Characterization of Lipid A,A Component of Lipopolysaccharides from Selenomonas ruminantium

Lipid components of a glycolipid, formerly designated as spot A, from the cells of Selenomonas ruminantium were investigated. The basic structure of this material had been previously shown to be β-glucosaminyl-l,6-glucosamine. The major component of O- and N-acyl side chains was β-OH C_13:0 acid when the cells were grown with added valerate. Approximately 85 % of the total amide linked fatty acids was this compound. A considerable amount of C_13:2 acid was also present as a component of O-acyl fatty acids. When the cells were grown in a glucose medium containing caproate, the major fatty acid component of the spot A compound was β-OH myristic and β-OH C_13:0: acids. ¹⁴C-Valerate or ¹⁴C-caproate, supplemented to the glucose medium, was incorporated into O- and N-acyl linked fatty acid moieties of the spot A compound. It was also shown that the spot A compound was the lipid A component of lipopolysaccharides of this organism.     

Antifungal garcinia acid esters from the fruits of Garcinia atroviridis

Two new garcinia acid derivatives, 2-(butoxycarbonylmethyl)-3-butoxycarbonyl-2-hydroxy-3-propanolide and 1',1"-dibutyl methyl hydroxycitrate, were isolated from the fruits of Garcinia atroviridis guided by TLC bioautography against the fungus Cladosporium herbarum. The structures of these compounds were established by spectral analysis. The former compound represents a unique beta-lactone structure and the latter compound is most likely an artefact of garcinia acid (= hydroxycitric acid). Both compounds showed selective antifungal activity comparable to that of cycloheximide (MID: 0.5 microg/spot) only against C herbarum at the MIDs of 0.4 and 0.8 microg/spot but were inactive against bacteria (Bacillus subtilis, methicillin-resistant Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli), other fungi (Alternaria sp., Fusarium moniliforme and Aspergillus ochraceous) including the yeast Candida albicans.     

Identification of the Basic Structure of a Glycolipid from Selenomonas ruminantium as β-Glucosaminyl-1,6-glucosamine

Lipid materials extracted by chloroform-methanol from solvent and acid treated cells of Selenomonas ruminantium grown with ¹⁴C-valerate were analyzed by thin-layer chromatography. At least 12 radioactive lipid materials were present in the extract. The major compound, designated as spot A, carried approximately 70% of radioactivity of this fraction. The spot A compound was purified by column and thin-layer chromatography and its chemical structure was studied. The basic structural unit of this material was tentatively identified as β-gIucosaminyl-1,6-glucosamine with O-acyl and amide linked fatty acids.     

Sinapine Leakage from Non-viable Cabbage Seeds

Seeds leak many compounds during the early phases of germinationand seed viability may be associated with differential leakageof specific compounds. Leakage of a fluorescent compound fromnon-viable cabbage (Brassica oleracca var. capitata L.) wasdocumented and studies were performed to identify the fluorescentcompound. Imbibed samples of both heat-killed (HK) and viablecabbage seeds were submerged in a viscous colloidal gel. After2 h to 4 h, a fluorescent halo was observed under U.V. lightaround the heat-killed seed but not around the viable seed.Viable and HK seeds were imbibed in water for 8 h and the pHof the leachate was adjusted to either 7 or 10. The absorptionspectra of leakage from HK seeds revealed peak values at 322nm and 388 nm at pH 7 and 10, respectively. This pattern wasnot observed from viable seed leakage. Two-dimensional paperchromatography was conducted on the HK seed leachate. Four fluorescentspots were observed after development first with BAW (n-butanol:acetic acid: water, 4: 1: 5 by vol.) followed by 6% acetic acid.One of the fluorescent spots (spot 3) was studied further dueto its observed intensity. Sinapine thiocyanate was preparedfrom rapeseed oilmeal and used as a reference sample. Absorptionspectra of spot 3 and sinapine thiocyanate were similar at bothpH 7 and 10. Spot 3 had identical R_F values using three solventsystems and identical colour reactions in five tests comparedto sinapine thiocyanate. It was concluded that sinapine wasthe major compound responsible for the fluorescent leakage fromHK cabbage seeds. Key words: Leachate, viability, Brassica     

Isolation and identification of β-citryl-L-glutamic acid from newborn rat brain

Ab unknwon compound containing glutamic acid residue was found in newborn rat brain. The compound occurred predominantly in brain. Its concentration was approx. 1 μmol/g tissue at birth and decreased to one-tenth 24 days after birth.The compound was isolated from newborn rat brains, and subjected to elementary analysis and to infrared and mass spectrometric analysis. Glutamic acid and citric acid were formed from the compound on acid hydrolysis. The compound was presumed to be a citryglutamic acid.Two isomers, α- and β-citrylglutamic acid, were sunthesized. The unknown compound was identified as β-citryl-L-glutamic acid. The occurrence of this compound has not been reported in nature.     

