Characteristics of major mutagenicity of instant coffee

Detailed quantitative studies on the mutagenicity of methylglyoxal showed that its contribution to the total mutagenicity of instant coffee on S. typhimurium TA100 was minor although we reported previously (Kasai et al., 1982) that its contribution to the mutagenicity of freshly brewed coffee was about 50%. Cysteine suppressed the mutagenicity of methylglyoxal and of methylglyoxal when added to instant coffee, but did not affect the mutagenicity of coffee itself. Catalase suppressed most of the mutagenicity of coffee, but not that of methylglyoxal or of methylglyoxal added to coffee.     

Involvement of colchicine-sensitive cytoplasmic element in premitotic nuclear positioning ofAdiantum protonemata

Summary When the red-light grown protonema ofAdiantum capillus-veneris was transferred to the dark, the nucleus ceased its migration ca. 5 hours before cell plate formation (Mineyuki andFuruya 1980). To see whether the nucleus was held by some cytoplasmic structure during nuclear positioning, protonemata were treated with various centrifugal forces at different stages of the cell cycle. Nuclei of G₁ phase were easily displaced by centrifugation at 360×g for 15 minutes, but those of G₂ or M phase were not displaced by it, suggesting that the nuclei were held by some cytoplasmic elements in G₂ or M phase. This nuclear anchoring was not detectable in protonemata that were treated with 5mM colchicine. With this treatment, the nucleus did not stop its migration at late G₂ and moved even in prophase. And the retardation of organelle movement which was observed in cytoplasm on the lateral side of the nucleus after the cessation of premitotic nuclear migration (Mineyuki andFuruya 1984) was not observed in the presence of colchicine. Thus the nuclei appear to be held by colchicine-sensitive structure in cytoplasm between the lateral surface of the nucleus and cell wall during the premitotic nuclear positioning. Electron micrographs showing cytoplasmic microtubules were consistent with the idea.Abbreviations PPN Premitotic positioning of the nucleus - L region Cytoplasm between the lateral surface of the nucleus and cell wall (seeMineyuki et al. 1984)     

Some Electrical Properties of a Nuclear Membrane Examined with a Microelectrode

Electrical potential and resistance were measured with microelectrodes in in situ and isolated nuclei of gland cells of Drosophila flavorepleta. The nucleus-cytoplasm boundary was found to be rather impermeable to ion diffusion. It presents a resistance of the order of 1 Ω cm² and sustains a "resting" potential, the nucleoplasm being about 15 mv negative with respect to the cytoplasm. Both the resistance and potential appear to be associated with the nuclear membrane: the potential declines to zero and the resistance to a fraction of its original value, when the membrane is perforated experimentally.     

Site-directed mutagenesis of glutamine residue of calmodulin. Activation of guanylate cyclase of Tetrahymena plasma membrane

Tetrahymena calmodulin (CaM) differs from mammalian CaM in its ability to activate Tetrahymena guanylate cyclase. Of 12 differences in amino acid sequence, two occur near the carboxyl terminus (Gln-143----Arg and Thr-146----deletion). To investigate the functional significance of the carboxyl-terminal region in activation of the guanylate cyclase, three mutated CaMs were engineered by using cassette mutagenesis of rat CaM cDNA: Gln-143----Arg (CaM.A), Thr-146----deletion (CaM.D), and Gln-143----Arg/Thr-146 deletion (CaM.AD). Recombinant wild type CaM (wCaM), CaM.A, CaM.D, and CaM.AD were indistinguishable in their ability to activate cyclic AMP phosphodiesterase. The two mutated CaMs (CaM.A and CaM.AD) with the Gln-143 replacement activated guanylate cyclase of Tetrahymena plasma membrane in the presence of Ca2+, with the maximal activation being half of that produced by Tetrahymena CaM. In contrast, neither CaM.D nor wCaM could stimulate the cyclase activity. A CaM antagonist, W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide), prevented the cyclase activation by either Tetrahymena CaM, CaM.A, or CaM.AD. Thus, we conclude that Arg-143 is in a region of the molecule involved in activation of Tetrahymena guanylate cyclase. The data also suggest that the cyclase activation by Tetrahymena CaM requires complex macromolecular interactions between the entire CaM molecule and the enzyme.     

Reversal by protein kinase C inhibitor of suppressive actions of phorbol-12-myristate-13-acetate on polyphosphoinositide metabolism and cytosolic Ca2+ mobilization in thrombin-stimulated human platelets

In the presence of 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7), a potent inhibitor of protein kinase C in vitro, phorbol-12-myristate-13-acetate (PMA) did not suppress the thrombin-induced increase of cytosolic Ca2+ concentration in human platelets. The H-7 reversal of the inhibitory action of PMA was also observed in thrombin-induced polyphosphoinositide breakdown by phospholipase C. These results provide additional support to the developing theory that the inhibition of PMA on Ca2+ mobilization and phosphoinositide turnover may be mediated by protein kinase C activation.     

