Distinct properties of wild-type and the amyloidogenic human cystatin C variant of hereditary cerebral hemorrhage with amyloidosis, Icelandic type

Variant human cystatin C (L68Q) is an amyloidogenic protein. It deposits in the cerebral vasculature of Icelandic patients with cerebral amyloid angiopathy, leading to stroke. Wild-type and variant cystatin C are cysteine proteinase inhibitors which form concentration dependent inactive dimers; however, variant cystatin C dimerizes at lower concentrations and has an increased susceptibility to a serine protease. We studied the effect of the L68Q amino acid substitution on cystatin C properties, utilizing full length cystatin C purified in mild conditions from media of cells stably transfected with either the wild-type or variant cystatin C genes. The variant cystatin C forms fibrils in vitro detectable by electron microscopy in conditions in which the wild-type protein forms amorphous aggregates. We also show by circular dichroism, steady-state fluorescence and Fourier-transformed infrared spectroscopy that the amino acid substitution modifies cystatin C structure by destabilizing alpha-helical structures and exposing the tryptophan residue to a more polar environment, yielding a more unfolded molecule. These spectral changes demonstrate that variant cystatin C has a three-dimensional structure different from that of the wild-type protein. The structural differences between variant and wild-type cystatin C account for the susceptibility of the variant protein to unfolding, proteolysis and fibrillogenesis.     

A possible balance of magnesium accumulations among bone, cartilage, artery, and vein in single human individuals

It is known that a large quantity of magnesium contains bones, and the magnesium contents in spongy bones decrease gradually with advancing age. To elucidate the relationships between a decrease of mineral contents in human bones and an accumulation of minerals in the other human tissues, the content of magnesium was analyzed by inductively coupled plasma-atomic emission spectrometry among human bones, arteries, veins, and cartilages in 27 subjects (17 men and 10 women). These were resected from the subjects who died in the age range 40–98 yr. Calcanei were chosen for analysis of magnesium contents in contrast with femoral, popliteal, and common carotid arteries, internal jugular and femoral veins, superior and inferior venae cavae, and pubic symphyses. The magnesium contents in the calcanei decreased gradually with aging, whereas they increased progressively in the arteries, veins, and pubic symphyses with aging. It was found that as the magnesium contents decreased in the calcanei, they increased in the arteries, such as the femoral, popliteal, and common carotid arteries, whereas they decreased inversely in the veins, such as the internal jugular and femoral veins and superior and inferior venae cavae. Furthermore, as the magnesium contents decreased in the calcanei, they hardly changed in the pubic symphyses. These suggest that magnesium released from bones is accompanied by accumulation of magnesium in the arteries.     

内源性一氧化氮在内毒素引起的肺动脉高压和肺损伤中的作用

本实验观察了家兔静脉内注入内毒素的主要成分脂多糖(LPS)后平均动脉血压(MAP)、肺动脉压(PAP)及入、出肺血NO含量的变化,并观察了静脉内预注入NO生成抑制剂Nω-硝基-L-精氨酸(L-NNA)及诱生型NO生成抑制剂氨基胍(AG)后PAP和肺损伤的变化.结果观察到:家兔LPS注入后,MAP均明显下降,LPS注入后0.5、1、1.5、2h PAP明显增高(P<0.05).LPS注入后PAP的高峰期(1h)入肺血NO含量明显降低,出肺血NO无明显变化.与对照组相比,LPS注入后3h出肺血NO含量和5h入、出肺血NO含量均明显增多.相关分析表明,兔LPS注入前和LPS注入后1h PAP与入肺血NO含量呈明显的负相关,而LPS注入后 3h和5h两者相关不明显.静脉预注入L-NNA后,LPS处理组的动物PAP明显增高,入、出肺血丙二醛(MDA)含量也明显增高,动物生存率明显降低.肺组织光镜下可见肺萎陷和小血管淤血加重,白细胞明显增加.静脉预注入AG后,LPS处理组的动物MAP在3～5h明显增高,此时PAP无明显改变,但5h时血中MDA含量明显减低,5h时与LPS组相比肺萎陷和小血管淤血减轻,白细胞也明显减少.以上结果提示,内毒素入血后较早期阶段可出现PAP的升高,此时入肺血NO的减少是参与肺动脉压增高(PAH)的机制之一.家兔内毒素进入血后较早期阶段NO对减轻内毒素引起的PAH和肺损伤起重要作用,而较晚的时期当诱生型NO合酶(iNOS)诱生后释放的NO则参与内毒素引起的肺组织炎症反应和肺损伤.     

