High Efficiency Gene Transfer by Multiple Transfection Protocol

A high efficiency transfection protocol employing a common polycationic lipid is described. Using LipofectAMINE, a widely used transfection reagent, we transfected 293T cells with a plasmid harboring the -galactosidase (-gal) gene. The transfection efficiency was determined by direct staining for X-gal. The conventional transfection protocol achieved an efficiency of <40% while our protocol, which employs the repetition of transfection a few times, achieved an efficiency of approximately 80%. Thus, a dramatic increase in transfection efficiency can be obtained by simply repeating transfection with the use of a common polycationic lipid. This method will be useful in many molecular biological experiments.     

Specific sandwich-type enzyme immunoassays for smooth muscle constricting novel 31-amino acid endothelins

We established highly sensitive and specific sandwich-enzyme immunoassays (EIAs) for three newly discovered bioactive 31-amino acid endothelins [ETs(1-31)], which can detect as little as 0.16 pg/well of ET-1(1-31), 0.39 pg/well of ET-2(1-31), and 0.16 pg/well of ET-3(1-31). The EIAs showed no crossreactivity with 21-amino acid endothelins [ETs(1-21)] or big ETs at the usual assay concentrations below 1-5 ng/ml. In reversed-phase HPLC, immunoreactive ETs(1-31) in the granulocytes of normal human subjects eluted at the exact positions of authentic ETs(1-31), except for the presence of one additional unknown immunoreactive ET-1(1-31). The results also indicate that ETs(1-31) exist in the granulocytes at levels higher than or similar to those of ETs(1-21). This study is the first to establish EIAs for novel bioactive ETs(1-31). These assays can be utilized to assess the pathophysiological roles of ETs(1-31).     

Protective immunity induced by vaccination with SAG1 gene-transfected cells against Toxoplasma gondii-infection in mice

To develop a vaccine by augmenting the protective cellular immunity against Toxoplasma gondii (T. gondii)-infection, T gondii SAG1 gene-transfectants were established by using RMA.S (H-2b), a murine transporter associated with the antigen processing (TAP) molecule-deficient lymphoma line, as a host antigen-presenting cell (APC). Immunization of C57BL/6 mice with the SAG1-transfected RMA.S induced CD8+ cytotoxic T lymphocytes (CTL) specific for not only SAG1-transfected RMA.S but also T gondii-infected RMA.S, and elicited protective responses to infection with a virulent T. gondii strain, RH.     

Dissociation of double-headed cytoplasmic dynein into single-headed species and its motile properties

Cytoplasmic dynein is a minus-end directed microtubule motor and plays important roles in the transport of various intracellular cargoes. Cytoplasmic dynein comprises two identical heavy chains and forms a dimer (double-headed dynein); the total molecular weight of the cytoplasmic dynein complex is about 1.5 million. The dynein motor domain is structurally very different from those of kinesin and myosin, and our understanding of the mechanisms of dynein energy transduction is limited mainly because of the difficulty in obtaining a sufficient quantity of purified and active cytoplasmic dynein. We purified cytoplasmic dynein, which was free from dynactin and other dynein-associated proteins. The purified cytoplasmic dynein was active in an in vitro motility assay. The controlled dialysis of the purified dynein against 4 M urea resulted in its complete dissociation into monomeric species (single-headed dynein). The separation of the dynein heads by the treatment was reversible. The MgATPase activities of the single-headed and reconstituted double-headed dynein were comparable to that of intact dynein. The double-headed dynein bundled microtubules in the absence of ATP; the single-headed dynein did not. The single-headed dynein produced in vitro microtubule-gliding motility at velocities very similar to those of double-headed dynein at various ATP concentrations. These results indicate that a single cytoplasmic dynein heavy chain is sufficient to produce robust microtubule motility. Application of the double- and single-headed dynein molecules in various assay systems will elucidate the mechanism of action of the cytoplasmic dynein.     

