Yau-huei Wei | National Yang Ming Chiao Tung University (original) (raw)
Papers by Yau-huei Wei
PloS one, 2015
Graves' ophthalmopathy (GO) is a disfiguring and sometimes blinding disease, which is charact... more Graves' ophthalmopathy (GO) is a disfiguring and sometimes blinding disease, which is characterized by inflammation and swelling of orbital tissues, with fibrosis and adipogenesis being predominant features. The aim of this study is to investigate whether the expression levels of fibrosis-related genes, especially that of connective tissue growth factor (CTGF), are altered in orbital fibroblasts of patients with GO. The role of oxidative stress in the regulation of CTGF expression in GO orbital fibroblasts is also examined. By a SYBR Green-based real time quantitative PCR (RT-QPCR), we demonstrated that the mRNA expression levels of fibronectin, apolipoprotein J, and CTGF in cultured orbital fibroblasts from patients with GO were significantly higher than those of age-matched normal controls (p = 0.007, 0.037, and 0.002, respectively). In addition, the protein expression levels of fibronectin, apolipoprotein J, and CTGF analyzed by Western blot were also significantly higher in ...
Translational research : the journal of laboratory and clinical medicine, Jan 15, 2015
Although restoration of mitochondrial function in mitochondrial diseases through peptide-mediated... more Although restoration of mitochondrial function in mitochondrial diseases through peptide-mediated mitochondrial delivery (PMD) has been demonstrated in vitro, the in vivo therapeutic efficacy of PMD in Parkinson's disease (PD) has yet to be determined. In this study, we compared the functionality of mitochondrial transfer with or without Pep-1 conjugation in neurotoxin (6-hydroxydopamine, 6-OHDA)-induced PC12 cells and PD rat models. We injected mitochondria into the medial forebrain bundle (MFB) of the PD rats after subjecting the nigrostriatal pathway to a unilateral 6-OHDA lesion for 21 days, and we verified the effectiveness of the mitochondrial graft in enhancing mitochondrial function in the soma of the substantia nigra (SN) neuron through mitochondrial transport dynamics in the nigrostriatal circuit. The result demonstrated that only PMD with allogeneic and xenogeneic sources significantly sustained mitochondrial function to resist the neurotoxin-induced oxidative stress ...
Journal of Biological Chemistry
The subunits of the cytochrome oxidase from bovine heart were isolated in large quantities suitab... more The subunits of the cytochrome oxidase from bovine heart were isolated in large quantities suitable for amino acid sequence studies. The preparation of subunits III, IV, V, VI, and VII for sequence determination can be achieved without employing sodium dodecyl sulfate. The method presented essentially involves pyridine extraction, pH fractionation, ammonium sulfate fractionation, and various types of column chromatography. However, subunits I and II can be prepared only in the presence of sodium dodecyl sulfate by molecular sieve chromatography; subunit III can also be isolated in this manner. The separation of subunits is found to be hindered by phospholipids associated with the enzyme and therefore the phospholipid-depleted preparation is used as the starting material. The molecular weights of subunits I, II, III, IV, V, VI, and VII are 40,000, 21,000, 14,800, 13,500, 11,600, 9,500, and 7,600, respectively. These values are based on the results of the conventional Weber and Osborn method of gel electrophoresis in the presence of sodium dodecyl sulfate. The amino termini of subunits I and II have been determined as N-formylmethionine, and those of subunits III, IV, V, VI, and VII are alanine, alanine, serine, alanine, and an N-acetyl-blocked residue, respectively. The carboxyl termini for subunits I to VII are lysine, leucine, lysine, histidine, valine, isoleucine, and valine, respectively. The complete amino acid sequence of some subunits has been published and that of other subunits will be reported elsewhere in collaboration with the Amino Acid Sequence Group of Cytochrome Oxidase at the University of Hawaii.
Journal of Biological Chemistry
The amino acid sequence of subunit VI derived from bovine heart cytochrome oxidase has been compl... more The amino acid sequence of subunit VI derived from bovine heart cytochrome oxidase has been completed and is a single polypeptide chain consisting of 85 amino acid residues. The sequence is: acetyl (formula: see text). The NH2 terminus of subunit VI is blocked with an acetyl group and the molecular weight of the native protein including the acetyl group is 10,067. From the sequence of subunit VI, it is obvious that this subunit corresponds to polypeptide VII of Steffens et al. (Steffens, G. C. M., Steffens, G. J., and Buse, G. (1979) Hoppe-Seyler's Z. Physiol. Chem. 360, 1641-1650).
