Volatile sulfur compounds in human expiration after eating raw or heat-treated garlic.

Volatile sulfur compounds arising from grated raw or heat-treated garlic in both in-vitro and in-vivo tests were gas-chromatographically analyzed. In in-vitro tests, the head-space vapor gas from garlic in a vial was analyzed. It was clarified that allyl mercaptan arising from raw garlic decreased with the passage of time and other volatile low-molecular sulfur compounds (LMSC) did not show remarkable changes. The change of LMSC from heat-treated garlic was also studied. Methyl mercaptan and allyl mercaptan from heat-treated garlic gradually increased to some extent. On the other hand, the quantities of somewhat high-molecular sulfur compounds (HMSC) were much less in heat-treated garlic compared to those of raw garlic. These compounds increased till approx. 60 min and then decreased gradually. In in-vivo tests, human expiration after eating garlic was analyzed. Allyl mercaptan, methyl mercaptan and allyl methyl sulfide in LMSC were detected in significant amounts. The quantities of these compounds arising from heat-treated garlic were smaller than those from raw garlic. These compounds had the tendency of decreasing with the passage of time. On the other hand, almost no HMSC was detected in both raw and heat-treated garlic. By sensory testing, raw garlic showed a stronger smell than heat-treated garlic in both in-vitro and in-vivo tests. GC analysis exhibited higher values of volatile sulfur compounds in raw garlic. That is, the higher the volatile sulfur compound level, the stronger the garlic flavor or malodor.

In vitro activity of the hairpin ribozyme derived from the negative strand of arabis mosaic virus satellite RNA.

The negative strand of the satellite RNA of tobacco ringspot virus [(-)sTRSV] is a self-cleaving RNA, of which self-cleaving domain is called the hairpin ribozyme. The negative strand of the satellite RNA of arabis mosaic virus [(-)sArMV] has been suggested to have a hairpin ribozyme-like secondary structure, and we have previously shown that this hairpin domain of (-)sArMV has ribozyme activity. Here we report characterization of the cleavage reaction of the (-)sArMV hairpin ribozyme. Mutagenesis analyses in a trans-acting system revealed, surprisingly, that the wild-type ribozyme was less active than almost all the other mutant ribozymes tested. In a cis-acting system (self-cleaving reaction), however, the reaction of the RNA containing the wild-type sequence proceeds highly efficiently. This result suggests that the inefficient cleavage of the wild-type substrate in trans-acting system may be due to low efficiency at the substrate-binding step but not at the chemical cleavage step in the reaction. We also constructed a chimeric ribozyme between the catalytic hairpin domain from (-)sArMV and the substrate-binding site from (-)sTRSV. This chimeric ribozyme had the highest activity among the trans-acting hairpin ribozymes tested.

Hairpin ribozyme-mediated cleavage of the full-length beta-glucuronidase (GUS) mRNA.

We designed three hairpin ribozymes to cleave Escherichia coli beta-glucuronidase (GUS) mRNA and tested those activities in vitro. One of the ribozymes was designed to form 9 base pairs in total with the target GUS mRNA, and the other two ribozymes had longer substrate binding sites. All ribozymes cleaved the model substrate (100 bases long) at the predicted target site. Two ribozymes containing longer substrate binding sites cleaved the substrate much more efficiently than the other ribozyme containing shorter substrate binding site. Also, the ribozymes with long substrate binding sites had high activity against the full-length GUS mRNA (1.9 kilobases) and maintained the activity even at a low temperature, 26 degrees C, a general growth condition of plant cells. Effects of the substrate binding site length of the ribozyme on cleavage activity are discussed.

Cis and trans reactions of hairpin ribozymes derived from the negative strand of arabis mosaic virus satellite RNA.

We have previously shown that the hairpin ribozyme-like structure of the negative strand of the satellite RNA of arabis mosaic virus [(- )sArMV] has indeed ribozyme activity. However, some mutagenesis analyses revealed surprisingly that the wild type ribozyme was less active than almost all the other mutant ribozymes tested. These results were derived from a trans-acting system. Here we tested this ribozyme activity in a cis-acting system. We show that the (-)s ArMV hairpin ribozyme has different target-site specificities between cis and trans cleavages. The wild type ribozyme has the highest self-cleaving activity among the ribozyme variants tested.

A novel fucosylated glycosphingolipid with a Gal beta 1-4Glc beta 1-3Gal sequence in plerocercoids of the parasite, Spirometra erinacei.

