抗t-PA单克隆抗体的制备及初步应用

用猪心t-PA和人黑色素瘤细胞分泌的t-PA作抗原,经腹腔及脾内免疫Bal b/c小鼠。用细胞融合技术制备杂交瘤细胞,细胞融合串达85％,获得4株抗t-PA杂交瘤细胞株,鼠腹水抗体效价达1∶10~5;Western Blot结果表明该单抗所结合的抗原分子量与t-PA相符,证明所获得的单抗为特异的抗t-PA单抗;对t-PA的纤溶活性中和抑制试验结果表明,4株单抗中的1株能完全抑制st-PA的纤溶活性,另外3株表现出程度不等的抑制;其中1株亚类为IgG,另外3株为IgM。杂交瘤细胞株无支原体污染;染色体数目正常。对该抗体进行初步纯化后,将其应用于rt-PA的研究中。     

Salt Dependence of DNA Structural Stabilities in Solution. Theoretical Predictions Versus Experiments

Abstract

The predictions of currently available theories for treating DNA-diffuse ionic cloud free energy contributions to conformational stability have been tested against experimental data for salt induced B-Z and B-A transitions. The theories considered are (i) Manning's counterion condensation approach (CC), (ii) the idealized Poisson-Boltzmann approximation (PB), and (iii) the potentials of mean force (PMF) approach proposed by Soumpasis. As far as we can judge from comparison with the set of experimental data currently available, it is found that only the latter theory yields satisfactory quantitative results for the dependence of the B-Z and B-A relative stabilities on monovalent salt concentration. The correct application of the PB and CC theories does not yield very low salt Z-B transitions, in contradiction to earlier assertions. At low salt concentrations the PB theory is qualitatively correct in predicting that the B form is electrostatically more favorable than both the A and B forms, whereas the CC theory is qualitatively wrong predicting that Z-DNA is more stable than both B and A DNA.     

Hypoxia-inducible factor 1alpha regulates lung adenocarcinoma cell invasion

We studied the role of hypoxia-inducible factor-1alpha (HIF-1alpha) in human lung adenocarcinoma cell invasion using a metastatic cell model composed of low invasive CL1 and highly invasive CL1-5 cells. We showed that HIF-1alpha was expressed in CL1-5 but not in CL1 cells under normoxic condition, and that inhibition of HIF-1alpha expression by a small interfering RNA decreased invasiveness of CL1-5 cells. Complementary, overexpression of HIF-1alpha increased the invasiveness of CL1 and gastric cancer SC-M1 cells. Subsequently, we showed that urokinase-type plasminogen activator receptor (uPAR), and matrix metalloproteinases (MMPs) 1 and 2 were critical in HIF-1alpha-induced invasion. Mechanistic studies revealed that HIF-1alpha overexpression could increase the expression of uPAR and MMP1, but not MMP2. However, ELISA assays on the conditioned media generated from control CL1 and CL1 cells overexpressing HIF-1alpha showed that overexpression of HIF-1alpha increased the levels of endogenous free active MMP2 and total free MMP2, and the former was blocked by inhibition of MMP1 expression. We conclude that (i) HIF-1alpha overexpression enhances lung cancer cell invasion at least through up-regulating the expression and activities of uPAR, MMP1, and MMP2; and (ii) induction of MMP1 participates in cell invasion and also plays an important role in HIF-1alpha-induced activation of MMP2.     

Plasminogen activator/plasmin system regulates formation of the hepatocyte spheroids

The isolated rat hepatocytes inoculated onto the surface of positively charged culture dishes are anchored initially and then begin to migrate and aggregate gradually to form multicellular spheroids detached from the dish. We studied the roles of fibrinolytic factors in the spheroid formation. The fibrinolytic factors, tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA), were increased in the course of spheroid formation. Then, we introduced fibrinolytic inhibitors into the spheroid cultures to determine functions of fibrinolytic factors. Plasmin inhibitor inhibited markedly the spheroid formation. Interestingly, the anti-plasmin antibody showed different effect depending on the timing of its administration. In summary, we demonstrated for the first time that induction of PAs and ensuing plasmin generation on the cell surface play important roles in hepatocyte spheroid formation, and that plasmin is involved in the different processes such as cell migration and cell detachment in the formation of hepatocyte spheroid.     

