The plateau zokor (Myospalax baileyi) and plateau pika (Ochotona curzoniae) are native species unique to the Qinghai-Tibetan Plateau with successful adaptation to the hypoxic environment. In this study, the number of red blood cells, hemoglobin concentration, mean hematocrit and mean volume of red blood cells were measured in plateau zokors and plateau pikas at different altitudes. Hemoglobin subtypes of two plateau animals were identified by mass spectrometry sequencing. The forward selection sites in two animals' hemoglobin subunits were analyzed by PAML4.8 program. Homologous modeling was used to analyze the effect of forward selection sites on the affinity of hemoglobin to oxygen. The adapting strategies of plateau zokors and plateau pikas to hypoxia at different altitudes were analyzed through comparing blood parameters between the two species. The results indicated that, with increasing altitudes, plateau zokors responded to hypoxia by increasing red blood cell count and decreasing red blood cell volume, while plateau pikas took the opposite strategies to plateau zokors. In erythrocytes of plateau pikas, both adult α₂β₂ and fetal α₂ε₂ hemoglobins were identified, while erythrocytes of plateau zokors only had adult α₂β₂ hemoglobin, however the affinities and the allosteric effects of the hemoglobin of plateau zokors were significantly higher than those of plateau pikas. Mechanistically, in the α and β subunits of hemoglobin of plateau zokors and pikas, the numbers and the sites of the positively selected amino acids as well as the side chain groups polarities and orientations of the amino acids differed significantly, which may result in the difference of the affinities to oxygen of hemoglobin between plateau zokors and pikas. In conclusion, the adaptive mechanisms to respond to hypoxia in blood properties of plateau zokors and plateau pikas are species-specific.
Animals ; Altitude ; Amino Acids ; Hemoglobins ; Hypoxia ; Lagomorpha

OBJECTIVE: To prepare and develop a GDH-2 air sampling tube for detecting 12 kinds of chlorobenzenes(CBs) in workplace air and to establish a matching detecting method. METHODS: The self-developed GDH-2 air sampling tube was filled with ion exchange resin and activated carbon, and the mass ratio was 10 ∶1. The GDH-2 air sampling tube was used to collect 12 kinds of CBs with coexistence of gaseous and aerosol in the air. After elution with toluene, they were separated on a chromatographic column and determined by microcell electron capture detector. RESULTS: The quantitative detecting range of the method was 0.51×10~(-3)-6 000.00 mg/L, with the correlation coefficients greater than 0.999 4. The minimum detection concentration was 0.02-61.99 μg/m~3, and the minimum quantitative concentration was 0.05-206.62 μg/m~3. The average desorption efficiency was 90.8%-104.0%. The within-run relative standard deviation(RSD) was 1.0%-5.7%, and the between-run RSD was 3.0%-7.3%. The samples can be stored at room temperature for at least 26 days. CONCLUSION: The self-developed GDH-2 air sampling tube and its matching measuring method can be used for the collection and determination of the 12 kinds of CBs in the air of workplace.

