Association Between the Polymorphism of Steroid Hormone Metabolism Genes and High-Altitude Pulmonary Edema in the Chinese Han Population.

PURPOSE: Steroid hormone metabolism plays an essential role in high-altitude pulmonary edema (HAPE) progression. This study aimed to investigate the association between polymorphism in seven steroid hormone metabolism genes (STAR, HSD3B1, HSD3B2, CYP17A1, CYP21A2, CYP11B1, and CYP11B2) and HAPE susceptibility among Han Chinese. PATIENTS AND METHODS: A total of 41 tagSNPs in the seven genes were genotyped using Sequenom MassARRAY SNP assays from 169 HAPE patients (HAPE-p) and 309 matched Han Chinese individuals resistant to HAPE (HAPE-r). The genotypic and allele frequencies, odds ratios (ORs), and 95% confidence intervals (95% CIs) were calculated. RESULTS: Four SNPs, including the allele C of rs6203 (p = 0.034, OR [95% CI] = 1.344 [1.022-1.767]) in HSD3B1, allele G of rs3740397 (p = 0.044, OR [95% CI] = 1.314 [1.007-1.714]) and allele C of rs10786712 (p = 0.039, OR [95% CI] = 0.751 [0.572-0.986]) in CYP17A1, and allele T of rs6402 (p = 0.006, OR [95% CI] = 0.504 [0.306-0.830]) in CYP11B1, were significantly associated with HAPE. The distribution of the genotypes of these SNPs also significantly differed between the HAPE-p and HAPE-r groups. Moreover, six haplotypes (the linkage disequilibrium block including rs10883783, rs4919686, rs3740397, rs3824755, and rs10786712) of CYP17A1 were also significantly associated with HAPE. CONCLUSION: The four SNPs located in HSD3B1 (rs6203), CYP17A1 (rs3740397 and rs10786712), and CYP11B1 (rs6402) and the six haplotypes of CYP17A1 are likely to have an effect on HAPE.

Adaptive Changes in Hemoglobin Function in High-Altitude Tibetan Canids Were Derived via Gene Conversion and Introgression.

A key question in evolutionary biology concerns the relative importance of different sources of adaptive genetic variation, such as de novo mutations, standing variation, and introgressive hybridization. A corollary question concerns how allelic variants derived from these different sources may influence the molecular basis of phenotypic adaptation. Here, we use a protein-engineering approach to examine the phenotypic effect of putatively adaptive hemoglobin (Hb) mutations in the high-altitude Tibetan wolf that were selectively introgressed into the Tibetan mastiff, a high-altitude dog breed that is renowned for its hypoxia tolerance. Experiments revealed that the introgressed coding variants confer an increased Hb-O2 affinity in conjunction with an enhanced Bohr effect. We also document that affinity-enhancing mutations in the ß-globin gene of Tibetan wolf were originally derived via interparalog gene conversion from a tandemly linked ß-globin pseudogene. Thus, affinity-enhancing mutations were introduced into the ß-globin gene of Tibetan wolf via one form of intragenomic lateral transfer (ectopic gene conversion) and were subsequently introduced into the Tibetan mastiff genome via a second form of lateral transfer (introgression). Site-directed mutagenesis experiments revealed that the increased Hb-O2 affinity requires a specific two-site combination of amino acid replacements, suggesting that the molecular underpinnings of Hb adaptation in Tibetan mastiff (involving mutations that arose in a nonexpressed gene and which originally fixed in Tibetan wolf) may be qualitatively distinct from functionally similar changes in protein function that could have evolved via sequential fixation of de novo mutations during the breed's relatively short duration of residency at high altitude.

The susceptibility gene screening in a Chinese high-altitude pulmonary edema family by whole-exome sequencing.

