Biomedical subjects

H T Chang

Publications and source records attributed to H T Chang.

At least 37 records · Page 2Linked to original sources

PubMed2001

Separation of dsDNA in the presence of electroosmotic flow under discontinuous conditions.

Separations of phiX-174/HaeIII DNA restriction fragments have been performed in the presence of electroosmotic flow (EOF) using five different polymer solutions, including linear polyacrylamide (LPA), poly(ethylene oxide) (PEO), hydroxypropylcellulose (HPC), hydroxyethylcellulose (HEC), and agarose. During the separation, polymer solutions entered the capillary by EOF. When using LPA solutions, bulk EOF is small due to adsorption on the capillary wall. On the other hand, separation is faster and better for the large DNA fragments (> 872 base pairs, bp) using derivative celluloses and PEO solutions. Several approaches to optimum resolution and speed by controlling EOF and/or altering electrophoretic mobility of DNA have been developed, including (i) stepwise changes of ethidium bromide (0.5-5 microg/mL), (ii) voltage programming (125-375 V/cm), (iii) use of mixed polymer solutions, and (iv) use of high concentrations of Tris-borate (TB) buffers. The DNA fragments ranging from 434 to 653 bp that were not separated using 2% PEO (8,000,000) under isocratic conditions have been completely resolved by either stepwise changes of ethidium bromide or voltage programming. Compared to PEO solutions, mixed polymer solutions prepared from PEO and HEC provide higher resolving power. Using a capillary filled with 600 mM TB buffers, pH 10.0, high-speed (< 15 min) separation of DNA (pBR 322/HaeIII digest, pBR 328/ Bg/l digest and pBR 328/Hinfl digest) has been achieved in 1.5% PEO.

Bacteriophage phi X 174↗

PubMed2001

Direct vertical electroelution of protein from a PhastSystem band for mass spectrometric identification at the level of a few picomoles.

An electroelution apparatus prototype of a new design was constructed. In that design, the electric field passes vertically through the protein band located on a horizontal (PhastSystem) minigel polymerized on a net of Gel-Fix (Serva). A simple, home-made apparatus allows for electroelution of protein bands at the level of a few picomoles and their identification, after concentration, by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. The technique is applicable to one-dimensional (1-D) or two-dimensional (2-D) gels of any size, but has been exemplified only by application to 1-D minigels to demonstrate the lower limits of protein load of the method. When in the course of further development of the prototype it will be combined with a modification to two dimensions of the electroelution mechanism under computer control of the high-performance gel electrophoresis apparatus (formerly of LabIntelligence), the new design appears uniquely qualified for an automated spot elution from 2-D gels under avoidance of gel sectioning.

Electrophoresis, Polyacrylamide Gel↗

PubMed2001

Effect of fluoxetine on intracellular Ca2+ levels in bladder female transitional carcinoma (BFTC) cells.

The effect of the antidepressant fluoxetine on Ca2+ signaling in cultured cells was largely unknown. The effect of various concentrations of fluoxetine on [Ca 2+] i in populations of bladder female transitional cancer (BFTC) cells was evaluated by using fura-2 as a Ca2+ probe. Fluoxetine increased [Ca 2+] i concentration dependently (20-100 microM) with an EC50 value of 30 microM. The response was inhibited by 50-60% on extracellular Ca2+ removal. In Ca2+ -free medium, pretreatment with 1 microM thapsigargin (an inhibitor of the endoplasmic reticulum Ca2+ pump) abolished 50 microM fluoxetine-induced Ca2+ release; whereas pretreatment with fluoxetine did not alter the thapsigargin-induced Ca2+ response. Addition of 3 mM Ca2+ increased [Ca 2+] i after pretreatment with 50 microM fluoxetine in Ca2+ -free medium, suggestive of fluoxetine-induced capacitative Ca2+ entry. Suppression of inositol 1,4,5-trisphosphate formation by 2 microM U73122 (a phospholipase C inhibitor) did not affect 50 microM fluoxetine-induced Ca2+ release. Collectively, this study shows that fluoxetine increased [Ca 2+] i in bladder cancer cells in a concentration-dependent fashion, by releasing Ca2+ from thapsigargin-sensitive Ca2+ stores in an IP3-independent manner, and by inducing Ca2+ influx from extracellular medium.

Calcium↗

PubMed2001

Mechanism underlying histamine-induced intracellular Ca2+ movement in PC3 human prostate cancer cells.

