一种朊蛋白错误折叠循环扩增方法的建立

目的建立一种类似于PCR的蛋白质扩增方法-蛋白错误折叠循环扩增技术(PMCA),用于朊病毒病脑组织中PrPSc的检测。方法将不同浓度的羊瘙痒因子263K毒株原液与正常仓鼠脑组织匀浆混合,经反复孵育/超声,共10~15个循环。WesternBlot检测扩增产物中蛋白酶K抗性PrPSc信号。结果在本研究试验体系下,263K毒株可以利用仓鼠脑组织为基质在体外迅速复制。所建立的PrPSc-PMCA技术可检测到10-5稀释的毒株原液中的PrPSc。与常规的脑组织免疫印记方法相比,敏感度提高了105~106倍。研究还显示PrPSc还可利用小脑和脑干为基质进行体外扩增复制。结论成功建立了PrPSc-PMCA技术,为朊病毒病的早期诊断和朊病毒生物学特性的研究提供了一种新的手段。     

Glucagon-induced somatostatin release from perifused rat hypothalamus: calcium dependency and effect of cysteamine treatment

Somatostatin (SRIF) release from rat hypothalamus was investigated in vitro with a perifusion system. Glucagon (1 microM) and high potassium concentrations (56 mM) stimulated SRIF release in a calcium-dependent manner. Pretreatment of the rat with cysteamine (30 mg/100 g body weight, 7 h earlier) significantly reduced SRIF release from the hypothalamus in glucagon- and high potassium-stimulated states as well as in the basal state. SRIF release from rat hypothalamus was also stimulated by both dibutyryl cyclic AMP (1 mM) and theophylline (3 mM). These results suggest that glucagon, acting in a calcium-dependent manner and possibly through the adenylate cyclase-cyclic AMP system, stimulates SRIF release from rat hypothalamus and that cysteamine treatment reduces releasable SRIF in the hypothalamus.     

Potential role of phosphodiesterase 7 in human T cell function: comparative effects of two phosphodiesterase inhibitors

Even though the existence of phosphodiesterase (PDE) 7 in T cells has been proved, the lack of a selective PDE7 inhibitor has confounded an accurate assessment of PDE7 function in such cells. In order to elucidate the role of PDE7 in human T cell function, the effects of two PDE inhibitors on PDE7A activity, cytokine synthesis, proliferation and CD25 expression of human peripheral blood mononuclear cells (PBMC) were determined. Recombinant human PDE7A was obtained and subjected to cyclic AMP-hydrolysis assay. PBMC of Dermatophagoides farinae mite extract (Df)-sensitive donors were stimulated with the relevant antigen or an anti-CD3 monoclonal antibody (MoAb). PBMC produced IL-5 and proliferated in response to stimulation with Df, while stimulation with anti-CD3 MoAb induced CD25 expression and messenger RNA (mRNA) synthesis of IL-2, IL-4 and IL-5 in peripheral T cells. A PDE inhibitor, T-2585, which suppressed PDE4 isoenzyme with high potency (IC50 = 0.00013 microM) and PDE7A with low potency (IC50 = 1.7 microM) inhibited cytokine synthesis, proliferation and CD25 expression in the dose range at which the drug suppressed PDE7A activity. A potent selective inhibitor of PDE4 (IC50 = 0.00031 microM), RP 73401, which did not effectively suppress PDE7A (IC50 > 10 microM), inhibited the Df- and anti-CD3 MoAb-stimulated responses only weakly, even at 10 microM. PDE7 may play a critical role in the regulation of human T cell function, and thereby selective PDE7 inhibitors have the potential to be used to treat immunological and inflammatory disorders.     

Effect of GABA-ergic agents on the analgesic effect of morphine in rats

In order to detect possible interaction between GABA and opiates, the effects of GABA-ergic drugs on analgesia induced by morphine were studied. The vocalization response to electrical stimulation of the tail in rats was used as an index of the action of morphine. Thiosemicarbazide, an inhibitor of glutamate decarboxylase, and bicuculline, which blocks GABA-ergic receptors, drugs which, it is suggested, can be considered as a group of GABA-negative compounds, weaken and shorten the effect of morphine. Depakine, an inhibitor of -ketoglutarate-GABA-transaminase, like GABA itself, given in large doses (GABA-positive effects) strengthens morphine analgesia and prolongs its effect. The possible causes of these relations between GABA and opiates are discussed.Laboratory of Pharmacology of the Nervous System, Institute of Pharmacology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR V. V. Zakusov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 88, No. 7, pp. 35–37, July, 1979.     

