Transient transfection of mammalian cells with DNA of the plant-pathogenic Ti-plasmid and expression of marker and resident sequences

Summary The large Ti-plasmid from Agrobacterium tumefaciens strain C58 has been used for transfection experiments with mammalian cells. In DNA from Tupaia baby fibroblasts Ti-plasmid sequences could be identified by filter hybridization as long as four weeks after transfection including two cell passages. The hybridization signals decreased rapidly after addition of the Ti-plasmid DNA-coprecipitate to the cells. The signals were often not detected any more after the first day, but were visible one week after transfection. Nuclei prepared from Ti-plasmid-transfected cells hybridized to pTi-specific RNA. With the chloramphenicol acetyl transferase-gene as marker no discrimination in DNA uptake was found between the Ti-plasmid and much smaller plasmids. According to the number of nuclei with homology to pTi-sequences it is assumed that about 0.2% of the cells carry Ti-plasmid DNA in the nucleus. Analysis of RNA isolated from cells transfected with cloned segments of the Ti-plasmid revealed that the TDNA region of the Ti-plasmid was predominantly transcribed.Abbreviations CAT Chloramphenicol Acetyl Transferase - NPT Neomycin Phosphotransferase - SDS Sodium Dodecylsulfate - TK Thymidine Kinase     

Antigenicity of carcinogen and viral induced sarcomas in inbred and random bred guinea pigs.

A tumor specific transplantation antigen (TSTA) has been detected in a methylcholanthrene (MCA) induced guinea pig tumor. It was possible to induce resistance to rechallenge with the tumor by immunization with irradiated cells in CFA. In contrast, the same technique failed to detect TSTA in two viral (Kirsten strain mouse sarcoma virus, Ki-MSV) induced guinea pig tumors; these results are similar to observations made with mouse Ki-MSV-induced tumors. Transplantation studies with these tumors in both inbred and random-bred guinea pigs showed a complexity of growth and rejection patterns. The B alloantigen, a major serologically defined antigen of the guinea pig histocompatibility complex, seemed to play a central role in acting as a guniea pig transplantation antigen. In all cases studied, the absence of B antigens in the recipient led to tumor rejection and anti-B antibody protection.     

Synthesis and biological evaluation of benzoisothiazole derivatives possessing N,N-dimethylformimidamide group as 5-HT₆ receptor antagonists

A series of novel N,N-dimethyl-N'-(5-(Ar-sulfonamido) benzo[d]isothiazol-3-yl)formimidamides was designed and synthesized as 5-HT(6) ligands. Here N,N-dimethyl formimidamides was used as a replacement for an aminoethyl moiety. In vitro functional assays demonstrated compounds 9b and 9i significantly inhibited the 5-HT-induced Ca(2+) increases (9b; IC(50)=0.36 μM and 9i; IC(50)=0.44 μM), indicating that 9b and 9i were potent 5-HT(6) receptor antagonists. Compounds 9i also showed good selectivity on the 5-HT(6) over 5-HT(4) and 5-HT(7) receptors.     

Establishment of the expression system for studying the function of active caspase-3 in zebrafish

Caspase-3, a key molecule in apoptosis, has been extensively studied in cell culture system; however, it has been less well characterized in vivo because certain mediators are required for the proteolytic activation of effector caspases, including caspase-3. In this study, various forms of caspase-3 with the C-terminal GFP tag were inserted into the pCS2+ plasmid, and the expression patterns of caspase-3 proteins were characterized in a zebrafish model system using microinjection of nucleic acids into zebrafish embryos. We have verified that active caspase-3 was generated by its autocatalytic activity under the condition of caspase-2 prodomain (C2P)-caspase-3-GFP overexpression, indicating that the C2P domain is crucial for the activation of caspase-3. We also confirmed that the C2P domain plays an important role in regulating the nuclear localization of the C2P-caspase-3 chimeric protein. We used this expression system to establish an animal model system suitable for the investigation of the functional characteristics of caspase-3 in vivo. Thus, our study provides a useful and specific tool for investigating the molecular mechanisms by which active caspase-3 regulates apoptosis during embryonic development.     

Modulation of Ca(v)3.1 T-type Ca2+ channels by the ran binding protein RanBPM

Recombinant antibody-toxins are constructed via the fusion of a “carcinoma-specific” antibody fragment to a toxin. Due to the high affinity and high selectivity of the antibody fragments, antibody-toxins can bind to surface antigens on cancer cells and kill them without harming normal cells [L.H. Pai, J.K. Batra, D.J. FitzGerald, M.C. Willingham, I. Pastan, Anti-tumor activities of immunotoxins made of monoclonal antibody B3 and various forms of Pseudomonas exotoxin, Proc. Natl. Acad. Sci. USA 88 (1991) 3358-3362]. In this study, we constructed the antibody-toxin, Fab-SWn-PE38, with SWn (n = 3, 6, 9) sequences containing n-time repeated (G₄S) between the Fab fragment and PE38 (38 kDa truncated form of Pseudomonas exotoxin A). The SWn sequence also harbored one cysteine residue that could form a disulfide bridge between two Fab-SWn-PE38 monomers. We assessed the cytotoxicity of the monovalent (Fab-SWn-PE38), and divalent ([Fab-SWn-PE38]₂) antibody-toxins. The cytotoxicity of the dimer against the CRL1739 cell line was approximately 18.8-fold higher than that of the monomer on the ng/ml scale, which was approximately 37.6-fold higher on the pM scale. These results strongly indicate that divalency provides higher cytotoxicity for an antibody-toxin.     

