Targeting NADPH Oxidase and Phospholipases A2 in Alzheimer’s Disease

Alzheimer’s disease (AD) is marked by an increase in the production of extracellular beta amyloid plaques and intracellular neurofibrillary tangles associated with a decline in brain function. Increases in oxidative stress are regarded as an early sign of AD pathophysiology, although the source of reactive oxygen species (ROS) and the mechanism(s) whereby beta amyloid peptides (Aβ) impact oxidative stress have not been adequately investigated. Recent studies provide strong evidence for the involvement of NADPH oxidase and its downstream oxidative signaling pathways in the toxic effects elicited by Aβ. ROS produced by NADPH oxidase activate multiple signaling pathways leading to neuronal excitotoxicity and glial cell-mediated inflammation. This review describes recent studies demonstrating the neurotoxic effects of Aβ in conjunction with ROS produced by NADPH oxidase and the downstream pathways leading to activation of cytosolic phospholipase A₂ (PLA₂) and secretory PLA₂. In addition, this review also describes recent studies using botanical antioxidants to protect against oxidative damage associated with AD. Investigating the metabolic and signaling pathways involving Aβ NADPH oxidase and PLA₂ can help understand the mechanisms underlying the neurodegenerative effects of oxidative stress in AD. This information should provide new therapeutic approaches for prevention of this debilitating disease.     

Control of type II transforming growth factor-beta receptor expression by integrin ligation

Ectopic expression of the alpha5 integrin subunit in cancer cells with little or no endogenous expression of this integrin often results in reduced proliferation as well as reduced malignancy. We now show that inhibition resulting from ectopic expression of alpha5 integrin is due to induction of autocrine negative transforming growth factor-beta (TGF-beta) activity. MCF-7 breast cancer cells do not express either alpha5 integrin or type II TGF-beta receptor and hence are unable to generate TGF-beta signal transduction. Ectopic expression of alpha5integrin expression enhanced cell adhesion to fibronectin, reduced proliferation, and increased the expression of type II TGF-beta receptor mRNA and cell surface protein. Receptor expression was increased to a higher level in alpha5 transfectants by growth on fibronectin-coated plates. Induction of type II TGF-beta receptor expression also resulted in the generation of autocrine negative TGF-beta activity because colony formation was increased after TGF-beta neutralizing antibody treatment. Transient transfection with a TGF-beta promoter response element in tandem with a luciferase cDNA into cells stably transfected with alpha5 integrin resulted in basal promoter activities 5-10-fold higher than those of control cells. Moreover, when alpha5 transfectants were treated with a neutralizing antibody to either TGF-beta or integrin alpha5, this increased basal promoter activity was blocked. Autocrine TGF-beta activity also induced 3-fold higher endogenous fibronectin expression in alpha5 transfectants relative to that of control cells. Re-expression of type II receptor by alpha5 transfection also restored the ability of the cells to respond to exogenous TGF-beta and led to reduced tumor growth in athymic nude mice. Taken together, these results show for the first time that TGF-beta type II receptor expression can be controlled by alpha5beta1 ligation and integrin signal transduction. Moreover, TGF-beta and integrin signal transduction appear to cooperate in their tumor-suppressive functions.     

Ubiquitin-proteasome pathway modulates mouse oocyte meiotic maturation and fertilization via regulation of MAPK cascade and cyclin B1 degradation

