Dehydroevodiamine.HCl prevents impairment of learning and memory and neuronal loss in rat models of cognitive disturbance

We previously reported that dehydroevodiamine.HCl (DHED) has anticholinesterase and antiamnesic activities. To verify the effects of DHED on cognitive deficits further, we tested it on the scopolamine-induced amnesia model of the rat using the passive avoidance and eight-arm radial maze tests. A single (20 mg/kg p.o.) and repeated (10 mg/kg p.o.) administrations of DHED could significantly reverse the latency time shortened by scopolamine (1 mg/kg i.p.) to control level. The impaired spatial working memory induced by scopolamine (1 mg/kg i.p.) was also improved significantly by a single injection (6.25 mg/kg i.p.) and repeated administrations of DHED (10 mg/kg p.o.) in the eight-arm radial maze test. In addition, we examined the effects of DHED on the memory impairment and the histological changes of the brain after unilateral electrolytic lesion of the entorhinal cortex (EC) and middle cerebral artery occlusion in rats. The cognitive deficits caused by EC lesion and middle cerebral artery occlusion were improved significantly by repeated administrations of DHED (6.25 mg/kg i.p.) after EC lesion or ischemic insult once a day for 7 days in the passive avoidance test. Histological analysis showed that the neuronal loss in the DHED-treated group was notably reduced in the hippocampal area (CA1) of ischemic rats and in the dentate gyrus and hippocampal area (CA1 and CA3) of EC-lesioned rats compared with the nontreated group. The infarction area was decreased significantly by a single administration of DHED (6.25 mg/kg i.p.) 30 min before ischemic insult for 6 h. These results suggest that DHED might be an effective drug for not only the Alzheimer's disease type, but also the vascular type of dementia.     

Oxidative stress causes mucin synthesis via transactivation of epidermal growth factor receptor: role of neutrophils

Oxidative stress has been implicated in the pathogenesis of inflammatory diseases of airways. Here we show that oxidative stress causes ligand-independent activation of epidermal growth factor receptors (EGFR) and subsequent activation of mitogen-activated protein kinase kinase (MEK)-p44/42 mitogen-activated protein kinase (p44/42mapk), resulting in mucin synthesis in NCI-H292 cells. Exogenous hydrogen peroxide and neutrophils activated by IL-8, FMLP, or TNF-alpha increased EGFR tyrosine phosphorylation and subsequent activation of p44/42mapk and up-regulated the expression of MUC5AC at both mRNA and protein levels in NCI-H292 cells. These effects were blocked by selective EGFR tyrosine kinase inhibitors (AG1478, BIBX1522) and by a selective MEK inhibitor (PD98059), whereas a selective platelet-derived growth factor receptor tyrosine kinase inhibitor (AG1295), a selective p38 MAPK inhibitor (SB203580), and a negative compound of tyrosine kinase inhibitors (A1) were without effect. Neutrophil supernatant-induced EGFR tyrosine phosphorylation, activation of p44/42mapk, and MUC5AC synthesis were inhibited by antioxidants (N-acetyl-cysteine, DMSO, dimethyl thiourea, or superoxide dismutase); neutralizing Abs to EGFR ligands (EGF and TGF-alpha) were without effect, and no TGF-alpha protein was found in the neutrophil supernatant. In contrast, the EGFR ligand, TGF-alpha, increased EGFR tyrosine phosphorylation, activation of p44/42mapk, and subsequent MUC5AC synthesis, but these effects were not inhibited by antioxidants. These results implicate oxidative stress in stimulating mucin synthesis in airways and provide new therapeutic approaches in airway hypersecretory diseases.     

A role for cell cycle proteins in the serum-starvation resistance of Epstein-Barr virus immortalized B lymphocytes.

Epstein-Barr virus (EBV) is a B-lymphotropic human herpes virus that infects B lymphocytes and is associated with a broad spectrum of benign and malignant diseases. B cell infection by EBV causes indefinite cell proliferation that results in the development of immortalized lymphoblastoid cell lines (LCLs). We found that SNU-1103, a latency type III EBV-transformed LCL developed from a Korean cancer patient, resisted the G1 arrest that was normally caused by serum starvation. Western blot analyses revealed several alterations in the expression of key regulatory cell cycle proteins involved in the G1 phase. High expression of cyclin D2 and time-dependent increases in cyclin-dependent kinase 6 (CDK6) and cyclin D3 were observed in SNU-1103 during serum starvation. Very unexpectedly, in SNU-1103, the key G1 phase CDK inhibitor p21CiP1 was expressed at a consistently high level, while p27KiP1 expression was increased. Of three pRb family proteins, pRb expression was reduced and it became hypophosphorylated in SNU-1103 during serum starvation. Instead, p107 and p130 were expressed at consistently high levels in SNU-1103 during serum starvation. In conclusion, compared with an EBV-negative BJAB cell line, multiple cell cycle regulatory proteins were abnormally or inversely expressed in SNU-1103 during serum starvation.     

