Yeast 1,3-beta-glucan synthase activity is inhibited by phytosphingosine localized to the endoplasmic reticulum

1,3-beta-D-Glucan, a major filamentous component of the cell wall in the budding yeast Saccharomyces cerevisiae, is synthesized by 1,3-beta-glucan synthase (GS). Although a yeast gene whose product is required for GS activity in vitro, GNS1, was isolated and characterized, its role in GS function has remained unknown. In the current study we show that Deltagns1 cells accumulate a non-competitive and non-proteinous inhibitor(s) in the membrane fraction. Investigations of inhibitory activity on GS revealed that the inhibitor(s) is mainly present in the sphingolipid fraction. It is shown that Deltagns1 cells contain phytosphingosine (PHS), an intermediate in the sphingolipid biosynthesis, 30-fold more than wild-type cells do. The membrane fraction isolated from Deltasur2 cells contains an increased amount of dihydrosphingosine (DHS) and also exhibits reduced GS activity. Among constituents of the sphingolipid fraction, PHS and DHS show striking inhibition in a non-competitive manner. The intracellular level of DHS is much lower than that of PHS in wild-type cells, suggesting that PHS is the primary inhibitor of GS in vivo. The localization of PHS to the endoplasmic reticulum in wild-type cells coincides with that of the inhibitor(s) in Deltagns1 cells. Taken together, our results indicate that PHS is a potent inhibitor of yeast GS in vivo.     

Complementing yeast rho1 mutation groups with distinct functional defects

Saccharomyces cerevisiae is a multifunctional molecular switch involved in establishment of cell morphogenesis. We systematically characterized isolated temperature-sensitive mutations in the RHO1 gene and identified two groups of rho1 mutations (rho1A and rho1B) possessing distinct functional defects. Biochemical and cytological analyses demonstrated that mutant cells of the rho1A and rho1B groups have defects in activation of the Rho1p effectors Pkc1p kinase and 1,3-beta-glucan synthase, respectively. Heteroallelic diploid strains with rho1A and rho1B mutations were able to grow even at the restrictive temperature of the corresponding homoallelic diploid strains, showing intragenic complementation. The ability to activate both of the essential Rho1p effector proteins was restored in the heteroallelic diploid. Thus, each of the complementing rho1 mutation groups abolishes a distinct function of Rho1p, activation of Pkc1p kinase or 1,3-beta-glucan synthase activity.     

Accumulation of anthranilic acid and N-glucosylanthranilic acid by a Corynebacterium glutamicum mutant resistant to DL-serine hydroxamate

During a study on the effect of DL-serine hydroxamate on Corynebacterium glutamicum (JCM1318, a wild strain), a mutant resistant to the drug, strain TO3002, was isolated. This mutant accumulated five Ehrlich's reagent positive fluorescent substances in the culture medium. Two major and one minor fluorescent products were isolated by preparative high-performance liquid chromatography following charcoal column chromatography from the culture supernatant. One major product was identified as anthranilic acid whose molecular ion was confirmed to be 137 by a measurement of liquid chromatography-mass spectrometry (LC-MS), and NMR spectrum coincided with that of anthranilic acid. LC-MS spectra of another major and the minor product showed that they had the same molecular weight of 299. This major product was supported to be N-glucosylanthranilic acid (N-o-carboxyphenyl-1-beta-glucosylamine) by two-dimensional (1)H and (13)C NMR analyses. The minor product was speculated to be an Amadori compound derived from N-glucosylanthranilic acid. N-Glucosylanthranilic acid accumulated in the early phase, then decreased in the late phase of the culture. In contrast, the accumulation of anthranilic acid increased remarkably in the late phase of the fermentation. Based on this phenomenon, it was assumed that N-glucosylanthranilic acid once accumulated was decomposed to form anthranilic acid, at least in large part, with the progress of fermentation. The strain TO3002 showed a leaky requirement for L-tryptophan or indole (but did not for anthranilic acid) and resistance to DL-serine hydroxamate.     