Stimulation of chloramphenicol acetyltransferase synthesis by cyclic AMP in bacterial cell systems

The effect of cyclic 3,5-adenosine monophosphate (cAMP) on production of the enzyme chloramphenicol acetyltransferase (CAT) by whole bacterial cells was studied in strainsEscherichia coli CSH-2/R222 and WZ-78/R222 (cya₈₅₅). CAT synthesis in strainE. coli WZ-78/R222 was shown to have an intensity only half as great as that of strainE. coli CSH-2/R222. The production of CAT by strainE. coli CSH-2/R222 was increased only very slightly by cAMP, but its effect on the production of this enzyme in strain WZ-78/R222 was appreciable.Research Laboratory of Experimental Immunobiology, Academy of Medical Sciences of the USSR, Moscow. (Presented by Academician of the Academy of Medical Sciences of the USSR, N. N. Zhukov-Verezhnikov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 80, No. 10, pp. 65–66, October, 1975.     

Effect of radioprotectors on cyclic AMP-dependent protein phosphorylation

The ability of radioprotectors (serotonin, aminoethylisothiouronium) in radioprotective doses to stimulate cyclic AMP-dependent phosphorylation of mouse liver cytosol and nuclear and spleen cytosol proteins in vivo was demonstrated. In experiments in vitro, the radioprotectors had no direct action on protein kinase activity or its stimulation by cyclic AMP. It is postulated on the basis of these results and those of previous investigations that activation of cyclic AMP-dependent phosphorylation is due to an increase in the intracellular cyclic AMP concentration under the influence of the radioprotectors.Laboratory of Radiation Biophysics, Department of Biophysics, Biological Faculty, Moscow State University. (Presented by Academician of the Academy of Medical Sciences of the USSR S. E. Severin.) Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 87, No. 3, pp. 230–232, March, 1979.     

Synaptic efficacy of inhibitory synapses in hypoglossal motoneurons after transection of the hypoglossal nerves

In cat hypoglossal motoneurons after axotomy the synaptic efficacy of inhibitory synapses made by the lingual nerve afferent fibers was studied. The amplitude of the short- and the long-lasting inhibitory postsynaptic potential produced in tongue protruder motoneurons 24 days after axotomy by stimulation of the lingual nerve was significantly reduced in size as compared with the control on the unoperated side. In most protruder motoneurons 40 days after axotomy a large excitatory postsynaptic potential and a spike was produced by stimulation of either the ipsilateral or the contralateral lingual nerve. We have demonstrated that the decline of synaptic efficacy of inhibitory synapses for the short-lasting inhibitory postsynaptic potential was more prominent than that for the long-lasting inhibitory potential in the motoneuron 24 days after axotomy. After the cut axons of protruder motoneurons were re-united to tongue muscles, we have demonstrated that the decline of synaptic efficacy of inhibitory synapses for the short-lasting inhibitory postsynaptic potential was less prominent than that in axotomized protruder motoneurons.     

内生致冷原对家兔内毒素性发热第二热相的影响

为验证内生致冷原(EC)能否影响内毒素(ET)性发热第二热相或热限水平,并确实脑脊液中cAMP水平是否与EC的降温作用有关,作者用90只新西兰兔进行实验。观察:①输注人尿或等量生理盐液对正常家免体温的影响,检测EC效应期血浆和脑脊液中cAMP的含量;②在第二热峰出现前输注人尿或生理盐液对第二热相的影响,检测EC效应期血浆及脑脊液中cAMP的含量。结果表明:①人尿明显降低正常家兔的直肠温度,而等量生理盐液则无此作用,且两者均引起血浆及脑脊液中cAMP浓度的明显下降,提示EC的降温作用与脑cAMP浓度下降可能无重要关系;②人尿(EC)抑制ET性发热第二热相的形成,从而降低热限水平,变双相热为单相热,同量生理盐水无此作用,两者都能降低血浆和脑脊液中cAMP的水平,但EC不及NS明显,表明EC抑制第二热相或降低热限水平的作用也与cAMP浓度变化无重要关系,作者推论cAMP不是ET性发热第二热相的唯一成因。     

Inhibition of heparin/protamine complex-induced complement activation by Compstatin in baboons

Complement activation products are major components of the inflammatory response induced by cardiac surgery and cardiopulmonary bypass which contribute to postoperative organ dysfunction, fluid accumulation, and morbidity. Activation of the complement system occurs during extracorporeal circulation, during reperfusion of ischemic tissue, and after the formation of heparin-protamine complexes. In this study we examine the efficacy of Compstatin, a recently discovered peptide inhibitor of complement, in preventing heparin/protamine-induced complement activation in baboons. The study was performed in baboons because Compstatin binds to baboon C3 and is resistant to proteolytic cleavage in baboon blood (similar to humans); Compstatin inhibits only the activation of primates' complement system. After testing various doses and administration regimens, Compstatin produced complete inhibition at a total dose of 21 mg/kg when given as a combination of bolus injection and infusion. Compstatin completely inhibited in vivo heparin/protamine-induced complement activation without adverse effects on heart rate or systemic arterial, central venous, and pulmonary arterial pressures. This study indicates that Compstatin is a safe and effective complement inhibitor that has the potential to prevent complement activation during and after clinical cardiac surgery. Furthermore, Compstatin can serve as the prototype for designing an orally administrated drug.