High-altitude pulmonary edema (HAPE) is one of idiopathic mountain sicknesses that occur in healthy lowlanders when they quickly ascend to altitudes exceeding 2500 m above sea levels within 1-7 days. Growing evidence suggests that genetics plays an important role in the risk of HAPE. In this study, we recruited a Chinese HAPE family and screened genetic variations in the 7 family members (including 6 family members with a medical history of HAPE and the propositus's mother) by whole-exome sequencing. The results showed 18 genetic variations (9 SNVs and 9 Indels) were related to HAPE. Two SNV sites (CFHR4 (p.L85F) and OXER1 (p.R176C)) were predicted to be damaging and alter protein functions by SIFT, PolyPhen-2 and PROVEAN software. The biological function of OXER1 was highly related to the hypoxia-inducible factor pathway. Therefore, those two sites were identified as candidate pathological variations. Moreover, other SNVs (NMB p.S150P, APOB p.I4194T, EIF4ENIF1 p.Q763P) and Indels (KCNJ12 p.EE333-334E, ANKRD31 p.LMN251-253LN, OR2A14 p.HFFC175-178HFC) were also predicted to be damaging as well, which also might be considered as potential candidate pathological variations related to HAPE. Collectively we firstly screened the susceptibility genes in a Chinese HAPE family by whole-exome sequencing, which will provide new clues for further mechanistic studies of HAPE.

Effects of the aqueous extract of a Tibetan herb, Rhodiola algida var. tangutica on proliferation and HIF-1α, HIF-2α expression in MCF-7 cells under hypoxic condition in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE: Rhodiola algida var. tangutica is a traditional Tibetan herb. Its root and rhizome have been successfully used as an effective clinical remedy for the prevention and treatment of cancer and high-altitude sickness. This study aimed to investigate the effect of Rhodiola algida var. tangutica on hypoxic MCF-7 breast cancer cells and the underlying mechanisms. MATERIALS AND METHODS: The antiproliferative effects of R. algida on MCF-7 breast cancer cells were compared in vitro under hypoxic and normal conditions by using MTT analysis. The influence of R. algida on cancer cell apoptosis was determined by flow cytometry. The expression levels of hypoxia-inducible factor (HIF)-1α and HIF-2α were evaluated by western blot analysis. RESULTS: R. algida inhibited the proliferation of MCF-7 breast cancer cells in a dose- and time-dependent manner. The results of flow cytometry indicated that the antiproliferative effect of R. algida was mediated by apoptosis induction. Pretreatment with R. algida significantly suppressed the hypoxia-induced proliferation and expression of HIF-1α and HIF-2α in MCF-7 breast cancer cells. CONCLUSIONS: R. algida might exert an anti-carcinogenic effect on MCF-7 breast cancer cells by decreasing the protein levels of HIF-1α and HIF-2α, which are overexpressed under hypoxic conditions. This effect might be elicited by inhibiting the hypoxia-induced proliferation of MCF-7 breast cancer cells.

CYP17A1 and CYP2E1 variants associated with high altitude polycythemia in Tibetans at the Qinghai-Tibetan Plateau.

Tibetans adapt to high altitude environments through low blood hemoglobin concentrations. Previous work has identified that CYP17A1 and CYP2E1 genes exhibit evidence of local positive selection for this Tibetan high-altitude adaptation. Nevertheless, despite this apparent genetic advantage, some Tibetans still develop high altitude polycythemia (HAPC) yet the reasons for this remain unknown. We sought to determine if polymorphisms in CYP17A1 and CYP2E1 genes were associated with susceptibility to HAPC in Tibetans at the Qinghai-Tibetan Plateau in China. We enrolled 63 Tibetan HAPC patients and 131 healthy, age- and gender-matched control Tibetans. All subjects are from the Yushu area of Qinghai where the altitude is over 3500 m. Three SNPs of the CYP17A1 including rs3781287, rs11191548 and rs1004467, and four SNPs of CYP2E1 gene, including rs1536836, rs3813865, rs3813867 and rs743535, were genotyped by the Sequenom MassARRAY SNP assays. We discovered that SNP rs1004467 of the CYP17A1 gene and SNP rs3813865 of the CYP2E1 gene were significantly associated with HAPC risk. Furthermore, we identified a positive correlation between these two SNPs and plasma hemoglobin levels. Thus, taken together, our study is the first to our knowledge to show that polymorphisms in the rs1004467 SNP of CYP17A1 and rs3813865 SNP of CYP2E1 correlate with susceptibility to HAPC.

EPAS1 Gene Polymorphisms Are Associated With High Altitude Polycythemia in Tibetans at the Qinghai-Tibetan Plateau.

