三氧化二砷对食管癌细胞增殖和热休克蛋白70表达的影响

目的：研究三氧化二砷（As2O3）对食管癌细胞增殖和热休克蛋白70（HSP70）表达的影响。方法：通过相差显微镜、流式细胞术、免疫细胞化学染色和免疫印迹分析等方法观察As2O3对人食管癌细胞株EC1的作用效果和作用机制。结果：与对照组相比,经2μmol/L和5μmol/Las2O3作用的细胞出现明显的生长抑制,G2/M期细胞比例增加;2μmol/Las2O3作用48h后经Ecl细胞HSP70（heat shock protein70）及HSC70(heat shock cognate protein70)表达均增加。结论：As2O3诱导食管癌细胞G2/M期阻滞抑制细胞增殖和生长;HSP70的升高是细胞对As2O3作用后出现的应激反应,并与细胞周期阻滞相关。     

5-HT(3) receptors mediate inflammation-induced unmasking of functional tachykinin responses in vitro.

Exogenously applied tachykinins produce no measurable electrophysiological responses in the somata of vagal afferent neurons [nodose ganglion neurons (NGNs)] isolated from naive guinea pigs. By contrast, after in vitro antigen challenge of nodose ganglia from guinea pigs immunized with chick ovalbumin, approximately 60% (53 of 89) of NGNs were depolarized an average of 13 +/- 1.2 mV by substance P (SP; 100 nM; n = 53). Receptor antagonists and enzyme inhibitors were utilized to screen a number of mast cell-derived mediators for their role in the uncovering or "unmasking" of functional tachykinin receptors after antigen challenge. Two chemically distinct 5-hydroxytryptamine-3-receptor antagonists significantly reduced the percentage of NGNs displaying depolarizing SP responses. Treatment with Y-25130 (1 or 10 microM) or tropisetron (1 microM) 15 min before and during antigen challenge reduced the percentage of SP-responsive neurons to approximately 20 and approximately 15% respectively. These results suggest that activation of 5-hydroxytryptamine-3 receptors plays an integral role in the unmasking of functional tachykinin receptors after specific antigen challenge of nodose ganglia. The mediator(s) underlying tachykinin-receptor unmasking in the remainder of the NGNs has yet to be characterized. However, it does not appear to be histamine, prostanoids, or peptidoleukotrienes.     

Mutations of the immunoglobulin heavy chain variable region gene in CD99-deficient BJAB cell line

Hodgkin's disease (HD) is a lymphoid neoplasm characterized by a low frequency of malignant giant tumor cells, known as Hodgkin's and Reed-Sternberg (HRS) cells. Sequence analysis of the immunoglobulin heavy chain hypervariable region (IgH V) genes of HRS cells revealed multiple nucleotide substitutions, indicating somatic mutations, and suggested that HRS cells originate from germinal center B cells or their progeny. We previously reported that CD99-antisense transfected B cell lines led to the generation of cells with a HRS phenotype. Because it is considered that HRS cells in HD carry somatic mutations of the IgH genes, we assume that somatic mutation may take place in the IgH genes of HRS-like cells which do not express CD99. Here we report that CD99 downregulated BJAB cell line has several mutations in IgH V genes. The frequency of mutation was 5.2 x 10(-4) mut.bp(-1) out of total sequenced cell clones. On the contrary, control vector transfected BJAB cell line or CD99 downregulated IM9 cell line did not show any mutations on single strand conformational polymorphism (SSCP) and sequence analysis. We expect that the analysis of the mutation pattern of the CD99-deficient BJAB cell line might be the basis for the understanding of the molecular and cellular mechanism that regulate somatic mutation and B cell selection.     

Modulation of the levels of NMDA receptor subunit mRNA and the bindings of [3H]MK-801 in rat brain by chronic infusion of subtoxic dose of MK-801

The effects of continuous infusion of NMDA receptor antagonist MK-801 on the modulation of NMDA receptor subunits NR1, NR2A, NR2B, and NR2C were investigated by using in situ hybridization study. Differential assembly of NMDA receptor subunits determines their functional characteristics. Continuous intracerebroventricular (i.c.v.) infusion with MK-801 (1 pmol/10 l/h) for 7 days resulted in significant modulations in the NR1, NR2A, and NR2B mRNA levels without producing stereotypic motor syndromes. The levels of NR1 mRNA were significantly increased (9-20%) in the cerebral cortex, striatum, septum, and CA1 of hippocampus in MK-801-infused rats. The levels of NR2A mRNA were significantly decreased (11-16%) in the CA3 and dentate gyrus of hippocampus in MK-801-infused rats. In contrast to NR2A, NR2B subunit mRNA levels were increased (10-14%) in the cerebral cortex, caudate putamen, and thalamus. However, no changes of NR2C subunits in cerebellar granule layer were observed. Using quantitative ligand autoradiography, the binding of NMDA receptor ligand [³H]MK-801 was increased (12-25%) significantly in almost all brain regions except in the thalamus and cerebellum after 7 days infusion with MK-801. These results suggest that region-specific changes of NMDA receptor subunit mRNA and [³H]MK-801 binding are involved in the MK-801-infused adult rats.     

