Proceedings of the National Science Council, Republic of China. Part B, Life sciences最新文献

The role of lipid peroxidation, intracellular glutathione and Ca2+ concentration in menadione-mediated toxicity was investigated in human hepatoma cell lines, Hep G2 and Hep 3B, and in human leukemia cell lines, CCRF-CEM and MOLT-3. Incubation of these cells with 80 microM menadione at 37 degrees C resulted in depletion of intracellular glutathione, increased intracellular Ca2+, and increased lipid peroxidation, events leading to cell degeneration. The sensitivity of these cells to menadione, in order, was: Hep G2 cells > Hep 3B cells > CCRF-CEM cells and MOLT-3 cells. The extent of menadione-induced lipid peroxidation in different cell types followed the same order as did their susceptibility to menadione-induced cell degeneration. The menadione-induced depletion in glutathione level was in the following sequence: Hep G2 cells > MOLT-3 and CCRF-CEM cells > Hep 3B cells. The extent of the menadione-induced increase in the intracellular Ca2+ concentration was: Hep G2 cells > Molt-3 cells > CCRF-CEM cells and Hep 3B cells. Pre-treatment of Hep G2 cells with 20 mM deferoxamine mesylate, an iron chelator, reduced both the menadione-induced cell degeneration and lipid peroxidation; however, it did not prevent the menadione-induced increase in intracellular Ca2+ nor the depletion of glutathione. These data suggest that menadione-induced cell degeneration is directly linked to lipid peroxidation, and that it is less related to the rise in intracellular Ca2+ and the depletion in glutathione content. Dicumarol (an inhibitor of DT diaphorase) enhanced the capacity of menadione to induce Hep 3B cell degeneration from 71.3% to 86.2% after 120 min of menadione treatment at 37 degrees C, but did not have this effect in Hep G2, CCRF-CEM or MOLT-3 cells. The activities of DT diaphorase were 52.4, 39.6, 1.5 and 1.8 nmol cytochrome c reduced/min/mg protein in Hep G2, Hep 3B, CCRF-CEM and MOLT-3 cells, respectively. The activity of DT diaphorase was much higher in Hep G2 cells than in the other cells. It seems that DT diaphorase may not, as suggested by others, protect against cell degeneration by quinones, such as menadione.

Tetramethylpyrazine, an inhibitor of phosphodiesterase, has been widely used for treatment of cardiovascular diseases in China. Here, we investigate the effects of tetramethylpyrazine on hypotension, vascular hyporeactivity to norepinephrine (NE), release of tumor necrosis factor-alpha (TNF alpha) and nitric oxide (NO) in a rat model of circulatory shock induced by bacterial endotoxin (E. coli lipopolysaccharide, LPS). Male Wistar-Kyoto rats were anesthetized and instrumented for the measurement of mean arterial pressure (MAP) and heart rate (HR). Injection of LPS (10 mg/kg, i.v.) resulted in a fall in MAP and an increase of HR. In contrast, animals pretreated with tetramethylpyrazine (10 micrograms/kg, i.p. at 30 min prior to LPS) maintained a significantly higher MAP, but tachycardia was further enhanced at 60 min and 120 min when compared to rats given only LPS (LPS-rats). The pressor effect of NE (1 microgram/kg, i.v.) was also significantly reduced after treatment of rats with LPS. Similarly, the thoracic aorta obtained from rats after in vivo studies showed a significant reduction in the contractile responses elicited by NE (1 microM). Pretreatment of LPS-rats with tetramethylpyrazine partially, but significantly, prevented this LPS-induced hyporeactivity to NE in vivo and ex vivo. The injection of LPS resulted in a significant increase in the plasma TNF alpha level at 60 min, whereas the effect of LPS on the plasma nitrate (an indicator of NO formation) level increased in a time-dependent manner. This increment of both TNF alpha and nitrate levels induced by LPS was significantly reduced in LPS-rats pretreated with tetramethylpyrazine. The early hypotension caused by LPS was slightly, but significantly, prevented by pretreatment with tetramethylpyrazine, suggesting that tetramethylpyrazine affects the endothelial constitutive NOS (eNOS). This was examined by the effect of tetramethylpyrazine on acetylcholine (ACh, 1 microM)-induced relaxation in rats treated with tetramethylpyrazine for 4 h. However, tetramethylpyrazine had no significant effects on the ACh-induced relaxation, indicating that tetramethylpyrazine does not affect the activity of eNOS. Thus, tetramethylpyrazine attenuates the early hypotension and the delayed circulatory failure caused by endotoxin in the rat. These effects may be due to inhibition of the release of circulation factors and TNF alpha, which usually reveal synergism upon the induction of iNOS.

