The discovery of the interaction between augmenter of liver regeneration and Na+, K+-ATPase with yeast two-hybrid system

AIM:To screen the proteins interacting with human augmenter of liver regeneration (hALR) by yeast two-hybrid system and to study the mechanism of hALR action. METHODS:hALR bait plasmid was constructed by ligating the gene of hALR into pGBKT7, then transformed into yeast AH109. The yeast strain AH109 containing pGBKT7-hALR was mated with yeast Y187 containing human liver cDNA library plasmid. Diploid yeast was plated on SD/-trp-leu-his-ade (QDO) for screening and on QDO containing X-α-gal for further selection.The AD/library inserts were amplified by PCR and the PCR products were characterized by digesting with Sau3AⅠ and HaeⅢ restriction enzyme to eliminate the duplicates. After sequencing, the positive clones were analysed by bioinformatics. RESULTS:Several positive clones were obtaind. The sequencing and analysis shown that one of them is 669 bp DNA fragment encoding β subunit of Na⁺, K⁺-ATPase. The 224 bp 3'terminal DNA fragment is non-encoder region, and the 445 bp 5'terminal DNA encodes C-terminal 147 amino acid residues of Na⁺, K⁺-ATPase β subunit. CONCLUSION:The results of screening proteins using yeast two-hybrid system showed that hALR could interact directly with Na⁺, K⁺-ATPase in the yeast cell.     

A review of augmenter of liver regeneration

Augmenter of liver regeneration (ALR) is a novel hepatic stimulator. Owing to its peculiar characteristic of gene structure and many kinds of biological functions, it is thought to be an important hepatotrophic factor. This review summarized the advance in ALR research in recent years including characteristics of ALR gene, molecular structure, physiochemical characteristics, biological functions and roles in liver injury.     

Mechanisms of augmenter of liver regeneration in promoting damaged hepatocyte proliferation

AIM: To investigate the mechanisms of augmenter of liver regeneration (ALR) in promoting damaged hepatocyte proliferation.METHODS: The effects of Kupffer cell condition medium (KCCM+) stimulated by ALR on damaged hepatocyte proliferation were studied by MTT. The localization of ALR binding to Kupffer cell membrane and in intact rat liver was studied by immunohistochemistry. The IL-6 expression in Kupffer cells stimulated with ALR was observed by immunohistochemistry. RESULTS: The proliferation of damaged hepatocytes stimulated with KCCM+ was increased significantly. ALR immunostaining particles in plasm of hepatocyte were found in intact liver. The rough immunostaining particles of ALR were seen on the surface of Kupffer cell membrane. Immunostaining particles of IL-6 in Kupffer cells induced by ALR increased. CONCLUSION: ALR promotes proliferation of damaged hepatocytes indirectly by stimulating Kupffer cells.     

柑橘全爪螨Na+-K+-ATPase的生化毒理学特性研究

为明确柑橘全爪螨Na+-K+-ATPase与其抗药性之间的关系,采用微量滴度酶标板法对柑橘全爪螨敏感品系(SS)、甲氰菊酯抗性品系(FeR)和阿维菌素抗性品系(AvR)的Na+-K+-ATPase的生化毒理学特性进行了比较研究.结果表明,柑橘全爪螨Na+-K+-ATPase比活力从高到低依次为AvR、SS和FeR;柑橘...     

Changes of BAEP,NO contents and Na+-K+ATPase activities in brain tissues in rats with hyperbilirubinemia

