Expression of hepatic thrombopoietin mRNA in primary cultured hepatocytes and in rats with acute liver injury or bone marrow suppression with or without cirrhosis

BACKGROUND AND AIMS: The main causes of thrombocytopenia in cirrhosis are thought to be platelet destruction and the reduction of thrombopoietin (TPO) expression in the liver. The mechanisms by which levels of TPO mRNA are regulated in cirrhosis have not been elucidated. In this study, we investigated some possible mechanisms. METHODS: We used three experimental models: bone marrow suppression, acute liver injury and primary cultured hepatocytes. We used northern blots to assess the kinetics of TPO mRNA expression in the livers of irradiated rats (with and without cirrhosis) in acute liver injury and in primary cultured hepatocytes treated with hepatotoxin or cytokines. RESULTS: Although the bone marrow was hypocellular, there was no apparent enhancement of TPO mRNA expression in the irradiated rats with cirrhotic livers compared with the unirradiated rats with cirrhotic livers. There were no conspicuous changes in hepatic TPO mRNA expression between the livers of the control rats and the three models of acute liver injury. There were no conspicuous changes in the levels of TPO mRNA between control hepatocytes and hepatocytes treated with hepatotoxin or cytokines. CONCLUSIONS: Our results suggest that bone marrow is not a regulator of hepatic TPO production in cirrhosis. The reduced TPO mRNA expression found in cirrhotic rats may not result merely from serious cellular damage; it may be associated with cirrhosis-specific regulatory mechanisms for the expression of the TPO gene. Further studies are needed to search for other factors that may induce reduced TPO expression.     

New staining methodology: Eastern blotting for glycosides in the field of Kampo medicines

Ginsenosides separated by silica gel TLC blotted to a polyvinylidene difluoride (PVDF) membrane treated with a NaIO₄ solution followed by bovine serum albumin (BSA) resulted in a ginsenoside–BSA conjugate on a PVDF membrane. The blotted spot were stained by antiginsenoside Rb1 (GRb1) and Rg1 (GRg1) monoclonal antibodies (MAbs). The newly established immunostaining method, Eastern blotting, was applied for the determination of ginsenosides possessing protopanaxadiol and/or protopanaxatriol in the Kampo medicines. In this method, we developed a new way to separate the ginsenoside molecule into two functional parts using a simple and well-known chemical reaction. The sugar parts were oxidized by sodium periodate to give dialdehydes, which reacted with amino groups of the protein and covalently bound to the adsorbent PVDF membrane. The MAb bound to the aglycone part of the ginsenoside molecule for immunostaining. Double staining of Eastern blotting for ginsenosides using anti-GRb1 and GRg1 MAbs promoted complete identification of ginsenosides in Panax species. This method was validated for immunocytolocalization of ginsenosides in fresh ginseng root. In addition, Eastern blotting for the detection of glycyrrhizin (GC) was also investigated. GC can be clearly determined by Eastern blotting in the Glycyrrhiza species. It is also possible for GC in rat serum to be surveyed by Eastern blotting by simple pretreatment as a further application.     

Gankyrin在肝癌细胞系中的表达

目的检测肝癌细胞系中癌基因Gankyrin的表达。为在肝癌细胞中对Gankyrin基因进行RNA干涉研究的体外实验筛选靶细胞。方法对肝癌细胞系HepG2、SMMC-7721、HHCC进行逆转录聚合酶链反应（RT—PCR）及Western blotting检测。结果Gankyrin基因在3种肝癌细胞系中均有表达。结论多种肝癌细胞系中均有Gankyrin基因的表达，HepG2、SMMC-7721、HHCC均可作为阳性靶细胞，进行Gankyrin基因的RNA干涉研究。     

High-level Expression and Purification of Human Zona Pellucida huZP3a^22-176 and huZP3b^177-348 Peptides in Escherichia coli

Objective To try making huZP3a^22-176 and huZP3b^177-348 polypeptides （representing an intact huZP^322-348 protein without its N-terminal signal peptide and C-terminal transmembrane domain ） express in E. coli at a higher level Methods The cDNAs encoding huZP3a and huZP3b were obtained with PCR method. The pBV221 plasmid was used to construct thermo-inducible recombinant expression vector. Purification of two target expression products employed an improved method of preparative gel polyacrylamide gel electrophoresis. Results Two polypeptides of recombinant huZP3a （rhuZP3a） and recombinant huZP3b （rhuZP3b） were all expressed respectively in an E. coli BL21（DE3）pLysS strain at a higher level, which were recognized by two specific polyclonal antisera in Western blotting test which recognize a linear B cell epitope present in rhuZP3a or rhuZP3b respectively. Using the shake-flask method, approximately 5 mg of rhuZP3a and rhuZP3b with more than 95% relative homogeneity were harvested from 1 L culture respectively. Conclusion The availability of two rhuZP3 polypeptides will help in detecting the immunogenicities of rhuZP3a and rhuZP3b through animal experiments and confirming the function domain of non-glycosylated huZP3 to induce acrosome reaction in vitro.     

