主在并[a] 龍財赤子愛腔蚓錢粒体編研基因衰退影蝸

The earthworm Eisenia fetida's benzo [a] pyrene (BaP) exposure experiments were carried out in artificial soil according to ISO 11268-1:1993. And then the upregulated and downregulated subtractive cDNA libraries were constructed by Clontech PCR-Select cDNA Subtration Kit. From the BaP exposure upregulated subtractive cDNA library, several cDNA segments matched mitochondrion-encoded genes were found, including cytochrome c oxidase subunit I (CO I), subunit II (CO II), subunit Ill (CO III), NADH dehydrogenase subunit 1 (NDH1), and ATP synthase subunit 6. The result indicated BaP and the subsequent oxidative stress disturbed the expression of mitochondrion-encoded genes, and this was potential biomarker for oxidative stress following xenobiotic exposure.

Uteroplacental insufficiency and reducing litter size alters skeletal muscle mitochondrial biogenesis in a sex-specific manner in the adult rat

Uteroplacental insufficiency has been shown to impair insulin action and glucose homeostasis in adult offspring and may act in part via altered mitochondrial biogenesis and lipid balance in skeletal muscle. Bilateral uterine vessel ligation to induce uteroplacental insufficiency in offspring (Restricted) or sham surgery was performed on day 18 of gestation in rats. To match the litter size of Restricted offspring, a separate cohort of sham litters had litter size reduced to five at birth (Reduced Litter), which also restricted postnatal growth. Remaining litters from sham mothers were unaltered (Control). Offspring were studied at 6 mo of age. In males, both Restricted and Reduced Litter offspring had reduced gastrocnemius PPAR γ coactivator-1α (PGC-1 α) mRNA and protein, and mitochondrial transcription factor A (mtTFA) and cytochrome oxidase (COX) III mRNA (P < 0.05), whereas only Restricted had reduced skeletal muscle COX IV mRNA and protein and glycogen (P < 0.05), despite unaltered glucose tolerance, homeostasis model assessment (HOMA) and intramuscular triglycerides. In females, only gastrocnemius mtTFA mRNA was lower in Reduced Litter offspring (P < 0.05). Furthermore, glucose tolerance was not altered in any female offspring, although HOMA and intramuscular triglycerides increased in Restricted offspring (P < 0.05). It is concluded that restriction of growth due to uteroplacental insufficiency alters skeletal muscle mitochondrial biogenesis and metabolic characteristics, such as glycogen and lipid levels, in a sex-specific manner in the adult rat in the absence of impaired glucose tolerance. Furthermore, an adverse postnatal environment induced by reducing litter size also restricts growth and alters skeletal muscle mitochondrial biogenesis and metabolic characteristics in the adult rat.

Effect of nitric oxide synthase inhibition on mitochondrial biogenesis in rat skeletal muscle

The purpose of this study was to determine whether nitric oxide synthase (NOS) inhibition decreased basal and exercise-induced skeletal muscle mitochondrial biogenesis. Male Sprague-Dawley rats were assigned to one of four treatment groups: NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME, ingested for 2 days in drinking water, 1 mg/ml) followed by acute exercise, no L-NAME ingestion and acute exercise, rest plus L-NAME, and rest without L-NAME. The exercised rats ran on a treadmill for 53 ± 2 min and were then killed 4 h later. NOS inhibition significantly (P < 0.05; main effect) decreased basal peroxisome proliferator-activated receptor-{gamma} coactivator 1beta (PGC-1beta) mRNA levels and tended (P = 0.08) to decrease mtTFA mRNA levels in the soleus, but not the extensor digitorum longus (EDL) muscle. This coincided with significantly reduced basal levels of cytochrome c oxidase (COX) I and COX IV mRNA, COX IV protein and COX enzyme activity following NOS inhibition in the soleus, but not the EDL muscle. NOS inhibition had no effect on citrate synthase or beta-hydroxyacyl CoA dehydrogenase activity, or cytochrome c protein abundance in the soleus or EDL. NOS inhibition did not reduce the exercise-induced increase in peroxisome proliferator-activated receptor-{gamma} coactivator 1{alpha} (PGC-1{alpha}) mRNA in the soleus or EDL. In conclusion, inhibition of NOS appears to decrease some aspects of the mitochondrial respiratory chain in the soleus under basal conditions, but does not attenuate exercise-induced mitochondrial biogenesis in the soleus or in the EDL.

