Adenosine A2A receptor antagonist SCH58261 attenuates hypoxic-ischemic brain damage in a fetal rabbit model

AIM: To study the effect of adenosine A_2A receptor antagonist SCH58261 on hypoxic-ischemic brain damage (HIBD) in a mature fetal rabbit model.METHODS: Pregnant New Zealand white rabbits at gestational day 29 were selected and were randomly divided into sham-operated group, hypoxic-ischemic group, SCH58261 0.04 mg/kg group, SCH58261 0.12 mg/kg group and DMSO group. The intrauterine rabbit HIBD model was established. All pregnant rabbits were subjected to cesarean section 24 h after the sham operation or experimental procedure to induce hypoxic-ischemic injury in the fetus. The survival neonatal rabbits were kept in a neonatal incubator at 35℃. The general conditions of the newborn rabbits were recorded. The degree of neurobehavioral damage in the newborn rabbits was estimated by a neurobehavioral scoring protocol. The concentration of SCH58261 in the serum of pregnant rabbits, the serum of neonatal rabbits and the brain tissues of neonatal rabbits was measured by mass spectrometry. The mRNA expression of Bcl-2/Bax and protein levels of p-P38 mitogen-activated protein kinase (MAPK) in the cortex, hippocampus and striatum area in the brain of the neonatal rabbits were determined by real-time PCR and Western blot. RESULTS: SCH58261 was detected in the serum and brain tissues of the newborn rabbits. The SCH58261 concentration was approximately 40 μg/L in the brain tissue of the newborn rabbits. The mRNA expression of Bcl-2 in the cortex, hippocampus and striatum of brain tissues in SCH58261 0.04 mg/kg group and SCH58261 0.12 mg/kg group was higher, and the mRNA expression of Bax was lower than those in HI group (P<0.05). The protein level of p-P38 MAPK in the cortex, hippocampus and striatum of brain tissues was reduced in SCH58261 0.04 mg/kg group and SCH58261 0.12 mg/kg group compared with HI group (P<0.05). The protein level of p-P38 MAPK in SCH58261 0.12 mg/kg group was a little lower than that in SCH58261 0.04 mg/kg group (P<0.05). CONCLUSION: Adenosine A_2A receptor antagonist SCH58261 attenuates hypoxia-ischemia induced neonatal brain injury by blocking adenosine A_2A receptor, subsequently inhibiting p-P38 MAPK phosphorylation to reduce neuronal apoptosis.     

Effects of SFKs in microglia on ATP-induced long-term potentiation in spinal dorsal horn

AIM: To investigate the effects of Src family kinases (SFKs) on adenosine 5'-triphosphate (ATP)-induced long-term potentiation (LTP) in the spinal dorsal horn. METHODS: Male Sprague-Dawley rats (250-280 g) were used in the experiments. Western blotting, electrophysiological recording in spinal dorsal horn in vivo and immunohistochemistry were used in the study. The C-fiber-evoked field potentials were recorded at the superficial layers of spinal dorsal horn at the lumbar enlargement and the phosphorylation level and location of SFKs in spinal dorsal horn were examined by Western blotting and immunohistochemistry. RESULTS: Thirty min and 60 min after ATP application, the levels of phosphorylated SFKs (p-SFKs) were significantly increased.The p-SFKs were expressed in microglia, but not in astrocytes or neurons. Spinal application of SFK inhibitors prevented ATP-induced LTP. CONCLUSION: Microglial SFKs may play an important role in ATP-induced LTP of C-fiber evoked field potentials in the spinal dorsal horn.     