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in PC3 human prostate cancer cells and the underlying mechanism were evaluated using fura-2 as a Ca2+ dye. Histamine at concentrations between 0.1 and 50 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 1 microM. The [Ca2+]i response comprised an initial rise and a slow decay, which returned to baseline within 3 min. Extracellular Ca2+ removal inhibited 50% of the [Ca2+]i signal. In the absence of extracellular Ca2+, after cells were treated with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 10 microM histamine did not increase [Ca2+]i. After pretreatment with 10 microM histamine in a Ca2+-free medium for several minutes, addition of 3 mM Ca2+ induced [Ca2+]i increases. Histamine (10 microM)-induced intracellular Ca2+ release was abolished by inhibiting phospholipase C with 2 microM 1-(6-((17 beta-3- methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), and by 10 microM pyrilamine but was not altered by 50 microM cimetidine. Collectively, the present study shows that histamine induced [Ca2+]i transients in PC3 human prostate cancer cells by stimulating H1 histamine receptors leading to Ca2+ release from the endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, and by inducing Ca2+ entry.

Calcium↗

PubMed2001

Tamoxifen-induced Ca2+ mobilization in bladder female transitional carcinoma cells.

This study examined the effect of tamoxifen, an anti-breast cancer drug, on Ca2+ handling in bladder female transitional cancer cells. Changes in cytosolic free Ca2+ levels were recorded by using the Ca2+-sensitive dye fura-2. In a dose-dependent manner, tamoxifen induced intracellular free Ca2+ concentrations ([Ca2+]i) increases between 5 and 20 microM with an EC50 of 10 microM. External Ca2+ removal reduced the response by 60+/-6%. Addition of 3 mM Ca2+ caused a [Ca2+]i increase after pretreatment with 10 microM tamoxifen in Ca2+-free medium. In Ca2+-free medium, pretreatment with 10 microM tamoxifen abolished the [Ca2+]i increase induced by 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 1 microM thapsigargin prevented tamoxifen from releasing more Ca2+. Inhibition of phospholipase C-dependent inositol 1,4,5-tris-phosphate formation with 2 microM U73122 did not alter 10 microM tamoxifen-induced Ca2+ release. The [Ca2+]i increase induced by 5 microM tamoxifen was not altered by 10 microM La3+, nifedipine, verapamil, and diltiazem. Collectively, it was found that tamoxifen increased [Ca2+]i in bladder cancer cells by releasing Ca2+ from the endoplasmic reticulum Ca2+ stores in a manner independent of phospholipase C activity, and by inducing Ca2+ entry from external medium.

Anticarcinogenic Agents↗

PubMed2001

Fendiline, an anti-anginal drug, increases intracellular Ca2+ in PC3 human prostate cancer cells.

BACKGROUND: The effects of the anti-anginal drug fendiline on intracellular Ca2+ concentrations ([Ca2+]i) in human PC3 prostate cancer cells were examined. METHODS: [Ca2+]i was measured using the fluorescent dye fura-2. RESULTS: Fendiline (0.5-100 microM) increased [Ca2+]i in a concentration-dependent manner. Ca2+ removal partly inhibited the Ca2+ signals. In Ca2+-free medium, pretreatment with 100 microM fendiline inhibited most of the [Ca2+]i increase induced by 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), and pretreatment with thapsigargin abolished the fendiline-induced [Ca2+]i increases. Adding 3 mM Ca2+ increased [Ca2+]i in cells pretreated with 0.5-200 microM fendiline in Ca2+-free medium. Pretreatment with 1 microM U73122 to block the formation of inositol-1.4.5-trisphosphate (IP3) did not alter fendiline-induced internal Ca2+ release. CONCLUSIONS: The anti-anginal drug fendiline induced internal Ca2+ release and external Ca2+ entry. Because prolonged increases in [Ca2+]i may lead to cell injury and death, the long-term effect of fendiline on the function of prostate cancer cells should be investigated.

Adenosine Triphosphate↗

PubMed2001

Fendiline mobilizes intracellular Ca2+ in Chang liver cells.

