中药固真方对一些与细胞增殖有关基因表达的影响

 中药固真方对一些与细胞增殖有关基因表达的影响姚明忠,顾文聪,丁卫,韩志芬,杜国光（上海中医药大学生物化学教研室,上海２０００３２）（北京医科大学生物化学与分子生物学系,北京１０００８３）中药固真方（ＶＲＦ）具有补肾益精、延缓衰老的作用［１］．能提高成...     

Forskolin对成骨样细胞蛋白激酶C及三磷酸肌醇的影响

Ｆｏｒｓｋｏｌｉｎ（ＦＳＫ）是一种植物二萜类化合物,为腺苷酸环化酶的特异激活剂,实验发现：ＦＳＫ和作为参照的诱导分化剂维甲酸（ＲＡ）单独或联合应用均可升高胞浆蛋白激酶Ｃ（ＰＫＣ）活性,并降低膜ＰＫＣ活性,ＦＳＫ可使表皮生长因子（ＥＧＦ）诱导的细胞内三磷酸肌醇（ＩＰ３－１,４,５）水平降低至对照组的４４．４％至６７％;ＦＳＫ与ＲＡ合用可显著降低成骨样细胞特征蛋白碱性磷酸酶（ＡＫＰ）的活性。以上结果表明,ＦＳＫ对成骨样细胞内磷脂酰肌醇信息传递体系有深刻影响,可能与其调节细胞的增殖分化有关。     

大鼠不同脑区突触体钙水平的年龄差异

本实验使用荧光指示剂Ｆｕｒａ－２与Ｔｂ~（３＋）,检测了不同年龄组大鼠的不同脑区（海马、皮层、间脑、小脑）突触体内游离钙与膜结合钙水平。结果显示,与青年对照组相比,老年大鼠大部分脑区（海马、皮层、间脑）突触体内游离钙水平显著增高,尤其是海马突触体内游离钙增高极为显著;其突触体膜结合钙水平表现为：海马、小脑两脑区明显升高,而皮层、间脑两脑区明显下降,呈现一种全脑范围内的钙水平失衡。提示动物的衰老与其脑内钙自体平衡失调有关。     

The 3-D structure of a zinc metallo-beta-lactamase from Bacillus cereus reveals a new type of protein fold.

The 3-D structure of Bacillus cereus (569/H/9) beta-lactamase (EC 3.5.2.6), which catalyses the hydrolysis of nearly all beta-lactams, has been solved at 2.5 A resolution by the multiple isomorphous replacement method, with density modification and phase combination, from crystals of the native protein and of a specially designed mutant (T97C). The current model includes 212 of the 227 amino acid residues, the zinc ion and 10 water molecules. The protein is folded into a beta beta sandwich with helices on each external face. To our knowledge, this fold has never been observed. An approximate internal molecular symmetry is found, with a 2-fold axis passing roughly through the zinc ion and suggesting a possible gene duplication. The active site is located at one edge of the beta beta sandwich and near the N-terminal end of a helix. The zinc ion is coordinated by three histidine residues (86, 88 and 149) and a water molecule. A sequence comparison of the relevant metallo-beta-lactamases, based on this protein structure, highlights a few well-conserved amino acid residues. The structure shows that most of these residues are in the active site. Among these, aspartic acid 90 and histidine 210 participate in a proposed catalytic mechanism for beta-lactam hydrolysis.     

A role for cAMP in angiotensin II mediated inhibition of cell growth in AT1A receptor-transfected CHO-K1 cells

