肝细胞生长素促进肝细胞再生与cAMP、cGMP关系的研究

cAMP,cGMP在细胞生化过程中起“第二信使”作用。本文通过体外原代培养肝细胞、观察肝细胞生长素(HGF)促进肝细胞再生,以及对肝细胞cAMP、cGMP水平的影响。实验结果表明HGF和胰岛素均可引起肝细胞cAMP、cGMP水平的升高,促进肝细胞再生;根据cAMP、cGMP比值的降低程度分析HGF促进肝细胞再生的能力较胰岛素强,表明HGF促进肝细胞再生可能与cAMP、cGMP系统有关。通过cAMP、cGMP两者的平衡,保持肝细胞不同发育阶段的完善代谢过程。     

Development of a whole cell pneumococcal vaccine: BPL inactivation,cGMP production,and stability

Pneumococcal infections impose a large burden of disease on the human population, mainly in developing countries, and the current pneumococcal vaccines offer serotype-specific protection, but do not cover all pathogenic strains, leaving populations vulnerable to disease caused by non-vaccine serotypes. The pneumococcal whole cell vaccine is a low-cost strategy based on non-capsular antigens common to all strains, inducing serotype-independent immunity. Therefore, we developed the process for the cGMP production of this cellular vaccine. Initially, three engineering runs and two cGMP runs were performed in 60-L bioreactors, demonstrating the consistency of the production process, as evaluated by the growth curves, glucose consumption and metabolite formation (lactate and acetate). Cell recovery by tangential filtration was 92 ± 13%. We optimized the conditions for beta-propiolactone (BPL) inactivation of the bacterial suspensions, establishing a maximum cell density of OD₆₀₀ between 27 and 30, with a BPL concentration of 1:4000 (v/v) at 150 rpm and 4 °C for 30 h. BPL was hydrolyzed by heating for 2 h at 37 °C. The criteria and methods for quality control were defined using the engineering runs and the cGMP Lots passed all specifications. cGMP vaccine Lots displayed high potency, inducing between 80 and 90% survival in immunized mice when challenged with virulent pneumococci. Sera from mice immunized with the cGMP Lots recognized several pneumococcal proteins in the extract of encapsulated strains by Western blot. The cGMP whole cell antigen bulk and whole cell vaccine product lots were shown to be stable for up to 12 and 18 months, respectively, based upon survival assays following i.p. challenge. Our results show the consistency and stability of the cGMP whole cell pneumococcal vaccine lots and demonstrate the feasibility of production in a developing country setting.     

Effects of various phosphodiesterase-inhibitors,forskolin, and sodium nitroprusside on porcine detrusor smooth muscle tonic responses to muscarinergic stimulation and cyclic nucleotide levels in vitro

The cyclic nucleotides cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) are second messengers involved in the regulation of contractility in various smooth muscle organs including detrusor smooth muscle. They are synthesized by activation of adenylate and guanylate cyclases, respectively, and inactivated by phosphodiesterases (PDEs). In order to delineate the intracellular regulation of porcine detrusor contractility by cyclic nucleotides and phosphodiesterases, functional organ bath studies and determinations of intracellular cyclic nucleotide contents were performed after incubation of porcine detrusor strips with forskolin (adenylate cyclase activator), sodium nitroprusside (guanylate cyclase activator), and various phosphodiesterase-inhibitors. Significant relaxant responses were achieved only by forskolin, the nonspecific phosphodiesterase-inhibitor papaverine, and the phosphodiesterase 1-inhibitor vinpocetine (62.4 ± 5.6%, 73 ± 4.3%, and 53 ± 7.9%, respectively). Sodium nitroprusside and the selective PDE-inhibitors milrinone, rolipram, zaprinast, and dipyridamole were significantly less efficacious (26.9 ± 3.9%, 15.5 ± 3.8%, 15.3 ± 3.0%, 13 ± 4.0%, and 13.2 ± 2.1%, respectively). Forskolin, papaverine, and vinpocetine elevated intracellular cAMP concentrations (7.3-, 1.9-, and 1.7-fold increase at 100 μM, respectively), whereas the other substances failed to enhance cAMP levels. cGMP levels were only increased by sodium nitroprusside (7.8-fold). The adenylate cyclase-cAMP system seems to be the more important signal transduction system involved in the relaxation of carbachol induced smooth muscle tone of the porcine detrusor. The role of the guanylate cyclase-cGMP system is less clear. In addition, the calcium/calmodulin-stimulated PDE I seems to be of major functional importance in regulating cAMP hydrolysis in the porcine detrusor smooth muscle in vitro. © 1996 Wiley-Liss, Inc.     

