p38 Mitogen-activated protein kinases is required for counteraction of 2-methoxyestradiol to estradiol-stimulated cell proliferation and induction of apoptosis in ovarian carcinoma cells via phosphorylation Bcl-2

INTRODUCTION: 2-Methoxyestradiol (2ME2), a natural endogenous product of estradiol (E2) metabolism, has been shown to be a selective apoptotic agent for cancer cells but not for normal cells. In this study, we determined that 2ME2 counteracts E2-stimulated cell growth and induces apoptosis in ovarian carcinoma cells. In addition, we demonstrate that 2ME2 induces apoptosis via p38 and phospho-Bcl2 pathway. METHODS: 2ME2 and/or E2 were administered to the OVCAR-3 (human ovarian cancer) cell line. Cell growth inhibition was analyzed by [3H] Thymidine incorporation assay and DNA fluorometric assay. Cell apoptosis was tested by DNA fragmentation analysis and FACS. The signaling pathway was determined by a series of biochemical assays. RESULTS: 2ME2 inhibited estradiol-stimulated cell growth and induced apoptosis in an ovarian carcinoma cell line. MAPK and p38, but not JNK, were found to be critical mediators in this process. Expression of a dominant negative mutant of p38 kinase or p38 specific inhibitor, SB 203580, almost completely blocked the process. Furthermore, Bcl-2 phosphorylation was required for 2ME2-induced effects. CONCLUSION: Our data suggest that 2ME2 inhibits E2-stimulated proliferation and induces apoptosis in ovarian carcinoma cells. Furthermore, activation of p38 and phosphorylation of Bcl-2 plays a critical role in the mechanism. 2ME2 therefore, may have a clinical application for the treatment of ovarian cancer.     

Comparing the whole-genome-shotgun and map-based sequences of the rice genome

The rice genome has now been sequenced using whole-genome-shotgun and map-based methods. The relative merits of the two methods are the subject of debate, as they were in the human genome project. In this Opinion article, we will show that the serious discrepancies between the resultant sequences are mostly found in the large transposable elements such as copia and gypsy that populate the intergenic regions of plant genomes. Differences in published gene counts and polymorphism rates are similarly resolved by considering how transposable elements affect the sequence analysis.     

Selective affinity chromatography with calmodulin fragments coupled to sepharose

Calmodulin tryptic fragments 78-148, 107-148, and 1-77 coupled to Sepharose 4B were used to test the ability of different calmodulin-regulated enzymes to recognize different domains of calmodulin. Fragment 107-148, which contains a single Ca2+-binding domain, does not interact with any of the calmodulin binding proteins. Fragments 1-77 and 78-148, each of which contains two Ca2+-binding domains, have preserved their ability to interact with several calmodulin-dependent enzymes. Most of the calmodulin-regulated enzymes in brain extracts, such as cAMP phosphodiesterase, cAMP-dependent protein kinase, and the calmodulin-stimulated protein phosphatase (calcineurin) interact with fragment 78-148 in a Ca2+-dependent fashion. An ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-sensitive, calmodulin-independent, p-nitrophenyl phosphatase does not bind to the affinity column and is resolved from calcineurin at this step. Although calmodulin-stimulated protein kinase(s) can interact with fragment 78-148, their interaction is prevented by increased ionic strength even in the presence of Ca2+. Fragment 1-77 exhibits a higher degree of selectivity than fragment 78-148. Only cAMP-dependent protein kinase and cAMP phosphodiesterase bind to fragment 1-77. These results confirm the multiple modes of interaction of calmodulin with its target proteins and provide the basis for a selective purification of calmodulin-regulated enzymes by affinity chromatography on specific calmodulin fragments coupled to Sepharose.     

Cleavage of nucleotides by Ce4+ and the lanthanide metal complexes

The cleavage of adenosine-5-monophosphate (5-AMP) and guanosine-5-monophosphate (5-GMP) by Ce⁴⁺ and lanthanide complex of 2-carboxyethylgermanium sesquioxide (Ge-132) in acidic and near neutral conditions was investigated by NMR , HPLC and measuring the liberated inorganic phosphate at 37°C and 50°C. The results showed that 5-GMP and 5-AMP was converted to guanine (G), 5-monophosphate (depurination of 5-GMP), ribose (depurination and dephosphorylation of 5-GMP), phosphate and adenine (A), 5-monophosphate (depurination of 5-AMP), ribose (depurination and dephosphorylation of 5-AMP), phosphate respectively by Ce⁴⁺. In presence of lanthanide complexes, 5-GMP and 5-AMP were converted to guanosine (Guo) and phosphate and adenosine (Ado) and phosphate respectively. The mechanism of cleaving 5-GMP and 5-AMP is hydrolytic scission     

Identification of a novel activation-inducible protein of the tumor necrosis factor receptor superfamily and its ligand