Degradation of proteins mediated by ubiquitin-proteasome pathway (UPP) plays important roles in the regulation of eukaryotic cell cycle. In this study, the functional roles and regulatory mechanisms of UPP in mouse oocyte meiotic maturation, fertilization, and early embryonic cleavage were studied by drug-treatment, Western blot, antibody microinjection, and confocal microscopy. The meiotic resumption of both cumulus-enclosed oocytes and denuded oocytes was stimulated by two potent, reversible, and cell-permeable proteasome inhibitors, ALLN and MG-132. The metaphase I spindle assembly was prevented, and the distribution of ubiquitin, cyclin B1, and polo-like kinase 1 (Plk1) was also distorted. When UPP was inhibited, mitogen-activated protein kinase (MAPK)/p90rsk phosphorylation was not affected, but the cyclin B1 degradation that occurs during normal metaphase-anaphase transition was not observed. During oocyte activation, the emission of second polar body (PB2) and the pronuclear formation were inhibited by ALLN or MG-132. In oocytes microinjected with ubiquitin antibodies, PB2 emission and pronuclear formation were also inhibited after in vitro fertilization. The expression of cyclin B1 and the phosphorylation of MAPK/p90rsk could still be detected in ALLN or MG-132-treated oocytes even at 8 h after parthenogenetic activation or insemination, which may account for the inhibition of PB2 emission and pronuclear formation. We also for the first time investigated the subcellular localization of ubiquitin protein at different stages of oocyte and early embryo development. Ubiquitin protein was accumulated in the germinal vesicle (GV), the region between the separating homologous chromosomes, the midbody, the pronuclei, and the region between the separating sister chromatids. In conclusion, our results suggest that the UPP plays important roles in oocyte meiosis resumption, spindle assembly, polar body emission, and pronuclear formation, probably by regulating cyclin B1 degradation and MAPK/p90rsk phosphorylation.     

从野皂荚提胶后的副产物中制备分离蛋白

采用碱提酸沉法从野皂荚提胶后的副产物中制备分离蛋白,研究了制备工艺条件对分离蛋白得率的影响。结果表明:当料液比1∶10~1∶12(m/V)、提取液pH8.0~9.0、浸提温度45~50℃、浸提两次、每次浸提45 m in时,分离蛋白得率可达78.45%。研究结果可为野皂荚提胶副产物中蛋白的综合利用奠定了基础。     

自发性高血压大鼠多组织炎症状态

高血压是一种慢性血管性疾病,易累及肾、肝、心、脑等组织,引起脑卒中和心、肾损害等并发症.本研究对高血压时肾、肝、心、脑等组织的炎症状态进行了观察.实验采用自发性高血压大鼠(spontaneously hypertensive rat,SHR)和正常血压的Wistar-Kyoto(WKY)大鼠,用RT-PCR和Western blot法观察肾、肝、心、脑等组织炎症相关因子IL-1p、TNFα、ICAM-1、iNOS、C/EBPδ和PPARγ的基因表达;紫外分光光度法观察蛋白质羰基化水平和FRAP法检测组织总抗氧化能力.结果显示(1)SHR组织炎症相关因子表达较对照WKY增强,除IL-1βmRNA在肝和脑的增加不明显外,其余均有显著性差异(P＜0.05);(2)SHR和WKY大鼠肾、心、脑蛋白质羰基化水平(nmol/mg蛋白)分别为8.93±1.08和2.27±0.43、2.23±0.23和0.17±0.02、13.42±1.10和5.72±1.01,SHR明显增加(P＜0.05);而肝脏蛋白质羰基化水平无明显变化;(3)SHR肾、肝、心、脑总抗氧化能力水平显著低于WKY大鼠(P＜0.05).以上结果表明,SHR多个组织(肾、肝、心和脑)均存在炎症因子被诱导和氧化应激反应等明显的炎症状态,提示炎症可能在高血压及其并发症的病理改变中起重要作用.     

鲤春病毒糖蛋白(G)基因的分离及同源性比较

通过RT-PCR和巢氏PCR方法从疑似鲤春病毒(SVCV)侵染的镜鲤肝组织中获得了鲤春病毒糖蛋白(G)基因。通过序列测定与分析,所获得的鲤春病毒糖蛋白(G)基因由606个核苷酸组成,编码一个由202个氨基酸组成的糖蛋白。通过NCBIblast与9个来自不同国家或地区的鲤春病毒糖蛋白(G)基因序列比对分析,发现获得的鲤鱼春季病毒糖蛋白G基因DNA序列与美国分离的AY527273株同源性最高为99.8%,与中国分离的AY842485株同源性次高为98.7%,与英国分离的两个序列SVI538065和SVI538066株的同源性为98.2%,而与其它国家的分离株如SVU18101、SVI318079、SVI538061、SVI538062、SVI538063的同源性最差,仅为89.4%~90.0%。氨基酸同源性分析结果与DNA同源性分析结果一致,所获得的鲤春病毒糖蛋白G基因氨基酸推导序列与其它9个分离株的氨基酸同源性在89.6%~99.5%之间。对SVCV不同分离株遗传变异和进化关系的分析为下一步开展SVCV快速诊断方法的研究和疫苗的研制奠定了基础。     