Proceedings of the National Science Council, Republic of China. Part B, Life sciences
The effect of ethanol intake on liver mitochondrial functions was investigated by feeding rats wi... more The effect of ethanol intake on liver mitochondrial functions was investigated by feeding rats with a liquid isocaloric diet containing various concentrations of ethanol. We found that after feeding the liquid diet for 2 to 3 months, the body weight of rats did not show a significant difference between treated and control groups. However, the mitochondrial respiration rate decreased significantly with the increase of ethanol concentration in the diet. We found that when the rats were fed on 10.8% ethanol, the average succinate-supported State 3 respiration rate decreased from 54.5 to 44.8 nmol O2/min/mg and the glutamate-malate-supported State 3 respiration rate decreased from 38.8 to 23.6 nmol O2/min/mg as compared with the control. Interestingly, we noted that ethanol intake caused a more drastic effect on State 3 respiration than on State 4 respiration, irrespective of the substrate utilized by the mitochondria. In addition, the respiratory control and ADP/O ratios were found to decrease concomitantly with the increase of ethanol level in the diet. Moreover, we found that the effect of ethanol on both respiratory control and ADP/O ratios of liver mitochondria was more pronounced in glutamate-malate-supported respiration than succinate-supported respiration. These results clearly demonstrate that ethanol intake by the rat can cause impairment of liver mitochondrial respiration and oxidative phosphorylation, and that these effects are exerted through damage to mitochondrial membranes.
Journal of the Formosan Medical Association
Proceedings of the National Science Council, Republic of China. Part B, Life sciences
The conventional microscopic methods for evaluating sperm motility of domestic animals are mostly... more The conventional microscopic methods for evaluating sperm motility of domestic animals are mostly inadequate due to their subjectivity and lack of precision. Recently, a trans-membrane migration method, originally developed for the examination of human sperm motility, has substantially overcome these problems. This study investigated the applicability of the method to boar sperm motility measurement. The apparatus used was simple and consisted only of syringe plungers, poriferous membranes, and modified multi-well culture plates. It measured the proportion of sperm in the semen that moved across the membrane after incubation at 37 degrees C for 3 hr. The sperm motility as measured by this method correlated well with that measured by direct microscopic examinations. The measurement was more reliable using an 8-microns instead of a 5-microns pore-size membrane. The method was found to work equally well for the sperm motility measurement of the semen with a sperm concentration between 1.5 x 10(8)/ml and 6.0 x 10(8)/ml. The results indicate that this method is a simple, objective, quantitative, and reproducible design for the measurement of boar sperm motility.
Journal of the Chinese Medical Association
Mitochondrial myopathies are a clinically and biochemically heterogeneous group of disorders shar... more Mitochondrial myopathies are a clinically and biochemically heterogeneous group of disorders sharing common features characterized by structural and functional abnormalities of the mitochondria in skeletal muscle biopsy of the patients. The genetic defects in some of the mitochondrial diseases have been reported recently. However, the mechanisms of pathogenesis of these disorders are still obscure. It has been hypothesized by Morgan-Hughes that there is an increased frequency of DNA polymorphism in the noncoding region of the mitochondrial genome in patients with mitochondriopathy. In this study, we analyzed the mtDNAs isolated from 62 healthy Chinese subjects and 34 blood samples of patients with various mitochondrial myopathies to search for disease-associated RFLPs by PCR technique and restriction pattern analysis. A comparison of the frequency of RFLPs in mtDNAs between normal Chinese subjects and patients was done by statistical analysis. Five new polymorphic sites in mtDNAs from patients were revealed by restriction endonucleases AluI, HaeIII, HinfI, and EcoRII, respectively. These results showed that the frequency of RFLPs in mtDNA increased in patients with mitochondriopathies. Although these results are in line with the hypothesis of Morgan-Hughes, we analyzed only 8.9% of the nucleotide sequence of human mtDNA. Further test of the hypothesis with more detailed analysis of mtDNAs from more patients and larger normal population size is warranted.
Biochemistry and molecular biology international
The general objective of this study was to examine the relationship between mitochondrial respira... more The general objective of this study was to examine the relationship between mitochondrial respiratory function and liver regeneration in the rat. The role that free radicals may play in the process was also evaluated. It was found that the respiratory control and ADP/O ratios were concomitantly decreased to the lowest level at 6 hr after hepatectomy and gradually recovered thereafter. Both ratios were significantly increased at 48 hr and quickly reached plateau levels. Assays of mitochondrial respiratory functions revealed that the activities of Complex I+III, Complex II+III and Complex IV all decreased drastically at 6 hr after hepatectomy and then gradually returned to the original level during 18-24 hr after hepatectomy. Interestingly, the activities of all these enzyme complexes continuously increased and were elevated significantly above the normal levels (145-200%). In contrast, the liver mitochondrial electron transport activities of sham-operated rats returned only to the original level after recovering from the operation-induced decline at 6 hr post-hepatectomy. We measured the superoxide dismutase (SOD) activity of liver mitochondria of the hepatectomized and sham-operated rats. The results showed that the Mn-SOD activity started to increase after hepatectomy, reached a maximum (900% of control) at 6 hr, and then returned to normal levels at 24 hr after operation. The Cu, Zn-SOD activity was increased 9-fold in hepatectomized rats and about 3-fold in sham-operated rats as compared with control rats. The maximum activity of Mn-SOD was found to be about 4 times higher than that of Cu, Zn-SOD after hepatectomy. The amount of lipoperoxides in the liver mitochondria was found to be increased to 140% in hepatectomized rats and to 120% in sham-operated rats as compared with that of the control rats. Taken together these results suggest that the changes in mitochondrial respiratory functions in the early phase of hepatectomy are due to tissue damage caused by the transient elevation of free radicals. These free radicals are then quickly disposed of by the ever-increasing activities of the Mn-SOD and Cu, Zn-SOD in the liver mitochondria, thereby protecting the liver from further damage and gearing the organ to the regeneration process.