Systematic spatiotemporal mapping reveals divergent cell death pathways in three mouse models of hereditary retinal degeneration

Calcium (Ca²⁺) dysregulation has been linked to neuronal cell death, including in hereditary retinal degeneration. Ca²⁺ dysregulation is thought to cause rod and cone photoreceptor cell death. Spatial and temporal heterogeneities in retinal disease models have hampered validation of this hypothesis. We examined the role of Ca²⁺ in photoreceptor degeneration, assessing the activation pattern of Ca²⁺-dependent calpain proteases, generating spatiotemporal maps of the entire retina in the cpfl1 mouse model for primary cone degeneration, and in the rd1 and rd10 models for primary rod degeneration. We used Gaussian process models to distinguish the temporal sequences of degenerative molecular processes from other variability sources.In the rd1 and rd10 models, spatiotemporal pattern of increased calpain activity matched the progression of primary rod degeneration. High calpain activity coincided with activation of the calpain-2 isoform but not with calpain-1, suggesting differential roles for both calpain isoforms. Primary rod loss was linked to upregulation of apoptosis-inducing factor, although only a minute fraction of cells showed activity of the apoptotic marker caspase-3. After primary rod degeneration concluded, caspase-3 activation appeared in cones, suggesting apoptosis as the dominant mechanism for secondary cone loss. Gaussian process models highlighted calpain activity as a key event during primary rod photoreceptor cell death. Our data suggest a causal link between Ca²⁺ dysregulation and primary, nonapoptotic degeneration of photoreceptors and a role for apoptosis in secondary degeneration of cones, highlighting the importance of the spatial and temporal location of key molecular events, which may guide the evaluation of new therapies.     

Anxiety‐like behavior in female mice changes by feeding,possible effect of guanylate cyclase C

Anxiety disorders are the most frequent mental disorders and are more prevalent in the female population. Up to date, an involvement of guanylate cyclase A and B in anxiety‐like behavior has been suggested. In this study, we showed an expression of guanylate cyclase C (GC‐C) in the amygdala which is regulated by feeding. Therefore, we further investigated sex differences of GC‐C effects on anxiety levels with special attention to female estrous cycle and feeding. The effects of estrous cycle and feeding were investigated by several behavior tests: elevated plus maze, home cage escape and novelty‐induced hypophagy. Possible changes in GC‐C expression in amygdala and hypothalamus during estrous cycle were established by qPCR. When GC‐C is activated (after a meal), the sex difference in all behavior tests used was abolished. As the expression of mRNA for GC‐C in the amygdala increases 2 hr after a meal only in female animals, the anxiety levels change after a meal again only in female animals. When the anxiety levels are investigated, it is very important to pay attention not only to estrous cycle in female animals but also when animals were fed compared to the time point of the experiments. Concluding from our results, the sex differences in the incidence of anxiety disorders in humans could be GC‐C dependent.     

NO—cGMP途径在嗜酸性粒细胞功能中作用的研究进展

一氧化氮是具有高度反应性的自由基 ,在体内广泛存在 ,是一种重要的细胞内信号转导分子 ,在细胞多种功能中起作用。NO主要通过提高环一磷酸鸟苷水平来实现其生物学功能。NO cGMP途径在嗜酸性粒细胞的功能中起重要作用 ,嗜酸性粒细胞在哮喘过程中能产生大量NO。两者之间存在密切联系。本文主要介绍NO cGMP信号转导通路、该通路在嗜酸性粒细胞趋化 ,凋亡等功能中作用以及NO释放在哮喘中意义的研究进展。     

吸入一氧化氮改善烟雾吸入性损伤犬肺通气功能的意义

目的评价吸入一氧化氮（ＮＯ）对犬烟雾吸入性损伤肺通气功能改善的效果。方法烟雾吸入伤后，将１７只犬随机分为２组，对照组（ｎ＝８）单纯吸氧（ＦｉＯ２，０．４５），治疗组（ｎ＝９）吸氧（ＦｉＯ２，０．４５）＋０．００４５％（４５ｐｐｍ）ＮＯ，连续监测１２小时血气变化；并按时相点抽血检测有关指标。数据行多个样本均数间方差分析。结果吸入ＮＯ治疗组ＰａＣＯ２、呼吸指数（ＲＩ）、肺泡死腔率（ＶＤ／ＶＴ）、肺动脉分流率（ＱＳ／ＱＴ）和碳氧血红蛋白（ＨｂＣＯ）含量比对照组均有不同程度的下降（Ｐ＜０．０５～０．０１），而动脉血浆亚硝酸盐（ＮＯ－２）水平则明显高于对照组（Ｐ＜０．０１）。结论吸入ＮＯ能明显改善肺通气功能，作为吸入性损伤的综合治疗，吸入ＮＯ疗法值得进一步研究。     