Adiponectin decreases pyruvate dehydrogenase kinase 4 gene expression in obese- and diabetic derived

Aim: To investigate the effects of globular adiponectin (gAd) on gene expression and whether these effects are mediated through 3',5'-cyclic monophosphate-activated protein kinase in skeletal muscle myotubes obtained from lean, obese and obese diabetic individuals.

Methods: Rectus abdominus muscle biopsies were obtained from surgical patients to establish primary skeletal muscle cell cultures. Three distinct primary cell culture groups were established (lean, obese and obese diabetic; n = 7 in each group). Once differentiated, these cultures were then exposed to gAd or 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (AICAR) for 6 h.

Results: Stimulation with gAd decreased pyruvate dehydrogenase kinase 4 (PDK4) gene expression in the obese and diabetic samples (p ≤ 0.05) and increased cytochrome c oxidase (COX) subunit 4 (COXIV) gene expression in the myotubes derived from lean individuals only (p < 0.05). AICAR treatment also decreased PDK4 gene expression in the obese- and diabetic-derived myotubes (p ≤ 0.05) and increased the gene expression of the mitochondrial gene, COXIII, in the lean-derived samples only (p < 0.05).

Conclusions: This study demonstrated distinct disparity between myotubes derived from lean compared with obese and obese diabetic individuals following gAd and AICAR treatment. Further understanding of the regulation of PDK4 in obese and diabetic skeletal muscle and its interaction with adiponectin signalling is required as this appears to be an important early molecular event in these disease states that may improve blood glucose control and metabolic flux.

Phylogenetic relationships among nine scallop species (Bivalvia : Pectinidae) inferred from nucleotide sequences of one mitochondrial and three nuclear gene regions

Current knowledge of the evolutionary relationships among scallop species (Mollusca: Bivalvia: Pectinidae) in the Indo-Pacific region is rather scanty. To enhance the understanding of the relationships within this group, phylogenies of nine species of scallops with the majority from coastal regions of Thailand, were reconstructed by maximum parsimony, maximum likelihood, and Bayesian methods using sequences of the 16S rRNA of the mitochondrial genome, and a fragment containing the ITS1, 5.8S and ITS2 genes of the nuclear DNA. The trees that resulted from the three methods of analysis were topologically identical, however, gained different levels of support at some nodes. Nine species were clustered into two major clades, corresponding to two subfamilies (Pectininae and Chlamydinae) of the three currently recognized subfamilies within Pectinidae. Overall, the relationships reported herein are mostly in accordance with the previous molecular studies that used sequences of the mtDNA cytochrome oxidase subunit I, and the classification system based on microsculpture of shell features and morphological characteristics of juveniles. Levels of divergences were different among genes (i.e., the 5.8S gene showed the lowest levels of nucleotide divergence at all levels, whereas the 16S rRNA showed the highest level of variation within species, and ITS2 gene revealed the highest level of divergence at higher levels).

CoQ10 conveys protection from oxidative stress in plasma but not skeletal muscle

Coenzyme Q10 (CoQ10) is commonly consumed as an antiaging supplement at doses of 30–210 mg/day. The aim of the study was to determine if CoQ10 alters markers of antioxidant status, oxidative damage, and gene expression in aging skeletal muscle. Female guinea pigs aged 26 months were supplemented for 6 weeks with CoQ10 at a human equivalent dose of 10 mg/kg/day. Body weight, plasma CoQ10 concentration, and WBC DNA abasic sites were measured at weeks 0, 2, 4, and 6 of the supplementation period. At the end of supplementation, concentrations of skeletal muscle CoQ10, glutathione, malondialdehyde, protein carbonyls, DNA abasic sites, activities of catalase and glutathione peroxidase, and the gene expression of cyctochrome c oxidase subunits were measured. Dietary supplementation with CoQ10 elevated plasma CoQ10 levels (pre 73 ± 3 nmol/L, post 581 ± 15 nmol/L, P < 0.05) and decreased abasic sites in WBC DNA (pre 16.8 ± 0.5 Ap/100000 bp, post 9.7 ± 0.4 Ap/100000 bp, P < 0.05). In contrast, all of the measures made in skeletal muscle were not different between groups (P > 0.05). These results indicate that dietary supplementation with CoQ10 at a dose of 10 mg/kg/day may be capable of increasing antioxidant protection and reducing oxidative damage in the plasma, but may have no effect in skeletal muscle.