Microtubule Severing by Katanin p60 AAA+ ATPase Requires the C-terminal Acidic Tails of Both α- and β-Tubulins and Basic Amino Acid Residues in the AAA+ Ring Pore

The microtubule (MT) network is highly dynamic and undergoes dramatic reorganizations during the cell cycle. Dimers of α- and β-tubulins rapidly polymerize to and depolymerize from the end of MT fibrils in an intrinsic GTP-dependent manner. MT severing by ATP-driven enzymes such as katanin and spastin contributes significantly to microtubule dynamics, and it has been shown that katanin p60, a AAA+ family protein, has ATPase and MT-severing activities. The mechanism of MT severing by katanin p60 is poorly understood, and the residues in katanin p60 and tubulins important for severing activity were therefore explored in this study. MT-severing activity, but not ATPase activity, was inhibited by mutations of the conserved aromatic residue and the flanking basic residues in the pore region of the katanin p60 hexameric ring. When the acidic residue-rich C-terminal unstructured segment of either α- or β-tubulin was removed, polymerized MTs were resistant to katanin p60 treatment. Interactions between katanin p60 and the mutant MTs, on the other hand, were unaffected. Taken together, these findings led us to propose that the interactions between the positively charged residues of katanin p60 and the acidic tails of both tubulins are essential for efficient severing of MTs.     

A rapid, homogeneous, fluorescence polarization binding assay for peroxisome proliferator-activated receptors alpha and gamma using a fluorescein-tagged dual PPARalpha/gamma activator

Peroxisome proliferator-activated receptors (PPARs) and other members of the nuclear hormone receptor family are important drug targets for the treatment of metabolic diseases. PPARalpha and PPARgamma play crucial roles in lipid and glucose metabolism, respectively. Therefore, screening methods that help to rapidly identify activators of these receptors should be of considerable value. A homogeneous fluorescence polarization (FP) ligand binding assay capable of rapidly identifying ligands that bind to both PPARalpha and PPARgamma has been developed using purified PPARalpha or PPARgamma ligand binding domains and a fluorescein-labeled analog (FLA) of a potent dual PPARalpha/gamma activator. FLA activator showed good binding affinity toward both PPARalpha (K(i)=0.7microM) and PPARgamma (K(i)=0.4microM). The binding of FLA activator was rapid and reached a plateau within 10 min. The resulting FP signal was stable for at least 18h. The FP binding assay performed robustly in a 384-well format, and the average Z' value was 0.77. There was a good correlation between the binding potency (IC(50) values) and rank order of binding potency for a panel of standard PPAR ligands obtained in FP binding assay and scintillation proximity assay or gel filtration binding assays using (3)H-labeled PPARalpha (r(2)=0.99) and PPARgamma (r(2)=0.99) ligands. There was also a good correlation of IC(50) values obtained by FP binding assay and scintillation proximity assay for the clinically used PPAR activators. Thus, the FP binding assay with a single fluorescein-labeled PPARalpha/gamma dual activator offers a homogeneous nonradioactive, sensitive, robust, and less expensive high-throughput assay for detecting compounds that bind to both PPARgamma and PPARalpha. Using this FP binding assay, we have identified a large number of PPARalpha/gamma dual activators. A similar assay platform may be easily adapted to other members of the nuclear hormone receptor family.     

Crosstalk of mTOR/PKM2 and STAT3/c-Myc signaling pathways regulate the energy metabolism and acidic microenvironment of gastric cancer

Cancer cells consume large amounts of glucose to produce lactate, even in the presence of ample oxygen. This phenomenon is called the Warburg effect. c-Myc is an important member of the Myc gene family and is involved in the development of various tumors. It plays an important role in the regulation of tumor energy metabolism, which can regulate glycolysis to promote the Warburg effect in a tumor. Our study aimed to improve the malignant biological behavior by controlling the energy metabolism of gastric cancer through the mTOR/PKM2 and signal transduction and activator 3 (STAT3)/c-Myc signaling pathways through a series of in vitro experiments. Human gastric cancer AGS and HGC-27 cells were treated with PKM2 and c-Myc lentivirus, and the effects of the knockdown of PKM2 and/or c-Myc were analyzed on cell proliferation, cell apoptosis, the ability of cell migration, and the growth signaling pathway in vitro. The expressions of PKM2, c-Myc, LDHA, STAT3, P-STAT3, GLUT-1 gene were identified by the quantitative real-time polymerase chain reaction and Western blot analysis. Lactate and glucose levels were tested by the corresponding kit. Our findings showed that PKM2 and c-Myc were upregulated in human gastric cancer. Knockdown of c-Myc in gastric cancer cells suppressed cell proliferation capacity and glycolysis level, and the inhibitory effects on gastric cancer cells upon co-knockdown of PKM2 and c-Myc were more obvious compared with knockout of PKM2 or c-Myc alone. And there was a correlation between the mTOR/PKM2 and the STAT3/c-Myc signaling pathways. Our results suggested that c-Myc might be considered a potential therapeutic target for gastric cancer and PKM2 combined with c-Myc could better inhibit the malignant biological behaviors of gastric cancer.