Eudesmane-type sesquiterpene lactones inhibit multiple steps in the NF-κB signaling pathway induced by inflammatory cytokines

Inflammatory cytokines, such as interleukin-1α (IL-1α) and tumor necrosis factor-α (TNF-α), induce the intracellular signaling pathway leading to the activation of nuclear factor κB (NF-κB). A series of eudesmane-type sesquiterpene lactones possessing an α-methylene γ-lactone group and/or an α-bromo ketone group were synthesized and evaluated for their inhibitory effects on the NF-κB-dependent gene expression and signaling pathway. Our present study reveals that eudesmane-type α-methylene γ-lactones and α-bromo ketones inhibit multiple steps in the NF-κB signaling pathway induced by IL-1α and TNF-α.     

An isoflavone conjugate-hydrolyzing beta-glucosidase from the roots of soybean (Glycine max) seedlings: purification, gene cloning, phylogenetics, and cellular localization

Soybeans (Glycine max (L.) Merr.) and certain other legumes excrete isoflavones from their roots, which participate in plantmicrobe interactions such as symbiosis and as a defense against infections by pathogens. In G. max, the release of free isoflavones from their conjugates, the latent forms, is mediated by an isoflavone conjugate-hydrolyzing beta-glucosidase. Here we report on the purification and cDNA cloning of this important beta-glucosidase from the roots of G. max seedlings as well as related phylogenetic and cellular localization studies. The purified enzyme, isoflavone conjugate-hydrolyzing beta-glucosidase from roots of G. max seedling (GmICHG), is a homodimeric glycoprotein with a subunit molecular mass of 58 kDa and is capable of directly hydrolyzing genistein 7-O-(6 '-O-malonyl-beta-d-glucoside) to produce free genistein (k(cat), 98 s(-1); K(m), 25 microM at 30 degrees C, pH 7.0). GmICHG cDNA was isolated based on the amino acid sequence of the purified enzyme. GmICHG cDNA was abundantly expressed in the roots of G. max seedlings but only negligibly in the hypocotyl and cotyledon. An immunocytochemical analysis using anti-GmICHG antibodies, along with green fluorescent protein imaging analyses of Arabidopsis cultured cells transformed by the GmICHG:GFP fusion gene, revealed that the enzyme is exclusively localized in the cell wall and intercellular space of seedling roots, particularly in the cell wall of root hairs. A phylogenetic analysis revealed that GmICHG is a member of glycoside hydrolase family 1 and can be co-clustered with many other leguminous beta-glucosidases, the majority of which may also be involved in flavonoid-mediated interactions of legumes with microbes.     

Effects of compressed hydrocarbon gases on the growth activity of Saccharomyces cerevisiae

The inhibitory action of compressed hydrocarbon gases on the growth of the yeast Saccharomyces cerevisiae was investigated quantitatively by microcalorimetry. Both the 50% inhibitory pressure (IP(50)) and the minimum inhibitory pressure (MIP), which are regarded as indices of the toxicity of hydrocarbon gases, were determined from growth thermograms. Based on these values, the inhibitory potency of the hydrocarbon gases increased in the order methane < ethane < propane < i-butane < n-butane. The toxicity of these hydrocarbon gases correlated to their hydrophobicity, suggesting that hydrocarbon gases interact with some hydrophobic regions of the cell membrane. In support of this, we found that UV absorbing materials at 260 nm were released from yeast cells exposed to compressed hydrocarbon gases. Additionally, scanning electron microscopy indicated that morphological changes occurred in these cells.     

Deciphering the molecular basis of the broad substrate specificity of alpha-glucosidase from Bacillus sp. SAM1606

