三种丙型肝炎病毒核蛋白在人肝癌细胞的表达

目的用基因芯片分析丙型肝炎病毒（HCV）三种基因型HCV—1b、HCV-2a和HCV-4d核蛋白在人肝癌细胞系（Huh-7）的基因表达,重新认识HCV核蛋白功能及其致病机制。方法建立表达HCV三种基因型核蛋白的Huh-7细胞系,提取总RNA制备探针。与Affymetrix人基因芯片HG-U133杂交。对基因表达改变≥3或1．5倍的基因,通过Affymetrix网站用NetAffx进一步分析,并按生物学功能分类。结果HCV—1b核蛋白引起16个基因表达改变,其中免疫反应基因PF4V1和SPP1分别上调3．4和4．4倍;HCV-2a核蛋白对免疫反应基因CXCL5和凋亡基因BTF分别下调3．4和3．1倍,但对凋亡基因HRK、LZTS1则能上调3．2和3．4倍;与HCV-1b和HCV-2a核蛋白相比,HCV-4d核蛋白可引起111个基因表达改变,对基因表达谱有更明显的影响。若设定基因表达改变≥1．5倍有意义,HCV—1b和HCV-2a核蛋白可引起若干相同基因的改变。结论HCV三种基因型核蛋白引起的基因表达谱各有特点,主要涉及免疫反应、凋亡、信号传导等基因,这对重新认识HCV核蛋白功能及致病机制均有重要意义。     

Inhibition of proteasome activity by various fruits and vegetables is associated with cancer cell death

There is a large amount of scientific evidence showing that fruits and vegetables lower the risk of cancer. However, the responsible molecular mechanisms remain poorly understood. Our previous studies have demonstrated that inhibition of proteasomal chymotrypsin-like activity is associated with cancer cell apoptosis, which may also be the major mechanism responsible for the anticancer effects of green tea polyphenols. In the current study, we tested the hypothesis that some fruits and vegetables inhibit tumor cell proteasome activity and that this inhibition contributes to their cancer-preventative activities. We report that the extracts of apple and grape are more potent than onion, tomato and celery in: (i) inhibiting the proteasomal chymotrypsin-like activity in leukemia Jurkat T cell extract; (ii) accumulating the polyubiquitinated proteins in intact Jurkat T cells; (iii) inducing activation of caspase-3/-7 and cleavage of poly(ADP-ribose) polymerase in intact Jurkat T cells; and (iv) inducing the appearance of spherical cells preferentially in prostate cancer PC-3 over the normal NIH 3T3 cell line. We also found that strawberry extract had some effect on Jurkat T cell extract and the prostate PC-3 cell line but not on intact Jurkat T cells. Our findings suggest that the proteasome is a cancer-related molecular target for, at least, the extracts of apple, grape and onion, and that the inhibition of proteasome activity by these fruits or vegetable may contribute to their cancer-preventative effects, although other molecular mechanisms may also be involved.     

Coregistration of quantitative proton magnetic resonance spectroscopic imaging with neuropathological and neurophysiological analyses defines the extent of neuronal impairments in murine human immunodeficiency virus type-1 encephalitis

Relatively few immune-activated and virus-infected mononuclear phagocytes (MP; perivascular macrophages and microglia) may affect widespread neuronal dysfunction during human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD). Indeed, histopathological evidence of neuronal dropout often belies the extent of cognitive impairment. To define relationships between neuronal function and histopathology, proton magnetic resonance spectroscopic imaging (1H MRSI) and hippocampal long-term potentiation (LTP) were compared with neuronal and glial immunohistology in a murine model of HIV-1 encephalitis (HIVE). HIV-1(ADA)-infected human monocyte-derived macrophages (MDM) were stereotactically injected into the subcortex of severe combined immunodeficient (SCID) mice. Sham-operated and unmanipulated mice served as controls. Seven days after cell injection, brain histological analyses revealed a focal giant cell encephalitis, with reactive astrocytes, microgliosis, and neuronal dropout. Strikingly, significant reductions in N-acetyl aspartate concentration ([NAA]) and LTP levels in HIVE mice were in both injected and contralateral hemispheres and in brain subregions, including the hippocampus, where neuropathology was limited or absent. The data support the importance of 1H MRSI as a tool for assessing neuronal function for HAD. The data also demonstrate that a highly focal encephalitis can produce global deficits for neuronal function and metabolism.     