RNA Interference-Mediated Gene Silencing of Vascular Endothelial Growth Factor

A ngiogenesis is a well-known process that is essential for tumor growth beyond 2 mm.[1] Although numerous growth factors are involved, vascular endothelial growth factor (VEGF), in particular VEGF-A, has been shown to play an important role in tumor angi…     

Regional distribution of nitric oxide synthase and NADPH-diaphorase activities in the central nervous system of teleosts

Neuronal nitric oxide synthase (nNOS) and NADPH-diaphorase activities were investigated in discrete areas of the central nervous system of goldfish and brown trout. Both species showed a similar distribution pattern of nNOS activity with regional differences in all examined areas. Telencephalon and hypothalamus showed the highest nNOS values, while in the goldfish cerebellum and its valvula nNOS was not detectable. In both species, NADPH-diaphorase activity showed a lower regional variability, compared to nNOS. The highest activity was measured in the olfactory bulbs where, conversely, low levels of nNOS activity were present. The non close correspondence between NOS and NADPH-diaphorase activities confirms the discrepancies indicated by morphological data. Western blot analysis revealed the presence of a nNOS isoform of about 150 kDa mol. wt. corresponding to that of mammals. The pattern of nNOS expression in the considered brain regions of the goldfish and trout was comparable to the levels of the nNOS activity.     

缩宫素受体在子宫内膜异位症的表达及与痛经的关系

目的探讨缩宫素受体（0TR）在子宫内膜异位症（EM）患者的异位内膜及在位子宫内膜组织中表达情况及与痛经的关系。方法应用蛋白免疫印迹法（Westernblotting）检测EM患者39例卵巢子宫内膜异位病灶，30例在位子宫内膜以及30例非EM患者的正常子宫内膜作为对照，比较不同组织中OTR蛋白的阳性表达率和表达强度的差异，以及与痛经的关系。结果OTR在EM组表达高于对照组（P〈0．05），在EM组在位内膜增生期和分泌期的表达无显著性差异（P〉0．05），OTR在EM组痛经者的表达高于非痛经者（P〈0．05）。结论EM患者OTR的高表达可能参与了EM发病机制，而且可能与EM的痛经有关，对OTR的深入研究将为EM痛经的治疗提供新的方向。     

The expression and localization of mRNA for colony-stimulating factor (CSF)-1 in human term placenta.

A 4-kb mRNA for colony-stimulating factor 1 (CSF-1) was detected in normal human placenta at term by Northern blot analysis. In-situ hybridization revealed that the mRNA for CSF-1 was localized in the mesenchymal cells of the chorionic villous stroma, but not in the trophoblasts or capillary epithelial cells. Because there are significant numbers of tissue macrophages (Hofbauer cells) in the placental stroma and because the receptor for CSF-1 (the c-fms proto-oncogene product) is known to be expressed by trophoblasts, our results suggest that CSF-1 produced by placental stromal cells may act as a growth and survival factor for human placental macrophages and trophoblasts.     

Increased levels of a lower molecular weight form of secretory component in cervical scrapes from women with cervical intraepithelial neoplasia

The protein composition of 'negative' and 'positive' cervical scrapes has been compared using electrophoresis to determine whether differences were present that could be used to pre-select specimens with negative cytology. Only one minor difference in the 75–80 kDa region of the silver-stained patterns was detected in extracts from two well-matched groups. Further studies using 2D-electrophoresis and Western blotting identified the major components in this region as two forms of secretory component (SC) and transferrin. Subsequent blotting of 13 extracts from patients with negative smears and 17 extracts from patients with positive smears indicated very significant differences in the expression of the two forms of SC ( P < 0.0002), the 'negatives' having more of the higher M _r form and the 'positives' having more of the lower M _r form. Measuring SC could prove useful for pre-screening cervical scrapes, but more investigations are required to establish the nature of the change and a quantitative method for its detection.     

Oridonin induces apoptosis of HeLa cells via altering expression of Bcl-2 ／ Bax and activating caspase-3 ／ ICAD pathway

INTRODUCTIONHerbal medicine, Donglingcao (rabdosiarubescens), has been traditionally used in China for thetreatment of various diseases such as leukemia.Oridonin (Fig 1) is a diterpenoid compound isolated fromRabdosia rubescens (hemsl). It has various pharmaco-logical and physiological effects such as anti-inflammation, anti-bacteria, anti-tumor[1-3] and has beenused for the treatment of human cancers, especiallyFig 1. Chemical structure of oridonin.esophageal carcinoma[4]. This comp…