Nutritional Prevention on Hypertension, Cerebral Hemodynamics and Thrombosis in Stroke-Prone Spontaneously Hypertensive Rats

1. Stroke-prone spontaneously hypertensive rats (SHRSP/Izm), which become severely hypertensive and exhibit a very high incidence of stroke (cerebral hemorrhage and/or infarction), are used widely for the study of the hypertension and stroke. In the previous study, we indicated that high thrombotic tendency of cerebral microvessels in SHRSP/Izm compared with stroke-resistant SHR (SHR/Izm) and normotensive Wistar Kyoto rats (WKY/Izm) at aged period. 2. L-arginine, a substrate of nitric oxide (NO), and voluntary exercise reduced blood pressure and thrombotic tendency in cerebral microvessels caused by highly production of NO in vivo. Furthermore, antioxidants show that the effects of antihypertensive and antithrombosis in SHRSP/Izm. 3. Although SHRSP/Izm become genetically hypertensive and exhibit stroke, a number of nutritional factors, particularly antioxydative nutrient, have preventive effects on hypertension, cerebral blood flow dysfunction, thrombus formation, and neuronal cell death in SHRSP/Izm. Our results indicate that those treatments are beneficial in the prevention of hypertension and stroke and that the nutritional science is very important for "prediction and prevention medicine."     

Extracting Information from the Power Spectrum of Synaptic Noise

In cortical neurons, synaptic "noise" is caused by the nearly random release of thousands of synapses. Few methods are presently available to analyze synaptic noise and deduce properties of the underlying synaptic inputs. We focus here on the power spectral density (PSD) of several models of synaptic noise. We examine different classes of analytically solvable kinetic models for synaptic currents, such as the "delta kinetic models," which use Dirac delta functions to represent the activation of the ion channel. We first show that, for this class of kinetic models, one can obtain an analytic expression for the PSD of the total synaptic conductance and derive equivalent stochastic models with only a few variables. This yields a method for constraining models of synaptic currents by analyzing voltage-clamp recordings of synaptic noise. Second, we show that a similar approach can be followed for the PSD of the the membrane potential (Vm) through an effective-leak approximation. Third, we show that this approach is also valid for inputs distributed in dendrites. In this case, the frequency scaling of the Vm PSD is preserved, suggesting that this approach may be applied to intracellular recordings of real neurons. In conclusion, using simple mathematical tools, we show that Vm recordings can be used to constrain kinetic models of synaptic currents, as well as to estimate equivalent stochastic models. This approach, therefore, provides a direct link between intracellular recordings in vivo and the design of models consistent with the dynamics and spectral structure of synaptic noise.     

Reduction of Glutamate Uptake into Cerebral Cortex of Developing Rats by the Branched-Chain Alpha-Keto Acids Accumulating in Maple Syrup Urine Disease

In the current study we investigated the effect of the branched-chain alpha-keto acids (BCKA) co-ketoisocaproic (KIC), alpha-keto-beta-methylvaleric (KMV), and alpha-ketoisovaleric (KIV) acids, which accumulate in maple syrup urine disease (MSUD), on the in vitro uptake of [3H]glutamate by cerebral cortical slices from rats aged 9, 21, and 60 days of life. We initially observed that glutamate uptake into cerebral cortex of 9- and 21-day-old rats was significantly higher, as compared to that of 60-day-old rats. Furthermore, KIC inhibited this uptake by tissue slices at all ages studied, whereas KMV and KIV produced the same effect only in cortical slices of 21- and 60-day-old rats. Kinetic assays showed that KIC significantly inhibited glutamate uptake in the presence of high glutamate concentrations (50 microM and greater). We also verified that the reduction of glutamate uptake was not due to cellular death, as evidenced by tetrazolium salt and lactate dehydrogenase viability tests of cortical slices in the presence of the BCKA. It is therefore presumed that the reduced glutamate uptake caused by the BCKA accumulating in MSUD may lead to higher extracellular glutamate levels and potentially to excitotoxicity, which may contribute to the neurological dysfunction of the affected individuals.     

大鼠局灶性脑缺血再灌注损伤后纹状体和海马区瞬时受体电位通道蛋白4的表达增加

实验在大鼠大脑中动脉阻塞性脑缺血(middle cerebral arterv occlusion,MCAO)模型上采用Western Blot方法检测脑缺血再灌注不同时程(6h、12h、1d、3d)脑组织中瞬时受体电位通道蛋白4(transient receptor potential channel4,TRPC4)的表达情况,并与正常对照组相比,结果显示,12 h、1 d、3 d组纹状体、海马区域TRPC4含量明显高于正常组(P<0.05)。采用免疫组织化学定位检测,显示TRPC4主要表达在神经元细胞膜上;免疫组化阳性细胞统计分析显示,在不同时程缺血组中纹状体、海马区域TRPC4的表达与正常组相比有所增加,其中纹状体、海马区缺血再灌注1 d、3 d组缺血同侧1RPC4阳性细胞升高显著(P<0.05)。脑缺血再灌注损伤后TRPC4相对含量增加,提示TRPC4可能参与脑缺血引起的急性和迟发性神经元损伤。     