电针至阳穴对急性心肌缺血家兔血浆cAMP、cGMP含量的影响

目的 观察电针至阳穴对急性心肌缺血家兔血浆环磷酸腺苷（cAMP）、环磷酸鸟苷（cGMP）含量的影响。方法 40只家兔随机分为4组：正常对照组（A组）、单纯造模组（B组）、造模＋电针内关穴组（C组）、造模＋电针至阳穴组（D组），采用垂体后叶素（2U／kg）造成家兔心肌急性缺血，在全过程中多次测定Ⅱ导联心电图，并用放射免疫法测定电针后家兔血浆cAMP、cGMP含量。结果 C组和D组家兔血浆中cAMP含量明显低于B组（P〈0．01），与A组比较无显著性差异（P〉0．05），C组与D组间亦无显著性差异（P〉0．05）；各组间cGMP含量无显著性差异（P〉0．05）。结论 电针至阳穴可提高家兔心肌对缺血缺氧的耐受力．改善心肌的缺血缺氧状态，与电针内关穴有同等效果。     

电针对抑郁模型大鼠海马一氧化氮-环磷酸鸟苷信号转导通路的影响

目的:观察电针对抑郁模型大鼠海马一氧化氮-环磷酸鸟苷(NO-cGMP)信号转导通路的影响,探讨电针治疗抑郁症的部分作用机制。方法:SD大鼠随机分为正常组、模型组和电针组,每组10只。用慢性不可预见性轻度刺激结合孤养的方法制造抑郁模型。造模同时针刺治疗电针组动物,选取"百会"印堂"穴,电针20min,每日1次,连续针刺21d。治疗结束后取大鼠海马组织,用免疫组化法检测神经细胞性一氧化氮合酶(nNOS)表达量,用放射免疫法测定cGMP含量。结果:模型组大鼠海马区的nNOS免疫反应阳性颗粒数目减少,电针组nNOS免疫反应阳性颗粒数目较模型组增多。各组测得的nNOS灰度值,模型组明显高于正常组(P<0.01),电针组明显低于模型组(P<0.01)。模型组大鼠海马中cGMP含量与正常组比较有降低的趋势,但差异无统计学意义(P>0.05);电针组大鼠海马中cGMP含量与模型组比较显著升高(P<0.01)。结论:电针"百会"印堂"穴能够提高抑郁模型大鼠海马区nNOS的表达水平,同时提高海马中cGMP的含量,维持NO-cGMP信号转导通路的信息传递功能,从而起到抗抑郁作用。     

Ammonia metabolism,the brain and fatigue; revisiting the link

This review addresses the ammonia fatigue theory in light of new evidence from exercise and disease studies and aims to provide a view of the role of ammonia during exercise. Hyperammonemia is a condition common to pathological liver disorders and intense or exhausting exercise. In pathology, hyperammonemia is linked to impairment of normal brain function and the onset of the neurological condition, hepatic encephalopathy. Elevated blood ammonia concentrations arise due to a diminished capacity for removal via the liver and lead to increased exposure of organs, such as the brain, to the toxic effects of ammonia. High levels of brain ammonia can lead to deleterious alterations in astrocyte morphology, cerebral energy metabolism and neurotransmission, which may in turn impact on the functioning of important signalling pathways within the neuron. Such changes are believed to contribute to the disturbances in neuropsychological function, in particular the learning, memory, and motor control deficits observed in animal models of liver disease and also patients with cirrhosis. Hyperammonemia in exercise occurs as a result of an increased production by contracting muscle, through adenosine monophosphate (AMP) deamination (the purine nucleotide cycle) and branched chain amino acid (BCAA) deamination prior to oxidation. Plasma concentrations of ammonia during exercise often achieve or exceed those measured in liver disease patients, resulting in increased cerebral uptake. In this article we propose that exercise-induced hyperammonemia may lead to concomitant disturbances in brain function, potentially through similar mechanisms underpinning pathology, which may impact on performance as fatigue or reduced function, especially during extreme exercise.