Biochemistry and molecular biology international
The gene encoding cytochrome c oxidase subunit II (COXII) was amplified by polymerase chain react... more The gene encoding cytochrome c oxidase subunit II (COXII) was amplified by polymerase chain reaction (PCR) using mitochondrial DNAs (mtDNAs) isolated from two genera of domesticated ducks, Anas platyrhnchos and Cairina muschata, and their intergeneric and intrageneric hybrids as templates. Comparison of the nucleotide sequences from the two different duck lineages shows that they have approximately 91% homology. The nucleotide substitution pattern in the duck COXII gene reveals a high transition/transversion ratio (8.8:1.0) while 93.2% of the nucleotide substitutions are silent. The duck COXII, which uses GUG as the initiation codon, contains 228 amino acid residues. Comparison of the deduced COXII amino acid sequences in duck, chicken, Xenopus laevis, mouse, bovine and human reveals five highly conserved domains. The evolutionary conservation of the primary structure of COXII implies its functional importance.
Chinese medical journal
By use of restriction fragment length polymorphism analysis, we examined the liver mitochondrial ... more By use of restriction fragment length polymorphism analysis, we examined the liver mitochondrial DNA amplified by polymerase chain reaction from 60 Chinese subjects of 31 to 78 years of age. We found nine specific mtDNA polymorphisms that had never been reported before. Eleven subjects had an Alu I polymorphic site in the subunit 2 gene of NADH dehydrogenase, five had a Hae III polymorphic site in the cytochrome oxidase subunit 2 gene, and five had a Hinf I polymorphic site in the subunit 3 gene of cytochrome oxidase. No polymorphic site was found in the structural genes coding for subunits 1, 3, 4, 4L and 6 of NADH dehydrogenase, cytochrome b, and subunit 8 of ATP synthase. Detailed analysis of the RFLP data did not show age-dependent mtDNA polymorphisms. In addition, the analysis of the restriction patterns of all the mtDNAs revealed 12 mtDNA haplotypes in all the Chinese subjects examined. Among them, type 1 mtDNA was found to be the most predominant and comprised 63.3% of the total study subjects. The restriction patterns of type 1 mtDNA generated by all restriction enzymes were identical to those deduced from the Cambridge sequence of human mtDNA. About 8.3% of the subjects exhibited type 2 mtDNA, and 5% had types 3, 5 and 8 mtDNA, respectively. Each of the rest seven mtDNA types comprised about 2% of the samples. Moreover, type 1 mtDNA was found in the platelets of three white Americans. These findings suggest that type 2 to type 12 mtDNAs have come into existence through the generation or loss of specific polymorphic restriction sites in the mtDNA of the Chinese.
The American Journal of Chinese Medicine
ABSTRACT
Journal of the Formosan Medical Association
Free radicals may be involved in various human disease processes. Cutaneous edema and acute pulmo... more Free radicals may be involved in various human disease processes. Cutaneous edema and acute pulmonary damage of thermal injury mediated by oxygen free radicals can be monitored by studying the levels of lipid peroxidation in blood plasma. In this study, we investigated lipid peroxidation induced by human skin burns. The subjects included 14 healthy subjects and eight burn patients. Healthy subjects included nine males and five females, aged 50.5 +/- 19.1 years. The burn patients included seven males and one female, aged 46.9 +/- 19.1 years, who had burns over 45.6% +/- 14.0% of their total body surface area. Plasma lipoperoxides were measured by high performance liquid chromatographic separation of malondialdehyde-thiobarbituric acid adduct. The average plasma lipoperoxide concentration (measured as malondialdehyde) of the 14 healthy subjects was 0.72 +/- 0.18 mumol/L. This concentration increased significantly (p < 0.0013) in the eight burn patients to a 3.75 +/- 1.34 mumol/L (peak level). Plasma lipoperoxides increased in the first few hours postburn. Plasma lipid peroxide peak levels were found on the third day postburn in 75% of burn patients. Moreover, patients with inhalation injuries had higher plasma lipid peroxide levels (5.27 +/- 5.30 mumol/L) than those without inhalation injury (2.84 +/- 2.88 mumol/L). These results suggest that both oxygen free radicals and lipid peroxidation play a major role in the injuries caused by skin burns and related inhalation [corrected].
Journal of the Formosan Medical Association
In forensic DNA typing, evidential samples generally involve limited amounts of DNA and so should... more In forensic DNA typing, evidential samples generally involve limited amounts of DNA and so should be carefully utilized. Although polymerase chain reaction (PCR) of variable number of tandem repeats (VNTR) alleles is the prevailing method for forensic identification, the fidelity of amplification of heterozygous VNTR alleles with large disparities in length needs to be carefully examined. Reports in the literature and our own observations have demonstrated that PCR artifacts, bogus alleles and allelic drop-out of VNTRs, are related to the amount of genomic DNA, the number of amplification cycles and the length of alleles amplified. Two small (< 1 kb) hypervariable VNTRs (Apo B and HVR-Ig) markers used for forensic identification were chosen to study these relationships. The results revealed that PCR amplification for the heterozygous VNTR alleles with wide disparity in length (> 400 bp) easily produced the allelic drop-out problem and therefore, led to the false results; and the allelic fragment of PCR products was preferentially lost after only 2 cycles of overamplification. We also further established the relationship between the optimal number of amplification cycles and the amount of genomic DNA in the reaction mixture. In our routine forensic screening this relationship has been successfully applied to determine the optimal number of amplification cycles and to avoid the allelic drop-out problem and achieve fidelity of PCR-VNTR amplification. It has also been used to investigate forensic casework.