Mitochondria and Neuronal Death/Survival Signaling Pathways in Cerebral Ischemia

Apoptotic cell death pathways have been implicated in acute brain injuries, including cerebral ischemia, brain trauma, and spinal cord injury, and in chronic neurodegenerative diseases. Experimental ischemia and reperfusion models, such as transient focal/global ischemia in rodents, have been thoroughly studied and suggest the involvement of mitochondria and the cell survival/death signaling pathways in cell death/survival cascades. Recent studies have implicated mitochondria-dependent apoptosis involving pro- and anti-apoptotic protein binding, the release of cytochrome c and second mitochondria-derived activator of caspase, the activation of downstream caspases-9 and –3, and DNA fragmentation. Reactive oxygen species are known to be significantly generated in the mitochondrial electron transport chain in the dysfunctional mitochondria during reperfusion after ischemia, and are also implicated in the survival signaling pathway that involves phosphatidylinositol-3-kinase (PI3-K), Akt, and downstream signaling molecules, like Bad, 14-3-3, and the proline-rich Akt substrate (PRAS), and their bindings. Further studies of these survival pathways may provide novel therapeutic strategies for clinical stroke.Special issue dedicated to Lawrence F. Eng.     

慢性间歇性低氧降低急性缺氧对大鼠肺动脉平滑肌细胞膜上电压门控性钾通道的抑制

为了从离子通道水平上探讨机体低氧适应的离子机制,本实验将雄性 SD 大鼠随机分为常氧对照组和慢性间歇性低氧组[氧浓度(10 ± 0.5) %, 间断缺氧每天 8 h]。用酶解法急性分离单个大鼠肺内动脉平滑肌细胞(pulmonary artery smoothmuscle cells, PASMCs),以全细胞膜片钳技术记录 PASMCs 膜上的电压门控性钾通道 (voltage-gated potassium channel, KV) 电流,观察急性缺氧对慢性间歇性低氧大鼠 PASMCs 的 KV 的影响, 为机体适应低氧能力提供实验依据。结果显示:⑴常氧对照组在电流钳下,急性缺氧可使膜电位明显去极化(由-47.2 ±2.6 mV 去极到 -26.7 ±1.2 mV ); 在电压钳下, 急性缺氧可显著抑制 KV电流( 60 mV 时, KV电流密度从 153.4 ± 9.5 pA/pF降到 70.1 ± 10.6 pA/pF), 峰电流的抑制率为(57.6 ± 3.3) %, 电流-电压关系曲线向右下移。⑵慢性间歇性低氧组KV电流密度随低氧时间延长而逐渐减少(慢性低氧10 d后就有显著性意义),电流- 电压关系曲线逐渐右下移。⑶急性缺氧对慢性间歇性低氧大鼠PASMCs KV电流的抑制作用随慢性间歇性低氧时间延长而逐渐减弱。上述观察结果提示慢性间歇性低氧减弱急性缺氧对 KV 的抑制, 这可能是机体低氧适应的一种重要机制。     

High accumulation of calcium in human uterine artery with aging

To elucidate compositional changes of the rami of the internal iliac artery with aging, the authors investigated age-related changes of the calcium content in the uterine, internal pudendal, umbilical, and obturator arteries by inductively coupled plasma-atomic emission spectrometry. After an ordinary dissection was finished, the uterine, internal pudendal, umbilical, and obturator arteries were resected from 10 female subjects, and the internal pudendal, umbilical, and obturator arteries were resected from 10 male subjects. The female subjects ranged in age from 52 to 96 yr, and the male subjects ranged in age from 63 to 88 yr. The calcium content in the uterine artery began to increase in the seventies and increased markedly in the nineties. In the internal pudendal artery, the calcium content hardly increased up to the eighties and increased significantly in the nineties. In contrast, the calcium content did not change in both the umbilical and obturator arteries with advancing age. It was found that the average content of calcium was the highest in the uterine artery and decreased in the order internal pudendal, umbilical, and obturator arteries. The average content of calcium in the uterine arteries corresponded to 46-fold the amount of the women's obturartor arteries, in which it was the lowest. In the cases of men, the average content of calcium was higher in the order of the internal pudendal, umbilical, and obturator arteries. Regarding the average content of calcium, the order internal pudendal, umbilical, and obturator arteries of the men was consistent with that of the women.