Bioanalytical experiments for the undergraduate laboratory : monitoring glucose in sports drinks

This paper describes two complementary bioanalytical experiments for analyzing the concentration of glucose in sports drinks. The first experiment is a spectrophotometric enzyme assay employing the enzymes glucose oxidase (GOx) and horseradish peroxidase (HRP). The glucose is oxidized by the GOx, producing hydrogen peroxide, which is the substrate for HRP. In the reduction of the H2O2 a chromogen is oxidized, causing a color change. In the partner experiment, the GOx is immobilized on a platinum electrode using a dialysis membrane. The hydrogen peroxide produced in the enzyme reaction is monitored amperometrically by oxidizing the hydrogen peroxide produced. The simple method of preparing the enzyme electrode is useful in demonstrating the important parameters in defining the response of enzyme electrodes. The same sports drinks are analyzed in both experiments. The two experiments together illustrate the advantage of bioanalysis in analyzing complex samples with minimal sample preparation.

Parameters important in fabricating enzyme electrodes using self-assembled monolayers of alkanethiols

The fabrication of enzyme electrodes using self-assembled monolayers (SAMs) has attracted considerable interest because of the spatial control over the enzyme immobilization. A model system of glucose oxidase covalently bound to a gold electrode modified with a SAM of 3-mercaptopropionic acid was investigated with regard to the effect of fabrication variables such as the surface topography of the underlying gold electrode, the conditions during covalent attachment of the enzyme and the buffer used. The resultant monolayer enzyme electrodes have excellent sensitivity and dynamic range which can easily be adjusted by controlling the amount of enzyme immobilized. The major drawback of such electrodes is the response which is limited by the kinetics of the enzyme rather than mass transport of substrates. Approaches to bringing such enzyme electrodes into the mass transport limiting regime by exploiting direct electron transfer between the enzyme and the electrode are outlined.

Molecular phylogenetic relationships between Albugo candida collections on the Brassicaceae in Australia

Recent phylogenetic analyses of Albugo candida using the mitochondrial cytochrome c oxidase subunit II (cox2) gene, the nuclear ribosomal RNA large subunit (LSU) gene and the nuclear ribosomal RNA internal transcribed spacer (ITS) gene region have revealed significant genetic variation and led to the description of new species in the A. candida complex. This study examined the genetic diversity within Australian collections of A. candida from various Brassicaceae species in a range of geographic locations. Phylogenetic analysis of 31 Australian A. candida collections from 11 hosts using the rDNA ITS region, rDNA LSU region and cox2 mtDNA showed that the majority of Australian A. candida collections were the common form of A. candida. One collection from a common weed host, hairy bitter cress (Cardamine hirsuta), was found to belong to a previously reported but undescribed species, while three collections, also from C. hirsuta, were found to belong to a new undescribed species.

Milk lactate determination with a rotating bioreactor based on an electron transfer mediated by osmium complexes incorporating a continuous-flow/stopped-flow system

The high sensitivity that can be attained using a bienzymatic system and mediated by the redox polymer [Os(bpy)₂ClPyCH₂NHpoly(allylamine)] (Os-PAA), has been verified by on-line interfacing of a rotating bioreactor and continuous-flow/stopped-flow/continuous-flow processing. When the hydrogen peroxide formed by LO_x layer reaches the inner layer, the electronic flow between the immobilized peroxidase and the electrode surface produces a current, proportional to lactate concentration. The determination of lactate was possible with a limit of detection of 5 nmol l⁻¹ in the processing of as many as 30 samples per hour. This arrangement allows working in undiluted milk samples with a good stability and reproducibility. Horseradish peroxidase [EC 1.11.1.7] and Os-PAA were covalently immobilized on the glassy carbon electrode surface (upper cell body), lactate oxidase [EC 1.1.3.x] was immobilized on a disk that can be rotated.

Conducting polymer enzyme alloys : electromaterials exhibiting direct electron transfer

Glucose oxidase (GOx) is an important enzyme with great potential application for enzymatic sensing of glucose, in implantable biofuel cells for powering of medical devices in vivo and for large-scale biofuel cells for distributed energy generation. For these applications, immobilisation of GOx and direct transfer of electrons from the enzyme to an electrode material is required. This paper describes synthesis of conducting polymer (CP) structures in which GOx has been entrained such that direct electron transfer is possible between GOx and the CP. CP/enzyme composites prepared by other means show no evidence of such “wiring”. These materials therefore show promise for mediator-less electronic connection of GOx into easily produced electrodes for biosensing or biofuel cell applications.

