见于湖南的一例血红蛋白G-台中家系

见于湖南江华县的一例慢泳血红蛋白α链变种,其先证者为汉族青年,属杂合子。家系调查发现其父、大姐和弟均携有同样的异常Hb。血液学常规检查一般正常。除先证者肝在肋缘下0.5cm扪及外,无任何临床症状。经一级结构分析,确证此例为HbG一台中[α74(EF3)Asp→His]。功能观测其氧平衡特征与HbA无差异。这是见于湖南的首例。     

豚鼠耳蜗Hensen细胞的分离、活性鉴定及其胞内静态游离Ca~(2+)分布

使用酶消化法及机械分离法对 Hensen细胞进行分离 ,置于倒置显微镜下用碘化丙啶及钙敏荧光染料 Fluo-3进行细胞活性鉴定并在激光扫描共聚焦显微镜下观察静息状态 Hensen细胞内的游离 Ca2 +的时空分布。结果证明 ,每个豚鼠耳蜗可以得到单离的 Hensen细胞 5～ 12个。细胞活性良好 ,可保持 5～ 6h。当细胞变性、坏死时可见一系列的形态学变化。对此得出几点判断 Hensen细胞活性的标准 :(1)细胞体呈卵圆形或椭圆形 ,大小可不一致 ;(2 )细胞膜完整 ,细胞边界清楚 ,折光现象明显 ;(3 )细胞浆清澈透明 ,无布朗运动 ,无溢出 ;(4 )细胞浆内的脂滴清晰可辨 ,折光现象明显 ;(5 )细胞无水肿 (即无气球样变 )。在静息状态下 ,Hensen细胞内的 Ca2 + 在细胞内分布不均匀 ,脂滴所在部位没有 Ca2 + 的分布。随时间延长 ,细胞内的 [Ca2 + ] i可小幅度振荡 ,但基本处于一种稳定状态。本工作为进一步对 Hensen细胞的其他特性进行研究打下了基础     

听觉诱发电位的时变滤波叠加

多数诱发电位具有突出的“时变”特性:它们属瞬态反应,在反应前、反应中、反应后各个阶段,电活动的频谱都不断在变化,时变滤波的技术要点:使滤波参数根据电反应的瞬时特性而变,从而更有效地提取有用信号,提高信噪比、缩短操作时间。本文对一般叠加平均处理和时变滤波叠加在动物和人各种听觉诱发电位的信噪比进行了比较。结果显示时变滤波叠加的诱发电位信噪比明显提高,使用时变滤波叠加处理,能达到背景噪声小的效果和反应波形清晰的优点。在大多数情况下,叠加次数可相应地减少,使听觉诱发电位测试效率有所提高。     

过氧化氢对豚鼠耳蜗功能及形态的影响

目的 在体观察过氧化氢(H2O2)对豚鼠耳蜗功能及形态的影响。方法 实验动物分为4组，分别灌流人工外淋巴液(artificial perilymph,APL)，50μMH2O2，100μMH2O2和200μMH2O2(H2O2均溶于APL中)，并用Pl和H033342双染色方法观察H2O2引起的内耳细胞损伤情况。结果 所有H2O2灌流组复合动作电位((CAP)阈移和耳蜗微音电位(CM)幅度变化与人工外淋巴液组比较差异有显著性，且各H2O2组的变化呈现出浓度依赖性；Pl和H033342双染色方法发现外毛细胞是H2O2攻击的主要靶细胞，而Hensen细胞未见任何损伤痕迹。结论 H2O2可导致耳蜗功能下降及耳蜗毛细胞损伤：Hensen细胞较毛细胞可能具有更强的抗氧化能力。     