表皮生长因子受体调控的人鼻咽癌细胞系CNE2分泌蛋白质的筛选

为筛选表皮生长因子受体(epidermalgrowthfactorreceptor,EGFR)调控的鼻咽癌(nasopharyngealcarcinoma,NPC)细胞的分泌蛋白质,揭示EGFR在NPC发病中的作用机制,采用无血清培养法培养NPC细胞系CNE2,并用转化生长因子(transforminggrowthfactor-α,TGF-α)刺激CNE2细胞24h作为实验组,对照组CNE2细胞不用TGF-α刺激.超滤法脱盐并浓缩两组细胞的培养上清制备分泌蛋白,采用双向凝胶电泳技术(two-dimensionalelectrophoresis,2-DE)分离两组细胞的分泌蛋白,PDquest图像分析软件识别差异表达的蛋白质点,基质辅助激光解吸电离飞行时间质谱(matrix-assistedlaserdesorption/ionizationtimeofflightmassspectrometry,MALDI-TOF-MS)鉴定差异表达蛋白.建立了实验组和对照组CNE2细胞分泌蛋白的2-DE图谱,图像分析识别了22个差异蛋白质点,质谱鉴定了8个非冗余蛋白质,其功能涉及肿瘤细胞侵袭转移、细胞凋亡和增殖,为进一步揭示EGFR在NPC发病中的作用及其机制奠定了基础.     

载脂蛋白A-Ⅰ通过PKA信号途径影响ABCA1的表达与功能

以THP-1巨噬细胞源性泡沫细胞为研究对象,观察载脂蛋白A-Ⅰ与三磷酸腺苷结合盒转运体A1(ATP binding cassette transporter A1,ABCA1)的相互作用,并探讨它们相互作用的机制,以便了解载脂蛋白A-Ⅰ和ABCA1在动脉粥样硬化发生发展中的作用.THP-1巨噬细胞源性泡沫细胞经各种因素处理后,采用油红“O”染色,观察细胞内的脂滴,运用液体闪烁计数器检测细胞内胆固醇流出,高效液相色谱分析细胞内总胆固醇、游离胆固醇和胆固醇酯含量,用逆转录-聚合酶链反应和蛋白质印迹分析法分别检测ABCA1 mRNA与ABCA1蛋白质的水平.实验结果显示,载脂蛋白A-Ⅰ和腺苷酸环化酶激动剂Forskolin(FRK)引起THP-1巨噬细胞源性泡沫细胞总胆固醇、游离胆固醇与胆固醇酯减少,而腺苷酸环化酶抑制剂SQ-22536引起THP-1巨噬细胞源性泡沫细胞总胆固醇、游离胆固醇与胆固醇酯增加.载脂蛋白A-Ⅰ引起THP-1巨噬细胞源性泡沫细胞ABCA1蛋白质水平和细胞内胆固醇流出增加.FRK引起THP-1巨噬细胞源性泡沫细胞ABCA1蛋白质水平和细胞内胆固醇流出呈时间和浓度依赖性增加.SQ-22536引起THP-1巨噬细胞源性泡沫细胞ABCA1蛋白质水平和细胞内胆固醇流出减少.结果提示,载脂蛋白A-Ⅰ可提高THP-1巨噬细胞源性泡沫细胞ABCA1蛋白质水平,增加细胞内胆固醇流出,降低细胞内胆固醇聚积.其机制可能是通过PKA信号途经使细胞ABCA1蛋白质水平增加.     

应用蛋白质组学和RNAi技术筛选鼻咽癌细胞中p53功能相关蛋白质

为了阐明鼻咽癌中高表达的p53蛋白聚集与失活的机制,高通量地检测与p53功能相关的蛋白质,首先采用RNA干扰(RNAi)技术稳定沉默鼻咽癌细胞系CNE2的p53基因表达,然后用蛋白质组技术研究稳定沉默该基因对鼻咽癌蛋白质表达谱的影响.通过对稳定干扰p53基因后鼻咽癌细胞系CNE2的蛋白质表达谱改变的研究,用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)分析和电喷雾串联质谱(ESI-Q-TOF-MS)验证鉴定了22个差异表达蛋白质.在这些差异表达蛋白质中,有些是已经报道的p53功能相关蛋白质,如热休克蛋白27(HSP27)、异质性胞核核糖核蛋白K(hnRNPK)、14-3-3σ等,其他可能是新的p53功能相关蛋白质,如eIF4B、TPT1、hnRNPH3、SFRS1等.部分差异表达蛋白质如HSP27、14-3-3σ和GRP75经蛋白质印迹分析技术进行了验证,同时pcDNA3.1-FLAG-p53质粒转染CNE2细胞引起了HSP27、14-3-3σ表达下调,GRP75表达上调.在鼻咽癌细胞中鉴定的22个差异表达蛋白质大致可以分为5类,包括信号传导相关蛋白质、分子伴侣、与转录和翻译相关蛋白质、代谢相关蛋白和细胞结构相关蛋白质,涉及到细胞周期的调控、分子基因表达调控、细胞黏附、细胞代谢等众多事件,它们可能作为p53功能相关蛋白质,为阐明鼻咽癌中p53蛋白聚集及失活的机制提供了重要依据和线索.     

