Na+/K+-ATPase is a membrane protein and plays a key role in osmotic regulation in living organisms. In the present study, a cDNA sequence encoding the Na+/K+-ATPase alpha subunit from the monogonont rotifer, Brachionus koreanus was cloned by rapid amplification of cDNA ends technique. To investigate the role of this enzyme in osmotic stress, enzymatic activities of Na+/K+-ATPase were measured after exposure to different salinities for 48 h. The full-length Bk Na+/K+-ATPase cDNA was 3069 bp-long, encoding a 1022-amino acid polypeptide. Bk Na+/K+- ATPase possesses eight membrane spanning regions and five conserved domains. Phylogenetic analysis showed that Bk Na+/K+-ATPase had high identity with those of other species, and was closely clustered with other Brachionus sp. These findings indicate that this protein was conserved both structurally and functionally. B. koreanus Na+/K+-ATPase activity was stimulated in both hyposaline (6 psu) and hypersaline (32 psu) conditions, suggesting that this protein may play a role in osmoregulation. This study would provide better understanding of the physiology of B. koreanus and this enzyme may be useful as a molecular marker for evaluation of osmotic stress in aquatic environment.

Tick salivary secretion during blood-feeding is crucial for successful tick feeding. Control of salivary secretion involves dopamine, which is the most potent inducer of tick salivation. Dopamine activates salivation by orchestrating two different physiological responses through two distinct dopamine receptors. In addition, the study demonstrated that two different types of cells in the salivary gland acini are responsible for each of the diverging physiological pathways: epithelial cells for inward fluid transport and myoepithelial cells for expelling fluid out through the acinar ducts. We were further interested in the downstream physiology of the dopamine receptors. A candidate gene (Na/K-ATPase), which is highly expressed in the salivary glands, was investigated. Immunoreactivity revealed that Na/K-ATPase is expressed in epithelial cells of acini. Ouabain, a Na/K-ATPase blocker, significantly suppressed both dopamine induced inward fluid transport and dopamine induced salivation in a dose-dependent manner. We measured the salivary contents to determine Na, K, and Cl ion, and protein concentrations. Treatment of ouabain at the low dose produced hyperosmolar saliva, but with same amount of protein as the control saliva. The results suggest that ouabain-sensitive Na/K-ATPase is the main downstream pathway for dopamine response in the epithelial cells of salivary gland for water transport, but not for protein secretion.

The ubiquitous Na, K-ATPase is a membrane-bound ion pump located in the plasma membrane in all animal cells and plays an essential role in a variety of cellular functions. Studies in several organisms have shown that this protein regulates different aspects of embryonic development and is responsible for the pathogenesis of several human diseases. Na, K-ATPase is an important factor for retinal development, and combinations of the isoforms of each of its subunits are expressed in different cell types and determine its functional properties. In this study, we performed RT-PCR assay to determine temporal expression and in situ hybridization to determine spatial expression of Na, K-ATPase β2 isoform (atp1b2) in Xenopus laevis. Focusing on retinal expression to distinguish the specific expression domain, we used retinal marker genes sox4, sox11, vsx1, and pax6. Xenopus atp1b2 was expressed from late gastrulation to the tadpole stage. Using whole mount in situ hybridization, we showed that Xenopus atp1b2 was expressed broadly in the eye, the whole surface ectoderm, and gills. In situ hybridization on sections revealed detailed and specific expression in the outer nuclear layer of the retina, which consists of two major classes of photoreceptors, rods and cones, surface ectoderm, pharyngeal epithelium, and gills. These findings indicate that atp1b2 may play an important role for the development of Xenopus retina.