OBJECTIVE: To test the hypothesis that the polymorphisms in the EPAS1 gene are associated with the susceptibility to high altitude polycythemia (HAPC) in Tibetans at the Qinghai-Tibetan Plateau. METHODS: We enrolled 63 Tibetan HAPC patients and 131 matched healthy Tibetans as a control group, from the Yushu area in Qinghai where the altitude is greater than 3500 m. Eight single-nucleotide polymorphisms (SNPs) of the EPAS1 gene, including rs12619696, rs13420857, rs2881504, rs4953388, rs13419896, rs4953354, rs10187368, and rs7587138, were genotyped by the Sequenom MassARRAY SNP assay. RESULTS: The frequencies of the G allele of EPAS1 SNP rs13419896 were significantly higher in the HAPC group than in the control group (P < .05). Moreover, the A alleles of rs12619696 and rs4953354 were prevalent in the HAPC group, and their counterpart homozygotes were prevalent in the normal Tibetan group (P < .05). CONCLUSIONS: Compared with normal Tibetans, Tibetans with HAPC are maladapted and have a different haplotype in EPAS1 SNPs rs12619696, rs13419896, and rs4953354.

Sleep disturbances in long-term immigrants with chronic mountain sickness: a comparison with healthy immigrants at high altitude.

The aim of this study was to examine sleep disturbances in patients with chronic mountain sickness (CMS). The sleep of 14 patients with CMS and 11 healthy controls with or without sleep disorders (control N: without sleep disorders; control D: with sleep disorders) was studied by polysomnography. Hypopnea was the sleep disorder most commonly suffered by CMS patients and control D subjects. No major differences were observed in sleep structure between CMS and control groups, with the exception of shorter rapid eye movement latency in controls and increased deep non-rapid eye movement in the control N group. Periodic breathing was observed in only two study participants, one each in the CMS and control D groups. The level of saturated oxygen was significantly lower in the CMS group during sleep than the control groups (P<0.05). CMS scores were positively correlated with the apnea-hypopnea index, and negatively correlated with saturated oxygen levels. These results demonstrate that sleep disorders and nocturnal hypoxia are important in the development of CMS.

[Effect of different altitudes on telomere length of rat peripheral blood leukocyte].

The present study was aimed to investigate the effect of different altitudes on telomere length of rat peripheral blood leukocyte and possible mechanism. Sixty male rats were randomly divided into three groups, lower altitude control group (10 m), moderate altitude group (2 260 m) and very high altitude group (simulated 5 000 m). The moderate altitude group and very high altitude group rats were transported to Xining and hypobaric chamber in Qinghai University, respectively. The peripheral blood specimens were extracted 30 d after the transportation. By means of real-time PCR, automatic blood cell analyzer, ELISA, TBA and WST-1 methods, the telomere lengths of blood leukocyte, the hemoglobin (Hb) contents, the plasma levels of telomerase reverse transcriptase (TERT) and hypoxia-inducible factor 1α (HIF-1α), the plasma content of malondialdehyde (MDA) and superoxide dismutase (SOD) activity were measured, respectively. The results showed that the telomere lengths of peripheral blood leukocyte in moderate altitude group were longer than those in control group and very high altitude group. The changes of TERT were compatible with the telomere length of peripheral blood leukocyte under different altitudes. The levels of HIF-1α in moderate altitude group and very high altitude group were higher than that of control group. The very high altitude group showed decreased SOD activities and increased level of MDA, compared with the other two groups. These results suggest that the telomere lengths of rat peripheral blood leukocyte in moderate altitude are elongated, and that the telomere-elongating effect is lost under very high altitude. The changes of HIF-1α, TERT and oxidative stress damage are the main mechanisms of telomere length changes. Moderate altitude living might be beneficial to increasing the life span in mammals.

Excision repair cross-complementation group 1 codon 118 polymorphism, micro ribonucleic acid and protein expression, clinical outcome of the advanced gastric cancer response to first-line FOLFOX-4 in Qinghai-Tibetan plateau population.