Nitric oxide stimulates gamma-aminobutyric acid release and inhibits glycine release in retina

Nitric oxide (NO) modulates the uptake and/or release of neurotransmitters through a variety of cellular mechanisms. However, the pharmacological and biochemical processes underlying these neurochemical effects of NO often remain unclear. In our study, we used immunocytochemical methods to study the effects of NO, cyclic guanosine monophosphate (cGMP), and peroxynitrite on the uptake and release of gamma-aminobutyric acid (GABA) and glycine in the turtle retina. In addition, we examined the involvement of glutamate receptors, calcium, and the GABA transporter in this GABA uptake and release. We also tested for interactions between the GABAergic and glycinergic systems. In general, we show that NO stimulated GABA release and inhibited glycine release. The NO-stimulated GABA release involved calcium-dependent or calcium-independent synaptic release or reversal of the GABA transporter. Some effects of NO on GABA release involved glutamate, cGMP, or peroxynitrite. NO promoted glycine uptake and inhibited its release, and this inhibition of glycine release was influenced by GABAergic modulation. These findings indicate that NO modulates the levels of the inhibitory transmitters GABA and glycine through several specific biochemical mechanisms in different retinal cell types and layers. Thus it appears that some of the previously described reciprocal interactions between GABA and glycine in the retina function through specific NO signaling pathways.     

Further investigations of morpholino pretargeting in mice—establishing quantitative relations in tumor

Purpose This laboratory has previously published on phosphorodiamidate morpholino (MORF) pretargeting of tumor in which an anti-tumor antibody conjugated with MORF (a DNA analogue) is first administered, followed at a later time by the radiolabeled complementary MORF (cMORF) as the effector. In the present study, the pharmacokinetics of the antibody and effector were measured under different conditions in mice to establish their quantitative relationships with tumor accumulations by pretargeting.Methods A cytosine-free 18 mer cMORF was conjugated with MAG₃ for ^99mTc labeling while the anti-CEA antibody MN14 was conjugated with DTPA for ¹¹¹In labeling and with MORF to impart binding affinity for radiolabeled cMORF. Mice bearing LS174T thigh tumors were used to study: (1) the pharmacokinetics of MN14-MORF by administering ¹¹¹In-MN14 at doses between 10 and 100 g with sacrifice at 2 days and at 30 g with sacrifice between 1 and 3 days; (2) the biodistribution of ^99mTc-cMORF following one to four injections (containing 0.15 g each and separated by 1 h) to animals having received 30 g of antibody–MORF 2 days earlier and with sacrifice at 3 h after the final injection; and (3) the influence on the biodistribution of ^99mTc-cMORF of a 2 to 4 day interval between the administration of 30 g of antibody–MORF and 0.30 g of ^99mTc-cMORF.Results (1) The biodistribution of antibody in percent accumulation (%ID or %ID/g) was largely independent of antibody dose but the absolute accumulation of antibody in tumor increased linearly with dose, showing no evidence of tumor saturation of CEA sites by MN14. Over 1–3 days post antibody administration, blood levels of radiolabeled antibody decreased as expected; however, tumor levels remained constant, thus showing an absence of antibody clearance in tumor over this period. (2) With fixed antibody–MORF dose and increasing number of injections of ^99mTc-cMORF, cumulative percent blood levels steadily decreased in agreement with the values calculated based on the antibody–MORF in blood. In contrast, cumulative percent tumor levels stayed fairly constant over the first two injections. Thus the antibody–MORF in tumor became saturated with cMORF more slowly than that in blood owing to delivery differences. (3) As expected, percent blood levels decreased with increasing interval between injections of antibody–MORF and ^99mTc-cMORF. The percent tumor accumulation, however, remained constant over the 3 day interval, thus demonstrating only slow loss of MORF expression in situ. The ^99mTc-cMORF accumulation in tumor after saturation was mathematically determined based on the antibody–MORF concentration in tumor while the blood levels of ^99mTc-cMORF were determined based on the concentration of antibody-MORF in blood.Conclusion Contrary to conclusions arrived at in our earlier study, the results of this study show that tumor CEA sites were not saturated even at the highest antibody dose investigated, that accessibility of MORF sites in tumor by ^99mTc-cMORF was unhindered and that the maximum percent tumor accumulation of ^99mTc-cMORF depended only on the tumor delivery efficiency of ^99mTc-cMORF.     