Isolation and identification of β-citryl-L-glutamic acid from newborn rat brain

Ab unknwon compound containing glutamic acid residue was found in newborn rat brain. The compound occurred predominantly in brain. Its concentration was approx. 1 μmol/g tissue at birth and decreased to one-tenth 24 days after birth.The compound was isolated from newborn rat brains, and subjected to elementary analysis and to infrared and mass spectrometric analysis. Glutamic acid and citric acid were formed from the compound on acid hydrolysis. The compound was presumed to be a citryglutamic acid.Two isomers, α- and β-citrylglutamic acid, were sunthesized. The unknown compound was identified as β-citryl-L-glutamic acid. The occurrence of this compound has not been reported in nature.     

Benzylideneacetone, an eicosanoid biosynthesis inhibitor enhances baculovirus pathogenicity in the diamondback moth, Plutella xylostella

Benzylideneacetone (BZA) is a monoterpenoid compound produced by an entomopathogenic bacterium, Xenorhabdus nematophila. BZA inhibits phospholipase A₂ to suppress biosynthesis of eicosanoids that mediate immune responses in insects. In response to per os infection of Autographa californica multiple nucleopolyhedrosis virus (AcMNPV), the diamondback moth, Plutella xylostella, developed red spots on the midgut epithelium. The midgut exhibiting red spot formation suffered abnormal cell integrity, such as genomic DNA fragmentation and condensed spots in the nucleoplasm. The number of red spots increased with viral dose and incubation time after the viral treatment. BZA inhibited the formation of the midgut red spots in a dose-dependent manner. However, the inhibitory effect of BZA on the red spot formation was reversed by addition of arachidonic acid, suggesting that the red spot response may be mediated by eicosanoids. BZA treatment resulted in significant enhancement of AcMNPV occlusion body (OB) pathogenicity to P. xylostella.     

Effect of kinetin and abscisic acid on the distribution of 2-amino (1-14C) isobutyric acid in leaves of Zea mays

2-Amino (1-¹⁴C) isobutyric acid (AIB) was applied to the leaf tips of thirteen-day-old primary leaves of Zea mays L. cv. Goldprinz. Simultaneously, the leaves were treated in the basal region with solutions of kinetin (KN), abscisic acid (ABA) and mixtures of both hormones. The distribution of the radioactive material was determined after 72 h. Treatment with KN caused an accumulation of radioactive material at the point of application (KN spot). In comparison to controls, the leaf zones between the KN spot and the leaf tip contained lower amounts of radioactive material. Treatment with ABA caused a pattern of distribution opposite to that induced by KN. The ABA point of application (ABA spot) was depleted of radioactive material, whereas the leaf area between the ABA spot and the leaf tip showed enhanched levels of radioactive AIB. The effect of ABA was reduced by application of ABA and KN in equimolar amounts. The results are discussed in relation to the possible role of ABA in hormone-directed transport processes.     

Rapid screening of triazines and quantitative determination in drinking water

A sensitive, rapid and inexpensive analysis method has been developed for the triazines most frequently used in Palestine; the method includes fluorodensitometric screening and densitometric determination of the individual substances. Terbutryn as a model substance was derivatized with dansyl chloride in sodium hydrogen-carbonate or phosphate buffer solution to yield a green-blue fluorescent compound. Derivatization occurred at 120 degrees C within maximum of 10-min reaction time. The fluorescent compound formed was separated from excess reagent and other by-products on silica gel TLC plates and was then determined fluorodensitometrically. A linearity range between 20 and 1200 pg/spot was achieved. The method was also applied to other triazine herbicides such as ametryn, atrazine, propazine, terbuthylazine and simazine. Drinking water samples spiked with triazines were extracted using RP-C18 polar plus cartridges, and the extract could be then dansylated as a total. Recoveries were between 88% and 95%; the detection limit was 10 pg/spot and could be further improved to 2 pg/spot by a dipping solution. For quantification, each of the six triazines can be separated on one of three different stationary phases after solid phase extraction and measured densitometrically. The LOD for each individual triazine was 100 ng/l.     

Water soluble products of UV-irradiated autoxidized linoleic and linolenic acids

The water-soluble products of the UV-initiated autoxidation of linoleic and linolenic acids emulsified in water were separated into volatile and relatively involatile components, each of which reacted with both thiobarbituric acid (TBA) and peroxidase. The volatile TBA-reactive compound is probably malonaldehyde and the volatile peroxidase-reactive compound is hydrogen peroxide. Additional compounds which absorb UV light were present in the volatile fraction. After thin-layer chromatography of the involatile fraction, reactivity toward TBA and peroxidase was found in the same spot. Approximate molar yields of hydrogen peroxide, malonaldehyde, "hydroperoxides", and other TBA-reactive compounds were estimated. The ratio of "hydroperoxide" to TBA reactivity was lower for linoleic than for linolenic acid. The mass of relatively involatile compounds was about 20 times greater than that predicted from either peroxidase or TBA assays of water extracts of oxidized linolenic acid. The properties of the water extract were similar to those shown by others for the products of prolonged autoxidation (without UV-irradiation) of emulsified methyl linoleate.     

