. Successful recordings of DEQ volume during preload reduction were obtained in only 50% of studies. End-diastolic volumes from the 2 methods were significantly correlated (mean slope 0.88 +/- 0.31, mean intercept 14 +/- 37 ml, average r2 = 0.89 +/- 0.11, all p <0.01), as were end-systolic volumes: mean slope 0.80 +/- 0.43, intercept = -20 +/- 26 ml, r2 = 0.67 +/- 0.18, all p <0.05). We conclude that automated border detection technique by DEQ is reliable for noninvasive, transthoracic, continuous tracking of LV volumes at steady state, but has limitations in use during preload reduction maneuvers in humans.

Adult↗
PubMed1997

Catfish thrombocytes express an integrin-like CD41/CD61 complex.

A thrombocyte-specific antigen was identified in two closely related catfish, Ictalurus punctatus and Ictalurus furcatus, by monoclonal antibodies 4-20 and 7-2. The antibodies immunoprecipitate two noncovalently associated glycoprotein chains of Mr 180,000 and Mr 95,000. Under reducing conditions the Mr 180,000 chain is resolved into Mr 150,000 and 32,000 subcomponents. Analysis of N-terminal amino acid sequences indicates homology of the Mr 95,000 chain with the beta3 integrin subunit and homology of the Mr 150,000 chain with the alphaIIb integrin subunit. These antibodies induce catfish thrombocyte aggregation and alteration of cell shape. The data indicate conservation of the megakaryocyte/platelet-restricted CD41/CD61 complex in bony fish.

Amino Acid Sequence↗
PubMed1997

The screening of 13 short tandem repeat loci in the Chinese population.

Population studies of 13 short tandem repeat (STR) loci were carried out on Chinese in Taiwan. The STR loci included HUMF13B, HUMF13A01, HUMFES/FPS, HUMFABP, HUMPLA2A1, HUMTPOX, HUMTH01, HUMVWFA31/A, HUMCSFIPO, HUMLPL, HUMGPP3A09, HUMCYAR04 and HUMCD4. DNA samples from 100 unrelated individuals were screened. The STR allele patterns were detected by the fluorescence detector of an automated DNA sequencer. Two PCR amplifications were performed for each STR locus in this study. The first PCR amplification strategy used 26 base pairs of the T7 sequence extension in the 5' end of the forward primer of each STR locus. The second PCR amplification used a dye-labeled T7 primer instead of the forward primer in the first PCR amplification, and the first PCR products as template to produce fluorescent dye-labeled PCR products. PCR products of different STR loci with overlapping allele sizes could be detected in the same lane of the polyacrylamide gel on an automated DNA sequencer using different colored dye-labeled T7 primers. There was no need to directly conjugate the fluorescent dye to individual STR primers. The PCR products were obtained using 2 ng of template DNA in 25 microliters of PCR reaction mixture. No deviations from the Hardy-Weinberg equilibrium were observed for the 13 STR loci. The distributions of these STR alleles were different from those of Caucasians or Blacks. The probability of matching from the combination of the 13 STR loci was 5.9 x 10(-10) for our Chinese population. However, HUMF13B, HUMLPL and HUMCD4 loci were not as highly polymorphic as observed in other populations.

Alleles↗
PubMed1997

An inhibitory fragment derived from protein kinase Cepsilon prevents enhancement of nerve growth factor responses by ethanol and phorbol esters.

We have studied nerve growth factor (NGF)-induced differentiation of PC12 cells to identify PKC isozymes important for neuronal differentiation. Previous work showed that tumor-promoting phorbol esters and ethanol enhance NGF-induced mitogen-activated protein (MAP) kinase activation and neurite outgrowth by a PKC-dependent mechanism. Ethanol also increases expression of PKCdelta and PKCepsilon, suggesting that one these isozymes regulates responses to NGF. To examine this possibility, we established PC12 cell lines that express a fragment encoding the first variable domain of PKCepsilon (amino acids 2-144), which acts as an isozyme-specific inhibitor of PKCepsilon in cardiac myocytes. Phorbol ester-stimulated translocation of PKCepsilon was markedly reduced in these PC12 cell lines. In addition, phorbol ester and ethanol did not enhance NGF-induced MAP kinase activation or neurite outgrowth in these cells. In contrast, phorbol ester and ethanol increased neurite outgrowth and MAP kinase phosphorylation in cells expressing a fragment derived from the first variable domain of PKCdelta. These results demonstrate that PKCepsilon mediates enhancement of NGF-induced signaling and neurite outgrowth by phorbol esters and ethanol in PC12 cells.