1. The effects of the antianginal drug fendiline (N-[3,3-diphenylpropyl]-alpha-methyl-benzylamine) on intracellular free Ca2+ levels ([Ca2+](i)) in Chang liver cells were evaluated using fura-2 as a fluorescent Ca2+ indicator. 2. Fendiline (1-100 micromol/L) increased [Ca2+](i) in a concentration-dependent manner, with an EC50 of 25 micromol/L. 3. The [Ca2+](i) response was composed of an initial rise and a slow decay to a sustained phase. Removal of extracellular Ca2+ partly reduced the [Ca2+](i) signals. 4. Fendiline (10 micromol/L)-induced release of intracellular Ca2+ was reduced by 65% following pretreatment with 1 micromol/L thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete Ca2+ stored in the endoplasmic reticulum. 5. After pretreatment with 10 micromol/L fendiline in Ca2+-free medium for several minutes, addition of 3 mmol/L Ca2+ induced an increase in [Ca2+](i) of a magnitude four-fold greater than control. This increase in [Ca2+](i) was not reduced by 10 micromol/L SKF96365, econazole, nifedipine or verapamil. 6. Fendiline (10 micromol/L)-induced release of intracellular Ca2+ was not altered by inhibition of phospholipase C with 2 micromol/L 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino) hexyl)-1H-pyrrole-2,5-dione (U73122). 7. The results of the present study show that fendiline induces an increase in [Ca2+](i) in Chang liver cells by releasing stored Ca2+ in an inositol 1,4,5-trisphosphate-independent manner and by causing extracellular Ca2+ influx.

Calcium↗

PubMed2001

Characterization of histamine-induced increases in intracellular free Ca2+ concentrations in Chang liver cells.

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in Chang liver cells were investigated by using fura-2 as a Ca2+ dye. Histamine (0.2-50 microM) increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 0.8 microM. The [Ca2+]i response comprised an initial rise, a slow decay, and a sustained phase. Extracellular Ca2+ removal inhibited 50% of the maximum [Ca2+]i signal and abolished the sustained phase. After pretreatment with 5 microM histamine in Ca2+-free medium for 4 min, addition of 3 mM Ca2+ induced a [Ca2+]i increase with a magnitude 7-fold greater than control. In Ca2+-free medium, after treatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 5 microM histamine failed to increase [Ca2+]i. Histamine (5 microM)-induced intracellular Ca2+ release was abolished

Calcium↗

PubMed2001

Effects of the antianginal drug fendiline on Ca2+ movement in hepatoma cells.

This study investigated the effect of the anti-anginal drug, fendiline, on intracellular free Ca2+ levels ([Ca2+]i) in HA/ 22 human hepatoma cells by using fura-2 as a fluorescent Ca2+ dye. Fendiline (1-100 microM) increased [Ca2+]i with an EC50 of 25 microM. Removal of extracellular Ca2+ reduced the [Ca2+]i signals by 51 +/- 5%. Fendiline (10 microM)-induced Ca2+ release was abolished by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Inhibition of phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122) did not alter 10 microM fendiline-induced Ca2+ release. Several other calmodulin antagonists, such as phenoxybenzamine (100-200 microM), trifluoperazine (5-50 microM), and fluphenazine-N-chloroethane (2-100 microM), had no effect on [Ca2+]i. Together, it was found that fendiline increased [Ca2+]i in human hepatoma cells by discharging Ca2+ from the endoplasmic reticulum in an inositol 1,4,5-trisphosphate-independent manner and by inducing Ca2+ entry. This effect of fendiline does not appear to be via antagonism of calmodulin.

Calcium↗

PubMed2000

Dynamic modification of the capillary wall for electrophoretic separations of small ions.

Separations of small ions were carried out under nonequilibrated conditions using capillaries treated with NaOH, HCl, or tris(hydroxymethyl)aminomethane (Tris) prior to analysis. For separations of benzoic acid isomers or acids and amines under weakly acidic conditions, capillaries flushed with 0.1 M NaOH and subsequently with running buffers prior to analysis were used. Separations of six benzoic acid isomers were accomplished in 4 min in 1 mM phosphate buffers, pH 4.01, containing 2.5 mM hydroxypropyl-beta-cyclodextrin. Without additives, the separation of biological amines and acids were also achieved in 10 min at pH 4.01. Capillaries treated with 0.1 M HCl prior to analysis were tested in separations of six phenols in 5 mM Tris solutions at pH 7.0. As a result of small electrophoretic mobilities of phenols against a small electroosmotic flow, resolution was optimized. We also found that reproducibility was improved using capillaries treated with HCl. The relative standard deviations of migration mobility of phenols were less than 1%, which were smaller than those obtained using capillaries treated with 0.1 M NaOH or Tris.

Electrophoresis, Capillary↗

PubMed2000

On-line concentration and separation of proteins by capillary electrophoresis using polymer solutions.