AIM: To explore the roles of brain-stem auditory evoked potential (BAEP) in early monitoring the hearing loss and brain damages in hyperbilirubinemia and nitric oxide(NO) in the pathogenesis of bilirubin-induced hearing loss and brain damages. METHODS: Different doses of bilirubin solution (30 mg/kg, 60 mg/kg, 90 mg/kg, 120 mg/kg and 150 mg/kg) were injected into the abdominal cavity of 15-day old SD rats to make the animal model of hyperbilirubinemia. The serum concentrations of bilirubin were detected by a micro-gauge. The bilirubin concentrations in the brain tissues were examined via a diazo method. The Na+-K+ATPase activities in the brain tissues were analyzed by rooting phosphorus. The NO contents in the brain tissues were assayed via the method of nitrate reductase. BAEP were recorded with an evoked potential recorder. RESULTS: After making the ejection, parts of the rats in the high dosage groups (120 mg/kg and 150 mg/kg) showed the abnormal neuro-behaviors. After 6 hours of the ejection, the bilirubin concentrations in serum and in brain tissues, and NO contents in the brain tissues were increased significantly. The Na+-K+ATPase activities in the brain tissues were decreased obviously, and the PL and IPL of BAEP were prolonged significantly in all the experimental rats except the ones in low dosage group (30 mg/kg). The changes of them were closely related to the dose of injected bilirubin. CONCLUSION: The PL and IPL of BAEP are the objective and sensitive indexes for early monitoring the hearing loss and brain damages in hyperbilirubinemia. NO may plays a certain role in the pathogenesis of bilirubin induced hearing loss and brain damages.     

LaCl3和CPZ对磷饥饿番茄液泡膜H+-ATPase和H+-PPase活性的影响

采用水培法研究了LaCl3和氯丙嗪(CPZ)对磷饥饿番茄幼苗液泡膜H+-ATPase和H+-PPase活性的影响。结果表明:LaCl3处理抑制了液泡膜H+-ATPase活性,但提高了H+-PPase活性;而CPZ处理既抑制了液泡膜H+-ATPase活性,又抑制了液泡膜H+-PPase活性。同时,CPZ处理引起磷饥饿番茄幼苗可溶性蛋白质含量下降,LaCl3处理引起可溶性蛋白质含量增加。     

水分胁迫对荔枝叶片H+-ATPase活性的影响及其与抗旱性的关系

研究了水分胁迫对荔枝(Litchi chinensis Sonn．)叶片细胞胞质以及以离子健和共价健与细胞壁结合的H^ -ATPase活性的影响，结果表明：(1)叶片RWC随水分胁迫程度的增加而降低，抗旱性强的东刘l号降低幅度小于抗旱性弱的陈紫；(2)水分胁迫下，荔枝叶片细胞胞质以及以离子健和共价健与细胞壁结合的H^ -ATPase活性(比活性)均上升，抗旱性强的品种上升幅度大于抗旱性弱的品种。     

Activity of H+-K+-ATPase in gastric parietal cells under stress in rats and analysis of electron microscopic enzyme cytochemistry

AIM: To demonstrate the changes of activity and electron microscopic enzyme cytochemistry staining of H⁺-K⁺-ATPase of gastric parietal cells under stress in rats. METHODS: Twenty-four male SD rats were randomly divided into normal group, stress group and stress+omeprazole (OM) group. Water immersion-restraint stress (WRS) model in SD rats was performed. The ulcer index (UI) of gastric mucosa and H⁺-K⁺-ATPase activity of gastric parietal cells were measured. The changes of ultrastructure and electron microscopic enzyme cytochemistry staining of parietal cells were observed under transmission electron microscope (TEM). RESULTS: Compared with control group, the UI of gastric mucosa and H⁺-K⁺-ATPase activity of gastric parietal cells increased (P<0.01 and P<0.05) in stress group. In stress+OM group, both UI and H⁺-K⁺-ATPase activity decreased (P<0.01) compared with stress group. Parietal cells were in a resting state in control group, and became active in stress group, where plenty of intracellular canaliculi were observed under the TEM. In stress+OM group, the dilated intracellular canaliculi lined with rare microvilli were founded. Enzyme cytochemistry staining showed that there was little black punctate enzyme reactive product scatted in intracellular canaliculi and the apical plasma membrane of parietal cells in control group, and there were large amounts of black enzyme reactive product accumulated at the intracellular canaliculi in stress group. Scarcely deposition of enzyme reactive product in intracellular canaliculi was observed in stress+OM group. CONCLUSION: The results indicate that the H⁺-K⁺-ATPase activity of gastric parietal cells increases under WRS, and is in accordance with ultrastructure changes. These findings suggeste that gastric acid might be one of the most important factors that result in stress ulcer.     