Clioquinol and pyrrolidine dithiocarbamate complex with copper to form proteasome inhibitors and apoptosis inducers in human breast cancer cells

Introduction

A physiological feature of many tumor tissues and cells is the tendency to accumulate high concentrations of copper. While the precise role of copper in tumors is cryptic, copper, but not other trace metals, is required for angiogenesis. We have recently reported that organic copper-containing compounds, including 8-hydroxyquinoline-copper(II) and 5,7-dichloro-8-hydroxyquinoline-copper(II), comprise a novel class of proteasome inhibitors and tumor cell apoptosis inducers. In the current study, we investigate whether clioquinol (CQ), an analog of 8-hydroxyquinoline and an Alzheimer's disease drug, and pyrrolidine dithiocarbamate (PDTC), a known copper-binding compound and antioxidant, can interact with copper to form cancer-specific proteasome inhibitors and apoptosis inducers in human breast cancer cells. Tetrathiomolybdate (TM), a strong copper chelator currently being tested in clinical trials, is used as a comparison.

Methods

Breast cell lines, normal, immortalized MCF-10A, premalignant MCF10AT1K.cl2, and malignant MCF10DCIS.com and MDA-MB-231, were treated with CQ or PDTC with or without prior interaction with copper, followed by measurement of proteasome inhibition and cell death. Inhibition of the proteasome was determined by levels of the proteasomal chymotrypsin-like activity and ubiquitinated proteins in protein extracts of the treated cells. Apoptotic cell death was measured by morphological changes, Hoechst staining, and poly(ADP-ribose) polymerase cleavage.

Results

When in complex with copper, both CQ and PDTC, but not TM, can inhibit the proteasome chymotrypsin-like activity, block proliferation, and induce apoptotic cell death preferentially in breast cancer cells, less in premalignant breast cells, but are non-toxic to normal/non-transformed breast cells at the concentrations tested. In contrast, CQ, PDTC, TM or copper alone had no effects on any of the cells. Breast premalignant or cancer cells that contain copper at concentrations similar to those found in patients, when treated with just CQ or PDTC alone, but not TM, undergo proteasome inhibition and apoptosis.

Conclusion

The feature of breast cancer cells and tissues to accumulate copper can be used as a targeting method for anticancer therapy through treatment with novel compounds such as CQ and PDTC that become active proteasome inhibitors and breast cancer cell killers in the presence of copper.     