Among members of the tumor necrosis factor receptor (TNFR) superfamily, 4-1BB, CD27, and glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) share a striking homology in the cytoplasmic domain. Here we report the identification of a new member, activation-inducible TNFR family member (AITR), which belongs to this subfamily, and its ligand. The receptor is expressed in lymph node and peripheral blood leukocytes, and its expression is up-regulated in human peripheral mononuclear cells mainly after stimulation with anti-CD3/CD28 monoclonal antibodies or phorbol 12-myristate 13-acetate/ionomycin. AITR associates with TRAF1 (TNF receptor-associated factor 1), TRAF2, and TRAF3, and induces nuclear factor (NF)-kappaB activation via TRAF2. The ligand for AITR (AITRL) was found to be an undescribed member of the TNF family, which is expressed in endothelial cells. Thus, AITR and AITRL seem to be important for interactions between activated T lymphocytes and endothelial cells.     

Identification and characterization of a novel siglec, siglec-7, expressed by human natural killer cells and monocytes

We describe the characterization of sialic acid-binding Ig-like lectin-7 (siglec-7), a novel member of the siglec subgroup of the immunoglobulin superfamily. A full-length cDNA encoding siglec-7 was isolated from a human primary dendritic cell cDNA library. Siglec-7 is predicted to contain three extracellular immunoglobulin-like domains that comprise an N-terminal V-set domain and two C2-set domains, a transmembrane region and a cytoplasmic tail containing two tyrosine residues embodied in immunoreceptor tyrosine-based inhibition motif-like motifs. Overall, siglec-7 exhibited a high degree of sequence similarity to genes encoding CD33 (siglec-3), siglec-5, OBBP1/siglec-6, and OBBP-like protein and mapped to the same region on chromosome 19q13.3. When siglec-7 was expressed on COS or Chinese hamster ovary cells, it was able to mediate high levels of sialic acid-dependent binding to human erythrocytes and soluble sialoglycoconjugates, suggesting that it may be involved in cell-cell interactions. Among human peripheral blood leukocytes, siglec-7 was found to be present at low levels on granulocytes, intermediate levels on monocytes, and relatively high levels on a major subset of natural killer cells and a minor subset of CD8(+) T cells. Immunoprecipitation experiments indicated that siglec-7 is expressed as a monomer of approximately 65 kDa.     

Concentration of serum lipids and aortic lesion size in female and male apo E-deficient mice fed docosahexaenoic acid

Apolipoprotein (apo) E-deficient mice were fed an atherogenic diet with either 1% ethyl ester docosahexaenoic acid (DHA) or safflower oil (SO) as a source of linoleic acid for 8 week. Both genders fed DHA had higher proportions of eicosapentaenoic acid and DHA, and lower proportions of linoleic and arachidonic acids in the liver and serum phospholipids than those fed SO. Males fed DHA had greater liver weight and tended to have higher concentrations of serum lipids and liver cholesterol than those fed SO, and there were opposite trends in females. Dietary fats and gender led to no significant effect on lesion sizes in aortic arch and thoracic plus abdominal aorta. These results indicate that the interactive action of sex-related factor(s) with dietary polyunsaturated fatty acids is involved in metabolic changes of serum lipids in apoE-deficient mice, and addition of DHA, compared with addition of SO, is not effective to abolish the atherosclerosis in this animal model.     

抑制素α亚基片段P33对大鼠离体培养黄体细胞凋亡的影响

我室先前的工作表明,抑制素α亚基片段Ｐ３３显著抑制离体培养大鼠黄体细胞的孕酮分泌,整体实验显示Ｐ３３促进黄体功能萎缩和细胞凋亡。本实验进一步在细胞水平探讨Ｐ３３促进黄体细胞凋亡的作用机制。应用ＤＮＡ电泳检测技术、ＤＮＡ荧光（ＡＯＥＢＰＩ）染色和流式细胞分析方法观察了Ｐ３３对ＰＭＳＧｈＣＧ假孕大鼠胶原酶ＤＮＡ酶分散的黄体细胞的自发凋亡的影响。结果三种方法一致显示,Ｐ３３（１μｇ／ｍｌ）促进黄体细胞的自发凋亡。阻断酪氨酸蛋白激酶活性（ｇｅｎｉｓｔｅｉｎ５０μｇ）则抑制Ｐ３３诱导的黄体凋亡;而阻断ＲＮＡ和蛋白质合成（Ｃｙｘ,５０μｇ／ｍｌ;ＡｃｔＤ,５０μｇ／ｍｌ）均不抑制Ｐ３３促进的黄体细胞凋亡。结果表明,Ｐ３３促进培养大鼠黄体细胞的自发凋亡,其作用机制可能与ＴＰＫ途径有关。本实验为抑制素α亚单位或其相关衍生物可能是卵巢局部调节因子之一的假说提供了又一证据。