Zebrafish smoothened functions in ventral neural tube specification and axon tract formation

Sonic hedgehog (Shh) signaling patterns many vertebrate tissues. shh mutations dramatically affect mouse ventral forebrain and floor plate but produce minor defects in zebrafish. Zebrafish have two mammalian Shh orthologs, sonic hedgehog and tiggy-winkle hedgehog, and another gene, echidna hedgehog, that could have overlapping functions. To examine the role of Hedgehog signaling in zebrafish, we have characterized slow muscle omitted (smu) mutants. We show that smu encodes a zebrafish ortholog of Smoothened that transduces Hedgehog signals. Zebrafish smoothened is expressed maternally and zygotically and supports specification of motoneurons, pituitary cells and ventral forebrain. We propose that smoothened is required for induction of lateral floor plate and a subpopulation of hypothalamic cells and for maintenance of medial floor plate and hypothalamic cells.     

Effect of endothelial cell polarity on beta-amyloid-induced migration of monocytes across normal and AD endothelium

During normal aging and amyloid beta-peptide (Abeta) disorders such as Alzheimer's disease (AD), one finds increased deposition of Abeta and activated monocytes/microglial cells in the brain. Our previous studies show that Abeta interaction with a monolayer of normal human brain microvascular endothelial cells results in increased adherence and transmigration of monocytes. Relatively little is known of the role of Abeta accumulated in the AD brain in mediating trafficking of peripheral blood monocytes (PBM) across the blood-brain barrier (BBB) and concomitant accumulation of monocytes/microglia in the AD brain. In this study, we showed that interaction of Abeta(1--40) with apical surface of monolayer of brain endothelial cells (BEC), derived either from normal or AD individuals, resulted in increased transendothelial migration of monocytic cells (HL-60 and THP-1) and PBM. However, transmigration of monocytes across the BEC monolayer cultivated in a Transwell chamber was increased 2.5-fold when Abeta was added to the basolateral side of AD compared with normal individual BEC. The Abeta-induced transmigration of monocytes was inhibited in both normal and AD-BEC by antibodies to the putative Abeta receptor, receptor for advanced glycation end products (RAGE), and to the endothelial cell junction molecule, platelet-endothelial cell adhesion molecule-1 (PECAM-1). We conclude that interaction of Abeta with the basolateral surface of AD-BEC induces cellular signaling, promoting transmigration of monocytes from the apical to basolateral direction. We suggest that Abeta in the AD brain parenchyma or cerebrovasculature initiates cellular signaling that induces PBM to transmigrate across the BBB and accumulate in the brain.     

电刺激下丘脑外侧区对大鼠胃缺血-再灌注损伤的影响

采用夹闭大鼠腹腔动脉30min,松开动脉夹血流复灌60min的胃缺血－再灌注损伤(gastric ischemia reperfusion injury,GI-RI)模型,用电和化学刺激,电损毁的方法观察了下丘脑外侧区（lateral hypothalamic area,LHA)对GI－RI的影响,并对其机制进行了初步分析,结果表明：（1）以0．2,0．4,0．6mA的电流强度刺激LHA,GI－RI均显著加重,且有强度－效应依赖关系,LHA内注射L－谷氨酸（L－Glu)后,对LI－RI的效应与电刺激相似,电损毁双侧LHA,GI－RI面积较电刺激组明显减小;（2）损毁双侧背侧迷走复合体（dorsal vagal complex,DVC)或切损毁是LHA,GI－RI面积较电刺激组明显减小;（2）损 侧背侧迷走复合体（dorsal vagal complex,DVC)或切断膈下迷走神经均能取消电刺激LHA加重GI－RI的作用。（3）电刺激LHA使缺血－再灌注（ischemia-reperfusion,I-R)的胃粘膜丙二醛（MDA）含量升高,超氧化物歧化酶（SOD）活性降低;（4）电刺激LHA使I－R的胃液量和总酸排出量增多,而酸度,胃蛋白酶活性和胃壁结合粘液量等无明显改变,结果提示;LHA是对GI－RI具有加重作用的中枢部位,其作用是通过DVC及迷走神经下传的,电刺激LHA加重GI－RI的作用与胃粘膜MDA含量增加,SOD活性降低,胃液量和总酸排出量增加等因素有关,而似与酸度,胃蛋白酶活性,胃壁结合粘液量等因素无关。     