NaCl胁迫对南瓜幼苗Na~+及Ca~(2+)含量的影响

采用19个不同类型南瓜品种,研究了300 mmol/L NaCl下,幼苗地上部和根系Na+和Ca2+离子含量和Na+/Ca2+比值的变化。结果表明:NaCl胁迫8 d后,不同南瓜品种幼苗Na+含量均明显增加,离子平衡被打破;南瓜幼苗体内的Na+含量、地上部的Na+/Ca2+比值的变化反映植物对盐离子和营养元素相对的吸收情况;青栗(Q1)南瓜幼苗根系Na+含量和Na+/Ca2+比值明显高于黑蜜南瓜(H2)和黑籽南瓜(H3)。由此可知:不同南瓜品种幼苗体内Na+含量、地上部Na+/Ca2+比值变化趋势与NaCI胁迫下不同南瓜品种幼苗的盐害指数的结果基本一致;进一步验证了Q1耐盐性强与NaCl胁迫下地上部Na+/Ca2+比值较低,Ca2+离子含量较高有关;而品种H2和H3对盐敏感与NaCl胁迫下地上部Na+/Ca2+比值较高,Ca2+离子含量较低有关。     

Protective mechanism of metallothionein on cultivated rat cardiomyocytes in hypoxic preconditioning

AIM: Studying the mechanism of protective role of metallothionein(MT) in hypoxic preconditioning(HPC) of cultivated rat cardiomyocytes. METHODS:Using the model of hypoxia/reoxygenation of cultivated rat cardiomyocytes. Determining the contents of MT, malonyldialdehyde (MDA)-metabolism product of lipid peroxidation and the activities of Na⁺-K⁺ATPase, Ca²⁺-Mg²⁺ATPase of cardiomyocytes 24h after HPC, also determining the relevant changes after using MT antibody. RESULTS: After 24 h in HPC, the contents of MT and activities of Na⁺-K⁺ATPase, Ca²⁺-Mg²⁺ATPase were obviously higher than those in the control and hypoxia/reoxygenation(P＜0.05), and the contents of MDA were decreased remarkedly (P＜0.01). Then after using MT antibody, the activities of two enzyme were progressively decreased and the contents of MDA were significantly higher than those in the control and MT antibody-free groups(P＜0.01). CONCLUSION: HPC may induce excessive synthesis of MT, and MT can protect myocardial reoxygenation injury by eliminating lipid peroxidation and rising the activities of Na⁺-K⁺ATPase and Ca²⁺-Mg²⁺ATPase.     

The reversal effects of recombinant human augmenter of liver regeneration in immunocomplex induced liver fibrosis

AIM: To observe the anti-fibrosis activity of recombinant human augmenter of liver regeneration (hALR). METHODS: Albumin induced rat model of liver fibrosis was established and hALR was given peritoneally after the model production. Serum concentration of alanine aminotransferase(ALT),aspartic aminotransferase(AST),lactate dehydrogenase(LDH), hepatic collagen contents and pathological examination were selected as observing parameter. RESULTS: Recombinant human augmenter of liver regeneration (hALR) could decrease ALT, AST, LDH concentration of fibrotic rats. The measurements of hepatic collagen contents showed that hepatic collagen contents in hALR treatment group was much lower than those of model group and negative control group. Pathological examination also indicated that the degree of liver fibrosis in hALR treatment group was attenuated in comparison with those of model group and negative control group. CONCLUSION: Recombinant human augmenter of liver regeneration (hALR) had reversal effects on immunocomplex induced rat liver fibrosis.control group. CONCLUSION: Recombinant human augmenter of liver regeneration (hALR) had reversal effects on immunocomplex induced rat liver fibrosis.     

盐胁迫对黄瓜不同耐盐品种植株K~+和Na~+含量的影响

用体积浓度225mmol/L的NaCl对耐盐性不同的2个黄瓜品种进行处理,研究黄瓜盛果期NaCl胁迫下不同品种叶片和根中K+和Na+含量的变化。结果表明,盐胁迫下,黄瓜不同耐盐品种植株根和叶片中Na+含量明显增加,K+含量明显下降;耐盐品种叶片中Na+含量显著低于盐敏感品种,耐盐品种K+含量降低幅度显著小于盐敏感品种;植株的Na+、K+比值提高,耐盐品种Na+、K+比值显著低于盐敏感品种;耐盐品种SNa+,K+显著低于盐敏感品种。     

