Release of Antifungal Phenolic Compounds from Cucumber Mosaic Virus-Infected and Noninfected Cowpea Protoplasts

Fungitoxic phenolic compounds were released from cucumber mosaic virus (CMV)-infected and noninfected cowpea protoplasts. These compounds were presumed by thin layer chromatography as similar compounds released into the leaf ambient fluids when CMV-infected cowpea leaves were incubated in water. Larger amounts of the compounds were released from CMV-infected cowpea protoplasts than from noninfected protoplasts.     

Chemical ionization and electron impact mass spectra of oligosaccharides derived from sphingoglycolipids

Chemical ionization (CI) mass spectra with isobutane and ammonia for the oligosaccharides obtained from sphingoglycolipids were compared with their electron impact (EI) mass spectra. The oligosaccahride moieties were liberated from the parent glycolipids and were further reduced with sodium borohydride. They were analyzed as their permethyl peracetyl and pertrimethylsilyl derivatives. In the CI spectra, peaks corresponding to QM+ and/or [M-59]+ were observed in all of the peracetylated oligosaccharides examined. In CI with ammonia as the reagent, H+ was transferred to nitrogen-containing saccharides to produce [MH]+ and NH4 was transferred to nitrogen-free saccharides to yield [M+NH4]+ as QM+. Non-reducing ends yielded very intense peaks in CI spectra. On the other hand, the reduced end, glucitol, produced rather prominent peaks in EI spectra. Fragment ions due to cleavage of glycosidic bonds were major ones under the CI conditions, and they could be used for elucidating the sugar sequence in the oligosaccharides. An additional characteristic feature in the CI spectra was that ions due to scission of hexosaminyl glycosidic linkages were observed with very high intensities.     

Experimental toxicoinfection in infant mice challenged with spores of Clostridium botulinum type E

Conventionally raised suckling mice were given 10(7) spores of a strain of Clostridium botulinum type E. Most but not all infant mice aged 8 through 19 days at the time of administration died after developing symptoms typical of botulism. However, none of the infant mice challenged with the spores at dose levels lower than 10(6) spores/mouse developed illness.     

Preparation of methyl glycosides of di- and higher oligosaccharides from glycosaminoglycuronans by solvolysis with dimethyl sulfoxide containing methanol

Solvolysis of chondroitin 4- or 6-sulfate (pyridinium salt) with dimethyl sulfoxide containing 10% of methanol for 18 h at 95° resulted in the cleavage of the 2-amino-2-deoxy-D-glucoside bonds together with initial desulfation to give methyl β-glycosides of N-acetylchondrosine as a main product and, in addition, higher oligosaccharides, without any loss of uronic acid. Dermatan sulfate was also depolymerized to yield methyl glycosides of di- and higher oligosaccharides under the same conditions. Hyaluronic acid (free acid) was depolymerized by the same solvent in the presence of an equimolar amount of pyridine-sulfur trioxide or pyridinium sulfate per disaccharide unit to give methyl glycosides of di- and higher oligosaccharides. In contrast N-desulfated, N-acetylated heparin was stable under these solvolytic conditions and did not yield heparin oligosaccharides.     

REGIONAL BRAIN LEVELS OF γ-HYDROXYBUTYRATE IN HUNTINGTON''S DISEASE

A highly sensitive electron capture gas chromatographic method was developed for quantitation of γ-hydroxybutyrate (GHB) in tissue. This method involves an improved, extraction and purification procedure and a one-step derivatization of GHB to the methyl ester-O-heptafluorobutyrate. As low as 5 ng of GHB in tissue was accurately quantitated by this method. By means of this improved method, endogenous levels of GHB in several regions of brains obtained post-mortem from patients with Huntington's disease were determined, and compared with brain samples obtained post-mortem from non-neurological controls. The levels of GHB found in the caudate and substantia nigra obtained from Huntington's patients were significantly higher than the GHB levels found in similar regions of brain obtained from a non-neurological control group. The content of GABA in the same choreic and control brain samples was also determined. No significant correlation between changes in GHB and GABA levels was observed although there was a trend towards an inverse relationship. The high level of GHR in Huntington's disease may be related to the decrease in succinate:oxidoreductase (EC 1.3.99.1) activity reported by Stahl & Swanson (1974). In two subjects (one control and one Huntington patient) the zonal distribution of GHB in substantia nigra was also determined. The zona reticulata from choreic brain contained a substantially higher level of GHB, whereas the zona compacta contained an amount similar to the level found in control brain.     

Genetic structure of the IncN plasmid N3

N3, a plasmid of incompatibility group N (IncN) was mapped by cleavage with restriction endonucleases. The restriction fragments were cloned into vector plasmids. All of the genes unique to IncN plasmids such as specific replication machinery, a restriction-modification system, and repair functions were located on a large portion which had no cleavage sites for many of the site-specific six base-identifying restriction endonucleases tested.     

