Wenxiang Gao

Mitochondrial genome analysis of Ochotona curzoniae and implication of cytochrome c oxidase in hypoxic adaptation.

Pikas originated in Asia and are small lagomorphs native to cold climates. The plateau pika, Ochotona curzoniae is a keystone species on the Qinghai-Tibet Plateau and an ideal animal model for hypoxic adaptation studies. Altered mitochondrial function, especially cytochrome c oxidase activity, is an important factor in modulation of energy generation and expenditure during cold and hypoxia adaptation. In this study, we determined the complete nucleotide sequence of the O. curzoniae mitochondrial genome. The plateau pika mitochondrial DNA is 17,131bp long and encodes the complete set of 37 proteins typical for vertebrates. Phylogenetic analysis based on concatenated heavy-strand encoded protein-coding genes revealed that pikas are closer to rabbit and hare than to rat. This suggests that rabbit or hare would be a good control animal for pikas in cold and hypoxia adaptation studies. Fifteen novel mitochondrial DNA-encoded amino acid changes were identified in the pikas, including three in the subunits of cytochrome c oxidase. These amino acid substitutions potentially function in modulation of mitochondrial complexes and electron transport efficiency during cold and hypoxia adaptation.

Rare Mitochondrial DNA Polymorphisms are Associated with High Altitude Pulmonary Edema (HAPE) Susceptibility in Han Chinese

BACKGROUND The role of genetics in determining an individuals susceptibility to high altitude pulmonary edema (HAPE) is unclear. However a number of genetic polymorphisms have recently been found to be overrepresented in patients with HAPE. Changes at the mitochondrial level may play an important role in the human bodys adaptation to hypoxia. Polymorphisms of mitochondrial DNA (mtDNA) have been shown to be responsible for differences in organelle function. Therefore, the frequency of mtDNA 3397A/G and 3552T/A polymorphisms were studied to determine their potential role in HAPE. OBJECTIVES To further study the role of mtDNA 3397A/G and 3552T/A variations of reduced nicotinamide adenosine dinucleotide dehydrogenase 1 in HAPE susceptibility. METHODS The single-nucleotide polymorphisms of mtDNA 3397 and 3552 in patients with HAPE (n = 132) and their matched control subjects (n = 233) were studied using polymerase chain reaction sequencing. RESULTS The frequency of mtDNA 3397G in the HAPE group (2.3%) was significantly higher than that of the control group (0%; P = .021; odds ratio, 2.806; 95% confidence interval, 2.443 to 3.223). The frequency of mtDNA 3552A in the HAPE group (6.8%) also was significantly higher than in the control group (1.7%; P = .012; odds ratio, 4.198; 95% confidence interval, 1.264 to 13.880). CONCLUSIONS In this study, we present the first evidence of differences in mtDNA polymorphism frequencies between HAPE victims and healthy Han Chinese. Genotypes of mtDNA 3397G and 3552A were correlated with HAPE susceptibility. This result could contribute to defining the role of the mitochondrial genome in the pathogenesis of HAPE.

Mitochondrial nt3010G-nt3970C haplotype is implicated in high-altitude adaptation of Tibetans

Tibetans are well adapted to living and thriving in high-altitude environments. Mitochondria are central links to oxygen consumption, and variations in mitochondrial DNA (mtDNA) could play a role in high-altitude adaptation. Alleles at several polymorphic sites in mtDNA define common haplotypes, or haplogroups, including some that have been implicated in the risk of developing certain diseases. However, few reports have determined whether relationships exist between haplogroups and high-altitude adaptation in the Tibetan population. The D4 haplogroup is a major haplogroup of the Han Chinese. In the present study, genotypes of 12 polymorphisms were determined in members of a Tibetan population (n = 72), low altitude-Han (la-Han, n = 144), and high altitude-Han (ha-Han, n = 227) populations using polymerase chain reaction–restriction fragment length polymorphism and polymerase chain reaction–ligase detection reaction assays. The mitochondrial haplogroup D4 was negatively associated with high-altitude adaptation in Tibetans (P = 0.001 vs. la-Han, OR = 0.166, 95% CI = 0.048–0.567; P = 0.009 vs. ha-Han OR = 0.232, 95% CI = 0.069–0.778). The frequency of the nt3010G-nt3970C haplotype was significantly higher in Tibetans than in la-Han (P = 0.000) and ha-Han (P = 0.001) subjects. Findings in the present study suggest that unique mitochondrial variations determine a genetic background that is associated with high-altitude adaptation in the Tibetan population.

Hypoxia affects mitochondrial protein expression in rat skeletal muscle.

Hypoxia affects mammalian mitochondrial function, as well as mitochondria-based energy metabolism. The detail mechanism has not been fully understood. In this study, we detected protein expression levels in mitochondrial fractions of Wistar rats exposed to hypobaric hypoxia by use of proteomic methods. Adult male Wistar rats were randomized into an hypoxic (4,500 m, 30 days) group and a normoxic control group (sea level). Gastrocnemius muscles mitochondria were extracted and purified. Mitochondrial oxygen consumption was measured with a Clark oxygen electrode; mitochondrial transmembrane potential was detected with Rhodamine 123 as a fluoresce probe. Using 2-DE and MALDI-TOF MS analysis, we identified eight mitochondrial protein spots that were differentially expressed in the hypoxic group compared with the normoxic control. These proteins included Chain A of F1-ATPase, voltage dependent anion channel 1 (VDAC), hydroxyacyl Coenzyme A dehydrogenase α-subunit, mitochondrial F1 complex γ-subunit, androgen-regulated protein and tripartite motif protein 50. Two of the spots, VDAC and ATP synthase α-subunit, were confirmed by Western blotting analysis. Oxygen consumption during State 3 respiration, as well as the respiratory control ratio (RCR) was significantly higher in the control than that in the hypoxic group; mitochondrial transmembrane potential was significantly higher in hypoxic group than that in the control. With successful use of multiple proteomic analysis techniques, we demonstrates that 30 days hypoxia exposure has effects on the expression of mitochondrial proteins involved in ATP production and lipid metabolism, decrease the stability of mitochondrial membrane, and affect the mitochondrial electron transport chain.