桥小脑角占位性病变的临床听力学表现

本文总结了经CT或MRI证实以及部分经手术证实的101例桥小脑角占位性病变的纯音测听，听性脑干反应（ABR），耳蜗电图（ECochG）以及前庭功能的表现。结果显示：ABR多表现为Ⅰ～Ⅴ间或延长（＞45ms），仅Ⅰ波存在或ABR各波均消失。未见波Ⅴ幅度小于波Ⅰ。当肿物较大时，可见时测ABR异常。极重度聋患者（35．5％），仍可引出异常ABR波形，故仍不可忽视ABR检查；听力轻度下降，甚至正常考ABR仍有改变。AP出现率随肿物增大而降低，－SP／AP比值≥0．4，可能是继发性伤及耳蜗所致。5例ABR表现正常者仍有半规管功能低下。提示前庭功能检查对桥小脑均占位性病变的诊断具有一定参考意义，临床应将ECochG和前庭功能检测列为诊断桥小脑角占位性病变的参考指标。     

白噪声暴露对豚鼠耳蜗毛细胞感受器电位非线性特性的影响

为了探讨正常及在噪声暴露过程中耳蜗毛细胞感受器电位非线性特性的变化规律，采用玻璃微电极在体毛细胞胞内记录的实验方法，记录了７只豚鼠（７耳）正常状态和白噪声暴露后耳蜗外毛细胞（ＯＨＣ）胞内交流感受器电位输入－输出曲线（Ｉ－Ｏ曲线）。正常豚鼠耳蜗ＯＨＣ交流感受器电位幅度在刺激声强度较低时呈线性增长，声强达到５０～７０ｄＢＳＰＬ时幅度增长变慢，在８０～１００ｄＢＳＰＬ时，幅度不再随刺激强度的增加而继续增加，出现饱和现象。测试耳在用１００ｄＢＳＰＬ时白噪声暴露１０～２０分钟后，感受器电位幅度普遍下降，Ｉ－Ｏ曲线的线性段延长，非线性段缩短，但高强度感受器电位幅度增大，出现“幅度重振现象”，与临床上观察的响度重振现象具有相似的特点。从而推测ＯＨＣ感受器电位非线性特性减弱，发生幅度重振，可能是临床上主观响度重振现象的客观来源之一。     

云南省鼠疫疫源地鼠疫菌质粒DNA种类及分子流行病学研究

用琼脂糖凝胶电泳技术，检查了分离自云南省的８２８株鼠疫菌质粒ＤＮＡ特征。结果表明，云南鼠疫菌可观察到分子量为３．９３、６．０５、２２．９７、３５．６５、４５．３５、６４．８２、７４．５９、１１１．３６和１２９．５５Ｍｄａｌ九种质粒，按质粒组成可划分为Ⅰ～Ⅹ种质粒图谱。被检菌株中，９９．１５％具有６．０５、４５．３５和６４．８２Ｍｄａｌ三种规范化质粒，还发现２１．６２％、４．５９％、１．４５％和３．５％的菌株分别尚存３．９３、２２．９７、３５．６５和１１１．３６Ｍｄａｌ质粒，这些质粒有特定的分布区域，具有重要的分子流行病学意义。根据质粒图谱，可将我省鼠疫现有疫区初步划分成滇西北山地、保山盆地、大盈江、陇川江、南定河、澜沧江下游和红河元江段流域七大相对独立的疫源区。本研究的结果结合既往流行病学资料，似可说明：①质粒组成可作为云南省鼠疫疫源地中疫源地划分的重要指标；②云南家、野鼠两型疫源地菌株具有共同的遗传变异性；③近几年之间鼠疫有复燃也存在异地传播；④云南西南部存在鼠疫疫源。     

Altered gray matter structural covariance networks at both acute and chronic stages of mild traumatic brain injury