Generation and characterization of a series of monoclonal antibodiesagainst recombinant human augmenter of liver regeneration

AIM:To generate monoclonal antibodies against human augmenter of liver regeneration (rhALR). METHODS:After BALB/C mice were immunized by the purified rhALR, the cells of spleen were fused with the cells of SP2/0; The titer and speciality were respectively fathomed from ascites or foster fluid by ELISA and Western-blot test. RESULTS:2 hybridoma cell lines were successfully obtained. The McAbs titer from ascites and foster fluid are respectively about 10^-3-10^-5 and 10^-2-10^-3. It is evident that the two McAbs were directed at different epitopes. CONCLUSIONS:The McAbs have higher speciality. It is significantly useful of the value that how hALR distribute in tissue organs, how the hALR signals the metabolism in the body and the control distribution of the hALR on cell growth on the translational level and so on is researched.     

Sca-1+ cells from mouse fetal liver can differentiate into neurons in vitro

AIM: To study whether Sca-1⁺ cells from fetal liver can be induced to differentiate into neuronal cells in vitro. METHODS:Sca-1⁺cells from 14 5-days-old murine fetal liver were isolated with a magnetic cell sorting kit, and were cultured in Dulbecco s modif ied Eagle s medium(DMEM)/F12 supplemented with 10%fetal bovine serum(FBS), and passaged at a rat io of 1 3 when cells reached more than 80%confluence.The 5 passage cells were induced by 10^-3mol/Lβ-mercaptoethanol(β-ME)and 5×10^-7 mol/L all-trans-retinoic acid(RA)for 24 hours, and then incubated in serum-free medium for 5 hours to 5 days.The characteristics of treated cel s were assayed by immunocytochemistry staining analysis at 5 hours, or 5 days.RESULTS: Cells treated with β-ME and RA exhibited neuronal phenotype and expressed neuron-specific protein such as neuron-specific nuclear protein (NeuN), neuronfilament-M, and neuron-specific tubulin-1 (TuJ-1) but not tau, MAP-2, or the astrocyte-specific marker glial fibrillary acidic protein (GFAP).CONCLUSION: Sca-1⁺ cells from fetal liver, of which most are regarded as hematopoietic stem cells, could differentiate into early immature neuronal cells in vitro. These findings suggest that Sca-1⁺ cells from fetal liver may be an alternative source in cell therapy and gene therapy of neural dysfunction.     

柑橘果实总钙、可溶性Ca~(2+)含量及Ca~(2+)-ATPase活性的季节性变化

以单性结实的龟井和自花结实的鄂柑1号柑橘品种为材料,对果实的整个发育期的子房(幼果)、果皮和果肉的总钙、可溶性Ca2+含量和Ca2+-ATPase活性进行了测定。结果表明龟井花前至花期子房总钙含量就已很高,花后趋下降,而鄂柑1号花前至花期子房总钙含量相对较低,花后有一显著上升;龟井可溶性Ca2+含量在花期及花后有一明显下降,而对应鄂柑1号在花后却有一显著上升;龟井花前至花期Ca2+-ATPase活性较高,花后下降,而对应鄂柑1号花前较低,花期即始上升;果实增大期内两品种果皮的总钙均趋上升,对应果肉总钙却趋下降;而果皮和果肉的可溶性Ca2+含量和Ca2+-ATPase活性在增大期内均有一明显增长过程或居相对较高水平。     

草莓果实成熟过程中Ca~(2+)-ATPase活性变化及酶学特性

以不同成熟度的草莓果实为试材,研究了草莓果实成熟过程中Ca2+-ATPase活性变化规律、最适pH值、动力学参数Km值和Vmax值。研究结果表明,草莓果实从开始成熟时(绿熟期)Ca2+-ATPase活性不断升高,果实完全成熟时(粉红期)达到最大值,随着成熟后的不断衰老,其活性逐渐降低;该酶的最适pH值为7.0;不同成熟阶段果实Ca2+-ATPase反应所需的Ca2+浓度有所不同,EGTA对各成熟阶段草莓果实的Ca2+-ATPase活性的抑制作用存在明显的差异。经对Ca2+-ATPase的动力学分析表明,不同成熟度果实Ca2+-ATPase的Km值和Vmax值不同,从果实开始成熟Km值逐渐降低,衰老时又逐渐上升,变化趋势与酶活性变化趋势相反;Vmax值变化趋势与酶活性变化趋势相同。2+     