Animals↗
PubMed1997

Elucidating mechanisms of thermostabilization of poliovirus by D2O and MgCl2.

To understand a significant reduction in the loss of poliovirus infectivity by D2O and a combination of D2O and MgCl2 at 37-45 degrees C, this paper attempts to elucidate the mechanisms underlying the thermostabilization of poliovirus. Three serotypes of Sabin oral poliovirus vaccine strains were investigated. Temperature-dependent fluorescence emission intensity studies showed that the effects of D2O and MgCl2 on the stability and conformation of poliovirus are correlated with those of the infectivity of poliovirus. Fluorescence steady-state polarization revealed that the conformation of poliovirus capsid is sensitive to D2O medium and MgCl2 salt, and that the rigidity of poliovirus conformation is increased in their presence. The exposure of poliovirus tryptophan residues to water is modified by D2O and MgCl2, as evidenced by changes in fluorescence emission intensity excited at 295 nm. The involvement of hydrogen bonding in the D2O effect was demonstrated by the greatly increased value of relative fluorescence intensity. Conformational alteration was also shown by changes in the positive band (193-230 nm) of circular dichroism spectra. D2O and MgCl2 were also found to reduce the interaction of virus with water as examined by differential scanning microcalorimetry, leading to a decline in the extent of water penetration into the poliovirus capsid. All these observations were found to be more profound in a combination of D2O with MgCl2 than D2O or MgCl2 alone. By inducing a conformation favorable to maintaining the poliovirus assembly and by reducing virus-water interaction to decrease water penetration into the poliovirus capsid, D2O, MgCl2, or D2O-MgCl2 is able to exert its thermostabilization effect. Thus, to maintain the virus assembly and conformation of the virus and to reduce the swelling of the virus capsid are key factors in increasing the thermostability of poliovirus. These two factors are mutually complementary. The latter can provide a favorable environment for the formers and the formers, in turn, lead to the latter.

Calorimetry, Differential Scanning↗
PubMed1997

Estimation of central aortic pressure waveform by mathematical transformation of radial tonometry pressure. Validation of generalized transfer function.

BACKGROUND: Central aortic pressures and waveform convey important information about cardiovascular status, but direct measurements are invasive. Peripheral pressures can be measured noninvasively, and although they often differ substantially from central pressures, they may be mathematically transformed to approximate the latter. We tested this approach, examining intersubject and intrasubject variability and the validity of using a single averaged transformation, which would enhance its applicability. METHODS AND RESULTS: Invasive central aortic pressure by micromanometer and radial pressure by automated tonometry were measured in 20 patients at steady state and during hemodynamic transients (Valsalva maneuver, abdominal compression, nitroglycerin, or vena caval obstruction). For each patient, transfer functions (TFs) between aortic and radial pressures were calculated by parametric model and results averaged to yield individual TFs. A generalized TF was the average of individual functions. TFs varied among patients, with coefficients of variation for peak amplitude and frequency at peak amplitude of 24.9% and 16.9%, respectively. Intrapatient TF variance with altered loading (> 20% variation in peak amplitude) was observed in 28.5% of patients. Despite this, the generalized TF estimated central arterial pressures to < or = 0.2 +/- 3.8 mm Hg error, arterial compliance to 6 +/- 7% accuracy, and augmentation index to within -7% points (30 +/- 45% accuracy). Individual TFs were only marginally superior to the generalized TF for reconstructing central pressures. CONCLUSIONS: Central aortic pressures can be accurately estimated from radial tonometry with the use of a generalized TF. The reconstructed waveform can provide arterial compliance estimates but may underestimate the augmentation index because the latter requires greater fidelity reproduction of the wave contour.

Abdomen↗
PubMed1997

Efficacy of organ preservation media enriched with nonlactate-generating substrate for maintaining tissue viability: a transplantation study.

