Lysis of Yeast Cells Showing Low Susceptibility to Zymolyase

Lysis of commercial baker’s yeast cells was examined using Zymolyase. The lysis was stimulated by the addition of sodium sulfite or potassium chloride or both. The effect of potassium chloride was less than that of sodium sulfite, but the two compounds acted synergistically. The cells were effectively lysed by Zymolyase in the presence of 0.1 M sodium sulfite and 0.8 M potassium chloride. The extent of lysis was similar to that of brewery yeast cells obtained from a brewhouse. Cells pretreated with sodium sulfite did not show much of an increase in susceptibility to Zymolyase, but were effectively lysed by the enzyme in the presence of potassium chloride. Potassium chloride stimulated lysis only in the presence of Zymolyase. Yeast cells treated with cupric ions in the presence of sodium sulfite became highly susceptible to Zymolyase, suggesting irreversible destruction of the sodium sulfite-sensitive and potassium chloride-sensitive structure of the cell wall.

Cells of Saccharomyces cerevisiae prepared under various culture conditions were completely lysed by Zymolyase in the presence of sodium sulfite or potassium chloride or both.     

Isolation and Some Physico-chemical Properties of the Acidic Subunits of Soybean 11S Globulin

Four kinds of acidic subunits and three kinds of basic subunits of 11S globulin were separated by polyacryl amide gel electrophoresis in the urea system. The four acidic subunits designated as A₁, A₂, A₃ and A₄ (R_m=0.35, 0.40, 0.46 and 0.56 respectively) were isolated by stepwise elution followed by repeating gradient elution with DEAE-Sephadex A-50 in the presence of 6 m urea at 5°C.

Subsequently, some physico-chemical properties of the subunits were determined. For example, N-terminal amino acids were determined as phenylalanine for both A₁ and A₂ and as leucine (or isoleucine) for both A₃ and A₄ by the DNP-amino acid method. The molecular weights of A₁, A₂ and A₃ were shown as 37,000 and 45,000 for A₄ by SDS-gel electrophoresis. The amino acid compositions of the acidic subunits were roughly similar to each other, but some remarkable differences were observed in the content of basic amino acids (lysine, histidine and arginine), serine and proline.     

Effect of Copper on Growth of Yeast

The effect of copper was tested on the growth of many strains of yeast. Plate culture on density gradient agar of copper was used for estimating the growth response to copper. Growth in many strains was more strongly inhibited by the copper-aquo complex than by the copper-amino acid complex. Debaryomyces hansenii IFO 023 was found a suitable strain for the present study, because it was not resistant, not producing H₂S, and copper absorption by this strain was similar to that of the resistant strain. Growth of yeast cells in medium containing copper was affected by pH and concentration of amino acid in medium. Absorption of copper into intact cells was almost saturated for the initial few minutes. It was also affected by the addition of amino acid to copper solution. Our results indicated that the growth response of yeast to copper was closely related to copper absorption into cells. About 60 percent of copper absorbed into cells was distributed in the soluble fraction of the cell homogenate which was obtained by centrifugation at 105,000 g for 60 min.     

Apoptosis-inducing activity of the actin-depolymerizing agent aplyronine A and its side-chain derivatives

Aplyronine A (1) and mycalolide B (2), which are cytotoxic actin-depolymerizing marine macrolides, were revealed to induce apoptosis in human leukemia HL60 cells and human epithelial carcinoma HeLa S₃ cells. Based on these results, actin-depolymerizing compounds were expected to exhibit apoptosis-inducing activity in cancer cells. Compounds 3–6, which were synthesized based on the side-chain structure of aplyronine A, were evaluated for their actin-depolymerizing activities in vitro and cytotoxicities against HL60 cells. The growth-inhibitory activities of 3–6 were well correlated with their actin-depolymerizing activities, and derivative 6 was shown to induce the disruption of actin filaments and apoptosis in HL60 cells. These results suggested that actin-depolymerizing agents 1, 2, and 6-induced apoptosis in HL60 cells may have been due to their actin-depolymerizing activity.     

Comparative properties of Aβ1–42, Aβ11–42, and [Pyr11]Aβ11–42 generated from O-acyl isopeptides

The use of water-soluble O-acyl isopeptides enabled us to investigate the biochemical properties of Aβ11–42 species, by preparing highly concentrated stock solutions after a pretreatment. Aβ11–42 and [Pyr¹¹]Aβ11–42 showed comparable aggregation capability and cytotoxicity, suggesting that the pyroglutamate modification at Glu¹¹ does not have a crucial role in these events. However, given that Aβ11–42 is converted to [Pyr¹¹]Aβ11–42 by a glutamyl cyclase in vivo, the potential aggregative and cytotoxic nature of [Pyr¹¹]Aβ11–42 that was observed in the present study provides valuable insights into the pathological functions of pyroglutamate-modified Aβ species in Alzheimer’s disease.     

