Biomedical subjects

Y S Yoo

Publications and source records attributed to Y S Yoo.

At least 19 recordsLinked to original sources

PubMed2004

Simulation and sensitivity analysis of phosphorylation of EGFR signal transduction pathway in PC12 cell model.

The epidermal growth factor receptor (EGFR) signalling pathway is a complex signalling process with a wide network of interactions. The activation of the mitogen-activated protein kinases (MAPKs) cascade by this activated EGFR has been well studied. MAPKs form a highly integrated network, which is essential for certain specialised cell functions. This paper presents a kinetic model for the MAPK pathway downstream of the EGFR using a biochemical simulator. The model includes 30 signalling events and 29 signalling molecules. The time course data were examined for the activation of each signalling component. The simulation provides a large volume of data, by monitoring the kinetics of the signalling components, which were compared experimentally using the PC12 cell line. The kinetic model corresponded well with the experimental results observed in the EGFR induced activation of proteins. An examination of the kinetic analysis of the multiple signalling events provides a quantitative framework for representing the EGFR signalling network.

Animals↗

PubMed2001

Competitive immunoassay for recombinant hirudin using capillary electrophoresis with laser-induced fluorescence detection.

A competitive immunoassay based on capillary electrophoresis (CE) with laser-induced fluorescence (LIF) has been developed for the determination of recombinant hirudin (r-hirudin) in biological mixtures. Hirudin, a thrombin inhibitor, is a polypeptide of 65 amino acids. To check purity levels and perform pharmacokinetic studies of (r-hirudin), specific and reproducible analysis methods are demanded. The work involved the development of separation conditions allowing for routine analysis of plasma samples. In this study, r-hirudin was labeled with fluorescein isothiocyanate (FITC), and FITC-labeled r-hirudin was purified using high-performance liquid chromatography. The purified product was then mixed with the sample followed with the addition of anti-hirudin antibody. Free, antibody-bound, and tagged r-hirudin could be separated within 5 min by CE analysis using uncoated fused-silica capillary with high reproducibility. The developed method can be used to determine r-hirudin with good precision and a detection limit lower than 20 nM. This result demonstrates the feasibility of the CE-LIF immunoassay method for the determination of r-hirudin in plasma samples.

Antibodies↗

PubMed2001

Measurement of testosterone and pregnenolone in nails using gas chromatography-mass spectrometry.

An efficient method for the determination of testosterone and pregnenolone in human nails using gas chromatography-mass spectrometry (GC-MS) with d3-testosterone as an internal standard is described. The method involves alkaline digestion and liquid-liquid extraction, with subsequent conversion to mixed pentafluoropenyldimethylsilyl-trimethylsilyl (flophemesyl-TMS) derivatives for sensitive analysis in the selected-ion monitoring (SIM) mode. The limit of detection (LOD) and limit of quantification (LOQ) were lowered to 0.1 and 0.2 pg/g, respectively, when 100 mg of nail-clippings were used. The mean recoveries of testosterone and pregnenolone were 89.8 and 86.7%, respectively, while good overall precision (% C.V.; 4.5-9.5) and accuracy (% bias; 3.9-8.4) were demonstrated. Linearity as a correlation coefficient was 0.9913 (testosterone) and 0.9965 (pregnenolone). When applied to fingernail and toenail samples from seven healthy men and nine healthy women, testosterone and pregnenolone were positively detected in the concentration range of 0.24-5.80 ng/g. The levels of two steroids studied in the nails were found to be higher in the male subjects than in the female subjects, and except for the toenails of the females, the levels of testosterone were higher than those of pregnenolone.

Adult↗

PubMed2001

Influence of buffer composition and sample pretreatment on efficiency separation for monitoring neuropeptides in plasma using capillary electrophoresis.

