G蛋白Rab3a协同神经生长抑制因子(GIF)抑制神经元的生长

为了研究G蛋白Rab3a与神经生长抑制因子 (growthinhibitoryfactor,GIF ,原称金属硫蛋白 3,MT 3)相互作用对神经元细胞生长的影响 ,以嗜铬细胞瘤株 (pheochromocytoma)PC1 2充当神经元模型 .将hMT 3(humanMT 3)和Rab3a基因分别克隆至真核表达载体pFlag CMV 2和pSV HA中 ,质粒共同转染PC1 2细胞 ,观察转染后细胞的生长状态 .以共转染pFlag CMV 2 hMT 1和pSV HA Rab3a的细胞组作为对照 ,验证hMT 3与Rab3a相互作用对PC1 2影响的特异性 .结果发现 ,共转染pFlag CMV 2 hMT 3和pSV HA Rab3a的PC1 2细胞生长明显受到抑制 ,细胞生长抑制率与GIF在脑提取物存在F的神经元生长抑制作用接近 ,但转染两基因中的任何单个基因以及共转染pFlag CMV 2 hMT 1和pSV HA Rab3a对PC1 2细胞生长无影响 .进一步构建重组表达质粒pGEX 4T 1 Rab3a和pGEX 4T 1 hMT 3,转化大肠杆菌BL2 1 ,经谷胱甘肽 Sepharose 4B亲和层析、凝血酶酶切和SephacrylS 1 0 0纯化 ,得到纯度 95 %以上的Rab3a和hMT 3蛋白 .体外细胞生物学活性检测表明 ,表达的Rab3a蛋白与重组hMT 3蛋白共培养PC1 2 ,对细胞的生长产生了明显的特异性协同抑制作用 ,抑制曲线与GIF在脑提取物存在下的神经元生长抑制曲线极为相似     

Identification and Characterization of a Chitin-binding Protein Purified from Coelomic Fluid of the Lugworm Arenicola marina Defining a Novel Protein Sequence Family

We have isolated a novel type of lectin named Arenicola marina lectin-1 (AML-1) from the lugworm A. marina. The lectin was purified from the coelomic fluid by affinity chromatography on a GlcNAc-derivatized column and eluted with GlcNAc. On SDS-PAGE, AML-1 showed an apparent molecular mass of 27 and 31 kDa in the reduced state. The N-terminal amino acid sequences were identical in these two bands. In the unreduced state, a complex band pattern was observed with bands from 35 kDa to more than 200 kDa. Two different full-length clones encoding polypeptides of 241 and 243 amino acids, respectively, were isolated from a coelomocyte cDNA library. The two clones, designated AML-1a and AML-1b, were 92% identical at the protein level and represent a novel type of protein sequence family. Purified AML-1 induced agglutination of rabbit erythrocytes, which could be inhibited by N-acetylated saccharides. Recombinant AML-1b showed the same band pattern as the native protein, whereas recombinant AML-1a in the reduced state lacked a 27 kDa band. AML-1b bound GlcNAc-derivatized columns and chitin, whereas AML-1a did not bind to these matrices. Immunohistochemical analysis revealed that AML-1 is expressed by coelomocytes in the nephridium and in round cells in the epidermis and in eggs. Moreover, AML-1 expression was up-regulated in response to a parasitic infection. We conclude that AML-1 purified from coelomic fluid is encoded by AML-1b and represents a novel type of protein family that binds acetylated components.     

Expression of the glucose transporter isoform GLUT 4 is insufficient to confer insulin-regulatable hexose uptake to cultured muscle cells.

Glucose transporter isoform expression was studied in the skeletal muscle-like cell line, C2C12. Northern and Western blot analysis showed that the insulin-responsive muscle/fat glucose transporter isoform, GLUT 4, was expressed in these cells at very low levels, whereas the erythrocyte isoform, GLUT 1, was expressed at readily detectable levels. Insulin did not stimulate glucose transport in this cultured muscle cell line. The C2C12 cells were then transfected separately with either GLUT 1 or GLUT 4, and stable cell lines expressing high levels of mRNA and protein were isolated. GLUT 1-transfected cells exhibited a 3-fold increase in the amount of the GLUT 1 transporter protein which was accompanied by a 2- to 3-fold increase in the glucose uptake rate. However, despite at least a 10-fold increase in GLUT 4 mRNA and protein detected after GLUT 4 cDNA transfection, the glucose uptake of these cells was unchanged and remained insulin-insensitive. By laser confocal immunofluorescence imaging, it was established that the transfected GLUT 4 protein was localized almost entirely in cytoplasmic compartments. In contrast, the GLUT 1 isoform was detected both at the plasma membrane as well as in intracellular compartments. These results suggest that acute insulin stimulation of glucose transport is not solely dependent on the presence of the insulin receptor and the GLUT 4 protein, and that the presence of some additional protein(s) must be required.     