Study on the ophthalmic diseases in ICR mice and BALB/c mice.

In pharmaceutical companies and research institutes, many toxicity tests are performed with laboratory animals. This study was performed to produce reference data for eye toxicity tests and to investigate the ophthalmic diseases of 408 ICR mice and 119 BALB/c mice, which are commonly used as subjects in toxicity tests. The experimental animals without clinical disorders were selected regardless of sex. The ophthalmic diseases were examined by using special ophthalmic instruments: direct ophthalmoscope, indirect ophthalmoscope, slit-lamp biomicroscope and focal illuminator. The most prevalent ocular variation within normal limits was hyaloid vessel remnant (ICR mice, 28.2%; BALB/c mice, 31.9%) and the incidence gradually decreased with age. The ocular diseases found in ICR mice were retinal degeneration (9.8%), corneal scar (4.2%), focal cataract (2.2%), anisocoria (1.2%), corneal ulcer (0.2%) and uveitis (0.2%). In BALB/c mice, corneal scar (9.2%), focal cataract (1.7%) and corneal ulcer (0.8%) were the ocular diseases found.     

Involvement of p38 kinase in hydroxyurea-induced differentiation of K562 cells.

Hydroxyurea is a differentiation-inducing agent of human erythroleukemia K562 cells. However, the cellular mechanisms by which hydroxyurea exerts its effects on tumor cells, leading to the inhibition of cell growth and the induction of differentiation markers, are largely unknown. This study examined the role of different mitogen-activated protein kinase signal transduction pathways in hydroxyurea-induced erythroid differentiation of K562 cells. Using a panel of anti-extracellular signal-related kinase (ERK), c-Jun NH2-terminal kinase (JNK), and p38 phosphospecific antibodies, we demonstrated that phosphorylation of ERK and JNK is decreased after the treatment of cells with hydroxyurea, whereas phosphorylation of p38 is increased. Moreover, inhibition of ERK activity by PD98059 induced erythroid differentiation, and it acted synergistically with hydroxyurea on hemoglobin synthesis, whereas inhibition of p38 activity by SB203580 inhibited induction of hemoglobin production by hydroxyurea. These findings suggest that the activation of p38 kinase may play important roles in the signal transduction mechanisms of hydroxyurea leading to erythroid differentiation.     

Diversity and evolution of a non-TIR-NBS sequence family that clusters to a chromosomal ’’hotspot” for disease resistance genes in soybean

In soybean, genes controlling resistance to numerous diseases have been shown to cluster to regions on several chromosomes. One such vital chromosomal region is on the soybean molecular linkage group (MLG) F flanked by the RFLP markers K644 and B212. Here, genes controlling resistance to bacterial blight, Phytophthora root rot, and several viral diseases, as well as QTLs conditioning resistance to corn earworm, root knot nematode, and white mold have been mapped. We have previously identified two classes (b and j) of disease resistance-related nucleotide binding site (NBS) sequences that localize to this cluster of genes. Using both cDNA and genomic analyses, we have studied one multi-gene family of sequences representing the previously reported class j NBS of soybean. This class of NBS resembles the RPS2-like NBS sequences. RPS2 and similar resistance genes are referred to as non-TIR because they do not encode motifs homologous to the Toll-Interleukin-1 region (TIR). By designing PCR primers that specifically target these non-TIR-NBS encoding sequences, we have amplified at least six class j sequence members from soybean. In addition, we have conducted genomic and cDNA library screenings to identify additional class j members. In all, we have characterized 12 class j NBS sequence members. These members have been mapped within a 2-cM region of the soybean F linkage group. We have also identified homoeologous chromosomal regions on linkage groups A2 and E that contain class j NBS sequences. A BLAST search of the GenBank database has shown that non-TIR NBS sequences are present across the legume family. We have compared these non-TIR sequences from other legumes with the soybean clones to assess the level of diversity within this class of disease resistance-related sequences.