In the present study, the composition and content of pulmonary surfactant (PS) were analyzed to explore the hypoxia adaptation mechanism in plateau zokors (Myospalax baileyi) and plateau pikas (Ochotona curzoniae). 36 plateau zokors and plateau pikas were trapped alive at the Laji Mountain in Guide County, Qinghai Province (at the altitude of about 3 600 m), and 36 Sprague-Dawley (SD) rats were purchased from the experimental animal center of Lanzhou University (at the altitude of about 1 500 m). All animals were lavaged after laboratory anesthesia, the blood in lung tissues was fully washed out and the lung tissues were then taken out to obtain the bronchoalveolar lavage fluid by bronchoalveolar lavage. The composition and content of phospholipids in the PS of three different kinds of animals were analyzed by using high performance liquid chromatography; the protein composition, content and types in the PS were analyzed by G-250 Coomassie brilliant blue method, polyacrylamide gel electrophoresis (PAGE) and mass spectrometry; the dissolved oxygen in the PS solutions were determined by using dissolved oxygen electrode. The results showed that the total contents of phospholipids in the PS were successively increased among plateau zokors, plateau pikas and SD rats (P < 0.05), while the total content of proteins successively decreased (P < 0.05). There were five phospholipids identified in the PS, including linoleic palmitoylphosphatidylcholine (LPPC), dipalmitoylphosphatidylcholine (DPPC), phosphatidylglyerol (PG), phosphatidylinositol (PI) and phosphatidylserine (PSe), but the relative contents of these phospholipids were different. The relative content of LPPC was successively increased among plateau zokors, plateau pikas and SD rats (P < 0.01). The relative contents of DPPC, PG and PI in the PS of plateau zokors were significantly higher than those of plateau pikas (P < 0.01), while insignificant differences between plateau pikas and SD rats (P > 0.05). The relative content of PSe had no significant differences between plateau zokors and plateau pikas (P > 0.05), but both were significantly higher than that of SD rats (P < 0.01). The serum albumin (SA) was identified in the PS of three kinds of animals, including homologous tetramer protein containing heme, which is composed of hemoglobin β subunit, in plateau zokors and plateau pikas. Immunoglobulin (Ig) heavy chain was found in PS of plateau zokors and SD rats. The content of Ig heavy chain in plateau zokor was significantly higher than that in SD rats (P < 0.01), and the content of protein containing heme was significantly higher than that in plateau pikas (P < 0.05). The amount of dissolved oxygen was successively decreased in the PS among plateau zokors, plateau pikas and SD rats (P < 0.01), but it was significantly higher than that in saline (P < 0.01). These results suggest that the total content of proteins in the PS of plateau zokors and plateau pikas was significantly higher, while the total content of phospholipids was significantly decreased. There were high content of homologous tetramer protein containing heme in the PS of plateau zokors and plateau pikas. The relative content of DPPC, the main component of phospholipids, was significantly increased in plateau zokors. The changes of PS component and content improve the adaptability of the two plateau animals in hypoxia environment.
Altitude ; Animals ; Hypoxia ; Lagomorpha ; Pulmonary Surfactants ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo explore the intergrating of N-isopropyl oxamate and serum protein and establish a high performance liquid chromatography (HPLC) detection method of N-isopropyl oxamate (specific inhibitor of testis-specific lactate dehydrogenase (LDH-C4)) in the blood of plateau pikas.

METHODSTwenty highland pika 150-200 g, were randomly divided into two groups (n = 10): control group and inhibitor group. Different concentrations of N-isopropyl oxamate were added to examine the intergrating of N-isopropyl oxamate and serum protein. In order to determine its concentration in the pika blood accurately, we used the method of adding trypsin to incubate the serum first, followed by trichloroacetic acid treatment and detecting by HPLC. Results: When the concentrations of N-isopropyl oxamate in the pika serum were added to 0.05 mmol/L, 0.1 mmol/L, 1 mmol/L, 10 mmol/L, 16.7 mmol/L, 33.3 mmol/L and 100 mmol/L, the intergrating rates between N-isopropyl oxamate and plateau pika serum were 100%, 100%, 100%, 86.84%, 54.11%, 40.10% and 20.18%, respectively. The method established in this paper was good on recovery rates, precision and stability. A good linearity was obtained in the range of 0.0125-0.25 mmol/L. When the concentrations of N-isopropyl oxamate in the serum were added to 0. 15 mmol/L,0.3 mmol/L and 1 mmol/L, the recovery rates were 98.05%, 98.98% and 98.12%, respectively; the precision relative standard deviation( RSD) of concentrations were 1.17%, 0.92% and 0.83%, respectively; the stability relative standard deviation (RSD) of concentrations were 1.38%, 1.40% and 0.88%, respectively. The repeatability RSD of the method was 1.76%. Quantitative limit was 0.0125 mmol/L.

CONCLUSIONN-isopropyl oxamate has a strong affinity with plateau pika serum protein that can't be accurately determined with common HIPLC method. It can be accurately determined in the blood by adding trypsinto digest the serum protein first, followed by adding trichloroacetic acid to precipitate the protein.

Animals ; Chromatography, High Pressure Liquid ; methods ; Lagomorpha ; Male ; Oxamic Acid ; analogs & derivatives ; blood