Journal of the Formosan Medical Association
By use of a simple, rapid and reliable polymerase chain reaction (PCR)-based method, we analyzed ... more By use of a simple, rapid and reliable polymerase chain reaction (PCR)-based method, we analyzed three hypervariable tandem repeats in the 3'-Apo B, 5'-HVR-Ig and 3'-COL2A1 loci. As accurate data of allele frequency of genetic markers is a prerequisite for forensic application, the allele frequency distribution of the three variable number of tandem repeats (VNTR) among the Chinese population in Taiwan were studied. In a total of 123 unrelated Chinese subjects, the Apo B VNTR demonstrated a heterozygosity of 68.2% with 9 alleles, 0.85 of the power of discrimination (PD) value and 0.74 of the allelic diversity (h) value. In a sample of 103 unrelated Chinese subjects, the COL2A1 VNTR showed 49.0% heterozygosity with six alleles, 0.79 of the PD value and 0.74 of the h value. In 106 unrelated subjects, the HVR-Ig VNTR showed 47.4% heterozygosity with seven alleles, 0.79 of the PD value and 0.59 of the h value. The data obtained in this study are not only useful for forensic identification, but will also be helpful for paternity testing, genetic linkage studies and the identification of the three VNTR loci associated with human genetic diseases. Some verifying examinations for the validity and reliability of the three VNTR were performed. The high sensitivity and inexpensive nature of this approach make it superior to the traditional method of DNA fingerprinting for forensic typing. With the use of this PCR-VNTR system, many forensic cases have been successfully identified. The value of this system is illustrated in the investigation of a rape and murder case.
The Chinese journal of physiology
The role of EGTA in stimulating human sperm motility (reported in Lancet i: 460-461, 1984) was in... more The role of EGTA in stimulating human sperm motility (reported in Lancet i: 460-461, 1984) was investigated by measuring the calcium buffering capacity of human seminal plasma. Human seminal plasma contains 9.5 +/- 1.1 mM (mean +/- SE) calcium of which 0.16 +/- 0.01 mM only exists as free Ca2+. The free Ca2+ concentration was not changed by the addition of either 1 mM CaCl2 or 1 mM EGTA. The ability of seminal plasma to bind calcium ions was determined by adding varying amounts of CaCl2. Scatchard analysis of the results indicates the presence of high amounts of high-affinity (Kd1 = 3.4 +/- 0.2 microM, Bm1 = 11.5 +/- 1.5 mM) and low-affinity (Kd2 = 0.55 +/- 0.04 mM, Bm2 = 30.4 +/- 1.7 mM) Ca2+-complexing agents. The low-affinity Ca2+-binding substance may be citrate. These results seem to suggest that human seminal plasma has a high Ca2+ buffering capacity and the stimulation of sperm motility by EGTA ought to be mediated via a mechanism other than the reduction of the free calcium concentration in semen.
Journal of the Formosan Medical Association
A rapid and simple method using restriction enzymes to detect the restriction fragment length pol... more A rapid and simple method using restriction enzymes to detect the restriction fragment length polymorphism (RFLP) pattern of hypervariable segment 1 in the D-loop region of human mitochondrial DNA (mtDNA) was developed. We first focused on the investigation of variations of DNA sequence in the D-loop region among Chinese subjects, as well as on the determination of RFLP patterns of each restriction enzyme. Seven restriction enzymes were used to digest a 618 bp polymerase chain (PCR) reaction product of the D-loop region of mtDNA. Frequency distribution of RFLP patterns of each restriction enzyme among 145 unrelated Chinese subjects in Taiwan was also established. For the purposes of practical forensic application, a routine typing system was designed on the basis of the RFLP data. Two short hypervariable, mtDNA fragments, which were contained within the 618 bp region, were selected for this purpose. In this haplotyping system, a 281 bp PCR-amplified DNA product was analyzed by five restriction enzymes: Mnl I, Nla III, Rsa l, Mse I and Hinf I, and a 237 bp fragment was analyzed by Kpn I. The RFLP patterns were determined by agarose gel electrophoresis of the restriction enzyme-digested DNA fragments. Six restriction enzymes. Mul I, Nla III, Rsa I. Msc I, Hinf I and Kpn 1, defined eight, four, four, five, two and four polymorphic patterns, respectively among the 145 Chinese subjects. The RFLP patterns of restriction fragments for each individual were systematically analyzed and the mtDNAs of the 145 Chinese subjects were grouped into 52 haplotypes. This PCR-RFLP haplotyping system revealed a high degree of variability and diversity of segment I in the D-loop region of human mtDNA. The power of discrimination and allelic diversity values were 0.923 and 0.929, respectively. Successful application of this haplotyping system in a murder case is also discussed.