Granulocyte-macrophage colony stimulating factor and interleukin-3 regulate the production of an interleukin-1 inhibitor by the differentiated AML-193 leukemic cell line

Treatment of the AML-193 leukemic cell line with phorbol myristate acetate (PMA) resulted in the loss of their ability to proliferate in response to GM-CSF or IL-3. This was not due to a change in number or affinity of GM-CSF receptors, but possibly resulted from an other cellular mechanism. The AML-193 differentiated cells acquired the ability to phagocytose glutaraldehyde-fixed E.coli in a similar fashion to mature macrophages. In addition the PMA-differentiated AML-193 cells now secreted a factor which specifically inhibited the binding of interleukin-1 (IL-1) to its receptor on the murine thymoma cell line EL-4.6.1C10. The synthesis of this inhibitor was further increased by the addition of GM-CSF or IL-3. Pulse labelling experiments showed that this activity was due to a 26 kDa protein that bound to the IL-1 receptor even in the presence of neutralizing antibodies against IL-1 alpha or IL-1 beta, and this binding was only antagonized by IL-1 alpha or IL-1 beta. In contrast, peripheral monocytes obtained from the blood of normal donors, when induced with either GM-CSF or IL-3, produced large quantities of inhibitor in the absence of PMA. This report clearly shows that a leukaemic cell line can respond to GM-CSF and IL-3 in different ways before and after in vitro differentiation.     

Antioxidant Response to NaCl Stress in a Control and an NaCl-Tolerant Cotton Cell Line Grown in the Presence of Paraquat,Buthionine Sulfoximine,and Exogenous Glutathione

A cotton (Gossypium hirsutum L.) control and NaCl-tolerant cell line (cv Coker 312) were grown on media with or without NaCl in the presence or absence of paraquat, buthionine sulfoximine, and oxidized glutathione. On medium with 150 mM NaCl the NaCl-tolerant cell line exhibited no reduction in growth, whereas a 96% reduction was observed in the control line. The NaCl-tolerant cell line that was grown on 150 mM NaCl exhibited significantly greater catalase (341%), peroxidase (319%), glutathione reductase (287%), ascorbate peroxidase (450%), [gamma]-glutamylcysteine synthetase (224%), and glutathione S-transferase (500%) activities than the intolerant control. The NaCl-tolerant cell line had a significantly lower dehydroascorbic acid/ascorbic acid ratio. Paraquat reduced growth by 20 and 53.7%, respectively, in the NaCl-tolerant and control cell line. The NaCl-tolerant cell line also showed a slight tolerance to buthionine sulfoximine. In the buthionine sulfoximine experiments reduced glutathione restored growth in both cell lines, whereas oxidized glutathione restored growth only in the NaCl-tolerant cell line. These data indicate that the NaCl-tolerant cell line exhibited a cross-tolerance to a variety of stress variables and had a more active ascorbate-glutathione cycle.     

The role of voltage gated T-type Ca2+ channel isoforms in mediating "capacitative" Ca2+ entry in cancer cells

The mechanism by which Ca2+ enters electrically non-excitable cells is unclear. The sensitivity of the Ca2+ entry pathway in electrically non-excitable cells to inhibition by extracellular Ni2+ was used to direct the synthesis of a library of simple, novel compounds. These novel compounds inhibit Ca2+ entry into and, consequently, proliferation of several cancer cell lines. They showed stereoselective inhibition of proliferation and Ca2+ influx with identical stereoselective inhibition of heterologously expressed Cav3.2 isoform of T-type Ca2+ channels. Proliferation of human embryonic kidney (HEK)293 cells transfected with the Cav3.2 Ca2+ channel was also blocked. Cancer cell lines sensitive to our compounds express message for the Cav3.2 T-type Ca2+ channel isoform, its delta25B splice variant, or both, while a cell line resistant to our compounds does not. These observations raise the possibility that clinically useful drugs can be designed based upon the ability to block these Ca2+ channels.     

Cytotoxicity with target DNA breakdown by rat basophilic leukemia cells expressing both cytolysin and granzyme A.

The noncytotoxic rat mast cell tumor line RBL was transfected with genes for the cytotoxic lymphocyte granule proteins cytolysin (perforin) and granzyme A, giving transfectants with mRNA and protein expression levels comparable with cloned cytotoxic T lymphocytes. Both RBL-cytolysin and RBL-cytolysin-granzyme A transfectants showed extremely potent killing of red cell targets and lysed 20%-60% of EL4 lymphoma targets at an effector-to-target ratio of 30. RBL transfectants expressing only granzyme A were not cytotoxic. Significant EL4 DNA breakdown accompanying lysis was observed only with RBL that was transfected with both cytolysin and granzyme A. These results support the granule-exocytosis model for lymphocyte cytotoxicity and show that effector granzyme A plays a role in target cell DNA breakdown.     