The most influential factor contributing to foot and shoe comfort is underfoot cushioning. The shock absorbing ability of footwear in the heel area is of particular importance in reducing the impact load during athletic activities and in therapeutic footwear prescribed for heel pain. Furthermore, foot care for foot problem patients is an important part of treatment and educational programs. Therefore, a well-designed sport shoe which can provide comfort and protection is essential. In order to design a functional shoe, biomechanics and other new technologies should be considered, and the design process should be examined in the biomechanics laboratory over and over. The design process requires too much time and effort since the entire experimental and test work can only be done after the prototype is manufactured. Therefore, this study tried to introduce the Finite Element Method (FEM) into the shoe design process by building a three-dimensional FE model with various shoe soles and loading conditions. The material properties of shoe materials were tested using an Instron Testing Machine. An in-shoe pressure insole was used to measure the plantar pressure in different ambulation conditions with various shoe constructions. The subject for this study was a healthy young male without any foot problem. The average plantar pressures obtained from approximately 50 steps in the heel region for each of the various conditions were collected. The results showed that the mean peak plantar pressure of the running situation was significantly higher than that of the walking situation as predicted, and that the insole could provide better cushioning compared to the other shoe constructions. The stress strain relationship for shoe materials was approximated better by a second-order nonlinear curve according to the Instron test. The results of the finite element method suggested that only the second-order nonlinear stress strain curve could correctly describe the shoe material, which also confirmed a potential valuable role for FEM in designing functional shoes.

Health damage and environmental pollution are serious problems caused by betel quid chewing in Taiwan. Many people acquire the habit of chewing betel quid due to its physiological effects, including increased stamina and a general feeling of well-being. In this study, a sympathetic model system of adrenal chromaffin cells and sensory evaluation were used to examine the physiological effects of betel quid and the interaction of all the ingredients (areca fruit, Piper betle inflorescence and red time paste) in betel quid. Physiological effects of cardioacceleration, a slightly drunk feeling, sweating and salivation occurred during the chewing of betel quid (a mixture of areca fruit, Piper betle inflorescence and red lime paste) and a mixture of areca fruit and red lime paste. Both induced much more basal catecholamine secretion from adrenal chromaffin cells than did other ingredients and combinations of ingredients. It was evident that the responses in the sympathetic model system were closely correlated with the physiological feeling of well-being. The inhibitory effects of all the chewing juices on catecholamine secretion evoked by carbachol and a high concentration of potassium (high K+) showed that they perhaps affected the calcium influx through voltage-sensitive channels or the steps involved in secretion after calcium entry to stimulate basal catecholamine secretion from chromaffin cells.

Betel quid (BQ) chewing shows strong correlation to the incidence of oral submucous fibrosis and oral cancer in Taiwan. Arecoline, the main areca alkaloid, is considered to be one of the etiologic factors in BQ. To elucidate the role(s) of arecoline in the pathogenesis of BQ chewing related oral mucosal lesions, we used oral mucosal fibroblasts to study the effects of serum concentration, cell density, and incubation time on the cytotoxic response to arecoline. At a concentration less than 0.2 mM, arecoline was not cytotoxic to oral mucosal cells after 1, 3, and 6 days of incubation. After 3 days of incubation, the cytotoxic and cytostatic effects of arecoline became evident when the cells were exposed to higher concentrations of arecoline (0.2 mM) and serum (10% FCS). Exposure of cells (1 x 10(4) cells/well) to 0.2 mM of arecoline in 0.5% FCS for 3 and 6 days led to a 20% and 23% decrease, respectively, in the cell number, whereas exposure of cells (1 x 10(4) cells/well) to 0.2 mM arecoline in 10% FCS led to a 38% and 53% decrease, respectively, in cell number. At a higher cell density (5 x 10(4) and 1 x 10(5) cells/well), 0.2 mM arecoline led to less cytotoxicity (38% and 21% of decreasing in cell number, respectively) after 6 days of incubation. Our results indicated that arecoline was not mitogenic to oral mucosal fibroblasts, and that the cytotoxic and cytostatic effects of arecoline on oral mucosal fibroblasts could be modulated by the changes in the cell density, serum concentrations, and incubation time.

Calcific degeneration of aortic valves were investigated in 10 patients with senile calcific aortic stenosis by means of high resolution scanning electron microscopy (HR-SEM) and transmission electron microscopy (TEM). All the specimens obtained during aortic valve surgery HR-SEM and TEM examinations consistently showed various degrees of pathological alterations and calcification involving surface endothelium, underlying basement membranes and deeper layers of interweaving networks of collagen fiber bundles in the pars fibrosa of the valve tissues. Calcific deposits in the valve tissues always occurred in the vicinity of the endothelial defects and in the subendothelial structures just beneath the defective endothelium. The amount of calcific deposits in the valve tissues increased in proportion to the severity of endothelial damage and gradually decreased from the defective endothelial surface to the deeper layer of collagen tissue. In addition, apoptotic cell death, particularly of the fibrocytes in the valve tissue, was closely related to the severity of endothelial injury. Cellular fragments derived from the apoptotic cells were always associated with calcium deposits. Based on the above findings, our results provide evidence that the alteration of endothelial integrity plays a contributory role in calcific degeneration in the aortic valve leading to the development of senile calcific aortic stenosis.