The alpha-glucosidase of Bacillus sp. strain SAM1606 is a member of glycosyl hydrolase family 13, and shows an extraordinarily broad substrate specificity and is one of very few alpha-glucosidases that can efficiently hydrolyze the alpha-1,1-glucosidic linkage of alpha,alpha'-trehalose (trehalose). Phylogenetic analysis of family-13 enzymes suggests that SAM1606 alpha-glucosidase may be evolutionally derived from an alpha-1,6-specific ancestor, oligo-1,6-glucosidase (O16G). Indeed, replacement of Pro(273*) and Thr(342*) of B. cereus O16G by glycine and asparagine (the corresponding residues in the SAM1606 enzyme), respectively, was found to cause 192-fold enhancement of the relative catalytic efficiency for trehalose, suggesting that O16G may easily "evolved" into an enzyme with an extended substrate specificity by substitution of a limited number of amino acids, including that at position 273* (an asterisk indicates the amino-acid numbering of the SAM1606 sequence). To probe the role of the amino acid at position 273* of alpha-glucosidase in determination of the substrate specificity, the amino acid at position 273 of SAM1606 alpha-glucosidase was replaced by all other naturally occurring amino acids, and the resultant mutants were kinetically characterized. The results showed that substitution of bulky residues (e.g., isoleucine and methionine) for glycine at this position resulted in large increases in the K(m) values for trehalose and maltose, whereas the affinity to isomaltose was only minimally affected by such an amino-acid substitution at this position. Three-dimensional structural models of the enzyme-substrate complexes of the wild-type and mutant SAM1606 alpha-glucosidases were built to explore the mechanism responsible for these observations. It is proposed that substitution by glycine at position 273* could eliminate steric hindrance around subsite +1 that originally occurred in parental O16G and is, at least in part, responsible for the acquired broad substrate specificity of SAM1606 alpha-glucosidase.     

Collagenolytic serine-carboxyl proteinase from Alicyclobacillus sendaiensis strain NTAP-1: purification,characterization, gene cloning,and heterologous expression

Enzymatic degradation of collagen produces peptides, the collagen peptides, which show a variety of bioactivities of industrial interest. Alicyclobacillus sendaiensis strain NTAP-1, a slightly thermophilic, acidophilic bacterium, extracellularly produces a novel thermostable collagenolytic activity, which exhibits its optimum at the acidic region (pH 3.9) and is potentially applicable to the efficient production of such peptides. Here, we describe the purification to homogeneity, characterization, gene cloning, and heterologous expression of this enzyme, which we call ScpA. Purified ScpA is a monomeric, pepstatin-insensitive carboxyl proteinase with a molecular mass of 37 kDa which exhibited the highest reactivity toward collagen (type I, from a bovine Achilles tendon) among the macromolecular substrates examined. On the basis of the sequences of the peptides obtained by digestion of collagen with ScpA, the following synthetic peptides were designed as substrates for ScpA and kinetically analyzed: Phe-Gly-Pro-Ala*Gly-Pro-Ile-Gly (k(cat), 5.41 s(-1); K(m), 32 micro M) and Met-Gly-Pro-Arg*Gly-Phe-Pro-Gly-Ser (k(cat), 351 s(-1); K(m), 214 micro M), where the asterisks denote the scissile bonds. The cloned scpA gene encoded a protein of 553 amino acids with a calculated molecular mass of 57,167 Da. Heterologous expression of the scpA gene in the Escherichia coli cells yielded a mature 37-kDa species after a two-step proteolytic cleavage of the precursor protein. Sequencing of the scpA gene revealed that ScpA was a collagenolytic member of the serine-carboxyl proteinase family (the S53 family according to the MEROPS database), which is a recently identified proteinase family on the basis of crystallography results. Unexpectedly, ScpA was highly similar to a member of this family, kumamolysin, whose specificity toward macromolecular substrates has not been defined.     

Substrate specificities of several prenyl chain elongating enzymes with respect to 4-methyl-4-pentenyl diphosphate

In order to develop synthetic methods for biologically active homoallylic terpene sulfates, we examined the applicability and substrate specificities of several prenyl chain elongating enzymes with respect to 4-methyl-4-pentenyl diphosphate (homoIPP). The reaction of dimethylallyl diphosphate with homoIPP by use of Bacillus stearothermophilus (all-trans)-farnesyl diphosphate synthase resulted in efficient yields of cis-(yield: 45.9%) and trans-4,8-dimethylnona-3,7-dien-1-ol (homoGOH, 25.5%), which has a carbon skeleton of 4,8-dimethylnona-3-en-1-sulfate, an antiproliferative compound from a marine organism (Aiello, A. et al., Tetrahedron, 53, 11489-11492 (1997)). The homoIPP was found to be also active as a homoallylic substrate in place of isopentenyl diphosphate for Sulfolobus acidocaldarius geranylgeranyl diphosphate synthase to give diphosphate of cis- and trans-4,8,12-trimethyltrideca-3,7,11-trien-1-ol, for Micrococcus luteus B-P 26 hexaprenyl diphosphate synthase to give cis- and trans-4,8,12,16-tetramethylheptadeca-3,7,11,15-tetraen-1-ol (homoGGOH), and for Micrococcus luteus B-P 26 undecaprenyl diphosphate synthase to give cis-homoGGOH exclusively.