Na+/K+-ATPase, an energy-transducing ion pump, is responsible for maintenance of relatively high concentrations of potassium ions but low concentrations of sodium ions in the cell by transport of these ions across the plasma membrane and participates in transport of various nutrients including glucose, amino acids. and ions. Na+/K+-ATPase consists of α, β, and FXYD subunits, but only α and β subunits are needed for basic functions. FXYD subunit is an auxiliary protein for αβ complex of Na+/K+-ATPase. Our recent study has shown that α (ATP1A1-4) and β (ATP1B1-3) subunits of Na+/K+-ATPase are expressed in the uterine endometrium during the estrous cycle and pregnancy in pigs. In this study, we further determined expression of FXYD (FXYD1-7) subunits of Na+/K+-ATPase in the uterine endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that mRNAs for all subtypes of FXYD subunit were expressed in the uterine endometrium during the estrous cycle and pregnancy in a pregnancy status- and stage-specific fashion. In situ hybridization analysis exhibited that transcripts of all subtypes of FXYD subunit were primarily localized to luminal (LE) and glandular epithelia (GE) during the estrous cycle and early pregnancy and to chorionic membrane (CM) during mid to term pregnancy. RT-PCR analysis showed that FXYD subunits were expressed in conceptuses on D12 and D15 of pregnancy. These results indicate that all subtypes of FXYD subunit are expressed in the uterine endometrium and conceptuses during the estrous cycle and pregnancy in a pregnancy status- and stagespecific manner. These suggest that FXYD may be involved in the establishment and maintenance of pregnancy by regulating the activity of Na+/K+-ATPase in nutrient transport at the maternal-fetal interface in pigs. * This work was supported by the Next Generation BioGreen 21 program (#PJ007997), RDA and the National Research Foundation (NRF #2010-0012304) funded by the Korean Government, Republic of Korea.

Na+/K+-ATPase, an energy-transducing ion pump, is responsible for maintenance of relatively high concentrations of potassium ions but low concentrations of sodium ions in the cell by transport of these ions across the plasma membrane. Na+/K+-ATPase consists of α, β, and γ subunits, but only α and β subunits are needed for basic functions. Na+/K+-ATPase is also involved in regulation of intracellular calcium ion concentration by coupling with Na+/Ca2+ exchanger involved in intracellular calcium extrusion. Our previous study showed that calcium regulatory molecules including Na+/Ca2+ exchanger are expressed in the uterine endometrium during the estrous cycle and pregnancy in pigs, however, expression of Na+/K+-ATPase in the uterine endometrium has not been determined. Thus, we examined expression of α1 (ATP1A1) and β1 (ATP1- B1) subunits of Na+/K+-ATPase in the uterine endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that levels of ATP1A1 m- RNA in the uterine endometrium during the estrous cycle and early pregnancy were higher than those during mid and term pregnancy, and that levels of ATP1B1 mRNA were highest on day (D) 12 of the estrous cycle. In situ hybridization analysis revealed that ATP1A1 and ATP1B1 mRNAs were localized to luminal (LE) and glandular epithelia (GE) in the endometrium. During mid to term pregnancy, localization of ATP1A1 mRNA was confined to LE, GE, and chorionic membrane (CM) of areolae and ATP1- B1 mRNA was localized to LE, GE and CM with the strongest intensity in LE of areolae. Signal intensity of ATP1B1 mRNA in LE was slightly stronger than that in GE. RT-PCR analysis showed that ATP1A1 and ATP1B1 mRNAs were expressed in conceptuses on D12 and D15 of pregnancy. These results showed that ATP1A1 and ATP1B1 were expressed in the uterine endometrium and conceptuses during the estrous cycle and pregnancy in a pregnancy status- and stage-specific manner. These suggest that Na+/K+-ATPase may play a key role in the establishment and maintenance of pregnancy by regulating intracellular concentrations of various ions including calcium at the maternal-fetal interface in pigs.

본 연구는 고온과 고습 조건 하에서 양파 화구의 총 단백질 발현과 원형질막 H+ATPase 영향을 조사하고자 조생종 '신선황'과 중생종 '맵시황'를 이용하여 실험을 수행하였다. '신선황'과 '맵시황' 품종의 양파 회구 개화전, 반개화, 그리고 만개 단계에서 단백질의 양생에는 차이를 보이지 않았지만 결실 단계에서는 유도 및 비유도되는 단백질이 현저하게 나타났다. 양파 화구 개화 후 실시한 고온과 고습처리 18일째의 양파두 품종의 단벡질도 현저하게 감소하였고 특히 고온처리구에서 더 감소되는 경향을 보였다. 원형절막 H+ATPase 발현을 western-blot으로 살펴본 결과, '신선황' 과 '맵시황'의 경우 대조구에서 원형질막 H+ATPase 단백질 발현이 유지 되는 반면에, 고온처리구에서는 두품종 모두 원형질막 H+ATPase가 발현 되지 않았다. 고습처리구에서 중생종 '맵시황'약 원형질막 H+ATPase는 발현 되었는데 조생종 '신선황'에서 발현되지 않았다. 이는 양파 종자 성숙단계에서 고온 조건을 조우하면 고습처리 보다는 단백질 및 원형질막의 H+ATPase피해가 더 심각함을 보여 주는 결과이다.

