Structural basis for small G protein effector interaction of Ras-related protein 1 (Rap1) and adaptor protein Krev interaction trapped 1 (KRIT1)

Cerebral cavernous malformations (CCMs) affect 0.1–0.5% of the population resulting in leaky vasculature and severe neurological defects. KRIT1 (Krev interaction trapped-1) mutations associate with ∼40% of familial CCMs. KRIT1 is an effector of Ras-related protein 1 (Rap1) GTPase. Rap1 relocalizes KRIT1 from microtubules to cell membranes to impact integrin activation, potentially important for CCM pathology. We report the 1.95 Å co-crystal structure of KRIT1 FERM domain in complex with Rap1. Rap1-KRIT1 interaction encompasses an extended surface, including Rap1 Switch I and II and KRIT1 FERM F1 and F2 lobes. Rap1 binds KRIT1-F1 lobe using a GTPase-ubiquitin-like fold interaction but binds KRIT1-F2 lobe by a novel interaction. Point mutagenesis confirms the interaction. High similarity between KRIT1-F2/F3 and talin is revealed. Additionally, the mechanism for FERM domains acting as GTPase effectors is suggested. Finally, structure-based alignment of each lobe suggests classification of FERM domains as ERM-like and TMFK-like (talin-myosin-FAK-KRIT-like) and that FERM lobes resemble domain “modules.”     

Proteomics of rat biological fluids--the tenth anniversary update

In addition to a remarkable sexual dimorphism of serum and urine proteomes, the rat is exceptional for the wide difference between the serum patterns during an acute phase reaction vs baseline conditions. This feature allows monitoring with high sensitivity onset and progression of any pathological state that involves an inflammatory component as well as assessing the outcome of any therapeutic intervention. Reference maps have been defined for the proteomes of serum, urine, cerebrospinal fluid and bronchoalveolar lavage fluid. For both serum and urine most of the proteomic investigations have dealt with toxicological testing, for BALF with allergic or irritative reactions, whereas with CSF the main aim was the characterization of rat models of neurological disorders. When surveying more than ten years of literature on rat biological fluid proteomics, it is puzzling to see how seldom a consistent analytical plan has been set up for the comparative investigation on two or more types of sample, whether to fully characterize a disease model or to evaluate pharmacological/toxicological effects of a drug. It is also regrettable that in several cases only a negligible part of the results is discussed at length whereas most data are not even made known to the scientific community.     

The role of co-activation in strength and force modulation in the elbow of children with unilateral cerebral palsy

To study the role of coactivation in strength and force modulation in the elbow joint of children and adolescents with cerebral palsy (CP), we investigated the affected and contralateral arm of 21 persons (age 8-18) with spastic unilateral CP in three tasks: maximal voluntary isokinetic concentric contraction and passive isokinetic movement during elbow flexion and extension, and sub-maximal isometric force tracing during elbow flexion. Elbow flexion-extension torque and surface electromyography (EMG) of the biceps brachii (BB) and triceps brachii (TB) muscles were recorded. During the maximal contractions, the affected arm was weaker, had decreased agonist and similar antagonist EMG amplitudes, and thus increased antagonist co-activation (% of maximal activity as agonist) during both elbow flexion and extension, with higher coactivation levels of the TB than the BB. During passive elbow extension, the BB of the affected arm showed increased resistance torque and indication of reflex, and thus spastic, activity. No difference between the two arms was found in the ability to modulate force, despite increased TB coactivation in the affected arm. The results indicate that coactivation plays a minor role in muscle weakness in CP, and does not limit force modulation. Moreover, spasticity seems particularly to increase coactivation in the muscle antagonistic to the spastic one, possibly in order to increase stability.     