Proceedings of the National Science Council, Republic of China. Part B, Life sciences
ABSTRACT
PloS one, 2015
Graves' ophthalmopathy (GO) is a disfiguring and sometimes blinding disease, which is charact... more Graves' ophthalmopathy (GO) is a disfiguring and sometimes blinding disease, which is characterized by inflammation and swelling of orbital tissues, with fibrosis and adipogenesis being predominant features. The aim of this study is to investigate whether the expression levels of fibrosis-related genes, especially that of connective tissue growth factor (CTGF), are altered in orbital fibroblasts of patients with GO. The role of oxidative stress in the regulation of CTGF expression in GO orbital fibroblasts is also examined. By a SYBR Green-based real time quantitative PCR (RT-QPCR), we demonstrated that the mRNA expression levels of fibronectin, apolipoprotein J, and CTGF in cultured orbital fibroblasts from patients with GO were significantly higher than those of age-matched normal controls (p = 0.007, 0.037, and 0.002, respectively). In addition, the protein expression levels of fibronectin, apolipoprotein J, and CTGF analyzed by Western blot were also significantly higher in ...
Translational research : the journal of laboratory and clinical medicine, Jan 15, 2015
Although restoration of mitochondrial function in mitochondrial diseases through peptide-mediated... more Although restoration of mitochondrial function in mitochondrial diseases through peptide-mediated mitochondrial delivery (PMD) has been demonstrated in vitro, the in vivo therapeutic efficacy of PMD in Parkinson's disease (PD) has yet to be determined. In this study, we compared the functionality of mitochondrial transfer with or without Pep-1 conjugation in neurotoxin (6-hydroxydopamine, 6-OHDA)-induced PC12 cells and PD rat models. We injected mitochondria into the medial forebrain bundle (MFB) of the PD rats after subjecting the nigrostriatal pathway to a unilateral 6-OHDA lesion for 21 days, and we verified the effectiveness of the mitochondrial graft in enhancing mitochondrial function in the soma of the substantia nigra (SN) neuron through mitochondrial transport dynamics in the nigrostriatal circuit. The result demonstrated that only PMD with allogeneic and xenogeneic sources significantly sustained mitochondrial function to resist the neurotoxin-induced oxidative stress ...
Journal of Biological Chemistry
The subunits of the cytochrome oxidase from bovine heart were isolated in large quantities suitab... more The subunits of the cytochrome oxidase from bovine heart were isolated in large quantities suitable for amino acid sequence studies. The preparation of subunits III, IV, V, VI, and VII for sequence determination can be achieved without employing sodium dodecyl sulfate. The method presented essentially involves pyridine extraction, pH fractionation, ammonium sulfate fractionation, and various types of column chromatography. However, subunits I and II can be prepared only in the presence of sodium dodecyl sulfate by molecular sieve chromatography; subunit III can also be isolated in this manner. The separation of subunits is found to be hindered by phospholipids associated with the enzyme and therefore the phospholipid-depleted preparation is used as the starting material. The molecular weights of subunits I, II, III, IV, V, VI, and VII are 40,000, 21,000, 14,800, 13,500, 11,600, 9,500, and 7,600, respectively. These values are based on the results of the conventional Weber and Osborn method of gel electrophoresis in the presence of sodium dodecyl sulfate. The amino termini of subunits I and II have been determined as N-formylmethionine, and those of subunits III, IV, V, VI, and VII are alanine, alanine, serine, alanine, and an N-acetyl-blocked residue, respectively. The carboxyl termini for subunits I to VII are lysine, leucine, lysine, histidine, valine, isoleucine, and valine, respectively. The complete amino acid sequence of some subunits has been published and that of other subunits will be reported elsewhere in collaboration with the Amino Acid Sequence Group of Cytochrome Oxidase at the University of Hawaii.
Journal of Biological Chemistry
The amino acid sequence of subunit VI derived from bovine heart cytochrome oxidase has been compl... more The amino acid sequence of subunit VI derived from bovine heart cytochrome oxidase has been completed and is a single polypeptide chain consisting of 85 amino acid residues. The sequence is: acetyl (formula: see text). The NH2 terminus of subunit VI is blocked with an acetyl group and the molecular weight of the native protein including the acetyl group is 10,067. From the sequence of subunit VI, it is obvious that this subunit corresponds to polypeptide VII of Steffens et al. (Steffens, G. C. M., Steffens, G. J., and Buse, G. (1979) Hoppe-Seyler&amp;#39;s Z. Physiol. Chem. 360, 1641-1650).
Proceedings of the National Science Council, Republic of China. Part B, Life sciences
The effect of ethanol intake on liver mitochondrial functions was investigated by feeding rats wi... more The effect of ethanol intake on liver mitochondrial functions was investigated by feeding rats with a liquid isocaloric diet containing various concentrations of ethanol. We found that after feeding the liquid diet for 2 to 3 months, the body weight of rats did not show a significant difference between treated and control groups. However, the mitochondrial respiration rate decreased significantly with the increase of ethanol concentration in the diet. We found that when the rats were fed on 10.8% ethanol, the average succinate-supported State 3 respiration rate decreased from 54.5 to 44.8 nmol O2/min/mg and the glutamate-malate-supported State 3 respiration rate decreased from 38.8 to 23.6 nmol O2/min/mg as compared with the control. Interestingly, we noted that ethanol intake caused a more drastic effect on State 3 respiration than on State 4 respiration, irrespective of the substrate utilized by the mitochondria. In addition, the respiratory control and ADP/O ratios were found to decrease concomitantly with the increase of ethanol level in the diet. Moreover, we found that the effect of ethanol on both respiratory control and ADP/O ratios of liver mitochondria was more pronounced in glutamate-malate-supported respiration than succinate-supported respiration. These results clearly demonstrate that ethanol intake by the rat can cause impairment of liver mitochondrial respiration and oxidative phosphorylation, and that these effects are exerted through damage to mitochondrial membranes.