More efficient and faster separation conditions for qualitative as well as quantitative analysis of neuropeptides in human plasma using capillary zone electrophoresis (CZE) have been developed. The analysis method for neuropeptides has been improved specifically to study thyroid hormone related neuropetides for the regulation of thyroid disease. In this study, we investigated the pretreatment methods, composition of the running buffer and rinsing procedures between runs in order to obtain more sensitive and faster separation of trace neuropeptides in plasma by CZE. The tested neuropeptides were somatostatin (SOMA), vasopressin (VP), neurotensin (NT), and thyrotropin-releasing hormone (TRH). Plasma samples were pretreated by deproteinization and solid-phase extraction method. The fraction of neuropeptides was reconstituted in 40% acetonitrile followed by ultrafiltration, and then analyzed by CZE. Resolution and sensitivity was improved using the separation buffer composition with 100 mM Tris-phosphate buffer (pH 2.0) while the sensitivity was further improved via a stacking method using the sample buffer of 40% acetonitrile. These sample pretreatment methods and buffer condition permit quantitative analysis on tested neuropeptides at the 20 ng/mL level. The rinsing procedures between runs using 90% ethanol dramatically shortened the rinsing time to 30 min.

Buffers↗

PubMed2001

Capillary electrophoresis of high-molecular chitosan: the natural carbohydrate biopolymer.

Due to its high resolving power and diverse application range, capillary electrophoresis (CE) has been successfully applied to the analysis of carbohydrates. In this paper, a method for the determination of high-molecular chitosan (Mr 200,000) using CE is presented. We studied the optimal condition of buffer pH and type, and column type for determination of chitosan. Optimal CE performance was found when employing 100 mM triethylamine (TEA)-phosphate buffer, pH 2.0 and untreated fused-silica capillary (50 microm x 27 cm) for the chitosan analysis. Under optimum conditions, excellent linear responses were obtained in the concentration range of 1.25-20 microM, with a linear correlation coefficient of 0.9983. The standard deviations of the migration time and peak area were found to be 2.5 and 6.4%, respectively. This method could be readily applied to chitosan determination in real biological samples and commercial products.

Animals↗

PubMed2001

Biochemical roles of testosterone and epitestosterone to 5 alpha-reductase as indicators of male-pattern baldness.

In establishing a theory to predict male-pattern baldness, we investigated the correlation of testosterone, epitestosterone, and dihydrotestosterone with 5alpha-reductase in hair using gas chromatography-mass spectrometry. One hundred milligram hair samples were obtained from a group of balding subjects and their sons, as well as from a corresponding aged-matched, nonbalding group. The ratio of testosterone to epitestosterone was significantly greater (mean 46.41, p < 0.001; mean 35.83, p < 0.001, respectively) in the hair of balding fathers (n = 19, age 28-50 y) and their sons (n = 16, age 8-16 y) than in the hair of the nonbalding control subjects (mean 9.17 and 10.47, respectively). These findings demonstrate that analysis of terminal hair may not only provide a basis for predicting baldness when the subject is still young, but also for preventing and treating male-pattern baldness by controlling the steroid hormone balance.

5-alpha Reductase Inhibitors↗

PubMed2000

Isolation and characterization of a tetrachloroethylene dechlorinating bacterium, Clostridium bifermentans DPH-1.

A tetrachloroethylene (PCE)-degrading gram-positive, endospore forming, anaerobic bacterium, strain DPH-1, was isolated from a contaminated site. The organism was identified as Clostridium bifermentans by 16S rRNA gene sequence analysis and based on its physiological characteristics. Strain DPH-1 could dechlorinate high concentrations of PCE (0.9 mM), via trichloroethylene (TCE) to cis-1,2-dichloroethylene (cDCE) at a rate of 0.43 micromol/h.mg protein, as well as a number of other halogenated aliphatic compounds.

Journal Article↗

PubMed2000

Characterization of epidermal growth factor receptor function in lysophosphatidic acid signaling in PC12 cells.