Proteins were separated in 0.6% poly(ethylene oxide) (PEO) solutions using a capillary filled with buffers prior to analysis and were detected by laser-induced native fluorescence using a pulsed Nd:YAG laser. PEO solutions entered the capillary by electroosmotic flow (EOF) during the separation. The composition and concentration of the buffer affected the adsorption of PEO molecules on the capillary surface and, consequently caused changes in the EOF. Short separation times (< 7 min) were achieved on a sample solution of five proteins in a 0.6% PEO solution containing 5 microg/mL ethidium bromide using a capillary pre-filled with 100 mM TRIS-borate (TB) buffers (pH 10,0). We also extended this method for on-line concentration and separation of proteins. Proteins dissolved in low-conductivity media stacked in both TB buffers and in PEO solutions. The peak height was proportional to the injection volume up to 2.1 microL using an 80-cm capillary filled with 400 mM TB buffers. Using large injection volumes (2.1 microL), we achieved a limit of detection (S/N = 3) of 31 pM for carbonic anhydrase, which was a 1696-fold sensitivity enhancement compared to a conventional injection method (1 kV for 10 s). In high-conductivity media (urine matrix), stacking occurred at the boundary between the sample zone and PEO solutions. A urine sample without any pretreatment was analyzed, and after stacking, several peaks were detected. Spiking the urine sample with human serum albumin (HSA) affected the fluorescent intensity of some analytes as a result of interaction with HSA.

Electrophoresis, Capillary↗

PubMed2000

Effect of ionic strength, pH and polymer concentration on the separation of DNA fragments in the presence of electroosmotic flow.

DNA separations in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions have been demonstrated. During the separations, PEO entered capillaries filled with Tris-borate (TB) free buffers by EOF and acted as sieving matrices. We have found that ionic strength and pH of polymer and free solutions affect the bulk EOF and resolution differently from that in capillary zone electrophoresis. The EOF coefficient increases with increasing ionic strength of the free TB buffers as a result of decreases in the adsorption of PEO molecules. In contrast, the bulk EOF decreases with increasing the ionic strength of polymer solutions using capillaries filled with high concentrations of free TB buffers. Although resolution values are high due to larger differential migration times between any two DNA fragments in a small bulk EOF using 10 mM TB buffers, use of a capillary filled with at least 100 mM TB free buffers is suggested for high-speed separations. On the side of PEO solutions, 1.5% PEO solutions prepared in 100 to 200 mM TB buffers are more proper in terms of resolution and speed. The separation of DNA markers V and VI was accomplished less than 29 min in 1.5% PEO solutions prepared in 100 mM TB buffers, pH 7.0 at 500 V/cm using a capillary filled with 10 mM free TB buffers, pH 7.0.

Buffers↗

PubMed2000

On-column preconcentration and separation of DNA fragments using polymer solutions in the presence of electroosmotic flow.

We demonstrated DNA preconcentration and separation in the presence of electroosmotic flow (EOF) using poly(ethylene oxide) (PEO) solutions. After injecting large volumes of DNA samples into a capillary filled with free tris(hydroxymethyl)aminomethane (Tris)-borate (TB) buffers, PEO solutions entered the capillary by EOF and acted as sieving matrices. In contrast to conventional methods (in the absence of EOF), controlling the EOF was also useful for resolution optimization. We have found that PEO adsorption on the capillary wall was more pronounced when low ionic strength buffers were used. Thus, the EOF decreased with increasing injection length, which led to longer migration times and changes in resolution and stacking efficiency. All resolution values were higher than 1.5 when 1.0 microg/mL DNA samples were injected at 240 V/cm for 60 s (0.67 microL). In addition, as low as 0.015 microg/mL DNA samples (an about 66-fold increase in sensitivity) were detected when the injection was performed at 250 V/cm for 60 s.

Animals↗

PubMed2000

Lipid-rich diffuse malignant mesothelioma: a case report.

An 80-year old man presented with shortness of breath and was found to have a large right pleural effusion. Cytology of the pleural fluid showed atypical papillary clusters of epithelioid cells. Multiple white-yellow nodules studding the pleural surfaces were seen at thoracoscopy, and biopsies showed solid and papillary clusters of large epithelioid cells with abundant cytoplasm filled with clear vacuoles. Special stains and electron microscopic findings indicated that the tumor was a diffuse malignant mesothelioma with numerous intracytoplasmic lipid vacuoles. Fat stain may be useful at time of frozen section for a pleural-based tumor with vacuolated cells, and the presence of lipid vacuoles in a pleural-based tumor does not exclude diffuse malignant mesothelioma.

Aged↗

PubMed2000

Risk factors of mortality in perforated peptic ulcer.