Double-edged sword of chemosensitizer: increase of multidrug resistance protein (MRP) in leukemic cells by an MRP inhibitor probenecid

The multidrug resistance protein (MRP) is a drug efflux membrane pump conferring multidrug resistance to tumor cells. Clinical trials have been undertaken to improve the effectiveness of chemotherapy by adding an MRP inhibitor to the treatment regimen. This study attempted not only to determine novel resistance mechanisms in MRP-overexpressing AML cells (AML-2/DX100) by chronic exposure to doxorubicin in the presence of an MRP inhibitor probenecid but also to find out whether probenecid could increase MRP levels. AML-2/DXPBA cultured in the presence of probenecid (600 microM) and doxorubicin (100 ng/ml) showed a higher level of the multidrug resistance (MDR) phenotype when compared to AML-2/DX100. AML-2/DXPBA showed increased levels of MRP compared to those of AML-2/DX100. Probenecid increased the MRP levels without an increase in MRP mRNA in AML-2/WT in both a time- and dose-dependent manner. Of the MRP inhibitors including probenecid, ofloxacin, erythromycin, and rifampicin used in this study, only probenecid showed a marked chemosensitizing effect in AML-2/DX100 but not in HL-60/Adr, suggesting that the chemosensitizing effects of the MRP inhibitors vary according to the type of resistant cells. The maximum noncytotoxic concentrations of these MRP inhibitors increased the MRP levels to various degrees in both AML-2/WT and HL-60/WT. However, the chemosensitizing effects of the MRP inhibitors were not correlated with their MRP-increasing effects. Altogether, MRP inhibitors such as probenecid have been shown to function as a double-edged sword, indicating that they are not only an effective chemosensitizer of MRP-associated MDR tumor cells but also an MRP activator. Therefore caution should be taken whenever using MRP inhibitors to reverse MRP-mediated multidrug resistance in clinical cancer chemotherapy as well as when used to inhibit MRP expression in vitro.     

Advantages of the doxorubicin-resistant, acute myelogenous leukemia cell line, AML-2/DX100, for the screening of multidrug resistance protein inhibitors and pro-oxidants

The doxorubicin-resistant, acute myelogenous leukemia cell line, AML-2/DX100, characterized by the over-expression of multidrug resistance protein (MRP) and the down-regulation of catalase, has advantages for the screening of MRP inhibitors as well as for cytotoxic substances producing potential reactive oxygen species. The screening power of AML-2/DX100 cells for an MRP inhibitor, probenecid, was approximately 4-fold stronger than that of another resistant cell line, HL-60/Adr, over-expressing MRP. AML-2/DX100 was approximately 2- to 5-fold more sensitive to pro-oxidants such as Paraquat, H₂O₂ and t-butyl hydroperoxide, when compared with its parental cells.     

Effect of leptin in proliferating and differentiated HC11 mouse mammary cells

Leptin and its receptors have been shown to be expressed in several tissues thus suggesting that this protein might be effective not only at the CNS level, but also peripherically. We demonstrated by RT-PCR analysis that leptin and its long isoform receptor are expressed in the mouse mammary epithelial cell line HC11, an in vitro cell model considered suitable to study the regulation of the functional development of the mammary epithelium. Furthermore, leptin secretion by HC11 cells was demonstrated by heterologous ELISA. Neither mRNA expression nor protein secretion changed throughout the different phases of differentiation of the cell line. Receptor mRNA was not modified when cells were induced to express beta-casein. High concentrations of leptin (between 1.5 and 15 microM) significantly (p<0.05) reduced cell growth as measured by MTT test. HC11 cells were transfected with pbetacCAT, a chimeric rat-beta casein gene promoter-CAT gene construct and CAT ELISA was used to determine gene expression. Leptin, from 1.5 nM to 15 microM, was shown to positively (p<0.05) influence beta-casein expression both in the presence or in the absence of prolactin. These data provide evidence that leptin, through its receptor, may be an important mediator in regulating mammary gland growth and development.     