The plateau pika (Ochotona curzoniae) has a strong adaptability to hypoxic plateau environment. We found that the sperm-specific lactate dehydrogenase (LDH-C4) gene Ldh-c expressed in plateau pika cardiac muscle. In order to shed light on the effect of LDH-C4 on the anaerobic glycolysis in plateau pika cardiac muscle, 20 pikas were randomly divided into the inhibitor group and the control group, and the sample size of each group was 10. The pikas of inhibitor group were injected with 1 mL 1 mol/L N-isopropyl oxamate, a specific LDH-C4 inhibitor, in biceps femoris muscle of hind legs, each leg with 500 μL. The pikas of control group were injected with the same volume of normal saline (0.9% NaCl). The mRNA and protein expression levels of Ldh-c gene in plateau pika cardiac muscle were determined by real-time PCR and Western blot. The activities of LDH, and the contents of lactate (LD) and ATP in cardiac muscle were compared between the inhibitor group and the control group. The results showed that 1) the expression levels of Ldh-c mRNA and protein were 0.47 ± 0.06 and 0.68 ± 0.08, respectively; 2) 30 min after injection of 1 mL 1 mol/L N-isopropyl oxamate in biceps femoris muscle, the concentration of N-isopropyl oxamate in blood was 0.08 mmol/L; 3) in cardiac muscle of the inhibitor group and the control group, the LDH activities were (6.18 ± 0.48) U/mg and (9.08 ± 0.58) U/mg, the contents of LD were (0.21 ± 0.03) mmol/g and (0.26 ± 0.04) mmol/g, and the contents of ATP were (4.40 ± 0.69) nmol/mg and (6.18 ± 0.73) nmol/mg (P < 0.01); 5) the inhibition rates of N-isopropyl oxamate to LDH, LD and ATP were 31.98%, 20.90% and 28.70%, respectively. The results suggest that Ldh-c expresses in cardiac muscle of plateau pika, and the pika cardiac muscle may get at least 28% ATP for its activities by LDH-C4 catalyzed anaerobic glycolysis, which reduces the dependence on oxygen and enhances the adaptation to the hypoxic environments.
Acclimatization ; Animals ; Glycolysis ; Hypoxia ; Isoenzymes ; genetics ; metabolism ; L-Lactate Dehydrogenase ; genetics ; metabolism ; Lactic Acid ; analysis ; Lagomorpha ; genetics ; Male ; Myocardium ; enzymology ; Oxamic Acid ; analogs & derivatives ; Oxygen ; RNA, Messenger

Plateau zokor (Myospalax baileyi) is a subterranean mammal. Plateau zokor has high learning and memory ability, and can determine the location of blocking obstacles in their tunnels. Forkhead box p2 (FOXP2) is a transcription factor implicated in the neural control of orofacial coordination and sensory-motor integration, particularly with respect to learning, memory and vocalization. To explore the association of foxP2 with the high learning and memory ability of plateau zokor, the cDNA of foxP2 of plateau zokor was sequenced; by using plateau pika as control, the expression levels of foxP2 mRNA and FOXP2 protein in brain of plateau zokor were determined by real-time PCR and Western blot, respectively; and the location of FOXP2 protein in the brain of plateau zokor was determined by immunohistochemistry. The result showed that the cDNA sequence of plateau zokor foxP2 was similar to that of other mammals and the amino acid sequences showed a relatively high degree of conservation, with the exception of two particular amino acid substitutions [a Gln (Q)-to-His (H) change at position 231 and a Ser (S)-to-Ile (I) change at position 235]. Higher expression levels of foxP2 mRNA (3-fold higher) and FOXP2 protein (>2-fold higher) were detected in plateau zokor brain relative to plateau pika brain. In plateau zokor brain, FOXP2 protein was highly expressed in the cerebral cortex, thalamus and the striatum (a basal ganglia brain region). The results suggest that the high learning and memory ability of plateau zokor is related to the high expression levels of foxP2 in the brain.
Amino Acid Sequence ; Amino Acid Substitution ; Animals ; Brain ; metabolism ; Forkhead Transcription Factors ; metabolism ; Lagomorpha ; physiology ; Learning ; Memory ; RNA, Messenger