Frequent overexpression of CADM1/IGSF4 in lung adenocarcinoma

We reported comprehensive screening for antigens (Ags) overexpressed on various carcinomas via isolation of human monoclonal antibodies (mAbs) that may be therapeutic in a previous paper (Proc. Natl. Acad. Sci. USA 105, 7287-7292, 2008). Twenty-one distinct Ags highly expressed on several carcinomas were identified and 356 mAbs with unique sequences turned out to bind to one of the 21 Ags. Among them CADM1/IGSF4 which had been originally referred to as tumor suppressor lung cancer 1 (TSLC1) was included. Therefore we examined the expression of CADM1 in lung cancers in this study. Eight different anti CADM1 mAbs were used for immunohistochemical analysis of 29 fresh lung cancer specimens. Staining patterns were categorized to six groups based on the extent of positive staining and the localization of stained portions. While overexpression of CADM1 was observed on the cell surface of adenocarcinomas at a high frequency, around 60%, positive stainings were rarely observed on that of other lung carcinomas including squamous cell carcinomas. Moreover, some clones among the eight mAbs gave different staining patterns from those by the other clones against the same fresh specimen, suggesting presence of variant forms of CADM1 differentiated by mAbs.     

A two-step mechanism for free cholesterol and phospholipid efflux from human vascular cells to apolipoprotein A-1

Smooth muscle and endothelial cells in vivo are quiescent yet exposed to high levels of lipoprotein lipids. Phospholipid (PL) and free cholesterol (FC) efflux maintain homeostasis. Smooth muscle cells (SMC) expressed high levels of ABC-1 transporter mRNA, and glyburide-dependent PL and FC efflux to apolipoprotein A-1 (apo A-1), the major protein of high-density lipoprotein. FC efflux was inhibited by vanadate and okadaic acid, while PL efflux was not. Phosphatidylcholine was the major PL transferred by both cell types. Stimulation of phosphatidylserine efflux, redistributed within the membrane by this transporter, was only minimally increased. Umbilical vein and aortic endothelial cells expressed little ABC-1 mRNA, nor did these cells promote either PL or FC efflux in response to the presence of apo A-1. To investigate the mechanism of ABC-1-dependent lipid efflux from these cells, apo A-1 was preincubated in the presence of unlabeled SMC or fibroblasts, and the conditioned medium was then transferred to endothelial cells. This medium catalyzed the efflux of FC but not of PL from endothelial cells. Such FC efflux was resistant to glyburide but inhibited by okadaic acid and vanadate. The data suggest that ABC-1-dependent PL efflux precedes FC efflux to apo A-1 and that the complex of apo A-1 and PL is a much better acceptor of FC than apo A-1 itself. Inhibition of FC but not PL efflux by vanadate and okadaic acid suggests these transfers involve different mechanisms.     

Mutagenic properties of 2-amino-N6-hydroxyadenine in Salmonella and in Chinese hamster lung cells in culture

The mutagenicity of the base analogue, 2-amino-N6-hydroxyadenine (AHA), was tested in Salmonella typhimurium TA100 and TA98 and in Chinese hamster lung (CHL) cells. AHA showed very potent mutagenicity in TA100 without S9 mix, inducing 25,000 revertants/micrograms. The mutagenicity increased about 2-fold upon addition of S9 mix containing 10 microliters S9. AHA was found to be one of the strongest mutagens for TA100. Addition of S9 mix containing 100 microliters S9 induced no significant increase of revertants with AHA at amounts up to 50 ng per plate. AHA was also mutagenic for the frameshift mutant, TA98, without S9 mix, the mutagenicity for TA98 being about 1/1000 of that for TA100. When the mutagenicity of AHA was tested in CHL cells, with diphtheria toxin resistance (DTr) as a selective marker in the absence of S9 mix with a 3-h treatment of cells, DTr mutants increased dose-dependently at concentrations of 2.5-15 micrograms/ml. When cells were incubated with AHA for 24 h, a 200-fold increase in the number of DTr mutants was observed; the mutagenicity was 500-fold higher than that of ethyl methanesulfonate. This marked increase of mutagenicity by prolonged incubation may indicate that AHA induces mutations mainly after incorporation into DNA. The addition of a small amount of S9 increased the mutagenicity obtained with a 3-h treatment 2-fold, but a larger amount of S9 decreased the mutagenicity as was found with S. typhimurium TA100.     

Basic pH-induced modification of excitation-energy dynamics in fucoxanthin chlorophyll a/c-binding proteins isolated from a pinguiophyte,Glossomastix chrysoplasta

Photosynthetic organisms have diversified light-harvesting complexes (LHCs) to collect solar energy efficiently, leading to an acquisition of their ecological niches. Herein we report on biochemical and spectroscopic characterizations of fucoxanthin chlorophyll a/c-binding protein (FCP) complexes isolated from a marine pinguiophyte Glossomastix chrysoplasta. The pinguiophyte FCP showed one subunit band in SDS-PAGE and one protein-complex band with a molecular weight at around 66 kDa in clear-native PAGE. By HPLC analysis, the FCP possesses chlorophylls a and c, fucoxanthin, and violaxanthin. To clarify excitation-energy-relaxation processes in the FCP, we measured time-resolved fluorescence spectra at 77 K of the FCP adapted to pH 5.0, 6.5, and 8.0. Fluorescence curves measured at pH 5.0 and 8.0 showed shorter lifetime components compared with those at pH 6.5. The rapid decay components at pH 5.0 and 8.0 are unveiled by fluorescence decay-associated (FDA) spectra; fluorescence decays occur in the 270 and 160-ps FDA spectra only at pH 5.0 and 8.0, respectively. In addition, energy-transfer pathways with time constants of tens of picoseconds are altered under the basic pH condition but not the acidic pH condition. These findings provide novel insights into pH-dependent energy-transfer and energy-quenching machinery in not only FCP family but also photosynthetic LHCs.