孕期饮用酒精对子代大鼠学习记忆及海马cdk5表达的影响

目的:研究孕期饮用酒精对子代大鼠学习记忆及海马细胞周期依赖性蛋白激酶5(cdk5)表达的影响。方法:建立大鼠孕期饮用酒精模型,子代成年后,Y-型迷宫测试学习记忆成绩;聚合酶链式反应(RT-PCR)分析海马组织cdk5mRNA的表达;免疫荧光法检测子鼠海马区cdk5蛋白表达。结果:学习记忆测试结果显示孕期饮用酒精组子鼠学习记忆成绩比正常对照组和饮酒对照组明显下降;RT-PCR结果表明孕期饮用酒精组子鼠海马组织cdk5mRNA表达较正常对照组和饮酒对照组明显上升;免疫荧光结果显示孕期饮用酒精组子鼠海马区cdk5蛋白表达明显增加。结论:孕期饮用酒精对子代大鼠的神经损伤可能与cdk5蛋白表达的上调有关。     

细胞外液酸碱度改变对自发性高血压大鼠脑动脉平滑肌细胞电生理特性的影响

目的:探讨细胞外液酸碱度(pHo)的改变对自发性高血压大鼠(SHR)脑动脉平滑肌细胞电生理特性的影响。方法:取200~250 g自发性高血压大鼠,应用全细胞膜片钳记录技术观察细胞外液酸碱度改变后对SHR脑动脉平滑肌细胞膜电流的作用,进一步揭示其离子机制。结果:①pHo酸化可电压依赖性的抑制SHR脑动脉平滑肌细胞的外向电流。其主要抑制SHR脑动脉平滑肌细胞0~+60 mV区间的电流幅度;②1 mmol/L TEA可以有效抑制pHo酸化对脑动脉平滑肌细胞外向电流的抑制作用。结论:pHo的改变引起SHR脑动脉平滑肌细胞外向电流变化,其可能与电压依赖性的抑制SHR脑动脉平滑肌细胞BKCa通道电流有关。     

黄芪注射液对2型糖尿病动物模型KKAy小鼠脑微血管病变的影响

目的：观察黄芪注射液对2型糖尿病动物模型KKAy小鼠脑微血管病变的影响,探讨黄芪注射液对糖尿病脑血管病变的保护作用。方法：饲养至14周龄的雄性KKAy小鼠随机分成模型组和黄芪注射液治疗组（每日腹腔给药,剂量为3mL／kg）,同龄雄性C57BL／6J小鼠作为对照组。血糖仪测量20、24、28周龄时各组小鼠的空腹血糖水平。28周龄时处死各组小鼠,放射免疫法检测血清6-酮-前列腺素-F1α（6-Keto-PGF1α）和血栓素B2（TXB2）的含量。透射电子显微镜观察脑组织超微结构变化。结果：模型组KKAy小鼠从20周龄开始血糖水平明显高于正常组小鼠（P〈0．01）;黄芪治疗组小鼠从20周龄开始血糖水平明显高于正常组小鼠（P〈0．01）,但低于模型组小鼠（P〈0．05或P〈0．01）。模型组小鼠血清6-Keto—PGF1α水平较正常组降低（P〈0．01）,TXB2含量增高（P〈0．01）;与模型组相比,黄芪注射液治疗组小鼠6-Keto—PGF1α水平升高（P〈O．01）,TXB：含量下降（P〈0．01）。透射电镜显示模型组小鼠神经细胞胞核染色质疏松,线粒体肿胀,粗面内质网缩小,核糖体减少;治疗组小鼠以上病变明显改善。结论：黄芪注射液可以有效改善2型糖尿病动物模型KKAv小鼠脑微血管病变,保护神经细胞结构。     