Amiloride slows down calpain-mediated ABCA1 degradation through inhibition of hypoxia-induced NHE1 expression

AIM: To examine the effects of hypoxia on sodium-hydrogen exchange 1(NHE1) expression, intracellular Ca²⁺ concentration ([Ca²⁺]_i) and calpain activity, and to explore the effect of amiloride on adenosine triphosphate-binding cassette transporter A1(ABCA1) degradation and its calpain-related mechanism. METHODS: RAW264.7 cells were exposed to hypoxia for 0 h, 12 h, 24 h and 48 h. The cell viability was measured by MTT assay and the expression of NHE1 at mRNA and protein levels was detected by real-time PCR and Western blot. [Ca₂₊]_i was analyzed by flow cytometry. Calpain activity was assessed by the method of Suc-LLVY-aminoluciferin. Furthermore, the protein levels of ABCA1 in the RAW264.7 cells exposed to hypoxia for 24 h were determined after 6 h or 12 h treatment with NHE1 inhibitor amiloride in the presence of cycloheximide. ABCA1 protein levels and calpain activity were detected after 12 h incubation with calpain inhibitor ALLN or intracellular calcium-chelating agent BAPTA. RESULTS: Hypoxia inhibited the cell viability in a time-dependent manner. Hypoxia up-regulated the mRNA and protein expression of NHE1, and increased [Ca²⁺]_i and calpain activity. Hypoxia increased the degradation of ABCA1 and amiloride slowed down the ABCA1 degradation. ALLN or BAPTA increased ABCA1 protein level and decreased calpain activity. CONCLUSION: NHE1 inhibitor amiloride attenuates the calpain-mediated degradation of ABCA1, indicating that hypoxia-induced NHE1 might, at least in part, participate in the ABCA1 degradation.     

Adenylates as an estimate of microbial biomass C in different soil groups

Adenylate (i.e. adenosine tri- (ATP), di- (ADP) and monophosphates (AMP)) and microbial biomass C data were collected over a wide range of sites including forest floor layers and forest, grassland and arable soils. Microbial biomass C was measured by fumigation extraction and adenylates after alkaline Na₃PO₄/DMSO/EDTA extraction and HPLC detection. Our aims were (1) to test whether the sum of adenylates is a better estimate for microbial biomass than the determination of ATP, (2) to compare our conversion values with those proposed by others, and (3) to analyse whether soil properties or land use form affect the relationships between ATP, adenylates and microbial biomass C. A close relationship was found between microbial biomass C and ATP (r=0.96), but also with the sum of adenylates (r=0.96) within all appropriately conditioned soil samples (n=112). In the mineral soil (n=98), the geometric means of the ATP-to-microbial biomass C ratio and the adenylates-to-microbial biomass C ratio were 7.4 and 11.4 μmol g⁻¹, respectively. The mean ratios did not differ significantly between the different texture classes and land use forms. In the forest floor, the ATP-to-microbial biomass C ratio and the adenylates-to-microbial biomass C ratio were both roughly two-thirds of those of the mineral soil. The average adenylate energy charge (AEC) of all soil samples was 0.79 and showed a strong negative relationship with the soil pH (r=−0.69). However, the AEC is presumably only indirectly affected by the soil pH.     

Effects of endothelial nitric oxide synthase gene transfection on cytokines and cAMP production in human phagocytic cells

AIM and METHOD: Human endothelial nitric oxide synthase (eNOS) gene was transfected into human phagocytic cell U937 and the effects of gene transfer on cytokines and cAMP production were observed. RESULTS: A functional eNOS was stably expressed in transfected U937 cells, but NO release was undetectable in intact transfectants. However, eNOS gene expression upregulated tumor necrosis factor-α release and downregulated interleukin-10 and cAMP production in either presence or absence of NOS inhibitor N^ω-monomethyl-L-arginine. CONCLUSION: The function of tranfected eNOS gene product showed cellular speciality. The effector molecule that changed the produced pattern of cytokines and cAMP in phagocytic cells seems not likely the nitric oxide.     

The effect of anti-digoxin antiserum on endoxin and membrane ATPase activity in hypoxic myocardium

AIM: To evaluate the antagonistic effect of anti-digoxin antiserum on hypoxic myocardium and its mechanism. METHODS: It was observed that different concentration of anti-digoxin antiserum effect on endoxin and cell membrane ATPase activity in hypoxic myocardium model.RESULTS: The endoxin level was much higher,cell membrane ATPase activity was much lower in hypoxic myocardium than those of normal; anti-digoxin antiserum can resume membrane ATPase activity. CONCLUSION: Rise of endoxin was basic in molecular biology of myocardial damage during myocardial hypoxia. Anti-digoxin antiserum decreased myocardial damage and has protective effect on hypoxic myocardium by antagonistic effect of endoxin.     