Interaction of basic oligo-L-amino acids with deoxyribonucleic acids. Oligo-L-arginines of various chain lengths and herring sperm DNA

The interaction of DNA and oligo-L-arginines having definite chain lengths of 1-17 residues was studied by precipitate formation and thermal denaturation of the complexes in order to obtain a better understanding of the roles of nuclear basic proteins. The results can be summarized as follows. 1. Those oligo-L-arginines, (Arg)n, in which n greater than or larger than 4 can bind with DNA irreversibly to form precipitates of the complexes. Among them, oligomers larger than (Arg)5 precipitate DNA completely in Arg/P input ratios below 1. The Arg/P ratios in the precipitates are between 0.6-0.8. 2. The thermal stability of the complexes depends on the method of complex formation, and complexes formed by the dialysis method are more stable than those formed by the mixing method. 3. The binding of (Arg)n to DNA was found to be reversible and in a equilibrium for n less than or equal to 6. In general, the longer the oligomer, the higher the stability of the complex at a definite Arg/P ratio. 4. For (Arg)7-10, three kinds of complexes with different stabilities are formed between DNA and oligopeptides. 5. For (Arg)14-17, only a restricted type of complexes can be formed between DNA and oligomers, as in the case with poly-L-arginine or protamines. 6. The interaction between basic nuclear proteins and DNA is discussed in the light of the basic region in protamine and histone molecules.     

A cytolysin, theta-toxin, preferentially binds to membrane cholesterol surrounded by phospholipids with 18-carbon hydrocarbon chains in cholesterol-rich region.

We have previously suggested the existence of two distinctive states of cholesterol in erythrocyte and lymphoma cell membranes as revealed by high- and low-affinity binding sites for theta-toxin of Clostridium perfringens [Ohno-Iwashita, Y., Iwamoto, M., Mitsui, K., Ando, S., & Nagai, Y. (1988) Eur. J. Biochem. 176, 95-101; Ohno-Iwashita, Y., Iwamoto, M., Ando, S., Mitsui, K., & Iwashita, S. (1990) Biochim. Biophys. Acta 1023, 441-448]. To understand factor(s) which determine membrane cholesterol heterogeneity, we analyzed toxin binding to large unilamellar liposomes composed of cholesterol and phospholipids (phosphatidylcholine/phosphatidylglycerol = 82:18, mol/mol). Liposomes containing phospholipids with 18-carbon hydrocarbon chains at both positions 1 and 2 of the glycerol have both high- and low-affinity toxin-binding sites with Kd values similar to those of intact erythrocytes, whereas liposomes with hydrocarbon chains containing 16 or fewer carbons at either position 1 or 2 have only low-affinity toxin-binding sites. The cholesterol/phospholipid ratio, in addition to the length of phospholipid hydrocarbon chain, also determines the number of toxin-binding sites, indicating that at least these two factors determine the topology of membrane cholesterol by creating distinctively different affinity sites for the toxin. Since theta-toxin binding detects specific populations of membrane cholesterol that are not detectable by the measurements of susceptibility to cholesterol oxidase and cholesterol desorption from membranes, the toxin could provide a unique probe for studying the organization of cholesterol in membranes.     

Structure of triphosphonoglycosphingolipid containing N-acetylgalactosamine 6-O-2-aminoethylphosphonate in the nervous system of Aplysia kurodai

A phosphonoglycosphingolipid, named F-21, was found in the nervous system of Aplysia kurodai by two-dimensional thin-layer chromatography (Abe, S., Araki, S., and Satake, M. (1986) Biomed. Res. (Tokyo) 7, 47-51). F-21 was isolated from the nervous tissue of Aplysia in this study, and its chemical structure was characterized as follows, where 2-AEP is 2-aminoethylphosphonate. (Formula; see text) The major aliphatic components of the ceramide portion were palmitic acid (75%), stearic acid (22%), octadeca-4-sphingenine (43%), and anteisononadeca-4-sphingenine (54%). Some information on the steric interactions in the sugar moiety was obtained by NMR spectroscopy. The ring protons of the internal galactose, H1, H3, and H4 and the H3 of the side chain galactose were shifted, as compared to the corresponding protons of dephosphonylated F-21. This may indicate the interactions between the 2-AEP residue of N-acetylgalactosamine and the internal galactose and between the N-acetyl group of N-acetylgalactosamine and the side chain galactose, implying a sterically restricted and unique structure that may relate to some biological functions of F-21.     

Expression of the GABAB receptor in Xenopus oocytes and inhibition of the response by activation of protein kinase C.

The functional GABAB receptor was expressed in Xenopus oocytes by injecting mRNA obtained from the cerebellum of the rat. Application of GABA in the presence of bicuculline induced a hyperpolarization under current-clamp conditions and an outward current under voltage-clamp conditions. Baclofen mimicked the effect of GABA in the presence of bicuculline, and the effect of baclofen was antagonized by phaclofen. The GABA-induced outward current was slightly inhibited by treatment with GDP-beta-S and was completely inhibited by treatment with GTP-gamma-S. The activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate (TPA), but not 4 alpha-phorbol-12,13-didecanoate, suppressed the GABAB receptor-mediated hyperpolarization, and the effect of TPA was antagonized by sphingosine. Thus, activation of protein kinase C inhibits the expressed GABAB receptor-mediated response.