The plateau pika (Ochotona curzoniae) and plateau zokor (Myospalax baileyi) are specialized native species of the Qinghai-Tibetan plateau. The goal of this study was to examine physiological differences in skeletal muscle glycolysis and hepatic lactate metabolism between these two species. The partial sequence of pyruvate carboxylase (PC) gene was cloned and sequenced. The mRNA expression levels of PC and lactate dehydrogenases (LDH-A, LDH-B) were determined by real-time PCR. The enzymatic activity of PC was measured using malic acid coupling method. The concentration of lactic acid (LD) and the specific activities of LDH in liver and skeletal muscle of two species were measured. The different isoenzymes of LDH were determined by native polyacrylamide gel electrophoresis (PAGE). The results showed that, (1) LDH-B mRNA level in skeletal muscle of plateau zokor was significantly higher than that of plateau pika (P < 0.01), but no differences was found at LDH-A mRNA levels between them (P > 0.05); (2) PC, LDH-A and LDH-B mRNA levels in liver of plateau pika were significantly higher than those of plateau zokor (P < 0.01); (3) The LDH activity and concentration of LD in skeletal muscle and liver, as well as the PC activity in liver of plateau pika were significantly higher than those of plateau zokor (P < 0.01); (4) The isoenzymatic spectrum of lactate dehydrogenase showed that the main LDH isoenzymes were LDH-A4, LDH-A3B and LDH-A2B2 in skeletal muscle of plateau pika, while the main LDH isoenzymes were LDH-AB3 and LDH-B4 in skeletal muscle of plateau zokor; the main isoenzymes were LDH-A3B, LDH-A2B2, LDH-AB3 and LDH-B4 in liver of plateau pika, while LDH-A4 was the only isoenzyme in liver of plateau zokor. These results indicate that the plateau pika gets most of its energy for sprint running through enhancing anaerobic glycolysis, producing more lactate in their skeletal muscle, and converting lactate into glucose and glycogen in the liver by enhancing gluconeogenesis. As a result, the plateau pika has a reduced dependence on oxygen in its hypoxic environment. In contrast, plateau zokor derives most of its energy used for digging activity by enhancing aerobic oxidation in their skeletal muscle, although they inhabit hypoxic underground burrows.
Animals ; Glycolysis ; Hypoxia ; metabolism ; Isoenzymes ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Lactic Acid ; metabolism ; Lagomorpha ; physiology ; Liver ; enzymology ; Muscle, Skeletal ; enzymology ; Oxygen ; metabolism ; RNA, Messenger

To explore the adaptive mechanisms of plateau zokor (Myospalax baileyi) to the enduring digging activity in the hypoxic environment and of plateau pika (Ochotona curzoniae) to the sprint running activity, the functional differences of malate-aspartate shuttle system (MA) in liver of plateau zokor and plateau pika were studied. The ratio of liver weight to body weight, the parameters of mitochondria in hepatocyte and the contents of lactic acid in serum were measured; the open reading frame of cytoplasmic malate dehydrogenase (MDH1), mitochondrial malate dehydrogenase (MDH2), and the partial sequence of aspartate glutamate carrier (AGC) and oxoglutarate malate carrier (OMC) genes were cloned and sequenced; MDH1, MDH2, AGC and OMC mRNA levels were determined by real-time PCR; the specific activities of MDH1 and MDH2 in liver of plateau zokor and plateau pika were measured using enzymatic methods. The results showed that, (1) the ratio of liver weight to body weight, the number and the specific surface of mitochondria in hepatocyte of plateau zokor were markedly higher than those of plateau pika (P < 0.01 or P < 0.05), but the content of lactic acid in serum of plateau pika was significantly higher than that of plateau zokor (P < 0.01); (2) MDH1 and MDH2 mRNA levels as well as their enzymatic activities in liver of plateau zokor were significantly higher than those of plateau pika (P < 0.01 or 0.05), AGC mRNA level of the zokor was significantly higher than that of the pika (P < 0.01), while no difference was found at OMC mRNA level between them (P > 0.05); (3) mRNA level and enzymatic activity of MDH1 was significantly lower than those of MDH2 in the pika liver (P < 0.01), MDH1 mRNA level of plateau zokor was markedly higher than that of MDH2 (P < 0.01), but the activities had no difference between MDH1 and MDH2 in liver of the zokor (P > 0.05). These results indicate that the plateau zokor obtains ATP in the enduring digging activity by enhancing the function of MA, while plateau pika gets glycogen for their sprint running activity by increasing the process of gluconeogenesis. As a result, plateau pika converts the lactic acid quickly produced in their skeletal muscle by anaerobic glycolysis and reduces dependence on the oxygen.
Adaptation, Physiological ; physiology ; Adenosine Triphosphate ; metabolism ; Altitude ; Animals ; Aspartic Acid ; metabolism ; Cloning, Molecular ; L-Lactate Dehydrogenase ; analysis ; metabolism ; Lactic Acid ; blood ; Lagomorpha ; classification ; physiology ; Liver ; anatomy & histology ; physiology ; Malate Dehydrogenase ; genetics ; metabolism ; Malates ; metabolism ; Membrane Transport Proteins ; genetics ; metabolism ; RNA, Messenger ; genetics ; metabolism

OBJECTIVETo investigate whether there are different stromal compositions in the prostate tissue of patients with benign prostatic hyperplasia (BPH) and evaluate their significance in the course of the disease.