Protective effects of adenosine on cultured rat hippocampal neurons after oxygen-glucose deprivation

AIM:To investigate the protective effects of adenosine on cultured rat hippocampal neurons after oxygen-glucose deprivation.METHODS:The control and adenosine-treated hippocampal neurons cultured for 12 d were exposed to oxygen-glucose deprivation environment for 0.5-4 h and then cultured with original medium in normoxia for 24 h. The soma area, survival rate, effluxes of lactate dehydrogenase (LDH)and apoptosis of neurons were observed.RESULTS:The soma area, effluxes of lactate dehydrogenase from neurons and apoptosis were increased while survival rate of neurons was decreased after oxygen-glucose deprivation compared with those pre-oxygen-glucose deprivation. Compared with the control, after oxygen-glucose deprivation the soma area, effluxes of lactate dehydrogenase from neurons and apoptosis were decreased, however, the survival rate of neurons was increased in the adenosine group.CONCLUSION:Oxygen-glucose deprivation can lead to the severe damage of cultured hippocampal neurons, and adenosine can reduce neuronal injury induced by oxygen-glucose deprivation.     

The relaxation of rabbit femoral artery induced by cAMP is partly dependent on nitric oxide

AIM:To determine whether vasorelaxation induced by cAMP is dependent on eNOS activation.METHODS:A constant perfusion model of rabbit femoral artery was used to observe the changes of perfusion pressure before and after intra-arterial infusing different concentrations of dbcAMP(a membrane-permeable cAMP analogue) or forskolin(a direct stimulator of adenyl cyclase) and the effects of NOS inhibitor L-NAME on their actions.RESULTS:Both dbcAMP and forskolin induced concentration-dependent relaxation of rabbit femoral artery bed and NOS inhibitor L-NAME partly inhibited this vasorelaxation.CONCLUSION:Vasorelaxation induced by cAMP is mediated partly through eNOS activation.     

Human mesenchymal stem cells differentiate into neuron-like cells by increase in intracellular cyclic AMP

AIM: To investigate the differentiation from human mesenchymal stem cells(hMSC) into neuron-like cells by the increase in intracellular cyclic AMP. METHODS: hMSC were separated from human marrow with Ficoll-Paque reagent and expanded in culture medium. hMSC were induced to differentiate into neurons with Forskolin and 3-isobutyl-1-methyl-xanthine (IBMX). Neuron-specific enolase(NSE), neurofilament(NF), glial fibrillary acaidic protein(GFAP) were detected by immunohistochemistry. RESULTS: hMSC were expanded as undifferentiated cells in culture for more than10passages. Forskolin/IBMX can induce hMSC to exhibit a neuronal phenotype, expressing NSE and NF-M in 5 hours. But the neuron-like cells didn't express the glial astrocyte marker GFAP. CONCLUSION: hMSC can be induced to differentiate into neurons by increase in the intracellular cAMP.     

虫草保健啤酒生产工艺研究

[目的]研究利用蛹虫草发酵液制取虫草保健啤酒的生产工艺过程及发酵液生物活性物质检测方法,为虫草保健啤酒的工业生产提供参考。[方法]首先筛选出合适的蛹虫草菌株,然后以蛹虫草一次发酵液与麦芽汁混合培养基作为发酵培养基,在30L发酵罐中采用单罐低温发酵方法,接着检测发酵液理化、感官指标以及应用HPLC法检测生物活性物质。[结果]该虫草啤酒的理化和感官指标均达到国家标准,其中酒精、总酸、双乙酰、真正发酵度分别达到4.6%vol、1.8mg/100ml、0.02mg/g、71%;获得了富含虫草生物活性成分的虫草保健啤酒。当发酵周期为240h时,发酵液中腺苷和虫草素含量达到最高,分别为122.3和11.8μg/ml;发酵周期为144h时,甘露醇含量达到最高,为19.75mg/L。[结论]该发酵方法可以使虫草啤酒各项指标均达到国家标准,并且赋予了啤酒虫草生物活性物质。