Genotyping mitochondrial DNA single nucleotide polymorphisms by PCR ligase detection reactions.

Abstract Background: The identification of human mitochondrial DNA (mtDNA) sequence variations, especially single nucleotide polymorphisms (SNPs), is important for many applications. The PCR-ligase detection reaction (LDR) method can reduce false-positives and eliminate the need for both post-PCR and post-ligation purifications in SNP analyses. In addition, it has been successfully employed to detect point mutations in various nuclear genes. In this study, we used the PCR-LDR platform to characterize mtDNA SNPs. Methods: Multiplex PCR-LDRs were used to genotype 19 mtDNA single nucleotide polymorphic sites from 812 samples. Performance of the method was assessed by direct sequencing of 44 samples. Results: We established an overall 97.4% success rate with 99.2% accuracy using the multiplex PCR-LDR methodology. Conclusions: The PCR-LDR mtDNA genotyping technique is simple, highly accurate, has high-throughput, and is cost-effective. Therefore, this method is applicable to mtDNA haplotyping in various applications. Clin Chem Lab Med 2010;48:475–83.

Altofrequency SNPs of mitochondrial DNA in 26 Han Chinese

Abstract Objective To explore the possible mitochondrial DNA (mtDNA) polymorphism in Han Chinese. Methods The complete mitochondrial genome of 26 unrelated healthy Han Chinese were extracted and sequenced. Results The mtDNA nucleotide sites (2 706, 7 028, 8 860. 11 719, and 15 326) were found totally different from the Revised Cambridge Reference Sequence (rCRS). These single nucleotide polymorphisms (SNPs) were 2 706 A→G, 7 028 C→T. 8 860 A→G. 11 719 G→A, 15 326 A→G. Conclusion These findings provide new insights into the characteristics of Han Chinese mitochondrial genetic diversity.

Altered expression of mitochondrial related genes in the native Tibetan placents by mitochondrial cDNA array analysis

Abstract Objective To explore the mechanism of native Tibetan fetuses adaptation to hypoxia, we tried to find the different expression genes about mitochondrial function in the native Tibetan placents. Methods In this study, the placents of native Tibetan and the high-altitude Han (ha-Han) were collected. After the total RNA extraction, the finally synthesized cDNAs were hybridized to mitochondrial array to find the altered expression genes between them. Then, the cytochrome c oxidase 17 (Cox17), dynactin 2 (DCTN2, also known as p50), and vascular endothelial growth factor receptor (VEGFR, also known as KDR) were chosen from the altered expression genes to further verify the array results using the SYBR Green real-time PCR. Because the altered expression genes (such as Cybb and Cox17) in the array results related to the activities of COXI and COXIV, the placental mitochondria activities of COXI and COXIV were measured to find their changes in the hypoxia. Results By a standard of ≥1.5 or ≤0.67, there were 24 different expressed genes between the native Tibetan and the ha-Han placents, including 3 up-regulated genes and 21 down-regulated genes. These genes were related to energy metabolism, signal transduction, cell proliferation, electron transport, cell adhesion, nucleotide-excision repair. The array results of Cox17, DCTN2 and KDR were further verified by the real-time RT-PCR. Through the mitochondria respiration measurements, the activity of COXI in the native Tibetan placents were higher than that of ha-Han, there was no difference in COXIV activity between them. Conclusion The altered mitochondrial related genes in the native Tibetan placents may have a role in the high altitude adaptation for fetuses through changing the activity of mitochondrial COX.

An improved formula for standard hypoxia tolerance time (STT) to evaluate hypoxic tolerance in mice

BackgroundHypoxia is a primary cause of mountain sickness and a common pathological condition in patients with heart failure, shock, stroke, and chronic obstructive pulmonary disease (COPD). Thus far, little advancement in countering hypoxic damage has been achieved, and one of the main reasons is the absence of an ideal algorithm or calculation method to normalize hypoxia tolerance scores when evaluating an animal model. In this study, we improved a traditional calculation formula for assessment of hypoxia tolerance.MethodsWe used a sealed bottle model in which the oxygen is gradually consumed by a mouse inside. To evaluate the hypoxia tolerance of mice, the survival time (ST) of the mouse is recorded and was used to calculate standard hypoxia tolerance time (STT) and adjusted standard hypoxia tolerance time (ASTT). Mice administered with methazolamide and saline were used as positive and negative controls, respectively.ResultsSince mice were grouped according to either body weight (BW) or bottle volume, we found a strongly negative correlation between STT and BW instead of between STT and bottle volume, suggesting that different BWs could cause false positive or negative errors in the STT results. Furthermore, both false positive and negative errors could be rectified when ASTT was used as the evaluation index. Screening for anti-hypoxic medicines by using mice as the experimental subjects would provide more credible results with the improved ASTT method than with the STT method.ConclusionASTT could be a better index than STT for the evaluation of hypoxia tolerance abilities as it could eliminate the impact of animal BW.