Overexpression of insulin receptors in fibroblast and ovary cells induces a ligand-mediated transformed phenotype.

To investigate whether overexpression of the insulin receptor results in altered cell growth we used NIH 3T3 cells transfected with a bovine papilloma virus/insulin receptor cDNA construct (3T3/HIR). These cells expressed high numbers of insulin receptors (mean +/- sd, 631.0 +/- 16.7 ng receptors/10(6) cells). Insulin significantly stimulated the growth of 3T3/HIR cells maintained in serum-free medium. Moreover, in these cells, insulin induced marked phenotypic changes, including alterations in cell shape, loss of contact inhibition, and focal growth. In contrast to 3T3/HIR cells, insulin was without effect in either wild-type 3T3 cells (3T3/wt), 3T3 cells transfected with the neomycin resistance gene (3T3/NEO), or the bovine papilloma virus (3T3/BPV). To assess the presence of anchorage-independent growth, cells were seeded in soft agar and inspected for colony formation. 3T3/HIR cells showed absent or minimal colony growth in the absence of insulin. However, there was a dose-dependent insulin-stimulated increase in both colony size and number. Insulin-stimulated colony formation was specifically inhibited by an insulin antagonist, monoclonal antibody MA-10. In the presence of 100 nM insulin, about 3% of cells formed large colonies. Insulin neither stimulated growth nor induced colony formation in 3T3/wt cells or 3T3/NEO cells. Insulin also stimulated colony formation in CHO cells transfected with an insulin receptor cDNA construct. In conclusion, overexpression of normal insulin receptors induces a ligand-dependent transformed phenotype. This phenomenon may have clinical relevance by conferring a selective growth advantage to tumor cells with high numbers of insulin receptors.     

Caffeic Acid Phenylethyl Ester and MG-132 Have Apoptotic and Antiproliferative Effects on Leukemic Cells But Not on Normal Mononuclear Cells

Chemotherapy aims to limit proliferation and induce apoptotic cell death in tumor cells. Owing to blockade of signaling pathways involved in cell survival and proliferation, nuclear factor κB (NF-κB) inhibitors can induce apoptosis in a number of hematological malignancies. The efficacy of conventional chemotherapeutic drugs, such as vincristine (VCR) and doxorubicine (DOX), may be enhanced with combined therapy based on NF-κB modulation. In this study, we evaluated the effect of caffeic acid phenylethyl ester (CAPE) and MG-132, two nonspecific NF-κB inhibitors, and conventional chemotherapeutics drugs DOX and VCR on cell proliferation and apoptosis induction on a lymphoblastoid B-cell line, PL104, established and characterized in our laboratory. CAPE and MG-132 treatment showed a strong antiproliferative effect accompanied by clear cell cycle deregulation and apoptosis induction. Doxorubicine and VCR showed antiproliferative effects similar to those of CAPE and MG-132, although the latter drugs showed an apoptotic rate two-fold higher than DOX and VCR. None of the four compounds showed cytotoxic effect on peripheral mononuclear cells from healthy volunteers. CAPE- and MG-132-treated bone marrow cells from patients with myeloid and lymphoid leukemias showed 69% (P < .001) and 25% decrease (P < .01) in cell proliferation and 42% and 34% (P < .01) apoptosis induction, respectively. Overall, our results indicate that CAPE and MG-132 had a strong and selective apoptotic effect on tumor cells that may be useful in future treatment of hematological neoplasias.     

Patterns of ribosomal RNA evolution in salamanders

Sequence comparisons are presented for four segments of the large subunit of ribosomal RNA, including divergent domains D7a and D7b, portions of the large divergent domains D2, D3, and D8, and evolutionarily conservative sequences flanking divergent domains. These results resolve phylogenetic relationships among exemplars of seven families of salamanders and the three amphibian orders. Phylogenetic analysis confirms the prediction that divergent domains feature the highest relative rates of base substitution and length variation within the ribosome, but the divergent domains evolve more slowly than nuclear noncoding DNA and the silent sites of structural genes. Base substitutions demonstrate approximately twice as many transitions as transversions and an uneven distribution among sites within the divergent domains but no apparent bias in base composition. Length mutations are primarily small insertions and deletions, with deletions predominating. The divergent domains appear to be a good source of phylogenetic information for evolutionary events occurring approximately 100-200 million years ago.      

Spontaneous release of lipopolysaccharide by Pseudomonas aeruginosa.

Pseudomonas aeruginosa PAO grown in glucose mineral salts medium released lipopolysaccharide which was chemically and immunologically similar to the cellular lipopolysaccharide. In addition, it possessed identical phage E79-inactivating properties. Through neutralization of phage activity and hemolysis inhibition assays, the organism was found to liberate lipopolysaccharide at a constant rate during log-phase growth equivalent to 1.3 to 2.2 ng/10(8) cells over a growth temperature range of 25 to 42 degrees C. At 19 degrees C, a lipopolysaccharide was released which was deficient in phage-inactivating activity but retained its immunological properties. Chemical analysis of lipopolysaccharide extracted from cells grown at 19 degrees C showed a deficiency in the O-side-chain component fucosamine. Gel exclusion chromatography of the polysaccharide fraction derived from lipopolysaccharide isolated from cells grown at 19 degrees C exhibited a decreased content of side-chain polysaccharide as well as a difference in the hexosamine:hexose ratio. The results of sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis confirmed these results as well as establishing that an essentially normal distribution of side-chain repeating unit lengths were to be found in the 19 degrees C preparation. These results suggest a decrease in the frequency of capping R-form lipopolysaccharide at 19 degrees C.     

Comparison of the effects of concentration, pH and anion species on astringency and sourness of organic acids

The separate effects of concentration, pH and anion species on intensity of sourness and astringency of organic acids were evaluated. Judges rated sourness and astringency intensity of lactic, malic, tartaric and citric acid solutions at three levels of constant pH varying in normality and at three levels of constant concentration varying in pH. To assess the comparative sourness and astringency of the organic acid anions of study, binary acid solutions matched in pH and titratable acidity were also rated. As pH was decreased in equinormal solutions, both sourness and astringency increased significantly (P < 0.001). By contrast, as the normality of the equi-pH solutions was increased, only sourness demonstrated significant increases (P < 0.001) while astringency remained constant or decreased slightly. At the lowest normality tested, all solutions were more astringent than sour (P < 0.05). Although lactic acid was found to be significantly more sour than citric acid (P < 0.05), no other sourness or astringency differences among the organic acid anions were noted. This study demonstrates for the first time that astringency elicited by acids is a function of pH and not concentration or anion species, and confirms that sourness is independently influenced by concentration, pH and anion species of the acid.      

Applications of direct detection device in transmission electron microscopy

A prototype direct detection device (DDD) camera system has shown great promise in improving both the spatial resolution and the signal to noise ratio for electron microscopy at 120–400 keV beam energies (Xuong et al., 2007. Methods in Cell Biology, 79, 721–739). Without the need for a resolution-limiting scintillation screen as in the charge coupled device (CCD), the DDD camera can outperform CCD based systems in terms of spatial resolution, due to its small pixel size (5 μm). In this paper, the modulation transfer function (MTF) of the DDD prototype is measured and compared with the specifications of commercial scientific CCD camera systems. Combining the fast speed of the DDD with image mosaic techniques, fast wide-area imaging is now possible. In this paper, the first large area mosaic image and the first tomography dataset from the DDD camera are presented, along with an image processing algorithm to correct the specimen drift utilizing the fast readout of the DDD system.