Proteinases participating in the processing and activation of prolegumain in primary cultured rat macrophages

The mammalian legumain is a recently identified lysosomal cysteine proteinase belonging to the clan CD and homologous to plant legumain. This enzyme has the characteristic of specifically hydrolyzing peptide bonds after asparagine residues. As in the case of papain-type cysteine proteinases, legumain is synthesized as an inactive zymogen, and processed into a mature form localized in lysosomes. However, the mechanism of its activation remains unclear. In this study, we analyze which types of proteinases may participate in the processing of legumain in rat primary cultured macrophages using various proteinase inhibitors after 24 h treatment with Bafilomycin A1, a vacuolar ATPase inhibitor. The processing of legumain in macrophages was accomplished by papain-type cysteine proteinases other than cathepsin B.     

Overexpression of S-adenosylmethionine decarboxylase.（SAMDC） in Xeno-pus embryos activates maternal program of apoptosm as a “fail-safe”mechanism of early embryogenesis

In Xenopus, injection of S-adenosylmethionine decarboxylase (SAMDC) mRNA into fertilized eggs or 2-cell stage embryos induces massive cell dissociation and embryo-lysis at the early gastrula stage due toactivation of the maternal program of apoptosis. We injected SAMDC mRNA into only one of the animalside blastomeres of embryos at different stages of cleavage, and examined the timing of the onset of theapoptotic reaction. In the injection at 4-and 8-cell stages, a considerable number of embryos developed intotadpoles and in the injection at 16-and 32-cell stages, all the embryos became tadpoles, although tadpolesobtained were sometimes abnormal. However, using GFP as a lineage tracer, we found that descendant cellsof the blastomere injected with SAMDC mRNA at 8-to 32-cell stages are confined within the blastocoel atthe early gastrula stage and undergo apoptotic cell death within the blastocoel, in spite of the continued development of the injected embryos. These results indicate that cells overexpressed with SAMDC undergo apoptotic cell death consistently at the early gastrula stage, irrespective of the timing of the mRNA injection.We assume that apoptosis is executed in Xenopus early gastrulae as a “fall-safe“ mechanism to eliminate physiologically-severely damaged cells to save the rest of the embryo.     

Direct binding of Toll-like receptor 2 to zymosan,and zymosan-induced NF-kappa B activation and TNF-alpha secretion are down-regulated by lung collectin surfactant protein A

The lung collectin surfactant protein A (SP-A) has been implicated in the regulation of pulmonary host defense and inflammation. Zymosan induces proinflammatory cytokines in immune cells. Toll-like receptor (TLR)2 has been shown to be involved in zymosan-induced signaling. We first investigated the interaction of TLR2 with zymosan. Zymosan cosedimented the soluble form of rTLR2 possessing the putative extracellular domain (sTLR2). sTLR2 directly bound to zymosan with an apparent binding constant of 48 nM. We next examined whether SP-A modulated zymosan-induced cellular responses. SP-A significantly attenuated zymosan-induced TNF-alpha secretion in RAW264.7 cells and alveolar macrophages in a concentration-dependent manner. Although zymosan failed to cosediment SP-A, SP-A significantly reduced zymosan-elicited NF-kappaB activation in TLR2-transfected human embryonic kidney 293 cells. Because we have shown that SP-A binds to sTLR2, we also examined whether SP-A affected the binding of sTLR2 to zymosan. SP-A significantly attenuated the direct binding of sTLR2 to zymosan in a concentration-dependent fashion. From these results, we conclude that 1) TLR2 directly binds zymosan, 2) SP-A can alter zymosan-TLR2 interaction, and 3) SP-A down-regulates TLR2-mediated signaling and TNF-alpha secretion stimulated by zymosan. This study supports an important role of SP-A in controlling pulmonary inflammation caused by microbial pathogens.     