Journal of the Formosan Medical Association
Proceedings of the National Science Council, Republic of China. Part B, Life sciences
The conventional microscopic methods for evaluating sperm motility of domestic animals are mostly... more The conventional microscopic methods for evaluating sperm motility of domestic animals are mostly inadequate due to their subjectivity and lack of precision. Recently, a trans-membrane migration method, originally developed for the examination of human sperm motility, has substantially overcome these problems. This study investigated the applicability of the method to boar sperm motility measurement. The apparatus used was simple and consisted only of syringe plungers, poriferous membranes, and modified multi-well culture plates. It measured the proportion of sperm in the semen that moved across the membrane after incubation at 37 degrees C for 3 hr. The sperm motility as measured by this method correlated well with that measured by direct microscopic examinations. The measurement was more reliable using an 8-microns instead of a 5-microns pore-size membrane. The method was found to work equally well for the sperm motility measurement of the semen with a sperm concentration between 1.5 x 10(8)/ml and 6.0 x 10(8)/ml. The results indicate that this method is a simple, objective, quantitative, and reproducible design for the measurement of boar sperm motility.
Journal of the Chinese Medical Association
Mitochondrial myopathies are a clinically and biochemically heterogeneous group of disorders shar... more Mitochondrial myopathies are a clinically and biochemically heterogeneous group of disorders sharing common features characterized by structural and functional abnormalities of the mitochondria in skeletal muscle biopsy of the patients. The genetic defects in some of the mitochondrial diseases have been reported recently. However, the mechanisms of pathogenesis of these disorders are still obscure. It has been hypothesized by Morgan-Hughes that there is an increased frequency of DNA polymorphism in the noncoding region of the mitochondrial genome in patients with mitochondriopathy. In this study, we analyzed the mtDNAs isolated from 62 healthy Chinese subjects and 34 blood samples of patients with various mitochondrial myopathies to search for disease-associated RFLPs by PCR technique and restriction pattern analysis. A comparison of the frequency of RFLPs in mtDNAs between normal Chinese subjects and patients was done by statistical analysis. Five new polymorphic sites in mtDNAs from patients were revealed by restriction endonucleases AluI, HaeIII, HinfI, and EcoRII, respectively. These results showed that the frequency of RFLPs in mtDNA increased in patients with mitochondriopathies. Although these results are in line with the hypothesis of Morgan-Hughes, we analyzed only 8.9% of the nucleotide sequence of human mtDNA. Further test of the hypothesis with more detailed analysis of mtDNAs from more patients and larger normal population size is warranted.
Biochemistry and molecular biology international
The general objective of this study was to examine the relationship between mitochondrial respira... more The general objective of this study was to examine the relationship between mitochondrial respiratory function and liver regeneration in the rat. The role that free radicals may play in the process was also evaluated. It was found that the respiratory control and ADP/O ratios were concomitantly decreased to the lowest level at 6 hr after hepatectomy and gradually recovered thereafter. Both ratios were significantly increased at 48 hr and quickly reached plateau levels. Assays of mitochondrial respiratory functions revealed that the activities of Complex I+III, Complex II+III and Complex IV all decreased drastically at 6 hr after hepatectomy and then gradually returned to the original level during 18-24 hr after hepatectomy. Interestingly, the activities of all these enzyme complexes continuously increased and were elevated significantly above the normal levels (145-200%). In contrast, the liver mitochondrial electron transport activities of sham-operated rats returned only to the original level after recovering from the operation-induced decline at 6 hr post-hepatectomy. We measured the superoxide dismutase (SOD) activity of liver mitochondria of the hepatectomized and sham-operated rats. The results showed that the Mn-SOD activity started to increase after hepatectomy, reached a maximum (900% of control) at 6 hr, and then returned to normal levels at 24 hr after operation. The Cu, Zn-SOD activity was increased 9-fold in hepatectomized rats and about 3-fold in sham-operated rats as compared with control rats. The maximum activity of Mn-SOD was found to be about 4 times higher than that of Cu, Zn-SOD after hepatectomy. The amount of lipoperoxides in the liver mitochondria was found to be increased to 140% in hepatectomized rats and to 120% in sham-operated rats as compared with that of the control rats. Taken together these results suggest that the changes in mitochondrial respiratory functions in the early phase of hepatectomy are due to tissue damage caused by the transient elevation of free radicals. These free radicals are then quickly disposed of by the ever-increasing activities of the Mn-SOD and Cu, Zn-SOD in the liver mitochondria, thereby protecting the liver from further damage and gearing the organ to the regeneration process.