First success of catalytic epoxidation of olefins by an electron-rich iron(III) porphyrin complex and H2O2: imidazole effect on the activation of H2O2 by iron porphyrin complexes in aprotic solvent

An electron-rich iron(III) porphyrin complex (meso-tetramesitylporphinato)iron(III) chloride [Fe(TMP)Cl], was found to catalyze the epoxidation of olefins by aqueous 30% H₂O₂ when the reaction was carried out in the presence of 5-chloro-1-methylimidazole (5-Cl-1-MeIm) in aprotic solvent. Epoxides were the predominant products with trace amounts of allylic oxidation products, indicating that Fenton-type oxidation reactions were not involved in the olefin epoxidation reactions. cis-Stilbene was stereospecifically oxidized to cis-stilbene oxide without giving isomerized trans-stilbene oxide product, demonstrating that neither hydroperoxy radical (HOO·) nor oxoiron(IV) porphyrin [(TMP)Fe^IV=O] was responsible for the olefin epoxidations. We also found that the reactivities of other iron(III) porphyrin complexes such as (meso-tetrakis(2,6-dichlorophenyl)porphinato)iron(III) chloride [Fe(TDCPP)Cl], (meso-tetrakis(2,6-difluorophenyl)porphinato)iron(III) chloride [Fe(TDFPP)Cl], and (meso-tetrakis(pentafluorophenyl)porphinato)iron(III) chloride [Fe(TPFPP)Cl] were significantly affected by the presence of the imidazole in the epoxidation of olefins by H₂O₂. These iron porphyrin complexes did not yield cyclohexene oxide in the epoxidation of cyclohexene by H₂O₂ in the absence of 5-Cl-1-MeIm in aprotic solvent; however, addition of 5-Cl-1-MeIm to the reaction solutions gave high yields of cyclohexene oxide with the formation of trace amounts of allylic oxidation products. We proposed, on the basis of the results of mechanistic studies, that the role of the imidazole is to decelerate the O–O bond cleavage of an iron(III) hydroperoxide porphyrin (or H₂O₂–iron(III) porphyrin adduct) and that the intermediate transfers its oxygen to olefins prior to the O–O bond cleavage.     

Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp. strain CK 11-4 screened from Chungkook-Jang.

Bacillus sp. strain CK 11-4, which produces a strongly fibrinolytic enzyme, was screened from Chungkook-Jang, a traditional Korean fermented-soybean sauce. The fibrinolytic enzyme (CK) was purified from supernatant of Bacillus sp. strain CK 11-4 culture broth and showed thermophilic, hydrophilic, and strong fibrinolytic activity. The optimum temperature and pH were 70 degrees C and 10.5, respectively, and the molecular weight was 28,200 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The first 14 amino acids of the N-terminal sequence of CK are Ala-Gin-Thr-Val-Pro-Tyr-Gly-Ile-Pro-Leu-Ile-Lys-Ala-Asp. This sequence is identical to that of subtilisin Carlsberg and different from that of nattokinase, but CK showed a level of fibrinolytic activity that was about eight times higher than that of subtilisin Carlsberg. The amidolytic activity of CK increased about twofold at the initial state of the reaction when CK enzyme was added to a mixture of plasminogen and substrate (H-D-Val-Leu-Lys-pNA). A similar result was also obtained from fibrin plate analysis.     

Generation of fusion genes carrying drug resistance, green fluorescent protein, and herpes simplex virus thymidine kinase genes in a single cistron

We generated new fusion genes carrying positive- and negative-selection markers, and a reporter gene in a single reading frame. The new genes were constructed by sequentially linking the coding sequences of drug-resistance genes (hygro, or puro), a green fluorescence protein (GFP) gene (gfp), and the thymidine kinase gene (tk). The new synthetic genes (hygro/gfp/tk and puro/ gfp/tk) were inserted into retroviral vectors to test their usefulness as selective markers and reporters. The genes were functional in a positive selection in the presence of hygromycin (hygro/gfp/tk) or puromycin (puro/gfp/ tk). In addition, cells expressing the new fusion genes were clearly identifiable by their green fluorescence emitted from GFP. At the same time, these cells were sensitive to a gancyclovir treatment, allowing efficient removal of the transduced cells. The presently described synthetic genes will be valuable tools in both gene therapy and basic gene transfer studies, where positive selection of the transduced cells, monitoring gene expression, and negative selection of the transduced cells are simultaneously required.     

ORE9, an F-box protein that regulates leaf senescence in Arabidopsis

Senescence is a sequence of biochemical and physiological events that constitute the final stage of development. The identification of genes that alter senescence has practical value and is helpful in revealing pathways that influence senescence. However, the genetic mechanisms of senescence are largely unknown. The leaf of the oresara9 (ore9) mutant of Arabidopsis exhibits increased longevity during age-dependent natural senescence by delaying the onset of various senescence symptoms. It also displays delayed senescence symptoms during hormone-modulated senescence. Map-based cloning of ORE9 identified a 693-amino acid polypeptide containing an F-box motif and 18 leucine-rich repeats. The F-box motif of ORE9 interacts with ASK1 (Arabidopsis Skp1-like 1), a component of the plant SCF complex. These results suggest that ORE9 functions to limit leaf longevity by removing, through ubiquitin-dependent proteolysis, target proteins that are required to delay the leaf senescence program in Arabidopsis.