Estrogenic activity of an environmental pollutant, 2-nitrofluorene,after metabolic activation by rat liver microsomes

In this study, the metabolic activation of 2-nitrofluorene (NF) to estrogenic compounds was examined. NF was negative in estrogen reporter assays using estrogen-responsive yeast and human breast cancer cell line MCF-7. However, the compound exhibited estrogenic activity after incubation with liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH. Minor estrogenic activity was observed when liver microsomes of untreated or phenobarbital-treated rats were used instead of those from 3-methylcholanthrene-treated rats. When the compound was incubated with the liver microsomes of 3-methylcholanthrene-treated rats in the presence of NADPH, 7-hydroxy-2-nitrofluorene (7-OH-NF) was formed as a major metabolite. However, little of the metabolite was formed by liver microsomes of untreated or phenobarbital-treated rats. Rat recombinant cytochrome P450 1A1 exhibited a significant oxidase activity toward NF, affording 7-OH-NF. Liver microsomes of phenobarbital-treated rats also enhanced oxidase activity toward NF. In this case, 9-hydroxy-2-nitrofluorene was formed. 7-OH-NF exhibited a significant estrogenic activity, while the activity of 9-hydroxy-2-nitrofluorene was much lower. These results suggest that the estrogenic activity of NF was due to formation of the 7-hydroxylated metabolite by liver microsomes.     

Effects of different beta73 amino acids on formation of 14-stranded fibers of Hb S versus double-stranded crystals of Hb C-Harlem

Hb S (alpha(2)beta(2)(6Glu-->Val)) forms polymers, while Hb C-Harlem (alpha(2)beta(2)(6Glu-->Val,73Asp-->Asn)) forms crystals upon oversaturation. Since the only difference between the two is the beta73 amino acid, it follows that this site is a critical determinant in promoting either polymerization or crystallization. Beta73 Asp in Hb S forms a hydrogen bond with beta4 Thr, while beta73 Asn in Hb C-Harlem may inhibit this interaction as well as increase the hydrophobicity at the EF helix beta6 Val acceptor sites. Two new beta73 Hb S variants (beta73 His and Leu) were constructed and analyzed to define other amino acids facilitating formation of Hb S-like polymers versus Hb C-Harlem-like crystals. The two variants that were chosen were expected to either (1) enhance formation of the beta73-beta4 hydrogen bond (beta73 His) or (2) inhibit it and increase the hydrophobicity of the EF helix beta6 Val acceptor sites (beta73 Leu). beta73 His Hb S formed fibers but at a lower concentration than Hb S, while beta73 Leu Hb S formed crystals but at a higher concentration than Hb C-Harlem. The solubility of beta73 His Hb S was (1)/(7) of that of Hb S, while the solubility of beta73 Leu Hb S was similar to that of Hb C-Harlem. The delay time prior to polymer or crystal formation depended on Hb concentration. The delay time for beta73 His Hb S was 10(5)-fold shorter than that for Hb S, while that for beta73 Leu Hb S was 10(5)-fold longer in 1.0 M phosphate buffer. NMR results indicate beta73 amino acid changes induce alteration in the beta-chain heme pocket region, while CD results indicate no change in the helical content of the variants. These results suggest that enhancing the beta73-beta4 hydrogen bond and/or induced changes in the heme pocket by the beta73 Asp to His change facilitate formation of Hb S-like fibers. Our results also suggest that removal of the beta73-beta4 hydrogen bond and enhancing the hydrophobicity of the EF helix beta6 Val acceptor sites by the beta73 Asp to Leu or Asn changes delay nuclei formation and facilitate formation of Hb C-Harlem-like crystals.     

Inhibitory effect of curcumin on the invasion of rat ascites hepatoma cells in vitro and ex vivo

Curcumin, a yellow pigment in turmeric, is a food factor withantioxidative activity. The effect of curcumin on the proliferation and invasion of the rat ascites hepatoma AH109Acells was studied in vitro and ex vivo assay systems. Especially, a co-culture system of the hepatoma cellswith mesothelial cells derived from rat mesentery was employed to investigate the invasive motility. Curcumin suppressed thehepatoma slipping motility in a dose-dependent manner up to 5 M and thereafter maintained the effect up to 20 M, whereas this substance exerted little influence on the proliferation of the hepatoma cells at the same concentrations. Sera obtained from rats orally given curcumin also inhibited the AH109A cellular invasive movement when added to the culturemedium. Hepatoma cells previously cultured with hypoxanthineand xanthine oxidase showed a highly invasive activity. Curcumin and curcumin-loaded rat sera suppressed this reactive oxygen species-potentiated invasive capacity by simultaneously treating AH109A cells with hypoxanthine, xanthine oxidase and either of curcumin samples. These resultssuggest that the antioxidative property of curcumin may beinvolved in its anti-invasive action.