CONTEXT: The excision repair cross-complementation group 1 (ERCC1) codon 118 C/T polymorphism has been associated with clinical outcome in cancer patients treated with platinum chemotherapy. Ethnic differences in the frequency of this polymorphism have been observed in Caucasian and African populations. AIM: The aim of this study was to evaluate the frequency and survival benefit of the ERCC1 codon 118 C/T polymorphism in a high-altitude population with advanced gastric cancer. MATERIALS AND METHODS: Polymerase chain reaction-restriction fragment length polymorphism was used to determine the frequency of ERCC1 118 codon C/T polymorphism in 206 advanced gastric cancer patients residing in the high-altitude Qinghai-Tibetan plateau. The influence of the ERCC1 codon 118 C/T polymorphism on its micro ribonucleic acid (mRNA) and protein expression, clinicopathological features; response to the platinum-based combination chemotherapy, and the outcome was evaluated. STATISTICAL ANALYSIS: The Kaplan-Meier method was used for survival analysis. The correlation of ERCC1 codon 118 polymorphism with ERCC1 mRNA and protein expression, clinicopathological characteristics, and first-line oxaliplatin, 5-fluorouracil, and leucovorin (FOLFOX-4) response was determined by χ(2)-test. RESULTS AND CONCLUSIONS: ERCC1 codon 118 C/T polymorphism was not associated with ERCC1 mRNA and protein expression, FOLFOX-4 response, and progression-free survival (PFS) or overall survival (OS). High ERCC1 mRNA and protein expression levels were associated with significantly lower FOLFOX-4 responses, PFS, and OS. ERCC1 codon 118 C/T polymorphism is not an important prognostic marker for advanced gastric cancer. Determination of ERCC1 mRNA and protein levels may be beneficial in predicting the response and outcome of FOLFOX-4 therapy in gastric cancer.

Endothelial PAS domain protein 1 Chr2:46441523(hg18) polymorphism is associated with susceptibility to high altitude pulmonary edema in Han Chinese.

OBJECTIVE: The purpose of this study was to test the hypothesis that polymorphisms in the endothelial PAS domain protein 1 (EPAS1) gene are associated with the susceptibility to high altitude pulmonary edema (HAPE) in Han Chinese. METHODS: This study enrolled 153 HAPE patients (HAPE-p), matched with Han Chinese resistant to HAPE (HAPE-r) and local highland Tibetans from Yushu earthquake construction population in Qinghai where the altitude is more than 3500 m above sea level. The polymorphism of EPAS1 chr2:46441523(hg18) was genotyped by polymerase chain reaction restriction fragment length polymorphism and confirmed by DNA sequencing. RESULTS: The frequencies of EPAS1 chr2:46441523(hg18) polymorphism C allele were significantly higher in the HAPE-p group than in the HAPE-r group (P < .001), but the frequencies of heterozygous C/G were significantly higher in the HAPE-r group than in the HAPE-p group (P < .001). Moreover, the frequencies of the EPAS1 chr2:46441523(hg18) polymorphism G allele were significantly higher in the highland Tibetan group than in the HAPE-p and HAPE-r groups. CONCLUSIONS: The EPAS1 chr2:46441523(hg18) polymorphism C is strongly associated with susceptibility to HAPE in Han Chinese, and the EPAS1 chr2:46441523(hg18) polymorphism G is present at high frequency and may be associated with high altitude adaptation in the Tibetans.

[Serum levels and significance of HIF-1α and HIF-2α in healthy Tibetan and Han residents at different altitudes].