Cognitive and emotional impairments observed in mild traumatic brain injury (mTBI) patients may reflect variances of brain connectivity within specific networks. Although previous studies found altered functional connectivity (FC) in mTBI patients, the alterations of brain structural properties remain unclear. In the present study, we analyzed structural covariance (SC) for the acute stages of mTBI (amTBI) patients, the chronic stages of mTBI (cmTBI) patients, and healthy controls. We first extracted the mean gray matter volume (GMV) of seed regions that are located in the default-mode network (DMN), executive control network (ECN), salience network (SN), sensorimotor network (SMN), and the visual network (VN). Then we determined and compared the SC for each seed region among the amTBI, the cmTBI and the healthy controls. Compared with healthy controls, the amTBI patients showed lower SC for the ECN, and the cmTBI patients showed higher SC for the both DMN and SN but lower SC for the SMN. The results revealed disrupted ECN in the amTBI patients and disrupted DMN, SN and SMN in the cmTBI patients. These alterations suggest that early disruptions in SC between bilateral insula and the bilateral prefrontal cortices may appear in amTBI and persist into cmTBI, which might be potentially related to the cognitive and emotional impairments.

     

血必净注射液对脓毒症大鼠Claudin4蛋白和辅助性T淋巴细胞1型表达的影响

目的探讨血必净注射液对脓毒症大鼠肺组织Claudin4蛋白表达的影响。方法健康成年Sprague-Dawley大鼠100只,体重(250±25)g,随机分入4组,每组25只:假手术组大鼠只开腹、关腹和复苏,不行盲肠结扎穿孔术(CLP);脓毒症组大鼠行CLP;血必净3h组大鼠于CLP后3h腹腔内注射血必净注射液2mL/kg;血必净12h组大鼠于CLP后12h腹腔内注射血必净注射液2mL/kg。每组随机留取8只大鼠进行生存率分析。假手术组、脓毒症组、血必净3h组大鼠在术后6、15和24h,血必净12h组大鼠在术后15和24h各随机处死5只,分别留取血液和肺组织标本,测定肺组织湿/干质量比(肺水肿指数),对肺组织进行病理学观察并进行肺损伤病理学评分,测定血清促炎细胞因子IL-6和TNF-α水平,采用免疫组织化学方法测定肺组织骨架蛋白Claudin4蛋白表达情况,采用流式细胞术检测辅助性T淋巴细胞1型(Th1)在外周血单核细胞中的表达情况。结果假手术组、脓毒症组、血必净3h组和血必净12h组大鼠的术后72h存活率分别为8/8、0、1/8和0,血必净3h组显著高于脓毒症组(P<0.01),血必净12h组与脓毒症组的差异无统计学意义(P>0.05)。假手术组大鼠术后各项检测指标均基本不变。脓毒症组、血必净3h组和血必净12h组大鼠术后15和24h的肺水肿指数,以及术后6、15和24h的肺损伤病理学评分、血清IL-6和TNF-α水平、肺组织Claudin4蛋白表达阳性率均显著高于假手术组同时间点(P值均<0.01);脓毒症组大鼠术后6、15和24h,以及血必净12h组大鼠术后15和24h的肺水肿指数、肺损伤病理学评分、血清IL-6和TNF-α水平、肺组织Claudin4蛋白表达阳性率均显著高于血必净3h组同时间点(P值分别<0.05、0.01);脓毒症组与血必净12h组术后同时间点间上述指标的差异均无统计学意义(P值均>0.05)。脓毒症组和血必净3h组大鼠术后各时间点的外周血单核细胞中Th1的表达率均显著高于假手术组同时间点(P值均<0.01);脓毒症组大鼠术后6和15h的外周血单核细胞中Th1的表达率均显著高于血必净3h组同时间点(P值均<0.01),术后24h的外周血单核细胞中Th1的表达率显著低于血必净3h组同时间点(P<0.05)。结论早期使用血必净治疗可以有效地抑制脓毒症大鼠肺组织的炎性反应,减轻肺损伤,提高大鼠存活率,其可能机制与抑制肺组织Claudin4蛋白表达,降低肺泡通透性有关。     

The structure of a minimum amyloid fibril core formed by necroptosis-mediating RHIM of human RIPK3