肠缺血/再灌注损伤后白介素1-β水平与磷脂酶A2激活的关系

为了探讨肠缺血/再灌注损伤后IL-1β基因表达和蛋白含量变化与磷脂酶A2抑制之间的关系,采用大鼠肠缺血/再灌注损伤模型,在对照组,损伤组和磷脂酶A2抑制剂处理组动物中收集血清,肺灌洗液,腹腔灌洗液及全身重要脏器组织样品,采用放射免疫法测定IL-1β含量,并且RT-PCR法测定肺组织中IL-1β和Ⅱ型PLA2基因表达,结果表明,损伤后6h血清中IL-1β含量明显高于对照组;损伤后1和3h,腹腔注保IL-1β也明显高于对照组;损伤后肝组织中IL-1β水平有明显增加,而肺,肾、肠组织中IL-1β没有明显变化。损伤后肺灌洗液中IL-1β也明显高于对照组水平,肺组织中IL-1βmRNA表达增加,而Ⅱ型PLA2mRNA在损伤后表达反而有所下降,采用磷脂酶A2抑制剂氯喹,环氧化物酶抑制剂消炎痛,血小板活化因子受体阻断剂SR27417后,IL-1β蛋白和基因表达有不同的改变,提示肠缺血/再灌注损伤后一定时间内,肝内IL-1βmRNA表达和血中IL-1β水平明显增高,但是否与磷脂酶A2激活或其代谢产物的释放有关尚需进一步证明。     

Antibacterial effect of Kampo herbal formulation Hochu-ekki-to (Bu-Zhong-Yi-Qi-Tang) on Helicobacter pylori infection in mice

Because Helicobacter pylori (H. pylori) infection is a major cause of gastroduodenal diseases in humans, the eradication of H. pylori using antibiotics is very effective for the treatment of gastroduodenal diseases. However, it has recently been reported that resistance to these antibiotics is developing. In the present study, the antibacterial effect of a Kampo (traditional Japanese medicine) herbal formulation, Hochu-ekki-to (RET; Formula repletionis animalis et supletionis medii), against H. pylori was examined in vitro and in vivo. HET inhibited the growth of antibiotic-resistant strains of H. pylori as well as antibiotic-sensitive strains at a dose of 2.5 mg/ml in vitro. When 1,000 mg/kg of HET was administered orally to C57BL/6 mice for 7 days before or after inoculation with H. pylori, H. pylori in the stomach was significantly reduced in the HET-pre-treatment group compared with the control group. Furthermore, HET in combination with antibiotics completely eradicated the bacteria in mice. The expression of interferon (IFN)-gamma was induced in the gastric mucosa of the mice pre-treated with HET. There were no significant differences between the colonization of H. pylori in the control and HET treatment groups in IFN-gamma gene-deficient mice. These results suggest that the antibacterial effect of HET may be partly due to IFN-gamma induction, and that HET may be clinically useful for treatment of H. pylori infection.     

Evidence that extrachromosomal double-strand break repair can be coupled to the repair of chromosomal double-strand breaks in mammalian cells