Hepatic ischemia-reperfusion injury (HIRI) is linked with high mortality rate. Several agents have been developed so far to reduce the risk of HIRI. In this study, we investigated the effects of Acanthopanax senticosus extract (AS) on hepatic ischemia-reperfusion. To explore the protective effects of A. senticosus extract injection (ASI) on hepatic ischemia-reperfusion injury rats animal model were used. After the development of HIRI by using clamping method rats were then randomly divided into five groups. Different doses of AS were administered in HIRI rat model. The level of ALT, AST, and MDA content in serum were detected in sham and HIRI groups. The activity of SOD, MPO and Ca2+-ATPase, content of MDA, and cAMP in hepatic tissue were also measured. Expression of Bcl-2 and Bax protein were detected by immunohistochemical staining method. Compared with sham group, ASI has the protective effect on the HIRI model in rats. Blood levels of ALT, AST, SOD, MPO, and MDA were significantly lower in ASI group compared with HIRI. Indeed SOD and Ca2+-ATPase activities, MDA content, and cAMP level were improved in ASI group. Furthermore, Bcl-2 and Bax protein were improved in ASI group compared with only HIRI group. These results suggest that AS may provide potential ameliorative therapy by inhibiting the damage signaling mechanism in hepatic ischemia/reperfusion injury model.

Na+/K+-ATPase, an energy-transducing ion pump, is responsible for maintenance of relatively high concentrations of potassium ions but low concentrations of sodium ions in the cell by transport of these ions across the plasma membrane and participates in transport of various nutrients including glucose, amino acids, and ions. In addition, Na+/K+-ATPase is also involved in regulation of intracellular calcium ion concentration by coupling with Na+/Ca+ exchanger expressed at the maternal-fetal interface in pigs. Na+/K+-ATPase consists of α, β, and FXYD subunits, but only α and β subunits are required for primary functions. FXYD subunit is an auxiliary protein for αβ complex of Na+/K+-ATPase. However, it has not been determined that subunits of Na+/K+-ATPase are expressed in the uterine endometrium during the estrous cycle and pregnancy in pigs. In this study, we determined expression of alpha (ATP1A1-4), beta (ATP1B1-3), and FXYD (FXYD1-7) subunits of Na+/K+-ATPase in the uterine endometrium during the estrous cycle and pregnancy in pigs. Real-time RT-PCR analysis showed that all alpha, beta, and FXYD subunits, except ATP1A3, were expressed in the uterine endometrium during the estrous cycle and pregnancy in a pregnancy status- and stage-specific fashion. In situ hybridization analysis exhibited that ATP1A1, ATP1A4, and ATP1B1 were localized to luminal (LE) and glandular epithelium (GE) during the estrous cycle and early pregnancy, and during mid to term pregnancy. ATP1A1 mRNA was localized to LE, GE, and areolae of the chorioallantois, especially at high levels to LE in areolae regions. ATP1B3 mRNA was detected only in LE during the estrous cycle and pregnancy with highest levels on day (D) 12 of pregnancy. Transcripts of all subtypes of FXYD subunit were primarily localized to LE and GE during the estrous cycle and early pregnancy and to chorionic membrane (CM) during mid to term pregnancy. RT-PCR analysis showed that all subtypes of Na+/K+-ATPase subunits, except ATP1A2, ATP1A3, and ATP1B2 mRNAs, were expressed in conceptuses on D12 and D15 of pregnancy. These results indicate that Na+/K+-ATPase subunits are expressed in the uterine endometrium and conceptuses during the estrous cycle and pregnancy in a pregnancy status- and stage-specific manner. These suggest that Na+/K+-ATPase subunits may be involved in the establishment and maintenance of pregnancy by coordinate regulation of absorption and secretion of nutrients such as glucose, amino acids, and ions at the maternal-fetal interface in pigs.