湘蕾金银花叶柄、茎段的离体培养与植株再生

为建立湘蕾金银花的高效离体培养及植株再生技术体系,并进行快速繁殖,以其叶柄、茎段为外植体,对其愈伤组织诱导、分化、继代培养与生根炼苗等关键技术环节进行研究。结果表明:诱导愈伤组织的适宜培养方案是MS+NAA1.0mg/L,继代培养的适宜培养方案是MS+6-BA1.5mg/L+NAA0.1mg/L,壮苗培养的适宜方案是MS+6-BA1.0mg/L,生根培养的适宜培养方案是1/2MS+IBA0.2mg/L+NAA0.2mg/L。在珍珠岩中炼苗成功的组培苗,移栽成活率达100%。     

寒富苹果叶片离体再生及四倍体诱导

为建立寒富苹果高效的离体再生体系和多倍体诱导体系,以试管苗叶片为外植体,研究了培养基中激素对寒富叶片再生芽的影响及适宜的四倍体诱导方法。结果表明,当培养基中BA质量浓度为0.5mg/L时,再生频率最高达35.7%;当培养基中BA质量浓度为2.5mg/L时,再生频率超过90%,平均再生芽数在4以上。在培养基中附加1.0mg/LTDZ,再生频率达100%,平均再生芽数达19.47。以附加15、30、60、120mg/L秋水仙素的液体再生培养基处理叶片5d,各个质量浓度处理均诱导出四倍体植株,诱变率在5.3%～22.2%之间;寒富苹果叶片在附加50mg/L秋水仙素固体再生培养基上处理5d亦获得了四倍体植株。研究结果表明寒富苹果叶片具有极强的不定芽再生能力,叶片再生过程中进行秋水仙素处理是获得其四倍体的一个有效途径。     

Effect of sorafenib on liver regeneration after partial hepatoectomy in liver cirrhotic rats

AIM: To study the effect of sorafenib on the liver regeneration after partial hepatectomy (PH) in cirrhotic rats. METHODS: Thirty Wistar rats with liver cirrhosis induced successfully with diethylnitrosamine (DEN) underwent 30% PH and then were randomly divided into 2 groups (n=15). The rats in experimental group were fed with sorafenib at dose of 30 mg·kg^-1·d^-1 from the 1st day to the 10th day after PH, while those in control group were fed with vehicle by gavage. The blood and liver tissues of the rats were collected after PH and at the end of the experiment. Liver regeneration rate (LRR) and proliferating cell nuclear antigen (PCNA) expression were assessed for determining the hepatocyte proliferation. The content of alanine transaminase (ALT), albumin (ALB), total bilirubin (TBIL), direct bilirubin (DBIL), angiogenesis related factors including vascular endothelial growth factor (VEGF), vascular endothelial growth factor receptor 2 (VEGFR-2), platelet-derived growth factor receptor β (PDGFR-β) and micro-vessel density (MVD) were measured in both groups. RESULTS: LRRs on day 10 after PH were 45.43%±3.36% and 44.21%±2.77% in experimental group and control group, respectively (P>0.05), and the expression of PCNA in hepatic tissues of the rats was not found by the method of immunohistochemistry in both groups. Liver function index had no significant difference between the 2 groups (P>0.05). However, other than VEGF, sorafenib resulted in inhibition of VEGFR-2 and PDGFR-β expression and reduction of MVD in experiment group, and significant difference between the 2 groups was observed (P<0.01). CONCLUSION: Sorafenib does not influence live regeneration after PH in liver cirrhotic rats.     

NaCl胁迫对枳椇各器官Na~+、K~+吸收的影响

以盆栽枳椇的1 a生实生苗为试材,分析了在不同的盐处理0、0.15%、0.3%、0.45%、0.6%下对其根、茎、叶的K+、Na+离子含量和Na+/K+的影响。结果表明:随着盐浓度的增加,植物叶片中Na+含量和Na+/K+明显升高,说明枳椇没有将Na+截留在根、茎中,而较多的运输到了叶片,使枳椇受害较重。