Synthesis of carbocyclic pyrimidine nucleosides and their inhibitory activities against Plasmodium falciparum thymidylate kinase

Plasmodium falciparum thymidylate kinase (PfTMK) is a promising antimalarial target due to its unique substrate specificity. Recently, we reported that 2′,3′-dideoxycarbocyclic thymidine showed moderate inhibitory activity and reported the related structure–activity relationship for inhibitors against PfTMK. In this study, we have designed and synthesized enantioselective 2′,3′-dideoxycarbocyclic pyrimidine nucleosides based on our previous results and screened them for inhibitory activity against PfTMK. The most potent inhibitor showed K_i^TMP and K_i^dGMP values of 14 and 20 μM, respectively. The fluorinated dideoxy derivative (-)-7, exhibited lower K_i^TMP and higher K_i^dGMP compared with that of the parent compound (K_i^TMP, K_i^dGMP equals 20 and 7 μM, respectively). The modification of carbocyclic pyrimidine nucleosides is a promising strategy for developing powerful PfTMK inhibitors.     

Inhibition of NF-κB by MG132 through ER stress-mediated induction of LAP and LIP

Proteasome inhibitor MG132 blocks activation of NF-κB by preventing degradation of IκB. In this report, we propose an alternative mechanism by which MG132 inhibits cytokine-triggered NF-κB activation. We found that MG132 induced endoplasmic reticulum (ER) stress, and attenuation of ER stress blunted the suppressive effect of MG132 on NF-κB. Through ER stress, MG132 up-regulated C/EBPβ mRNA transiently and caused sustained accumulation of its translational products liver activating protein (LAP) and liver-enriched inhibitory protein (LIP), both of which were identified as suppressors of NF-κB. Our results disclosed a novel mechanism underlying inhibition of NF-κB by MG132.     

A map-based cloning strategy employing a residual heterozygous line reveals that the GIGANTEA gene is involved in soybean maturity and flowering

Flowering is indicative of the transition from vegetative to reproductive phase, a critical event in the life cycle of plants. In soybean (Glycine max), a flowering quantitative trait locus, FT2, corresponding to the maturity locus E2, was detected in recombinant inbred lines (RILs) derived from the varieties "Misuzudaizu" (ft2/ft2; JP28856) and "Moshidou Gong 503" (FT2/FT2; JP27603). A map-based cloning strategy using the progeny of a residual heterozygous line (RHL) from the RIL was employed to isolate the gene responsible for this quantitative trait locus. A GIGANTEA ortholog, GmGIa (Glyma10g36600), was identified as a candidate gene. A common premature stop codon at the 10th exon was present in the Misuzudaizu allele and in other near isogenic lines (NILs) originating from Harosoy (e2/e2; PI548573). Furthermore, a mutant line harboring another premature stop codon showed an earlier flowering phenotype than the original variety, Bay (E2/E2; PI553043). The e2/e2 genotype exhibited elevated expression of GmFT2a, one of the florigen genes that leads to early flowering. The effects of the E2 allele on flowering time were similar among NILs and constant under high (43°N) and middle (36°N) latitudinal regions in Japan. These results indicate that GmGIa is the gene responsible for the E2 locus and that a null mutation in GmGIa may contribute to the geographic adaptation of soybean.     

Gibberellin biosynthesis and signalling during development of the strawberry receptacle

The enlargement of receptacle cells during strawberry (Fragaria × ananassa) fruit development is a critical factor determining fruit size, with the increase in cell expansion being one of the most important physiological processes regulated by the phytohormone gibberellin (GA). Here, we studied the role of GA during strawberry fruit development by analyzing the endogenous content of bioactive GAs and the expression of key components of GA signalling and metabolism. Bioactive GA(1) , GA(3) and GA(4) were monitored during fruit development, with the content of GA(4) being extremely high in the receptacle, peaking at the white stage of development. ?Genes with high homology to genes encoding GA pathway components, including receptors (FaGID1(GIBBERELLIN-INSENSITIVE DWARF1)b and FaGID1c), DELLA (FaRGA(REPRESSOR OF GA) and FaGAI(GA-INSENSITIVE)), and enzymes involved in GA biosynthesis (FaGA3ox) and catabolism (FaGA2ox), were identified, and their expression in different tissues and developmental stages of strawberry fruit was studied in detail. The expression of all of these genes showed a stage-specific pattern during fruit development and was highest in the receptacle. FaGID1c bound GA in vitro, interacted with FaRGA in vitro and in vivo, and increased GA responses when ectopically expressed in Arabidopsis. This study thus reveals key elements of GA responses in strawberry and points to a critical role for GA in the development of the receptacle.