본 연구는 간이 수경재배법을 이용하여 보리의 알루미늄 스트레스 내성과 민감성 품종을 간편하고 빠르게 screen하는 방법을 소개하고, 선별된 품종간의 뿌리의 생장, 뿌리 조직의 염색, 알루미늄 함량, 원형질막의 H+ -ATPase의 발현 변화를 조사하여 분석하였다. l. 보리 65가지 품종을 간이 수경재배법을 이용하여 20uM 알루미늄을 24시간 처리 후 뿌리생장의 차이로 내성 세 품종(자예2, 자예6, 모치무기)과 민감성 세 품종(흰쌀, 올쌀, 품2)을 선별하였다. 2. 알루미늄에 내성 품종은 알루미늄 처리 농도(0, 5, 10, 20uM )에 따라 뿌리 생장 감소폭이 적었으나, 민감성 세 품종은 상대적으로 낮은 5uM 농도에서부터 80%의 생장이 억제되었다. 3. 내성인 자예2와 민감성인 품2의 알루미늄 처리 후, 농도별(0, 5, 10, 20uM ), 시간별(3, 6, 12, 24시간)로 0.2% hematoxylin으로 염색 시 주로 apex에 3시간 이후부터 염색되었으며, 민감성 품2가 내성인 자예2에 비해 농도와 시간에 따라 그 피해 정도가 매우 심각하였다. 4. 20uM 로 24 시간 처리된 뿌리 apex(10 mm)의 알루미늄 함량을 측정한 결과, 내성인 자예2는 주당 47.1 nmol의 함량을 보여 주었으나, 민감성인 품2는 주당 64.9 nmol의 높은 함량을 보여 주었다. 5. 24시간 동안 20uM 알루미늄을 처리한 뿌리 원형질막 H+ -ATPase 발현을 western blotting을 통해 분석한 결과, 내성인 자예2는 차이가 없었으나, 민감성 품2는 현저히 억제되었다. 이로 보아 원형질막 H+ -ATPase가 알루미늄의 내성 기작에 관여하는 것으로 보인다. 6. 본 연구를 통해 간이 수경재배와 hematoxylin을 이용한 염색으로 간단하고 빠르게 보리의 알루미늄 내성과 민감성 품종의 screening을 할 수 있었고, 보리뿐 아니라 쌀, 밀 등의 다른 종자에도 적용할 수 있을 것이다.

There are many species as bioenergy crops and have different cold sensitivity in each. Cold-tolerant camelina and rapeseed, -sensitive jatropha, were used to investigate the cold stress response. Various physiological parameters such as leaf length, width, electrolyte leakage, stomatal conductance and chlorophyll fluorescence were measured to determine the growth rate treated with cold (2℃) for 5 days. Cold treated jatropha was damaged seriousiy but camelina and rapeseed were withstand. In order to investigate the cold-response on plasma membrane H+-ATPase activity isolated from leaves and roots of camelina, rapeseed and jatropha crops were exposed to cold stress. There were an increase in the activity of leaves and roots plasma membrane in cold-tolerant crops (camelina, rapeseed) while decreased the activity in cold-sensitive crop (jatropha). By western-blot analyses, the protein expression of plasma membrane H+-ATPase isolated from leaves and roots of camelina and rapeseed was increased in the presence of cold stress, but not in jatropha. These results may suggest that increased plasma membrane H+-ATPase of crops are closely related with cold-tolerant.

Aluminum (Al) toxicity in plants is one of the major limitations to crop growth on acid soils. The Al-induced change of H+-ATPase expression has been regarded as an important mechanism for Al tolerance in soybean. To investigate whether translational regulation of plasma membrane H+-ATPase is involved in the response to Al stress, we conducted western - blot of this protein. The results show that western - blot of plasma membrane H+-ATPase in the "Sowon" (Al tolernace) significantly increased in translational expression level, while citric acid (50 μM) with Al (50 μM) treatment has not effected. In contrast, Al sensitive cultivar "Poongsannamool" inhibited expression level of plasma membrane H+-ATPase with Al treatment. Two - dimensional gel analysis were performed to determine the protein induction patterns of control and Al (50 μM, 24 h) treated soybean. There are many changes of plasma membrane proteins in both cultivars under Al stress. Especially "Sowon" was significantly enhanced the expression of the plasma membrane H+-ATPase in Al treatment. But protein expression of "Poongsannamool" was less than "Sowon". These results suggest that the regulatory role of plasma membrane H+-ATPase may involved the tolerance mechanism in soybean roots. At the present, transcriptional level of H+-ATPase is under investigation.