常用大鼠发热模型研究

目的探讨干酵母、2,4-二硝基酚、脂多糖（LPS）、细菌内毒素引起SD大鼠实验性发热的过程和特点,比较不同浓度外致热原对大鼠发热过程的影响。方法建立大鼠干酵母（2 g/kg、1 g/kg）、2,4-二硝基酚（30mg/kg、15 mg/kg）、LPS（100μg/kg、20μg/kg）、细菌内毒素（120 EU/kg、60 EU/kg）发热模型,记录不同时间点大鼠升温值,绘制各模型平均升温曲线,比较不同大鼠发热模型的发热特点。结果皮下注射干酵母混悬液,注射后2～3 h开始升温,6～7 h达峰值,升温持续20 h;皮下注射2,4-二硝基酚溶液,注射后20 min开始升温,1～1.5 h达峰值,升温持续3～5 h;腹腔注射LPS、细菌内毒素,注射后30 min开始升温,此后升温曲线表现为双相热或三相热,升温持续5～8 h。结论不同外致热原所致SD大鼠发热的过程和特点不同;外致热原浓度不同,所致大鼠发热过程和特点不同。解热试验中,应根据受试药物本身特点选用合适的大鼠发热模型。     

葛根素预防雌激素缺乏性骨质疏松的机制探讨

目的观察葛根素对去卵巢大鼠股骨骨密度、颌骨骨密度和血清中肿瘤坏死因子（TNF-α）、C反应蛋白（CRP）及雌二醇（E2）水平的影响。方法 3月龄雌性SD大鼠随机分为假手术组（sham group）、单纯去势组（OVX group）、去势＋雌二醇治疗组（OVX-estrogen group）和去势＋葛根素治疗组（OVX-puerarin group）。给药3个月后测量各组大鼠股骨和颌骨骨密度,血清中TNF-α和CRP及雌二醇（E2）水平。结果 OVX-puerarin组较sham组体重明显增加（P〈0.01）;较OVX-estrogen组的TNF-α（P〈0.01）、CRP（P〈0.05）及E2（P〈0.01）水平显著下降;其下颌骨和股骨远端骨密度与OVX组、sham组之间无显著性差异,但其股骨近端骨密度较OVX-estrogen组明显增加（P〈0.05）。结论葛根素作为抗炎因子,能够显著降低TNF-α和CRP的水平,但并不会提高雌激素的水平;葛根素对卵巢切除所致的骨质疏松症有一定的治疗作用,但会明显提高机体体重。     

兔脑微栓塞模型脑血流动力学的CT灌注动态变化

目的探讨兔脑微栓塞模型CT灌注成像（CT perfusion imaging,CTPI）脑血流动力学的动态变化规律。方法 30只新西兰兔,随机分成两组,A组：假手术对照组5只,B组：微栓塞组25只。经颈外动脉向颈内动脉注入直径约0.5 mm的SiO2颗粒10枚,分别于栓塞后30 min、3 h、6 h、12 h及24 h行CTPI,24 h处死动物取脑组织行HE染色。根据HE染色结果将模型分为缺血组和梗死组,分别观察其脑血流量（cerebral blood flow,CBF）、脑血容积（cerebral blood volume,CBV）和平均通过时间（mean transit time,MTT）的动态变化规律。结果 A组CTPI及HE染色均未见明显异常。B组3只因实验意外死亡,1只因下肢静脉穿刺失败导致CTPI失败,21只行CTPI,其中18只灌注异常,3只未见明显异常。18只灌注异常的兔中,HE染色10只脑梗死,7只脑缺血,1只未见明显异常。30 min时7只缺血兔脑不同程度低灌注,表现为CBF降低,MTT延长,CBV无显著变化,3～6 h低灌注进一步加重,CBV值略降低,12 h低灌注不同程度恢复,24 h进一步恢复。30 min时10只梗死兔脑明显低灌注,表现为CBF及CBV显著降低,MTT显著延长,3只兔低灌注分别在3 h、6 h及12 h不同程度恢复,然后下一时间又迅速降低并随着时间延长进一步加剧,其余7只兔低灌注程度随时间延长逐渐加剧或在一定水平上波动。结论脑缺血3～6 h低灌注最明显,12～24 h低灌注不同程度恢复,而脑梗死随时间延长低灌注程度不断加重或一过性恢复后再次加重。脑缺血的特征是CBF和CBV的不匹配,缺血组织CBF显著降低,CBV无显著变化,而脑梗死则表现为这两个参数的一致性下降。