Receptor-interacting protein kinases 3 (RIPK3), a central node in necroptosis, polymerizes in response to the upstream signals and then activates its downstream mediator to induce cell death. The active polymeric form of RIPK3 has been indicated as the form of amyloid fibrils assembled via its RIP homotypic interaction motif (RHIM). In this study, we combine cryogenic electron microscopy and solid-state NMR to determine the amyloid fibril structure of RIPK3 RHIM-containing C-terminal domain (CTD). The structure reveals a single protofilament composed of the RHIM domain. RHIM forms three β-strands (referred to as strands 1 through 3) folding into an S shape, a distinct fold from that in complex with RIPK1. The consensus tetrapeptide VQVG of RHIM forms strand 2, which zips up strands 1 and 3 via heterozipper-like interfaces. Notably, the RIPK3-CTD fibril, as a physiological fibril, exhibits distinctive assembly compared with pathological fibrils. It has an exceptionally small fibril core and twists in both handedness with the smallest pitch known so far. These traits may contribute to a favorable spatial arrangement of RIPK3 kinase domain for efficient phosphorylation.

Necroptosis is an important form of regulated necrotic cell death, dysregulation of which is closely associated with a variety of human diseases, including neurodegenerative diseases (1, 2), inflammatory disorders (3–5), and cancers (6, 7). RIPK3 (receptor-interacting protein kinase 3) serves as the central node to converge multiple upstream signals to induce necroptosis (8–11). RIPK3 is activated via interactions with proteins that contain the RIP homotypic interaction motif (RHIM) such as RIPK1 (receptor-interacting protein kinase 1), TRIF (TIR-domain-containing adapter-inducing interferon-β), and ZBP1/DAI (Z-DNA-binding protein 1/DNA-dependent activator of IFN-regulatory factors). RIPK1 mediates RIPK3 activation downstream of death receptors, such as TNFR1 (12). TRIF links RIPK3 to the TLR3 and TLR4 signaling pathway (8). ZBP1/DAI mediates RIPK3 activation in response to certain viruses, such as influenza A virus (9, 10). RIPK3 is composed of a well-defined N-terminal kinase domain and a RHIM-containing C-terminal domain (CTD) (13). Previous studies show that RHIM plays an important role in the interactions of RIPK3 with its upstream mediators and amyloid fibrillation of RIPK3 (9, 10, 14, 15). A previous solid-state NMR (ssNMR) study has revealed the structure of a heterofibril core formed by the CTDs of RIPK3 and RIPK1, where the RHIM domains of both proteins adopt a serpentine fold and stack alternatively along the fibril axis (15). The structure provides insights into how RIPK1 recruits and activates RIPK3 for signaling transduction. However, it remains unknown how RIPK3 assemblies into fibril in the absence of RIPK1.In this work, by using cryo-EM and ssNMR, we determined the structures of two amyloid fibrils formed by RIPK3-CTD. Despite the different fibril preparation, the RIPK3-CTD fibrils present a nearly identical structure. The fibril core exhibits an exceptionally small S-shaped fold of RHIM, which is distinct from that in the heterofibril of RIPK1 and RIPK3 CTDs. The consensus tetrapeptide VQVG forms the central strand 2 of the S-shaped structure and forms heterosteric zipper interfaces with the adjacent strands 1 and 2 within the same subunit. Intriguingly, the RIPK3-CTD fibril presents in both left and right handedness and features a minimum fibril core among the 50 different cryo-EM fibril structures reported previously and also represents the smallest fibril pitch and largest twist angle. By analyzing the reported cryo-EM fibril structures, we observed a strong positive correlation between the size of fibril core and the fibril pitch. Furthermore, we discussed how the small RIPK3 fibril core leads to a highly twisted fibril, which may display the N-terminal kinase domains in a favorable geometry to increase the efficiency of RIPK3 phosphorylation.