BACKGROUND: Previously, isotonic solution (IS) enriched with beta-hydroxybutyrate, a nonlactate-generating substrate, was shown to be efficacious in maintaining tissues' ATP and metabolic activity under cold storage. The objective of this study was to verify these findings in terms of tissue viability in vivo. METHODS: Using a rabbit cornea transplantation model, efficacy of IS was studied and compared with McCarey-Kaufman medium, with or without beta-hydroxybutyrate, and other known media (Optisol and Likorol), with different properties and without beta-hydroxybutyrate. After storage at 4 degrees C for 6, 7, and 11 days, the corneas were grafted, and postoperative changes in corneal thickness, and endothelial cell density and morphology were monitored with a pachymeter and specular microscope. RESULTS: The experiments revealed that before grafting, IS effectively controlled tissue swelling for 7 days or longer, comparable to Optisol. When grafted, the IS-stored corneas deturgesced at high rates, with a short half-time (t1/2) for the de-swelling process, and the grafts remained thin and clear with an intact endothelium. This reflects excellent tissue viability. In the controls, the deturgescence process was extremely sluggish, with significantly lower rates and longer t1/2 (P= >0.01-0.05). Immediate swelling for 3-5 days after grafting was also observed with Optisol. These grafts had significant endothelium cell loss of 24-30% after 12 weeks (P= >0.01-0.02). CONCLUSIONS: IS is more efficacious than currently used media for sustaining the viability of donor corneas under cold storage. These findings may serve as a useful basis for future tests of IS in other organ storage systems.

3-Hydroxybutyric Acid↗
PubMed1997

Further evidence of no association between Ser9Gly polymorphism of dopamine D3 receptor gene and schizophrenia.

Dopamine D3 receptor (DRD3) was demonstrated to have important implications in schizophrenia, because it binds antipsychotic drugs and is abundant in the limbic system of the brain. Several groups attempted to find an association between a serine-to-glycine polymorphism at codon 9 of the DRD3 gene (Ser9Gly) and schizophrenia; however, the results were inconsistent. We conducted a case-control association study in Han Chinese schizophrenic patients from Taiwan, to examine the relationship of this serine-to-glycine polymorphism and schizophrenia. We noted no significant differences of genotype distribution, allele frequencies, or homozygosity proportion of this polymorphism between schizophrenic patients (N = 178) and controls (N = 100). When patients were divided according to sex, or presence or absence of family history, the differences were still not significant. Our study does not support the contention that the Ser9Gly polymorphism of the DRD3 gene plays a major role in schizophrenia.

Alleles↗
PubMed1997

Matrix-assisted laser desorption/ionization for sequencing single-stranded and double-stranded DNA.

The DNA sequence of a single-stranded and double-stranded template was determined. The templates were sequenced using the chain termination method and cycle sequencing method and detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The sequencing products were analyzed successfully without the laborious and expensive methods for removal of the template. Direct sequencing of the double-stranded template was achieved with minimal post-reaction purifications, which could be extremely important for mutation analysis and clinical diagnosis. A systematic study of the mechanisms and kinetics of sequencing reactions was also performed. The details of this analysis and directions for future improvements of the quality of sequencing are presented.

Base Sequence↗
PubMed1997

Possible overlapping potentials of the auditory P50 in humans: factor analysis of middle latency auditory evoked potentials.

The auditory P50 in humans may consist of overlapping potentials. To test this hypothesis, we manipulated the conditions of stimulus discrimination and motor response difficulty and evaluated the data by factor analysis. Twenty right-handed males (mean age 27 years) performed the following 4 tasks: (1) a counting task, (2) an easy Go, No-Go task, (3) a difficult Go, No-Go task, and (4) a choice reaction task. Middle latency auditory evoked potentials were obtained with 100 times summation triggered by the onset of the auditory stimulus. Four factors were extracted by factor analysis for a 0-100 ms time period. Factor 1, the maximum factor loading at 91 ms, corresponded to N1, and factor 4, the maximum factor loading at 23 ms, appeared to correspond to P30. The latency of the maximum factor loading in factor 2 was adjacent to that in factor 3, the latency of factor 2 being 12 ms earlier than that of factor 3. Factor 2 and factor 3 latencies were approximately 55 ms which corresponded to the P50. Factor 3 started rising at the point that factor 2 reached the maximum factor loading, and the factor score demonstrated a significant group difference only when analyzed by motor response criteria. These results suggest that the P50 in humans consists of overlapping potentials and that a part of the potential might relate to a motor response process.

Adult↗