二型谷氨酸羧肽酶口服基因疫苗的制备及其降低大鼠麻醉药用量的初步研究

目的　研制携带二型谷氨酸羧肽酶 (GCPⅡ )的口服疫苗 ,探讨该疫苗对大鼠麻醉药用量的影响。方法　采用聚合酶链反应、酶切连接和蓝白筛选的方法 ,构建GCPⅡ的表达载体 ;用磷酸钙共沉淀的方法转染HEK2 93细胞 ,G4 18筛选阳性细胞克隆 ;逆转录聚合酶链反应和免疫荧光细胞染色的方法鉴定阳性细胞株 ;氯化钙法制备携带GCPⅡ表达载体的减毒鼠伤寒沙门氏菌口服疫苗(SL GCPⅡ ) ;采用原代培养的巨噬细胞吞噬SL GCPⅡ的方法 ,观察疫苗的体外表达情况 ;将 5 0只雄性SD大鼠随机分为A、B 2组 ,每组 2 5只 ,2组鼠的体重分别为 ( 174 g±13g)和 ( 172 g± 11g) ,差异无显著意义 (P =0 0 72 )。A组胃内灌注 6 0 0 μL、OD2 60 0 6SL GCPⅡ ,两周内 4次给药 ;B组为对照组 ,给予SL32 6 1,剂量和方法与A组相同。采用免疫荧光的方法检测大鼠循环中GCPⅡ抗体滴度 ,观察麻醉药戊巴比妥钠的用量。结果　扩增GCPⅡ基因并构建了含有GCPⅡ基因的表达载体———pCDNA3 1 GCPⅡ ;建立了细胞株HEK 2 93 GCPⅡ ;体外实验证明培养的巨噬细胞吞噬SL GCPⅡ后 ,能够有效表达GCPⅡ基因。体内实验发现口服疫苗能够激活 2 2 / 2 5只SD大鼠产生GCPⅡ抗体 ,实验组麻醉药戊巴比妥钠的用量 ( 36 9mg/kg± 1 6mg/kg)显著低于对照组 ( 4 0 8m     

脑梗死后神经干细胞的原位增殖及其可塑性

目的研究成年大鼠脑梗死后自体神经干细胞的原位增殖,并探讨其可塑性.方法雄性Wistar大鼠共68只,分对照组(n=8),脑梗死后1、3、7、14、28 d组,每组12只.免疫组织化学方法动态检测大鼠脑内5-溴脱氧尿苷嘧啶(BrdU)、Brdu/多唾液酸神经细胞黏附分子(PSA-NCAM)的表达.结果与对照组相比,室下区和海马BrdU阳性细胞在脑梗死后1 d开始增加(P＜0.05),7 d达到高峰,14 d后开始下降,但仍高于对照组(P＜0.05),28 d后接近正常水平;室下区和海马BrdU/PSA-NCAM阳性细胞在脑梗死后7 d开始增加(P＜0.05),14 d达到高峰,28 d后开始下降,但仍高于对照组(P＜0.05),大约占同期BrdU阳性细胞数60%.结论脑梗死可激活自体神经干细胞原位增殖,并且大多数增殖的神经干细胞具有可塑性.     

硬膜外阻滞用于分娩镇痛的临床研究

目的:研究罗哌卡因配伍小剂量芬太尼用于硬膜外腔阻滞分娩镇痛的临床意义及其应用价值。方法:对100例足月妊娠,单胎头位,自然临产的初产妇采用0.125%罗哌卡因及2μg/ml芬太尼复合液于L2~3行硬膜外阻滞,并与150例常规分娩者进行对比观察。结果:观察组第1、2产程镇痛效果良好,分别为94%、84%;第1产程明显缩短;母婴均无不良反应。结论:低位硬膜外阻滞,应用罗哌卡因配伍小剂量芬太尼在分娩镇痛中效果良好,安全、可靠。     

多重耐药性大肠埃希菌质粒谱与ESBLs基因型分析

目的分析临床耐药大肠埃希菌质粒谱与基因型间关系，确定超广谱β-内酰胺酶（extended spectrum bata lactarnase ESBLs）基因在细菌质粒谱上的座位。方法分离鉴定菌株，通过K-B法药敏实验确认产ESBLs菌株。凝胶电泳检测菌体质粒，并应用PCR技术扩增ESBLs基因。结果分离到产ESBLs细菌128株，检出率为78．0％，针对12种抗生素，产ESBLs细菌的耐药率在29％．71％之间；128株大肠埃希菌含11类不同谱型的质粒群，其中较大质粒（23kb、9．4kb）出现频率高（71．1％、26．6％）；PCR分型结果显示，单独携带TEM型ESBLs基因的菌株比例为24．2％，SHV型为19．5％，CTX-M型为53．1％。携带两种基因型以上的占18．8％。CTX-M型中三个亚型CTX-M-24、CTX-M-22型和CTX-M-3型比例分别是32．0％、28．1％和17．2％。结论质粒谱中，某些分子量相同或相近的较大质粒能稳定存在于菌体内并能导致相应耐药性的产生和播散。定位这类质粒上ESBLs基因的差异导致了菌株耐药性的不同。