OBJECTIVE: To assess the risk factors that influence mortality from perforated peptic ulcer. DESIGN: Retrospective study. SETTING: General hospital, Taiwan. SUBJECTS: 179 patients who had their perforated peptic ulcers operated on and who had minimum follow-up of one year. MAIN OUTCOME MEASURES: Mortality. RESULTS: The overall mortality was 15% (26/179). Of the 26 patients who died, the cause of death was uncontrolled systemic infection in 21 (81%), hypovolaemic shock in 2, and fatal arrhythmia and heart failure in 1 each. 15 of the patients who died of sepsis did not have fulminant abdominal sepsis. Most deaths occurred early after operation, (range 1-96 days). Old age, preoperative shock, and type of operation seemed to be related to these deaths on univariate analysis, but multivariate analysis showed that coexisting medical illness, delayed treatment, and low albumin concentration were independent risk factors for mortality. CONCLUSIONS: To improve the result of treatment of perforated peptic ulcer, the diagnosis and treatment should not be delayed, the associated medical illnesses should be treated, and nutritional support should be given.

Adolescent↗

PubMed1999

Stepwise capillary electrophoretic separation of DNA fragments using poly(ethylene oxide) solutions in the presence of electroosmotic flow.

Single-base resolution in the separation of DNA markers V and VI was achieved in the presence of electroosmotic flow (EOF), using poly(ethylene oxide) (PEO) solutions containing ethidium bromide (EtB) under isocratic conditions. Furthermore, a new approach called stepwise capillary electrophoresis (SCE) has been developed for DNA analysis, including stepwise changes in PEO concentration, EtB concentration as well as both PEO and EtB concentrations, wherein the EOF was used to introduce different PEO solutions into the capillary during the separation. DNA fragments smaller than 80 bp were both detected under isocratic conditions using 20 micrograms/ml EtB, and SCE using 1 and 20 micrograms/ml EtB, but not under isocratic conditions using 1 microgram/ml EtB. Resolution and speed of the DNA separation in SCE were different from those obtained from isocratic means, indicating that DNA underwent different concentrations of PEO and EtB in SCE. For example, DNA fragments with 458 and 504 base pairs (bp) were partially resolved in SCE, but not under isocratic conditions. The results further suggest that it is worth developing gradient techniques for widening the separation range and enhancing resolution in DNA analysis.

Buffers↗

PubMed1999

Indirect detection of organic acids in non-aqueous capillary electrophoresis.

Non-aqueous capillary electrophoresis (NACE) with indirect detection has been applied to the determination of fatty acids (FAs) and ascorbic acid (AA), respectively. C2-C18 FAs have been separated in less than 12 min using 8-hydroxy-7-iodoquinoline sulfonic acid as chromophores in NACE with indirect absorbance. The dissociation constant (pKa) values of C8-C18 FAs obtained from the slope of the linear plot -log [(mu 0/mu)-1] vs. pH, using 20% isopropanol and 40% acetonitrile as the organic modifier in NACE, are all above about two units than those obtained in aqueous solution. NACE with indirect laser-induced fluorescence, using merocyanine 540 (MC540) as fluorophores, has been performed to the analysis of AA and its stereoisomer, isoascorbic acid (IAA), and the limits of detection of AA and IAA are 0.30 microM and 0.17 microM, respectively. This method has been applied to the determination of AA in a lemon juice spiked with IAA as the internal standard in less than 3 min and its concentration is 76.7 +/- 0.4 mM.

Ascorbic Acid↗

PubMed1999

Electrophoretic separation of small DNA fragments in the presence of electroosmotic flow using poly(ethylene oxide) solutions.

A new and simple method was demonstrated for separating phi X-174/Hae III DNA restriction fragments and DNA markers V and VI, respectively, without filling capillaries with polymer solutions prior to analysis. Using this novel method, poly(ethylene oxide) (PEO) solutions containing ethidium bromide migrated into capillaries by electroosmotic flow (EOF) during the separation. Two DNA fragments (123 and 124 bp) in markers V and VI were well-resolved. RSD values for the separation of phi X-174/Hae III DNA restriction fragments were less than 0.52% for 3 runs using a single 75-micron capillary and less than 3.96% using three different 75-micron capillaries. A highly viscous polymer solution prepared from 3% PEO was also used for separation of DNA markers V and VI. Theoretical plates up to 11.91 million/m and separation times of less than 7 min were achieved in the separation of phi X-174/Hae III DNA restriction fragments using a 10-micron capillary and a 2% PEO solution. Advantages of this method include simplicity, short separation times, the ability to use highly viscous polymer solutions for separating small DNA fragments, and the possibility of introducing several different polymer solutions into capillaries to extend the DNA separation range.

DNA↗