Paraquat resistance associated with reduced NADPH reductase in an energy-dependent paraquat-accumulating cell line

Despite intensive investigation into paraquat toxicity, neither the final cytotoxic mechanism nor a clinically useful antidote has been discovered. In vitro screening of potential antidotes that act by blocking paraquat uptake requires a cell line that accumulates paraquat by an energy-dependent mechanism. We screened various lymphoblastoid cell lines until we found a line accumulating paraquat by an energy-dependent mechanism. During study of this cell line, a marked resistance to paraquat developed in a clone. The resistance was associated with a reduction in NADPH reductase activity, confirming the original report (using microsomal preparations) that intracellular reduction of paraquat occurs primarily by this enzyme. One-half of the NADPH-P450 reductase activity, as well as one-half of the NADPH-dependent paraquat-inducible superoxide production, was decreased. This suggests that the decrease is secondary to a genetic alteration in one of the genes encoding for the enzyme. Other antioxidant enzymes and proteins were not affected. Despite the loss of only 50% of the activity, the relative resistance to paraquat exceeded previous reports involving marked increases in antioxidant enzymes. Most exogenous enhancers or inhibitors alter the activity of more than one enzyme, thereby making selective changes in any one enzyme difficult. Thus, this cell line will be useful for studying other toxins where the involvement of NADPH reductase is suspected, but not proven.     

Drug concentration-dependent expression of multidrug resistance-associated protein and P-glycoprotein in the doxorubicin-resistant acute myelogenous leukemia sublines.

The multidrug resistance of cancer cells can be mediated by an overexpression of the human MDR1 and MRP genes, which encode the transmembrane efflux pumps, the 170 kDa P-glycoprotein (Pgp) and the 190 kDa multidrug resistance-associated protein (MRP), respectively. In this study, we investigate which protein is preferentially overexpressed in the function of doxorubicin concentrations in the acute myelogenous leukemia cell line (OCI/AML-2). Multidrug-resistant AML-2 sublines were isolated in doxorubicin concentrations of 20, 100, 250, and 500 ng/ml. MRP was at first expressed at low concentrations of less than 5 x IC50 (100 ng/ml) of doxorubicin followed by the overexpression of Pgp with concentrations of more than 12.5 x IC50 (250 ng/ml) of doxorubicin. In addition, it appeared that increased amounts of MRP and its mRNA in AML-2/DX20 and /DX100 decreased gradually in both AML-2/DX250 and /DX500 overexpressing Pgp. In conclusion, it is thought that the overexpression of MRP or Pgp is dependent upon drug concentrations. It could be implicated that the overexpression of MRP might be negatively related to that of Pgp.     

Dimerization of two novel apoptosis-inducing proteins and its function in regulating cell apoptosis

Apoptosis (or programmed cell death) was firstly described by Kerr[1] in 1972. Since bcl-2 cDNA was cloned by Cleary et al.[2] in 1986, many apoptosis-related genes have been found in human or mammalian cell lines. The bcl-2 family[35] containing 23 genes, the caspase family[68] bearing 14 members and the TNF family[9,10] are the most clearly elucidated ones. With the study of apoptosis going deeper, people have realized that cell apoptosis is impor-tant in development and homeostasis of mu…     

Prohormone convertase-1 is essential for conversion of chromogranin A to pancreastatin.

The purpose of this study was to test the hypothesis that the endoprotease, prohormone convertase-1 (PC-1), is involved in the processing of the precursor protein chromogranin A (CGA) to a smaller peptide called pancreastatin (PST). A human pancreatic carcinoid cell line (BON) that expresses PC-1, CGA and PST was stably transfected with antisense PC-1 mRNA. BON cells expressing antisense PC-1 mRNA showed nearly complete abolishment of PC-1 protein (approximately 95% reduction) and an 80% reduction in cell content of PST immunoreactivity (PST-IR) as assessed by high-performance liquid chromatography in combination with measurement of PST-IR. These findings indicate that PC-1 is essential for processing CGA to PST.     

siRNA对乳腺癌细胞Cyclin E表达和生长抑制作用

研究siRNA对乳腺癌MCF-7细胞株cyclin E表达的抑制及对细胞生长的影响。化学合成针对cyclin E基因的小干扰RNA（siRNA）,转染MCF-7细胞株;分别应用荧光定量PCR和免疫印迹测定cyclin E mRNA和蛋白质的表达,CCK-8测定细胞的增殖活性,流式细胞仪检测细胞周期,软琼脂培养检测细胞克隆形成能力。10、50、100nmol／L siRNA-cyclin E分别使MCF-7细胞cyclin E基因表达降低了24．7％、62．5％和71．0％,蛋白质表达降低了40．8％、66．5％和71．3％。转染siRNA-cyclin E后,G1期细胞增多,S期减少,增殖受到抑制,软琼脂克隆形成率降低。结果提示,在MCF-7细胞株中,导入针对cyclin E的siRNA,可有效抑制cyclin E的表达,进而使细胞增殖减缓,逆转其恶性表型。