An in vivo integration assay using the expressed human immunodeficiency virus type 1 (HIV-1) integrase (IN) protein and plasmids carrying a copy of the infectious Moloney murine leukemia virus (MuLV) provirus genome as substrates is presented. The HIV-1 IN gene was taken from vector pINSD and cloned into vector pXT1 to give pXT1-IN. Two and three nucleotides from the circle junction on one pair of U3 and U5 attachment (att) sequences on an infectious MuLV provirus vector pMLV-K were changed by means of site-directed mutagenesis to that of the corresponding HIV-1 att sequences to generate vector pMLV*(U3U5). The MuLV IN sequence was partially deleted for vectors pMLV-K and pMLV*(U3U5) to generate vectors pMLV delta IN and pMLV*(U3U5) delta IN. Integration of these wild type and MuLV IN partially deleted or att mutated MuLV provirus vectors in the transfected cells by the expressed HIV-1 IN was monitored by means of a non-radioactive reverse transcriptase (RT) assay for released and collected virions. No RT activity was detected for the NIH/3T3 cell singly transfected with vector pMLV delta IN. However some RT activities were observed for the HIV-1 IN expressing cell transfected either with vectors pMLV delta IN or pMLV*(U3U5) delta IN. This indicated that in the absence of other HIV-1 proteins expressed the MuLV provirus genome was integrated by the expressed HIV-1 IN protein. The integration of these MuLV provirus genomes was further confirmed by polymerase chain reaction analysis on the genomic DNA extracted from the transfected cells using the MuLV IN sequence remained from partial deletion as a target.

In the Escherichia coli K-12 chromosome, six copies of IS1 (IS1A - IS1F) have been identified and characterized. According to their nucleotide (nt) sequences, the six IS1 copies can be classified into four types, IS1A(IS1E), IS1B(IS1C), IS1D and IS1F type. Zuber and Schumann (1993) identified the seventh IS1 copy at 49.6 minutes on the E. coli W3110 genetic map. Unfortunately, only the end 21-bp sequence as well as the neighboring 120-bp E. coli sequence were reported. We therefore designed two oligonucleotide primers to specifically amplify the seventh IS1 copy by PCR. One primer is homologous to the first sixteen bases of the IRL sequence of IS1A(IS1E), IS1B(IS1C) and IS1D. The other primer is complementary to the eighteen bases of E. coli sequence adjacent to IRR of the seventh IS1 copy. An 800-bp PCR fragment was obtained and its nt sequence determined, revealing an identical nucleotide sequence to that of IS1A(IS1E). A plasmid system was then used to isolate insertion mutations caused by insertions of the chromosomal insertion sequences. Of the 142 plasmid insertion mutants isolated, thirty-eight were insertions of chromosomal IS5, ten were IS30, and ninety-four were IS1. Detailed restriction mapping indicates that all ninety-four plasmid IS1 insertions were insertions of IS1 of the IS1A(IS1E) type.

Spinocerebellar ataxia type 1 (SCA 1) is an autosomal dominant disorder characterized by neurodegeneration of the cerebellum, spinal cord and brainstem. This neurodegeneration disease is associated with expansion of unstable CAG repeats within the coding region of the gene. We are conducting a local survey of the normal population and candidate patients to analyze the CAG repeats in SCA 1 gene. So far, we have collected peripheral blood from 78 normal individuals and 10 patients with dominant inherited ataxia disorders, and assayed the SCA1 CAG trinucleotide repeat using genomic polymerase chain reaction (PCR). Even though no local SCA 1 patients have been identified, we have established the distributions of the CAG repeat units of SCA 1 gene in the normal population in Taiwan. The normal range of CAG repeats is from 22 to 33 repeats, with the most common being 30 repeats. The range is relatively narrow compared to that reported for other ethnic groups. In addition, direct genomic PCR analysis of the SCA 1 gene from villous DNA has been successful in our laboratory. Screening of SCA 1 patients from patients with dominant inherited ataxia is currently underway in our laboratory. Here, we demonstrate that our molecular analysis technique makes possible the quick and accurate diagnosis of SCA1 patients and prenatal screening for SCA 1 families.

Alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) are the major enzymes responsible for the metabolism of ethanol in the body. Both exhibit genetic polymorphism in racial populations. To determine hepatic ethanol metabolizing activities in relation to genetic polymorphism, a total of 23 surgical specimens were investigated. The expression patterns of ADH and ALDH isoenzymes were identified by means of agarose isoelectric focusing, and the activities were assayed spectrophotometrically. At 33 mM ethanol, pH 7.5, the activities in the liver with the homozygous phenotype ADH2 1-1 and ADH2 2-2 and the heterozygous phenotype ADH2 1-2 were determined to be 2.9 +/- 0.7, 16.0 +/- 2.5, and 13.6 +/- 1.0 U/g tissue, respectively. The activities of the ALDH2-active and ALDH2-inactive phenotypes at 200 microM acetaldehyde were determined to be 1.06 +/- 0.13 and 0.71 +/- 0.07 U/g tissue, respectively. These findings indicate that human hepatic ethanol-metabolizing activities differ significantly with respect to polymorphism at both the ADH2 and ALDH2 loci. The results suggest that this genetically determined differential hepatic activity may influence drinking behavior and the development of alcoholism among Orientals.