Vacuolar (H)-ATPases in Caenorhabditis elegans: What can we learn about giant H pumps from tiny worms?

Vacuolar (H⁺)-ATPases, also called V-ATPases, are ATP-driven proton pumps that are highly phylogenetically conserved. Early biochemical and cell biological studies have revealed many details of the molecular mechanism of proton pumping and of the structure of the multi-subunit membrane complex, including the stoichiometry of subunit composition. In addition, yeast and mouse genetics have broadened our understanding of the physiological consequences of defective vacuolar acidification and its related disease etiologies. Recently, phenotypic investigation of V-ATPase mutants in Caenorhabditis elegans has revealed unexpected new roles of V-ATPases in both cellular function and early development. In this review, we discuss the functions of the V-ATPases discovered in C. elegans.     

Role of protein kinase C-dependent A-kinase anchoring proteins in lysophosphatidic acid-induced cAMP signaling in human diploid fibroblasts

Previously, we reported that lysophosphatidic acid (LPA)-induced adenosine 3',5'-cyclic monophosphate (cAMP) production by human diploid fibroblasts depends on the age of the fibroblasts. In this study, we examined the role of A-kinase anchoring proteins (AKAP) in the regulation of LPA-stimulated cAMP production in senescent fibroblasts. We found that levels of protein kinase C (PKC)-dependent AKAPs, such as Gravin and AKAP79, were elevated in senescent cells. Co-immunoprecipitation experiments revealed that Gravin and AKAP79 do not associate with adenylyl cyclase type 2 (AC2) but bind to AC4/6, which interacts with calcium-dependent PKCs alpha/beta both in young and senescent fibroblasts. When the expression of Gravin and AKAP79 was blocked by small interference RNA transfection, the basal level of cAMP was greatly reduced and the cAMP status after LPA treatment was also reversed. Protein kinase A showed a similar pattern in terms of its basal activity and LPA-dependent modulation. These data suggest that Gravin and to a lesser extent, AKAP79, may play important roles in maintaining the basal AC activity and in coupling the AC systems to inhibitory signals such as Gialpha in young cells, and to stimulatory signals such as PKCs in senescent cells. This study also demonstrates that Gravin is especially important for the long-term activation of PKC by LPA in senescent cells. We conclude that LPA-dependent increased level of cAMP in senescent human diploid fibroblasts is associated with increases in Gravin levels resulting in its increased binding with and activation of calcium-dependent PKC alpha/beta and AC4/6.     

Prevention of ginsenoside-induced desensitization of Ca2+-activated Cl- current by microinjection of inositol hexakisphosphate in Xenopus laevis oocytes: involvement of GRK2 and beta-arrestin I

We demonstrated that ginsenosides, the active ingredient of Panax ginseng, enhance endogenous Ca(2+)-activated Cl(-) currents via Galpha(q/11)-phospholipase C-beta3 pathway in Xenopus laevis oocytes. Moreover, prolonged treatment of ginsenosides induced Cl(-) channel desensitization. However, the molecular mechanisms involved in ginsenoside-induced Cl(-) channel desensitization have not yet been determined precisely. To provide answers to these questions, we investigated the changes in ginsenoside-induced Cl(-) channel desensitization after intraoocyte injection of inositol hexakisphosphate (InsP(6)), which is known to bind beta-arrestins and interfere with beta-arrestin-induced receptor down-regulation, and cRNAs coding beta-arrestin I/II and G-protein receptor kinase 2 (GRK2), which is known to phosphorylate G protein-coupled receptors and attenuate agonist stimulations. When control oocytes were stimulated with ginsenosides, the second, third, and fourth responses to ginsenosides were 69.6 +/- 4.1, 9.2 +/- 2.3, and 2.6 +/- 2.2% of the first responses, respectively. Preintraoocyte injection of InsP(6) before ginsenoside treatment restored ginsenoside effect to initial response levels in a concentration-, time-, and structurally specific manner, in that inositol hexasulfate had no effect. The EC(50) was 13.9 +/- 8.7 microM. Injection of cRNA coding beta-arrestin I but not beta-arrestin II blocked InsP(6) effect on prevention of ginsenoside-induced Cl(-) channel desensitization. Injection of cRNA coding GRK2 abolished ginsenoside effect enhancing Cl(-) current. However, the GRK2-caused loss of ginsenoside effect on Cl(-) current was prevented by coinjection of GRK2 with GRK2-K220R, a dominant-negative mutant of GRK. These results indicate that ginsenoside-induced Cl(-) channel desensitization is mediated via activation of GRK2 and beta-arrestin I.