G-protein coupled Angiotensin II receptors (AT_1A), mediate cellular responses through multiple signal transduction pathways. In AT_1A receptor-transfected CHO-K1 cells (T3CHO/AT_1A), angiotensin II (AII) stimulated a dose-dependent (EC₅₀=3.3 nM) increase in cAMP accumulation, which was inhibited by the selective AT₁, nonpeptide receptor antagonist EXP3174. Activation of protein kinase C, or increasing intracellular Ca²⁺ with ATP, the calcium ionophore A23187 or ionomycin failed to stimulate cAMP accumulation. Thus, AII-induced cAMP accumulation was not secondary to activation of a protein kinase C- or Ca²⁺/calmodulin-dependent pathway. Since cAMP has an established role in cellular growth responses, we investigated the effect of the AII-mediated increase in cAMP on cell number and [³H]thymidine incorporation in T3CHOA/AT_1A cells. AII (1 M) significantly inhibited cell number (51% at 96 h) and [³H]thymidine incorporation (68% at 24 h) compared to vehicle controls. These effects were blocked by EXP3174, confirming that these responses were mediated through the AT₁ receptor. Forskolin (10 M) and the cAMP analog dibutyryl-cAMP (1 mM) also inhibited [³H]thymidine incorporation by 55 and 25% respectively. We extended our investigation on the effect of AII-stimulated increases in cAMP, to determine the role for established growth related signaling events, i.e., mitogen-activated protein kinase activity and tyrosine phosphorylation of cellular proteins. AII-stimulated mitogen-activated protein kinase activity and phosphorylation of the 42 and 44 kD forms. These events were unaffected by forskolin stimulated increases in cAMP, thus the AII-stimulated mitogen-activated protein kinase activity was independent of cAMP in these cells. AII also stimulated tyrosine phosphorylation of a number of cellular proteins in T3CHO/AT_1A cells, in particular a 127 kD protein. The phosphorylation of the 127 kD protein was transient, reaching a maximum at 1 min, and returning to basal levels within 10 min. The dephosphorylation of this protein was blocked by a selective inhibitor of cAMP dependent protein kinase A, H89-dihydrochloride and preexposure to forskolin prevented the AII-induced transient tyrosine phosphorylation of the 127 kD protein. These data suggest that cAMP, and therefore protein kinase A can contribute to AII-mediated growth inhibition by stimulating the dephosphorylation of substrates that are tyrosine phosphorylated in response to AII.     

氯喹导致大鼠肌组织载脂蛋白E大量表达

载脂蛋白E(ApoE)与迟发的家族性及孤发性阿尔茨海默(Alzheimer)病密切相关. 氯喹慢性中毒可诱发某些肌病理改变, 出现β淀粉样蛋白(βAP)与tau蛋白等的沉积, 与Alzheimer脑中见到的病理改变类似. 为分析这一改变的机制, 用逆转录结合多聚酶链反应技术(RT-PCR)对氯喹处理的大鼠肌肉中ApoE表达的改变进行了研究. 在PCR定量中采用了一种稳定表达的内源性甘油醛-3-磷酸脱氢酶mRNA作为内部参照. PCR扩增在很宽的循环数范围内成线性, 且靶mRNA与参照mRNA的扩增效率相当. 氯喹处理后大鼠肌肉中ApoE mRNA的表达从第6周开始增加, 第8周后超过对照组的20多倍. 结果提示, ApoE在氯喹慢性中毒所致的大鼠肌病理改变中发挥某些作用.     

A role for natural simian immunodeficiency virus and human immunodeficiency virus type 1 nef alleles in lymphocyte activation.

A T-lymphoid cell line termed 221 was derived from a rhesus monkey infected with herpesvirus saimiri. Growth of 221 cells was dependent on the addition of interleukin-2 (IL-2) to the culture medium. In the absence of IL-2, 221 cells arrested in G0-G1 but did not die. Simian immunodeficiency virus (SIV) replicated efficiently in IL-2-stimulated 221 cells whether or not the nef gene was present. In the absence of IL-2, nef-containing SIV replicated 8 to 100 times more efficiently in 221 cells than did the same virus lacking nef. nef-containing virus preferentially stimulated the production of IL-2 from 221 cells. HIV-1 nef and v-ras genes, but not the c-ras gene, were shown to substitute functionally for SIV nef when tested as recombinant viruses in this assay system. These results demonstrate a role for natural nef in causing lymphoid cell activation, and they provide a system for delineating the biochemical mechanisms responsible for this activation.