METHODSForty-three surgical or bioptic prostatic specimens of BPH and 5 autoptic normal prostatic specimens were stained by the Masson method to display the elements of the muscle fiber and collagen. The relationship of the changes in the prostatic stromal composition was analyzed with the degree of bladder outlet obstruction (BOO) , IPSS and medication results.

RESULTSThe mean ratio of muscle fiber to collagen in the normal prostate tissue was (3.2 +/- 0.2):1, significantly higher than that of the BPH patients (1: [4.7 +/- 3.1] ) (P < 0.01); that in the BPH patients with BOO was 1: (5.4 +/- 3.7) markedly lower than in those without BOO (1: [2.5 +/- 1.1] ) (P = 0.02); that in the BPH patients with severe prostatic symptoms was 1: (9.1 +/- 2.9), remarkably lower than in those with moderate (1: [5.3 +/- 3.4]) and mild prostatic symptoms (1: [2.8 +/- 1.7]) (P < 0.01); and that in the BPH patients with satisfactory medicinal therapeutic results was 1:(2.3 +/- 1.9), significantly higher than in those with poor therapeutic results (1: [7.6 +/- 4.3]) (P < 0.01).

CONCLUSIONThe stromal composition in the prostatic tissue of BPH patients undergoes different degrees of changes. More obvious BPH symptoms and poorer therapeutic results are associated with a bigger proportion of collagens and a smaller proportion of muscle fibers in the prostatic tissue. These changes may play an important role in the development and progression of BPH.

Aged ; Aged, 80 and over ; Case-Control Studies ; Fibrosis ; Humans ; Male ; Middle Aged ; Prostate ; pathology ; Prostatic Hyperplasia ; pathology ; Urinary Bladder Neck Obstruction ; pathology

Vascular endothelial growth factor (VEGF) plays an important role in tissues angiogenesis. The adaptation of animals to hypoxic environment is relative to the microvessel density (MVD) in tissues. To further explore the adaptation mechanisms of plateau zokor (Myospalax baileyi) to the hypoxic-hypercapnic burrows, the VEGF mRNA and the MVD in cerebral tissues of the plateau zokor were studied. Total RNA was isolated from liver, and VEGF cDNA was obtained by RT-PCR, then the VEGF cDNA was cloned and sequenced. The coding sequence of plateau pika (Ochotona curzniae), rat (Rattus norvegicus) and mouse (Mus musculus) VEGF cDNA are obtained from GenBank, and the nucleotide and amino acid sequence homology of plateau zokor VEGF cDNA coding sequence with that of plateau pika, rat and mouse were analyzed and compared by using of bioinformatics software. The VEGF mRNA was detected by real-time PCR, and the MVDs in cerebral tissues of the plateau zokor, plateau pika and Sprague-Dawley (SD) rat were measured by immunohistochemical staining. The results showed that the open reading frame of the plateau zokor VEGF was 645 bp, and the coding sequence of the plateau zokor VEGF cDNA shared 92.1%, 93.6% and 93.8% nucleotide sequence homology to that of the plateau pika, rat and mouse, respectively. The deduced amino acid sequence of the plateau zokor VEGF cDNA was composed of 188 amino acids and the amino acids from 1 to 26 were signal peptide sequence. The plateau zokor VEGF188 was 90.2%, 94.9% and 94.4% homologous to that of plateau pika, rat and mouse. The level of VEGF mRNA in brain of the plateau zokor was significantly lower than that of SD rat, but there was no obvious difference in VEGF mRNA level between plateau zokor and plateau pika. The MVD in brain of the plateau zokor was markedly higher than that of plateau pika and SD rat. In conclusion, plateau zokor enhances its adaptation to the hypoxic environment by increasing the MVD. The level of VEGF mRNA in the brain of plateau zokor is lower than that of SD rat, which may be as a result of inhibition by the higher concentration of carbon dioxide in the burrow.
Adaptation, Physiological ; physiology ; Amino Acid Sequence ; Animals ; Arvicolinae ; physiology ; Base Sequence ; Brain ; blood supply ; metabolism ; Hypoxia ; physiopathology ; Microvessels ; anatomy & histology ; Molecular Sequence Data ; RNA, Messenger ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Species Specificity ; Vascular Endothelial Growth Factor A ; genetics ; metabolism