Structural and functional characterization of the oxidoreductase a-DsbA1 from wolbachia pipientis

The α-proteobacterium Wolbachia pipientis is a highly successful intracellular endosymbiont of invertebrates that manipulates its host's reproductive biology to facilitate its own maternal transmission. The fastidious nature of Wolbachia and the lack of genetic transformation have hampered analysis of the molecular basis of these manipulations. Structure determination of key Wolbachia proteins will enable the development of inhibitors for chemical genetics studies. Wolbachia encodes a homologue (α-DsbA1) of the Escherichia coli dithiol oxidase enzyme EcDsbA, essential for the oxidative folding of many exported proteins. We found that the active-site cysteine pair of Wolbachia α-DsbA1 has the most reducing redox potential of any characterized DsbA. In addition, Wolbachia α-DsbA1 possesses a second disulfide that is highly conserved in α-proteobacterial DsbAs but not in other DsbAs. The α-DsbA1 structure lacks the characteristic hydrophobic features of EcDsbA, and the protein neither complements EcDsbA deletion mutants in E. coli nor interacts with EcDsbB, the redox partner of EcDsbA. The surface characteristics and redox profile of α-DsbA1 indicate that it probably plays a specialized oxidative folding role with a narrow substrate specificity. This first report of a Wolbachia protein structure provides the basis for future chemical genetics studies.

Determination of intracellular glutathione and glutathione disulfide using high performance liquid chromatography with acidic potassium permanganate chemiluminescence detection

Measurement of glutathione (GSH) and glutathione disulfide (GSSG) is a crucial tool to assess cellular redox state. Herein we report a direct approach to determine intracellular GSH based on a rapid chromatographic separation coupled with acidic potassium permanganate chemiluminescence detection, which was extended to GSSG by incorporating thiol blocking and disulfide bond reduction. Importantly, this simple procedure avoids derivatisation of GSH (thus minimising auto-oxidation) and overcomes problems encountered when deriving the concentration of GSSG from ‘total GSH’. The linear range and limit of detection for both analytes were 7.5 × 10−7 to 1 × 10−5 M, and 5 × 10−7 M, respectively. GSH and GSSG were determined in cultured muscle cells treated for 24 h with glucose oxidase (0, 15, 30, 100, 250 and 500 mU mL−1), which exposed them to a continuous source of reactive oxygen species (ROS). Both analyte concentrations were greater in myotubes treated with 100 or 250 mU mL−1 glucose oxidase (compared to untreated controls), but were significantly lower in myotubes treated with 500 mU mL−1 (p < 0.05), which was rationalised by considering measurements of H2O2 and cell viability. However, the GSH/GSSG ratio in myotubes treated with 100, 250 and 500 mU mL−1 glucose oxidase exhibited a dose-dependent decrease that reflected the increase in intracellular ROS.

The effect of short-term canola oil ingestion on oxidative stress in the vasculature of stroke-prone spontaneously hypertensive rats

Background: This study aimed to determine if 25 days of canola oil intake in the absence of excess dietary salt or together with salt loading affects antioxidant and oxidative stress markers in the circulation. A further aim was to determine the mRNA expression of NADPH oxidase subunits and superoxide dismutase (SOD) isoforms in the aorta of stroke-prone spontaneously hypertensive (SHRSP) rats.

Methods: Male SHRSP rats, were fed a defatted control diet containing 10% wt/wt soybean oil or a defatted treatment diet containing 10% wt/wt canola oil, and given tap water or water containing 1% NaCl. Blood was collected at the end of study for analysis of red blood cell (RBC) antioxidant enzymes, RBC and plasma malondialdehyde (MDA), plasma 8-isoprostane and plasma lipids. The aorta was removed and the mRNA expression of NOX2, p22phox, CuZn-SOD, Mn-SOD and EC-SOD were determined.

Results: In the absence of salt, canola oil reduced RBC SOD and glutathione peroxidase, and increased total cholesterol and LDL cholesterol compared with soybean oil. RBC glutathione peroxidase activity was significantly lower in both the salt loaded groups compared to the soybean oil only group. In addition, RBC MDA and plasma HDL cholesterol were significantly higher in both the salt loaded groups compared to the no salt groups. Plasma MDA concentration was higher and LDL cholesterol concentration lower in the canola oil group loaded with salt compared to the canola oil group without salt. The mRNA expression of NADPH oxidase subunits and SOD isoforms were significantly reduced in the canola oil group with salt compared to canola oil group without salt.

Conclusion: In conclusion, these results indicate that canola oil reduces antioxidant status and increases plasma lipids, which are risk factors for cardiovascular disease. However, canola oil in combination with salt intake increased MDA, a marker of lipid peroxidation and decreased NAPDH oxidase subunits and aortic SOD gene expression.