Biochemistry and molecular biology international
The gene encoding cytochrome c oxidase subunit II (COXII) was amplified by polymerase chain react... more The gene encoding cytochrome c oxidase subunit II (COXII) was amplified by polymerase chain reaction (PCR) using mitochondrial DNAs (mtDNAs) isolated from two genera of domesticated ducks, Anas platyrhnchos and Cairina muschata, and their intergeneric and intrageneric hybrids as templates. Comparison of the nucleotide sequences from the two different duck lineages shows that they have approximately 91% homology. The nucleotide substitution pattern in the duck COXII gene reveals a high transition/transversion ratio (8.8:1.0) while 93.2% of the nucleotide substitutions are silent. The duck COXII, which uses GUG as the initiation codon, contains 228 amino acid residues. Comparison of the deduced COXII amino acid sequences in duck, chicken, Xenopus laevis, mouse, bovine and human reveals five highly conserved domains. The evolutionary conservation of the primary structure of COXII implies its functional importance.
Chinese medical journal
By use of restriction fragment length polymorphism analysis, we examined the liver mitochondrial ... more By use of restriction fragment length polymorphism analysis, we examined the liver mitochondrial DNA amplified by polymerase chain reaction from 60 Chinese subjects of 31 to 78 years of age. We found nine specific mtDNA polymorphisms that had never been reported before. Eleven subjects had an Alu I polymorphic site in the subunit 2 gene of NADH dehydrogenase, five had a Hae III polymorphic site in the cytochrome oxidase subunit 2 gene, and five had a Hinf I polymorphic site in the subunit 3 gene of cytochrome oxidase. No polymorphic site was found in the structural genes coding for subunits 1, 3, 4, 4L and 6 of NADH dehydrogenase, cytochrome b, and subunit 8 of ATP synthase. Detailed analysis of the RFLP data did not show age-dependent mtDNA polymorphisms. In addition, the analysis of the restriction patterns of all the mtDNAs revealed 12 mtDNA haplotypes in all the Chinese subjects examined. Among them, type 1 mtDNA was found to be the most predominant and comprised 63.3% of the total study subjects. The restriction patterns of type 1 mtDNA generated by all restriction enzymes were identical to those deduced from the Cambridge sequence of human mtDNA. About 8.3% of the subjects exhibited type 2 mtDNA, and 5% had types 3, 5 and 8 mtDNA, respectively. Each of the rest seven mtDNA types comprised about 2% of the samples. Moreover, type 1 mtDNA was found in the platelets of three white Americans. These findings suggest that type 2 to type 12 mtDNAs have come into existence through the generation or loss of specific polymorphic restriction sites in the mtDNA of the Chinese.
The American Journal of Chinese Medicine
ABSTRACT
Journal of the Formosan Medical Association
Free radicals may be involved in various human disease processes. Cutaneous edema and acute pulmo... more Free radicals may be involved in various human disease processes. Cutaneous edema and acute pulmonary damage of thermal injury mediated by oxygen free radicals can be monitored by studying the levels of lipid peroxidation in blood plasma. In this study, we investigated lipid peroxidation induced by human skin burns. The subjects included 14 healthy subjects and eight burn patients. Healthy subjects included nine males and five females, aged 50.5 +/- 19.1 years. The burn patients included seven males and one female, aged 46.9 +/- 19.1 years, who had burns over 45.6% +/- 14.0% of their total body surface area. Plasma lipoperoxides were measured by high performance liquid chromatographic separation of malondialdehyde-thiobarbituric acid adduct. The average plasma lipoperoxide concentration (measured as malondialdehyde) of the 14 healthy subjects was 0.72 +/- 0.18 mumol/L. This concentration increased significantly (p < 0.0013) in the eight burn patients to a 3.75 +/- 1.34 mumol/L (peak level). Plasma lipoperoxides increased in the first few hours postburn. Plasma lipid peroxide peak levels were found on the third day postburn in 75% of burn patients. Moreover, patients with inhalation injuries had higher plasma lipid peroxide levels (5.27 +/- 5.30 mumol/L) than those without inhalation injury (2.84 +/- 2.88 mumol/L). These results suggest that both oxygen free radicals and lipid peroxidation play a major role in the injuries caused by skin burns and related inhalation [corrected].
Journal of the Formosan Medical Association
In forensic DNA typing, evidential samples generally involve limited amounts of DNA and so should... more In forensic DNA typing, evidential samples generally involve limited amounts of DNA and so should be carefully utilized. Although polymerase chain reaction (PCR) of variable number of tandem repeats (VNTR) alleles is the prevailing method for forensic identification, the fidelity of amplification of heterozygous VNTR alleles with large disparities in length needs to be carefully examined. Reports in the literature and our own observations have demonstrated that PCR artifacts, bogus alleles and allelic drop-out of VNTRs, are related to the amount of genomic DNA, the number of amplification cycles and the length of alleles amplified. Two small (< 1 kb) hypervariable VNTRs (Apo B and HVR-Ig) markers used for forensic identification were chosen to study these relationships. The results revealed that PCR amplification for the heterozygous VNTR alleles with wide disparity in length (> 400 bp) easily produced the allelic drop-out problem and therefore, led to the false results; and the allelic fragment of PCR products was preferentially lost after only 2 cycles of overamplification. We also further established the relationship between the optimal number of amplification cycles and the amount of genomic DNA in the reaction mixture. In our routine forensic screening this relationship has been successfully applied to determine the optimal number of amplification cycles and to avoid the allelic drop-out problem and achieve fidelity of PCR-VNTR amplification. It has also been used to investigate forensic casework.