Taurine inhibits apoptosis by preventing formation of the Apaf-1/caspase-9 apoptosome

Cardiomyocyte apoptosis contributes to cell death during myocardial infarction. One of the factors that regulate the degree of apoptosis during ischemia is the amino acid taurine. To study the mechanism underlying the beneficial effect of taurine, we examined the interaction between taurine and mitochondria-mediated apoptosis using a simulated ischemia model with cultured rat neonatal cardiomyocytes sealed in closed flasks. Exposure to medium containing 20 mM taurine reduced the degree of apoptosis following periods of ischemia varying from 24 to 72 h. In the untreated group, simulated ischemia for 24 h led to mitochondrial depolarization accompanied by cytochrome c release. The apoptotic cascade was also activated, as evidenced by the activation of caspase-9 and -3. Taurine treatment had no effect on mitochondrial membrane potential and cytochrome c release; however, it inhibited ischemia-induced cleavage of caspase-9 and -3. Taurine loading also suppressed the formation of the Apaf-1/caspase-9 apoptosome and the interaction of caspase-9 with Apaf-1. These findings demonstrate that taurine effectively prevents myocardial ischemia-induced apoptosis by inhibiting the assembly of the Apaf-1/caspase-9 apoptosome. ischemia; cultured cardiomyocytes     

Isolation of putative glycoprotein gene from early somatic embryo of carrot and its possible involvement in somatic embryo development

Somatic embryogenesis is a unique process in plant cells. For example, embryogenic cells (EC) of carrot (Daucus carota) maintained in a medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) regenerate whole plants via somatic embryogenesis after the depletion of 2,4-D. Although some genes such as C-ABI3 and C-LEC1 have been found to be involved in somatic embryogenesis, the critical molecular and cellular mechanisms for somatic embryogenesis are unknown. To characterize the early mechanism in the induction of somatic embryogenesis, we isolated genes expressed during the early stage of somatic embryogenesis after 2,4-D depletion. Subtractive hybridization screening and subsequent RNA gel blot analysis suggested a candidate gene, Carrot Early Somatic Embryogenesis 1 (C-ESE1). C-ESE1 encodes a protein that has agglutinin and S-locus-glycoprotein domains and its expression is highly specific to primordial cells of somatic embryo. Transgenic carrot cells with reduced expression of C-ESE1 had wide intercellular space and decreased polysaccharides on the cell surface and showed delayed development in somatic embryogenesis. The importance of cell-to-cell attachment in somatic embryogenesis is discussed.     

Delimitation of the chromosomal region for a quantitative trait locus, qUVR-10, conferring resistance to ultraviolet-B radiation in rice (Oryza sativa L.)

Wide variation in ultraviolet-B (UVB) resistance is observed among rice varieties. In a previous study, three quantitative trait loci (QTLs) controlling UVB resistance were detected by QTL analysis, using backcross inbred lines (BILs) derived from a cross between a japonica cultivar, Nipponbare, and an indica cultivar, Kasalath. Among them, qUVR-10, a QTL for UVB resistance on chromosome 10, showed the largest effect. Plants homozygous for the Nipponbare allele at qUVR-10 were resistant to UVB, unlike those homozygous for the Kasalath allele. To determine more precisely the chromosomal location of qUVR-10, we performed a linkage mapping of qUVR-10 as a single Mendelian factor using advanced backcross progeny. Advanced progeny testing of F₄ families enabled us to determine the genotype classes of the qUVR-10 locus with high reliability. As a result, qUVR-10 was mapped between RFLP markers C60755S and C1757S, and co-segregated with C913A. In addition, a sequence showing high similarity to the Arabidopsis cyclobutane pyrimidine dimer (CPD) photolyase gene, which has been found to be involved in sensitivity to UV radiation in Arabidopsis and rice, was mapped in the candidate genomic region of qUVR-10. This result suggests that the CPD photolyase gene is a positional candidate for qUVR-10.Communicated by D.J. Mackill