Anti-HCMV and KSHV effect of a trapping ligand antagonist for herpesvirus-encoded GPCR

We have found and synthesized a trapping ligand peptide H22-LP (the conservative sequence is NAHCALL) from a random phage library according to the broad-spectrum trapping receptor H22, which derived from the residue 14-35 near the N-terminal region of receptor US28 on HCMV. In this study, we will evaluate its potential as an efficient antagonist of US28 and the anti-virus activity, acting as a broad spectrum chemokine receptors antagonist. Stable expression of US28 and ORF74 in NIH/3T3 cells were successfully constructed in vitro. Flow cytomety was used to determine the concentration of Ca2+ induced by H22-LP, and the binding of H22-LP and US28 was confirmed by enzyme-linked immunosorbent assay (ELISA). Antivirus activity of H22-LP on HCMV and KSHV was evaluated by anti-virus experiments. Our data suggest that H22-LP is an effectual antagonist of receptor US28 of HCMV and ORF74 of KSHV in the transfection assay, and it has potential to inhibit infection of HCMV and KSHV. These results provide support for the development of anti-virus strategies based on targeted inhibiting the infection of herpesvirus.     

Impaired long-term potentiation and long-term memory deficits in xCT-deficient sut mice

xCT is the functional subunit of the cystine/glutamate antiporter system xc-, which exchanges intracellular glutamate with extracellular cystine. xCT has been reported to play roles in the maintenance of intracellular redox and ambient extracellular glutamate, which may affect neuronal function. To assess a potential role of xCT in the mouse hippocampus, we performed fear conditioning and passive avoidance for long-term memories and examined hippocampal synaptic plasticity in wild-type mice and xCT-null mutants, sut mice. Long-term memory was impaired in sut mice. Normal basal synaptic transmission and short-term presynaptic plasticity at hippocampal Schaffer collateral-CA1 synapses were observed in sut mice. However, LTP (long-term potentiation) was significantly reduced in sut mice compared with their wild-type counterparts. Supplementation of extracellular glutamate did not reverse the reduction in LTP. Taken together, our results suggest that xCT plays a role in the modulation of hippocampal long-term plasticity.     

Phosphorylation of cardiac troponin I by mammalian sterile 20-like kinase 1

Mst1 (mammalian sterile 20-like kinase 1) is a ubiquitously expressed serine/threonine kinase and its activation in the heart causes cardiomyocyte apoptosis and dilated cardiomyopathy. Its myocardial substrates, however, remain unknown. In a yeast two-hybrid screen of a human heart cDNA library with a dominant-negative Mst1 (K59R) mutant used as bait, cTn [cardiac Tn (troponin)] I was identified as an Mst1-interacting protein. The interaction of cTnI with Mst1 was confirmed by co-immunoprecipitation in both co-transfected HEK-293 cells (human embryonic kidney cells) and native cardiomyocytes, in which cTnI interacted with full-length Mst1, but not with its N-terminal kinase fragment. in vitro phosphorylation assays demonstrated that cTnI is a sensitive substrate for Mst1. In contrast, cTnT was phosphorylated by Mst1 only when it was incorporated into the Tn complex. MS analysis indicated that Mst1 phosphorylates cTnI at Thr(31), Thr(51), Thr(129) and Thr(143). Substitution of Thr(31) with an alanine residue reduced Mst1-mediated cTnI phosphorylation by 90%, whereas replacement of Thr(51), Thr(129) or Thr(143) with alanine residues reduced Mst1-catalysed cTnI phosphorylation by approx. 60%, suggesting that Thr(31) is a preferential phosphorylation site for Mst1. Furthermore, treatment of cardiomyocytes with hydrogen peroxide rapidly induced Mst1-dependent phosphorylation of cTnI at Thr(31). Protein epitope analysis and binding assays showed that Mst1-mediated phosphorylation modulates the molecular conformation of cTnI and its binding affinity to TnT and TnC, thus indicating functional significances. The results of the present study suggest that Mst1 is a novel mediator of cTnI phosphorylation in the heart and may contribute to the modulation of myofilament function under a variety of physiological and pathophysiological conditions.