Lysophosphatidic acid (LPA) is a lipid metabolite that induces the activation of mitogen-activated protein kinase (MAPK) through binding to the G protein-coupled receptor in a number of cell lines and cultures. Recent studies have revealed that LPA is able to rapidly induce the phosphorylation of MAPK through an epidermal growth factor (EGF) receptor-dependent pathway. We investigated the role of the EGF receptor in the signaling pathway initiated by LPA stimulation in nerve growth factor (NGF)-responsive PC12 cells well known to transiently retract their own neurites upon LPA stimulation. LPA-stimulated MAPK signaling was suppressed by the selective EGF receptor inhibitor and in the dominant negative mutant EGF receptor cell line. As in the EGF signaling pathway, the complex of EGF receptor with adapter proteins Shc and Sos was formed in response to LPA stimulation, suggesting there is an intracellular mechanism for transactivation. A neurite retraction assay was also performed to examine the role of the EGF receptor in PC12 cell differentiation, which related to the involvement of LPA-induced neurite retraction. These results suggest that the receptor tyrosine kinase can be activated in a ligand-independent manner through intracellular crosstalk between the signaling pathways.

Animals↗

PubMed1999

Two interaction modes of the gp41-derived peptides with gp41 and their correlation with antimembrane fusion activity.

Peptides derived from gp41 effectively block the gp41-mediated cell fusion or HIV infection. A 36-mer (naDP178), 51-mer (C51) and 27-mer peptide (C27) from the membrane proximal region of gp41 have been examined their interaction modes with the coiled-coil motif of gp41 presented in thioredoxin (Trx-N) or the bacterially expressed ectodomain of gp41 (Ec-gp41ec). All of these peptides effectively inhibited the gp41-mediated membrane fusion, however, they showed distinct interaction modes with Ec-gp41ec or Trx-N. C51 peptide bound tightly to Trx-N, and it increased the solubility of Ec-gp41ec. naDP178 showed very weak binding affinity to Trx-N, however, it effectively solubilized Ec-gp41ec. In contrast, C27 peptide showed significant binding to Trx-N; however, it did not affect the solubility of Ec-gp41ec. These interaction modes of C-peptides were assumed to be related to their different inhibitory mechanism against gp41-mediated cell fusion.

Amino Acid Motifs↗

PubMed1999

Cyclodextrin-mediated micellar electrokinetic chromatography and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for the enantiomer separation of racemorphan in human urine.

Micellar electrokinetic chromatography (MEKC) was successfully and conveniently applied to the chiral separation with the addition of cyclodextrins (CDs) as chiral selector to the running buffer. Chiral separation depended on the type of CD; in particular, beta-CD was effective for the chiral separation of racemorphan. We investigated the optimal conditions of type and concentration of CD as chiral selector for the routine enantiomeric separation of racemorphan with good reproducibility. The effects of other parameters such as buffer pH and detection wavelength were also investigated to obtain the optimum conditions for the enantiomeric separation of racemorphan. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was used for confirmation of racemorphan. The optimal conditions for enantiomeric separation of the racemorphan were as follows: 50 mM borate buffer at pH 9.4 with 50 mM SDS, 10 mM beta-CD and 20% 1-propanol, 57 cm x 50 microns fused-silica capillary column, and UV detection at 192 nm. Based on the developed method, racemorphan in human urine was also separated and determined using solid-phase extraction and MEKC.

Chromatography, Micellar Electrokinetic Capillary↗

PubMed1999

Determination of enzymatic activity and properties of secretory phospholipase A2 by capillary electrophoresis.

A capillary electrophoretic (CE) system coupled with a diode array UV detector was used for the assay of secretory phospholipase A2 (sPLA2) activity. This method is based on monitoring both the breakdown of substrates and the formation of products simultaneously using micellar electrokinetic chromatographic techniques. Under our developed separation conditions, we analyzed the substrates and products quantitatively, and investigated enzyme activity as a function of reaction time and presence of enzyme activator or inhibitor. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was also utilized to confirm the phosphatidylcholine, a substrate of sPLA2. In order to test the feasibility of the developed method for measurement of enzymatic activity, we compared it to the conventional radioactive assay method for sPLA2. On the basis of our results, the conventional method can be complemented, or even replaced, by this new CE method which possesses the advantages of short analysis time, use of non-radiolabeled and inexpensive substrates, simple measurement of enzymatic activity, and exact quantitation of substrate and product.

Arachidonic Acid↗

PubMed1999

Proliferation not apoptosis as a prognostic indicator in retinoblastoma.