Transfected linear DNA molecules are substrates for double-strand break (DSB) repair in mammalian cells. The DSB repair process can involve recombination between the transfected DNA molecules, between the transfected molecules and chromosomal DNA, or both. In order to determine whether these different types of repair events are linked, we devised assays enabling us to follow the fate of linear extrachromosomal DNA molecules involved in both interplasmid and chromosome-plasmid recombination, in the presence or absence of a pre-defined chromosomal DSB. Plasmid-based vectors were designed that could either recombine via interplasmid recombination or chromosome-plasmid recombination to produce a functional beta-galactosidase (betagal) fusion gene. By measuring the frequency of betagal+ cells at 36 h post-transfection versus the frequency of betagal+ clones after 14 days, we found that the number of cells containing extrachromosomal recombinant DNA molecules at 36 h (i.e., betagal+), either through interplasmid or chromosome-plasmid recombination, was nearly the same as the number of cells integrating these recombinant molecules. Furthermore, when a predefined DSB was created at a chromosomal site, the extrachromosomal recombinant DNA molecules were shown to integrate preferentially at that site by Southern and fiber-FISH (fluorescence in situ hybridization) analysis. Together these data indicate that the initial recombination event can potentiate or commit extrachromosomal DNA to integration in the genome at the site of a chromosomal DSB. The efficiency at which extrachromosomal recombinant molecules are used as substrates in chromosomal DSB repair suggests extrachromosomal DSB repair can be coupled to the repair of chromosomal DSBs in mammalian cells.     

BRCA1-induced apoptosis involves inactivation of ERK1/2 activities

Mutation in the BRCA1 gene is associated with an increased risk of breast and ovarian cancer. Recent studies have shown that the BRCA1 gene product may be important in mediating responses to DNA damage and genomic instability. Previous studies have indicated that overexpression of BRCA1 can induce apoptosis or cell cycle arrest at the G(2)/M border in various cell types. Although the activation of JNK kinase has been implicated in BRCA1-induced apoptosis, the role of other members of the mitogen-activated protein kinase family in mediating the cellular response to BRCA1 has not yet been examined. In this study, we monitored the activities of three members of the MAPK family (ERK1/2, JNK, p38) in MCF-7 breast cancer cells and U2OS osteosarcoma cells after their exposure to a recombinant adenovirus expressing wild type BRCA1 (Ad.BRCA1). Overexpression of BRCA1 in MCF-7 cells resulted in arrest at the G(2)/M border; however, BRCA1 expression in U2OS cells induced apoptosis. Although BRCA1 induced JNK activation in both cell lines, there were marked differences in ERK1/2 activation in response to BRCA1 expression in these two cell lines. BRCA1-induced apoptosis in U2OS cells was associated with no activation of ERK1/2. In contrast, BRCA1 expression in MCF-7 cells resulted in the activation of both ERK1/2 and JNK. To directly assess the role of ERK1/2 in determining the cellular response to BRCA1, we used dominant negative mutants of MEK1 as well as MEK1/2 inhibitor PD98059. Our results indicate that inhibition of ERK1/2 activation resulted in increased apoptosis after BRCA1 expression in MCF-7 cells. Furthermore, BRCA1-induced apoptosis involved activation of JNK, induction of Fas-L/Fas interaction, and activation of caspases 8 and 9. The studies presented in this report indicate that the response to BRCA1 expression is determined by the regulation of both the JNK and ERK1/2 signaling pathways in cells.     

Synthesis and biological evaluation of 3'-carboranyl thymidine analogues

Boron neutron capture therapy (BNCT) is a chemoradio-therapeutic method for the treatment of cancer. It depends on the selective targeting of tumor cells by boron-containing compounds. One category of BNCT agents with potential to selectively target tumor cells may be thymidine derivatives substituted at the 3'-position with appropriate boron moieties. Thus, several thymidine analogues were synthesized with a carborane cluster bound to the 3'-position either through an ether or a carbon linkage. The latter are the first reported carborane-containing nucleosides in which the carboranyl entity is directly linked to the carbohydrate portion of the nucleoside by a carbon-carbon bond. Low but significant phosphorylation rates in the range of 0.18% that of thymidine were observed for the carbon-linked 3'-carboranyl thymidine analogues in phosphoryl transfer assays using recombinant preparations of thymidine kinases 1 (TK1) and thymidine kinases 2 (TK2). Some of the ether-linked 3'-carboranyl thymidine analogues appeared to be slightly unstable under acidic as well as phosphoryl transfer assay conditions and were, if at all, poor substrates for TK1.