OBJECTIVE: To explore the serum levels and significance of hypoxic inducible factor (HIF)-1α and HIF-2α in Tibetan and Han residents living at different altitudes. METHODS: A total of 100 healthy adults of Han in plain (altitude 5 m, group P) and Tibetan and Han at moderate altitude (2260 m, groups M1 & M2) and high altitude (4380 m, groups H1 & H2) (n = 20 each) were selected from June 2010 to August 2010. Venous blood samples were collected in the morning. The serum concentrations of HIF-1α and HIF-2α were measured with enzyme-linked immunosorbent assay (ELISA) to analyze the correlations of HIF-1α and HIF-2α with arterial partial pressure of oxygen (PaO2) and hemoglobin (Hb). RESULTS: The level of HIF-1α in group H2 ( (6.06 ± 1.85) µg/L) was higher than that in groups P ( (4.56 ± 0.85) µg/L, P < 0.05) , M1 ((4.41 ± 1.05) µg/L, P < 0.01) and M2 ( (4.59 ± 1.03) µg/L, P < 0.05) . The level of HIF-1α in group H1 ( (5.27 ± 0.98) µg/L) was higher than that in group M1 (P < 0.05) . The level of HIF-2α in group H2 ((0.83 ± 0.48) µg/L) was higher than that in groups P ((0.33 ± 0.11) µg/L, P < 0.01), M1 ((0.14 ± 0.06 )µg/L, P < 0.01) and M2( (0.24 ± 0.11) µg/L, P < 0.01) . The level of HIF-2α in group M1 was lower than that in groups P (P < 0.01) and M2 (P < 0.01). The level of HIF-2α in group H1((0.18 ± 0.16)µg/L) was lower than that in groups P (P < 0.05) and H2 (P < 0.01) . The levels of HIF-1α and HIF-2α were negatively correlated with PaO2 (r = -0.475, -0.551, both P < 0.01) and positively with Hb in Han groups (r = 0.433, 0.463, all P < 0.01). The levels of HIF-1α and HIF-2α had no correlation with PaO2 in Tibetan groups (r = -0.270, -0.198, both P > 0.05). No correlation existed between HIF-1α and Hb in Tibetan. A positive correlation existed between HIF-2α and Hb in Tibetan (r = 0.325, P < 0.01). CONCLUSIONS: The changes of HIF-1α and HIF-2α vary for Tibetan and Han residents at different altitudes. And the level of HIF-2α is consistent with the change of Hb. As compared with HIF-1α, HIF-2α may play a more important role in regulating Hb.

[Genome-wide association study of high-altitude pulmonary edema in Han Chinese].

High-altitude pulmonary edema (HAPE) is a non-cardiogenic pulmonary edema that is always found among unacclimatized persons after rapid ascent to high altitude, and HAPE is caused by the interaction of genetic and environmental factors. To screen and analyze the susceptibility genes and single nucleotide polymorphisms (SNPs) of HAPE in Han Chinese, the DNA samples of 40 patients with HAPE and 33 healthy controls, who performed the reconstruction tasks from the plain region in Yushu area of Qinghai province during May of 2010 to July of 2012, were scanned by Affymetrix SNP Array 6.0 Chips in this study. Genome-wide association study (GWAS, by PLINK software) was used to screen the susceptibility genes and genetic markers, and a total of 57 SNPs were found to be significantly different between case and control groups (adjust P < 0.05). GO and Pathway enrichment analysis of 74 genes around the 57 SNPs indicated that these genes were significantly correlated with prostanoid metabolic process, arachidonic acid metabolism and nitrogen metabolism (adjust P < 0.05), which were involved in the physiopathologic mechanism of HAPE. Our studies suggest that these genetic polymorphisms and genes were associated with HAPE.

No relation between ACE-I/D polymorphism and high altitude pulmonary edema in the Han Chinese.

OBJECTIVES: To explore whether the angiotensin I -converting enzyme (ACE) I/D (insertion/ deletion) polymorphism is associated with the susceptibility to high altitude pulmonary edema (HAPE) in the Han Chinese. METHODS: One hundred and forty-seven HAPE-p (HAPE patients) and 193 HAPE-r (HAPE resistants) were enrolled from the Yushu earthquake reconstruction workers in Qinghai province where the altitude is over 3 500 m above sea level. Blood samples were collected from each of the HAPE-p and HAPE-r groups. Information about physiological phenotypes was obtained via fieldwork investigation. The ACE-I/D polymorphism in HAPE-p and HAPE-r was detected by polymerase chain reaction (PCR). RESULTS: The SaO2 was significantly lower while HR was significantly higher in HAPE-p group than those in HAPE-r group. The genotype frequencies of ACE-I/D for II, ID, DD in HAPE-r and HAPE-p groups were 0.430, 0.446, 0.124 and 0.435, 0.469, 0.095, respectively, the allelic frequencies of I and D were 0.650, 0.350 and 0.670, 0.330, respectively. The OR of ID, DD and D alleles relative to II for HAPE was 0.961 (0.610-1.514), 1.322 (0.634-2.758) and 1.080 (0.783-1.489). There was no significant difference of the genotypic and the allelic frequencies in ACE-I/D polymorphism between HAPE-p and HAPE-r groups. CONCLUSIONS: There is no relation between ACE-I/D polymorphism and HAPE in the Han Chinese.