The balance between proliferation and cell death is the major determinant of tumour growth. We analysed the proliferative and apoptotic indices (PI and AI, respectively) of 33 children with retinoblastoma. PI and AI were assessed by immunohistochemistry for Ki-67 antigen and TUNEL staining, respectively. The mean PI was 21.0+/-21.1%, and higher PI was associated with more advanced tumour stage (P<0.0001) and poor clinical outcome (P<0.05). Patients in whom amplified N-myc oncogene was found (n=6) determined by the multiplex polymerase chain reaction tended to have a higher PI (37.6+/-27.2%) than those without amplified N-myc (n=27; PI=17.3+/-18.1). A PI value of over 40% was clearly associated with an unfavourable prognosis. The AI, however, did not correlate with any of the other variables analysed. The findings suggest that proliferation, but not apoptosis, is of critical significance in retinoblastoma biology. PI, as determined by the Ki-67 antigen labelling index, seems to be a relevant histopathological parameter that can predict the clinical outcome of retinoblastoma.

Apoptosis↗

PubMed1997

Application of capillary electrophoresis to determination of enzyme activity and other properties of phosphatidylinositol-specific phospholipase C.

This paper describes a method for monitoring enzymatic activity using micellar electrokinetic chromatography (MEKC). MEKC is used to analyze the activity of phosphatidylinositol-specific phospholipase C (PI-PLC) with convenience and precision. PI specific PLC enzyme converts phosphatidylinositol (PI) to diacylglycerol (DAG) and inositol 1,2-cyclic phosphate (IP). The assay system developed is based on monitoring both the breakdown of substrate and the formation of products simultaneously. To obtain the best separation for the substrate PI and product DAG, we investigated changes in concentration of electrolyte buffer and SDS as well as in pH of the electrolyte buffer. Since the structures of the substrate and products are different, the reaction could be monitored easily by MEKC mostly based on hydrophobicity. Under the separation conditions developed, we investigated the enzymatic activity of PI-PLC depending on the concentrations of phosphatidylinositol, various divalent cations and the reaction temperature. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) was used to confirm the molecular masses of the substrate PI and products DAG and IP. The results of this study show that capillary electrophoresis can be widely applied to analyze and characterize many other enzymes since capillary electrophoresis has several advantages as follows, simplicity, speed, no need for radiolabelled substrate, small sample volumes and sufficient accuracy.

Cations, Divalent↗

PubMed1993

Separation of gangliosides using cyclodextrin in capillary zone electrophoresis.

Gangliosides are glycosphingolipids containing sialic acid. These glycolipids have been suggested to play important roles in biological processes such as cell growth, differentiation and malignant transformation. Based on these proposed biological functions, gangliosides can be used as diagnostic tools and therapeutics for various human diseases. In this study, capillary zone electrophoresis (CZE) was used to determine the major gangliosides GM1, GD1a, GD1b and GT1b in mammalian brains, in addition to GM3 and Lac. Enhancement of selectivity and efficiency of separation was obtained by using 50 mM borate-phosphate buffer containing 16.5 mM alpha-cyclodextrin (alpha-CD). Under this condition, several forms of gangliosides were successfully separated from extracts of deer antler, apricot seed and rat brain. The results demonstrate that the CD-modified CZE is a useful method for detecting glycolipids from various biological matrices.

Animals↗

PubMed1993

Neural regulation of phenylethanolamine N-methyltransferase in vivo: transcriptional and translational changes.