A novel glycosphingolipid (SEGLx) has been isolated from the plerocercoids of a tapeworm, Spirometra erinacei. From the results of compositional analysis, methylation analysis, exoglycosidase hydrolysis, acid hydrolysis, fast atom bombardment mass spectrometry, and proton nuclear magnetic resonance (NMR) analysis, its structure was concluded to be [formula: see text] This is the first report of a glycosphingolipid with a novel carbohydrate structure which is characterized by i) the occurrence of a penultimate glucose molecule attached to the reducing end galactose through a beta 1-3 linkage and ii) the presence of a fucose attached to a glucose through an alpha 1-3 linkage. The ceramide contained sphinganine or 4-D-hydroxy-sphinganine, and either a nonhydroxy fatty acid with 16, 18, 26, or 28 carbon atoms, or hydroxystearic acid. Proton NMR analysis revealed that the chemical species of both the long chain base and fatty acid moieties affect the chemical shifts of the anomeric proton resonances of not only the reducing terminal galactose but also the penultimate glucose.

Acquirement of hairpin ribozyme activity by the long substrate-binding site.

Three hairpin ribozymes were designed to cleave Escherichia coli beta-glucuronidase (GUS) mRNA at a target site. Two of the designed ribozymes (HG10L and HG10L2) had long substrate binding sites and the other (HG10) had short substrate binding site. All three ribozymes cleaved the substrate, and HG10L and HG10L2 cleaved it efficiently. However, HG10 had very low activity. Effect of length of substrate binding site of the ribozyme will be discussed.

Relationship between adenylate cytokinin production and Ti plasmid of Agrobacterium tumefaciens.

To know whether the tumor-inducing plasmid of Agrobacterium tumefaciens carries genetic information of the biosynthesis of cytokinins, the levels of 6-(3-methyl-2-butenyl-amino)purine (iPAde) and its 4-hydroxy derivative trans-zeatin (trans-Z) and its p-beta-D-ribofuranoside (trans-ZR) produced in media by wild-type virulent strain, plasmid-cured avirulent strain and the deletion mutant were compared. The highest levels of iPAde and trans-Z were found in the culture filtrate of late-log phase growth of plasmid-containing virulent strain, then the levels of iPAde and trans-Z were reduced rapidly at stationary phase. The plasmid-cured avirulent strain and deletion mutant had low levels of iPAde and trans-Z throughout the growth. Results obtained here showed Ti plasmid plays an important role in cytokinin biosynthesis.

Proteins conferred by the virulence-specifying plasmid of Agrobacterium tumefaciens C-58.

Membrane-associated and periplasmic proteins of Agrobacterium tumefaciens C-58 were compared with those from avirulent (nontumrigenic) derivative strains by slab and two-dimensional gel electrophoresis. Two proteins (Per-I and Per-2), with a molecular weight of 37,500 and 37,300, respectively, were detected in the supernatant fraction of cells of strain C-58 treated with EDTA and lysozyme in which a 117-megadalton plasmid confers virulence on the organism. The same proteins are missing in an avirulent plasmid-free derivative of C-58. When this derivative is mated with C-58, the resulting transconjugants regain the large C-58 plasmid together with the restoration of virulence and the expression of Per-1 and Per-2 proteins. When the transconjugants were cured of their plasmid, they concomitantly lost their virulence and Per-1 and Per-2. The functional roles of these proteins are unknown, but they are associated with the outer membrane and periplasmic fraction of the Agrobacterium cell. If directly involved in tumorigenesis, these proteins are not the sole determinants of tumorigenicity because they are synthesized in an avirulent derivative of C-58 that carries a deletion in the plasmid in the region conferring the tumorigenic phenotype. These results strongly suggest that the Per-1 and Per-2 proteins are plasmid-coded gene products. The possible roles of these proteins in specifying host range and host-cell attachment are also discussed.

Effect of phospholipase C hydrolysis of membrane phospholipids on acyltransferase systems in rat liver microsomes.