[Study on the content of myoglobin and the activity of lactate dehydrogenase and malate dehydrogenase in skeletal muscle of tibetan antelope].

OBJECTIVE: To explore the adaptive mechanism to hypoxia in skeletal muscle of tibetan antelope. METHODS: Tibetan sheep which living at the same altitude (4 300 m) with tibetan antelope and low altitude (1 800 m) sheep as control, the content of myoglobin (Mb) and lactic acid (LA), the activity of lactate dehydrogenase (LDH) and malate dehydrogenase (MDH) in skeletal muscles among three animals were analyzed by spectrophotometer. RESULTS: The content of myoglobin in skeletal muscle of tibetan antelope significantly higher than that of tibetan sheep and low altitude sheep (P < 0.05). And the content of LA in skeletal muscle of tibetan antelope significantly lower than that of tibetan sheep and low altitude sheep (P < 0.05), activity of LDH and MDH in skeletal muscle was significantly lower and higher respectively than that of tibetan sheep and low altitude sheep (P < 0.05). There was no significant difference between tibetan sheep and low altitude sheep. CONCLUSION: Tibetan antelope may improve their ability to get oxygen under hypoxia by increasing the content of myoglobin in skeletal muscle, and the proportion of aerobic metabolism is high in skeletal muscle, it may be relate that with high myoglobin content in skeletal muscle, we suppose that high myoglobin content in skeletal muscle of tibetan antelope might be one of the molecular basis to adapt hypoxia.

[Effect of high altitude hypoxia on the activity and protein expression of CYP2C9 and CYP2C19].

This study is to investigate the effect of high altitude hypoxia on the activity and protein expression of CYP2C9 and CYP2C19. Rats from plain (P) and rats with acute middle altitude hypoxia (AMH), chronic middle altitude hypoxia (CMH), acute high altitude hypoxia (AHH) and chronic high altitude hypoxia (CHH) were administered orally phenytoin sodium (PHT) and omeprazole (OMZ) to evaluate the activity of CYP2C9 and CYP2C19, separately. The serum concentrations of PHT and metabolite 4'-hydroxyphenytoin (HPPH) at 12 h after treatment and the serum concentrations of OMZ and metabolite 5-hydroxy omeprazole (5-OHOMZ) at 3 h after treatment were determined by RP-HPLC. The activity of CYP2C9 and CYP2C19 was evaluated by the ratio of HPPH to PHT and the ratio of 5-OHOMZ to OMZ, respectively. The protein expressions of CYP2C9 and CYP2C19 were determined by ELISA method. The activities of CYP2C9 (HPPH/PHT) in P, AMH, CMH, AHH and CHH were 0.67 +/- 0.31, 0.75 +/- 0.29, 0.76 +/- 0.23, 0.79 +/- 0.31 and 0.75 +/- 0.18, respectively, and the activities of CYP2C19 (5-OHOMZ/OMZ) in P, AMH, CMH, AHH and CHH were 0.17 +/- 0.06, 0.20 +/- 0.10, 0.11 +/- 0.05, 0.37 +/- 0.13 and 0.19 +/- 0.05, respectively. The protein expressions of CYP2C9 in P, AMH, CMH, AHH and CHH were 4.20 +/- 1.27, 3.95 +/- 0.81, 3.93 +/- 1.11, 4.32 +/- 1.03 and 4.12 +/- 0.86 ng x g(-1), respectively, and the protein expressions of CYP2C19 in P, AMH, CMH, AHH and CHH were 3.91 +/- 1.82, 3.63 +/- 2.07, 2.55 +/- 0.85, 4.78 +/- 2.37 and 3.51 +/- 1.03 ng x g(-1), respectively. The activities and protein expressions of CYP2C9 in AMH, CMH, AHH and CHH were not significantly different with those of P. The protein expressions of CYP2C19 in AMH, CMH, AHH and CHH were not significantly different with those of P, but the activity of CYP2C19 in AHH was significantly higher than that of P. This study found significant changes in the activity of CYP2C19 under the special environment of acute high altitude hypoxia.

[Neurological adaptations to hypoxia in Tibetan antelope (Pantholops hodgsonii) with a view of molecular biology of respiratory globin-neuroglobin].