Journal of the Formosan Medical Association
By use of a simple, rapid and reliable polymerase chain reaction (PCR)-based method, we analyzed ... more By use of a simple, rapid and reliable polymerase chain reaction (PCR)-based method, we analyzed three hypervariable tandem repeats in the 3'-Apo B, 5'-HVR-Ig and 3'-COL2A1 loci. As accurate data of allele frequency of genetic markers is a prerequisite for forensic application, the allele frequency distribution of the three variable number of tandem repeats (VNTR) among the Chinese population in Taiwan were studied. In a total of 123 unrelated Chinese subjects, the Apo B VNTR demonstrated a heterozygosity of 68.2% with 9 alleles, 0.85 of the power of discrimination (PD) value and 0.74 of the allelic diversity (h) value. In a sample of 103 unrelated Chinese subjects, the COL2A1 VNTR showed 49.0% heterozygosity with six alleles, 0.79 of the PD value and 0.74 of the h value. In 106 unrelated subjects, the HVR-Ig VNTR showed 47.4% heterozygosity with seven alleles, 0.79 of the PD value and 0.59 of the h value. The data obtained in this study are not only useful for forensic identification, but will also be helpful for paternity testing, genetic linkage studies and the identification of the three VNTR loci associated with human genetic diseases. Some verifying examinations for the validity and reliability of the three VNTR were performed. The high sensitivity and inexpensive nature of this approach make it superior to the traditional method of DNA fingerprinting for forensic typing. With the use of this PCR-VNTR system, many forensic cases have been successfully identified. The value of this system is illustrated in the investigation of a rape and murder case.
The Chinese journal of physiology
The role of EGTA in stimulating human sperm motility (reported in Lancet i: 460-461, 1984) was in... more The role of EGTA in stimulating human sperm motility (reported in Lancet i: 460-461, 1984) was investigated by measuring the calcium buffering capacity of human seminal plasma. Human seminal plasma contains 9.5 +/- 1.1 mM (mean +/- SE) calcium of which 0.16 +/- 0.01 mM only exists as free Ca2+. The free Ca2+ concentration was not changed by the addition of either 1 mM CaCl2 or 1 mM EGTA. The ability of seminal plasma to bind calcium ions was determined by adding varying amounts of CaCl2. Scatchard analysis of the results indicates the presence of high amounts of high-affinity (Kd1 = 3.4 +/- 0.2 microM, Bm1 = 11.5 +/- 1.5 mM) and low-affinity (Kd2 = 0.55 +/- 0.04 mM, Bm2 = 30.4 +/- 1.7 mM) Ca2+-complexing agents. The low-affinity Ca2+-binding substance may be citrate. These results seem to suggest that human seminal plasma has a high Ca2+ buffering capacity and the stimulation of sperm motility by EGTA ought to be mediated via a mechanism other than the reduction of the free calcium concentration in semen.
Journal of the Formosan Medical Association
A rapid and simple method using restriction enzymes to detect the restriction fragment length pol... more A rapid and simple method using restriction enzymes to detect the restriction fragment length polymorphism (RFLP) pattern of hypervariable segment 1 in the D-loop region of human mitochondrial DNA (mtDNA) was developed. We first focused on the investigation of variations of DNA sequence in the D-loop region among Chinese subjects, as well as on the determination of RFLP patterns of each restriction enzyme. Seven restriction enzymes were used to digest a 618 bp polymerase chain (PCR) reaction product of the D-loop region of mtDNA. Frequency distribution of RFLP patterns of each restriction enzyme among 145 unrelated Chinese subjects in Taiwan was also established. For the purposes of practical forensic application, a routine typing system was designed on the basis of the RFLP data. Two short hypervariable, mtDNA fragments, which were contained within the 618 bp region, were selected for this purpose. In this haplotyping system, a 281 bp PCR-amplified DNA product was analyzed by five restriction enzymes: Mnl I, Nla III, Rsa l, Mse I and Hinf I, and a 237 bp fragment was analyzed by Kpn I. The RFLP patterns were determined by agarose gel electrophoresis of the restriction enzyme-digested DNA fragments. Six restriction enzymes. Mul I, Nla III, Rsa I. Msc I, Hinf I and Kpn 1, defined eight, four, four, five, two and four polymorphic patterns, respectively among the 145 Chinese subjects. The RFLP patterns of restriction fragments for each individual were systematically analyzed and the mtDNAs of the 145 Chinese subjects were grouped into 52 haplotypes. This PCR-RFLP haplotyping system revealed a high degree of variability and diversity of segment I in the D-loop region of human mtDNA. The power of discrimination and allelic diversity values were 0.923 and 0.929, respectively. Successful application of this haplotyping system in a murder case is also discussed.
Proceedings of the National Science Council, Republic of China. Part B, Life sciences
ABSTRACT