Three kinds of phospholipase C [EC 3.1.4.3] were used to selectively hydrolyze phospholipids in rat liver microsomes, and their effects on the acyl-CoA: glycerophosphate and acyl-CoA: lysophospholipids acyltransferase systems were examined. The glycerophosphate acyltransferase [EC 2.3.1.15] system was inactivated rapidly by treatment with phospholipase C of Ps. aureofaciens or B. cereus and the loss of activity paralleled the degradation of phosphatidylcholine and phosphatidylethanolamine. The 1-acylglycerylphosphorylcholine acyltransferase [EC 2.3.1.23] system was only partially inactivated under the same conditions, whereas the 1-acylglycerophosphate acyltransferase [EC 2.3.1.51] system retained most of its activity even when more than 95% of phosphatidylcholine and phosphatidylethanolamine had been hydrolyzed. The results demonstrate the heterogeneity of acyltransferase systems with respect to their dependence on the intact membrane phospholipids. Hydrolysis of more than 80% of phosphatidylinositol by phosphoinositidase of B. cereus did not significantly affect these acyltransferase systems. The specificity for various acyl-CoA's of 1-acylglycerophosphate acyltransferase in microsomes treated with phospholipase C of Ps. aureofaciens was apparently different from that in untreated microsomes, while the specificity of 1-acylglycerylphosphorylcholine acyltransferase was unchanged. Saturation profiles of the acceptors were significantly different between the acyltransferase systems in phospholipase C-treated and untreated microsomes. These results suggest that 1-acylglycerophosphate and 1-acylglycerylphosphorylcholine acyltransferase systems do not require specific phospholipids such as phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol for their catalytic activities, but the integrity of these phospholipids is necessary for the proper functioning and stability of the enzymes.

Studies on phospholipase C from Pseudomonas aureofaciens. II. Further studies on the properties of the enzyme.

Phospholipase C [EC 3.1.4.3] from Pseudomonas aureofaciens was found to be inhibited by chelating reagents such as ethylenediaminetetraacetate [EDTA] and o-phenanthroline. The inhibition was reversed by the addition of Zn2+ and, to a lesser extent, by Co2+ and Mn2+. On isoelectric focusing, the isoelectric point of this enzyme proved to be 6.3--6.5, with a single peak. The enzyme reaction with the substrate was followed in media containing an organic solvent such as diethyl ether or diethyl ether-ethyl alcohol. When ethyl alcohol was added (up to 2%) to the reaction mixture in ether, there were no marked changes in the hydrolytic rates of phosphatidylcholine and phosphatidylethanolamine. However, the enzyme activity was inhibited when the alcohol concentration was increased above 2%. In 98% diethyl ether-2% ethyl alcohol, phosphatidylcholine was hydrolyzed more rapidly than phosphatidylethanolamine, in contrast with the result obtained in water. In the single micelle state, phosphatidylethanolamine was hydrolyzed more rapidly than phosphatidylcholine or lysophatidylcholine. Acidic phospholipids and sphinogomyelin were not hydrolyzed. When the enzyme was incubated with phospholipid mixture extracted from Ps aureofaciens and rat liver, both phosphatidylethanolamine and phosphatidylcholine were hydrolyzed more rapidly than in the single micelle state of these substrates.

Studies on phospholipase C from Pseudomonas aureofaciens. I. Purification and some properties of phospholipase C.

Phospholipase C (phosphatidylcholine cholinephosphohydrolase, EC 3.1.4.3) from Pseudomonas aureofaciens was purified 3600-fold from the culture filtrate with a recovery of 1.6%. Purification was performed with the useof (NH4)2SO4 precipitation, Sephadex G-100 gel filtration and by ion-exchange chromatography on DEAE-Sephadex A-50 and CM-Sephadex C-50. The purified enzyme appeared to be homogeneous as revealed by polyacrylamide disc gel electrophoresis at pH 9.3. The molecular weight was estimated to be 35 000 by gel filtration on Sephadex G-75. Under our experimental conditions, phosphatidylethanolamine was more rapidly hydrolysed than phosphatidylcholine. Lyso forms of these two phosphatides were poor substrates. Phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, cardiolipin and sphingomyelin were not hydrolysed. The enzyme activity with phosphatidylcholine as substrate was slightly stimulated by Ca2+, Mg2+, and Mn2+. However, these cations inhibited the activity with phosphatidylethanolamine as substrate. An anionic detergent, sodium deoxycholate, slightly enhanced the activity when phosphatidylcholine and phosphatidylethanolamine were used as substrates. A cationic detergent, cetyltrimethylammonium bromide, inhibited enzyme activity. EDTA and o-henanthroline inhibited the activity of the enzyme to a marked degree.