The hypothesis that neural regulation of rat adrenal medullary phenylethanolamine N-methyltransferase (PNMT, EC 2.1.1.28) occurs through transcriptional control is examined by following temporal changes in PNMT mRNA expression using paradigms of acute and chronic reserpine treatment. Concommitant changes in PNMT activity and protein were also measured to determine if reserpine induced changes depend solely on gene activation. Further, changes in adrenal corticosterone were measured to examine whether mRNA and enzyme responses might be mediated via reserpine induced changes in ACTH, and hence, corticosterone. Steady-state levels of PNMT mRNA peaked at approximately 8-fold normal by 6 h after a single reserpine injection (10 mg/kg i.p.), and then declined to control values. With continued treatment, a second, slower rise occurred after three alternate day injections (approximately 3-fold basal levels). Enzyme activity and protein rose simultaneously but were attenuated in magnitude and time course by comparison to message. With both acute and chronic treatment, activity increased 2.0-fold, peaking at 12 h after a single dose of reserpine and again after four doses of the drug. Protein, as measured by immunotitration, was elevated 1.2-and 1.4-fold, respectively. Adrenal corticosterone rose approximately 8-fold at 6 h, declined slightly at 12 h, rose again, and remained elevated thereafter. Comparison of the time courses for the various indices demonstrated that the early parallel bursts in PNMT mRNA and corticosterone are consistent with an increase in transcriptional activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenal Medulla↗

PubMed1993

The effect of thermal biofeedback and progressive muscle relaxation training in reducing blood pressure of patients with essential hypertension.

In order to assess the effectiveness of the thermal biofeedback training combined with the progressive muscle relaxation therapy in the treatment of patients with essential hypertension, blood pressure decline was measured on the treatment group who had the combined thermal biofeedback and progressive muscle relaxation training (N = 11), and on the control group who had only the progressive muscle relaxation training (N = 8). Baseline blood pressure was measured four times for two weeks on both groups. For the treatment group, blood pressure was measured twice before and after each of eight sessions of thermal biofeedback training for four weeks. For the control group, blood pressure was measured every two visits to a clinic for progressive muscle relaxation self-training twice before and after the self-training. A significant decline of the systolic blood pressure by 20.6 mmHg and of the diastolic blood pressure by 14.4 mmhg was observed in the treatment group. There was a tendency for both blood pressures to increase in the control group.

Biofeedback, Psychology↗

PubMed1991

Probing the function of conserved RNA structures in the 30S subunit of Escherichia coli ribosomes.

Ribosomes play an active role in protein biosynthesis. Ribosomal RNA conformation in ribosomal subunits, intramolecular interactions between different rRNA sequences within the confinement of the particles, and intermolecular interactions are presumed necessary to support efficient and accurate protein synthesis. Here we report an analysis of the disposition of 16S rRNA conserved zones centered about positions 525, 1400, and 1500 in 30S subunits. Complementary oligodeoxyribonucleotides in conjunction with nuclease S1 digestion were used to do this. All of the sequences examined in 30S subunits are accessible to DNA probes of 9 to 12 nucleotide residues in length. However, the kinetic characteristics of the respective DNA interactions with 30S particles vary significantly. In addition to the investigation of normal 30S particles, a four base deletion within the 1400 region of 16S rRNA was analyzed. The deletion was made by using synthetic DNAs to target the deletion site for RNase H digestion. The direct in vitro procedure for manipulating rRNA conserves nucleotide modifications. The alteration causes a significant change in the disposition of 16S rRNA in 30S subunits, suggesting a reduction in the freedom of movement of the altered zone in the particle. In a factor-dependent in vitro protein synthesis system primed with MS2 mRNA and altered 30S subunits, there was a 50% decrease in phage coat protein synthesis. The reduction could be due to a decrease in the rate of translation or premature termination of translation. We present evidence here, based on isotopic studies, which supports the latter possibility.

Base Sequence↗

PubMed1991

Genotoxicity of beryllium, gallium and antimony in short-term assays.

The genotoxicity of beryllium, gallium and antimony compounds was studied with the rec, Salmonella mutagenicity and SCE assays. In the rec assay, all the salts of the metals, BeCl2, Be(NO3)2, GaCl3, Ga(NO3)3, SbCl3, SbCl5, and an oxide, Sb2O3, had DNA-damaging activity. None of the compounds was mutagenic to Salmonella. In the SCE assays using V79 cells, 2 antimony(III) compounds, SbCl3 and Sb2O3, and 2 beryllium compounds, BeCl2 and Be(NO3)2, induced SCEs significantly. Sb2O3, slightly soluble in water, was positive in both the rec assay and the SCE assay at very low doses.

Animals↗