Neuroglobin (Ngb) is a respiratory protein that is preferentially expressed in brain of mouse and man. In this article, Tibetan antelope, living at altitude of 3 000-5 000 m for millions of years, was selected as the model of hypoxia-tolerant adaptation species. Using reverse transcription polymerase chain reaction (RT-PCR) and Western blot techniques, expression of Ngb gene was amplified and analyzed in antelope brain tissue. Our results showed that Ngb homology protein in Tibetan antelope was identified with more sequence similarity with cattle (96%), sheep (95%), and human (95%). We detected that there were some mutations occurred in the Open Reading Frame of Ngb in Tibetan antelope compared with sheep. Phylogenetic analysis of Ngb chain showed that it was closer to cattle than the others. This study suggests possible roles of central nervous system enriched Ngb in adaptation of Tibetan antelope to extremely high altitude.

[Genetic cloning and expression of hypoxia inducible factor 1 alpha in high altitude hypoxic adaptation species Tibetan antelope (Pantholops hodgsonii)].

In order to investigate the role of the hypoxia inducible factor 1 alpha (HIF-1α) in the adaptation mechanism to high altitude hypoxia, the cloning of the HIF-1α gene cDNA of Tibetan antelope (Pantholops hodgsonii), using RT-PCR and RACE, was applied, and the comparative analysis of the tissue-specific expressions of HIF-1α among Tibetan antelope, Tibetan sheep and plain sheep was performed using real-time PCR and Western blot. The sequence analysis indicated that the cDNA sequences acquired by cloning from the HIF-1α gene of Tibetan antelope comprised a 2 471-bp open reading frame (ORF) and a 1 911-bp 3'UTR. The similarity between its coding sequence, predicted amino acid sequence and HIF-1α of other mammals exceeded 87%, in which the similarity with cow was up to more than 98%, which showed that this sequence was the cDNA of HIF-1α of Tibetan antelope. The results of real-time PCR and Western blot showed that expressions of HIF-1α mRNA and protein appeared in Tibetan antelope's lung, cardiac muscle and skeletal muscle, with the highest expression in lung. HIF-1α mRNA and protein had obvious differential expression in these tissues. Further research showed that Tibetan antelope and Tibetan sheep possessed higher expressions of HIF-1α protein in the three tissues above-mentioned compared with plain sheep, and the expressions of HIF-1α mRNA and protein in Tibetan antelope's lung, cardiac muscle and skeletal muscle were higher than those of Tibetan sheep. It illustrates that the hypoxic HIF-1α-specific expression is one of the molecular bases of high altitude hypoxia adaptation in Tibetan antelope.

[Comparisons of endocrine hormones levels between Tibetan antelope and Tibetan sheep].

The Tibetan antelope, a prototype mammal, has developed a unique adaptation to extreme high altitude-associated hypoxia. To investigate the role of the endocrine system in adaptation to high altitude in the Tibetan antelope, comparisons of endocrine hormones levels between Tibetan antelope (n = 9) and Tibetan sheep (n = 10) were performed. Both two kinds of animals were captured at an altitude of 4 300 m and then transported to experimental base at 2 800 m altitude. The blood samples were drawn from right external jugular vein in the next morning, and the 20 hormones in hypothalamus-adenohypophysis-peripheral hormonal axis were measured with radioimmunoassay or enzyme-linked immunosorbent assay. Heart rate (HR), systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean pulmonary arterial pressure (mPAP) were recorded using catheterization. Moreover, hemoglobin (Hb) content was measured by blood analyser. The results showed that, the levels of FT(3), FT(4) and Ang II in Tibetan antelope were significantly lower than those in Tibetan sheep, whereas TRH, CRH, GHRH, F, E(2), Ald, ACTH and CGRP levels were significantly greater in Tibetan antelope than those in the Tibetan sheep. Compared with Tibetan sheep, Tibetan antelope showed lower HR, mPAP, SBP, DBP and Hb content. In Tibetan antelope and Tibetan sheep, both Hb and Ang II were correlated positively with respective mPAP. In Tibetan antelope, FT(3) level was correlated positively with GH and negatively with ACTH. These results suggest that the endocrine system of Tibetan antelope is characterized by low energy expenditure and high stress, which may be one of the mechanisms underlying the Tibetan antelope adaptation to chronic hypoxia.