Sterol-like compound from sensory ganglia. Effect of a nerve growth factor and insulin on its biosynthesis

1. Sensory ganglia from 8-day-old chick embryos were incubated with a specific nerve growth factor and with insulin. 2. From the total lipid extract of the ganglia a compound with steroid characteristics was isolated. 3. The synthesis of this compound, measured spectrophotometrically, diminished after addition of the nerve growth factor and insulin to the incubation medium. 4. The incorporation of sodium [2-(14)C]acetate and dl-[2-(14)C]mevalonic acid into total lipids of the sensory ganglia was stimulated by the nerve growth factor and insulin, but the radioactivity of the sterol-like compound was slightly lower. The incorporation of labelled mevalonic acid either into total lipids or into the sterol-like compound was about 25% lower. 5. About 20% of the acetate incorporated into total lipids and about 87% of the mevalonic acid were recovered in the sterol-like compound.     

Fluorescent tracer method for protein SH groups: III. Use ofN-(7-dimethylamino-4-methyleoumarinyl) malelmide as a tracer of cysteine-containing peptides

We studied whetherN-(7-dimethylamino-4-methyleoumarinyl) maleimide (DACM) could be used as a fluorescent tracer for the purification and analysis of cysteine-containing peptides. An addition product of DACM with SH compound was stable at room temperature in the solution of pH 9.0 and in 50% acetic acid. However, it was hydrolyzed when heated at 110°C for 48 h in 6n HCl. On filter paper, 1 pmol of the addition product in a spot 3 mm in diameter was visible under ultraviolet illumination. The addition product was also stable on filter paper for at least several months after spotting. The elution velocity of the addition products with low molecular weight SH compounds in the Sephadex G-25 column was low considering their molecular weights. However, in general, the elution velocity increased with an increase in the molecular weight of the addition product. The addition product with a peptide having cysteinyl residue at an N-terminal showed another abnormally retarded peak in elution profile which presumably corresponded to a cyclic compound, a thiazane derivative. However, it was shown on the C-terminal tryptic peptide of actin (Cys Phe) that the conversion to a thiazane derivative could be avoided by hydrolyzing the succinimide portion of DACM at pH 9.0 before digestion. The R_f values on paper chromatography for the addition products also depended on their molecular weights. However, the hydrophobicity of the coumarin portion of DACM and of the side chain of amino acid residues also affected the value. It was concluded that DACM was a valuable reagent for the purification and analysis of cysteine-containing peptides.     

New antifungal proteins from sugar beet (Beta vulgaris L.) showing homology to non-specific lipid transfer proteins

Two novel, nearly identical antifungal proteins, IWF1 and IWF2, were isolated from the intercellular washing fluid (IWF) of sugar beet leaves. The proteins were purified to homogeneity and their amino acid sequences were determined. They are basic, monomeric proteins of 91 amino acid residues, 89 of which are identical. Both proteins show strongin vitro antifungal activity againstCercospora beticola, the casual agent of leaf spot disease in sugar beet. Based on primary sequence homology, including the presence of 8 conserved cysteine residues, IWF1 and IWF2 are related to the family of plant non-specific lipid transfer proteins (nsLTPs). Antibodies were raised against IWF2 after conjugation to diphtheria toxoid. The amino acid sequence data was used to generate a polymerase chain reaction (PCR) clone, employed for the isolation of a cDNA clone encoding a closely related isoform IWFA, which differs from IWF1 by two amino acid substitutions only. The induction and subcellular localization of these proteins were studied by western and northern blotting analyses after treatment with 2,6-dichloroisonicotinic acid (INA), a compound capable of inducing resistance againstC. beticola, and after fungal infection. The following observations were made: (1) the proteins were present in leaves of non-INA-treated and uninfected control plants, (2) they were only slightly induced by INA treatment and during infection withC. beticola, and (3) they were present both intra- and extracellularly. However, their strong antifungal potentials together with immunohistological investigations, the proteins accumulating in contact with the fungus and in autolysing cells, suggested a role of these proteins in plant defence. Finally, immunohistology revealed a remarkable expression pattern of the IWF1 and IWF2 proteins, or serologically related proteins, in sugar beet styles, in that single or a few scattered papillae and a few cells in the lower transmitting tissue strongly and specifically reacted with the antibody.     

A new PGE1 analogue (CL115,574) III. Effects on gastric acid and mucus secretion in man

CL115,574, an analogue of PGE1, is a potent inhibitor of gastric acid secretion in animals. The effects of this compound on gastric acid and mucus secretion were studied in 8 male volunteers. The compound was well tolerated, and its maximally effective antisecretory dose (750 micrograms) inhibited pentagastrin stimulated acid secretion by approximately 40% over a 2-hour period, with stimulation beginning one hour after the drug was orally administered. CL115,574 proved to have a significant and sustained effect upon the stimulation of mucus secretion into gastric juice. Considering the possible role that mucus may play in mucosal cytoprotection, CL115,574 because of its antisecretory and mucogenic actions may prove to be an important clinical anti-ulcer compound.     

A C-glucoside benzoic acid derivative from the leaves of Peltophorum dubium

From the methanolic extract of the leaves of Peltophorum dubium Taub (Leguminosae) was isolated after successive chromatographic procedures a new C-glucoside benzoic acid derivative, 3αC-glucopyranosil-4,5-dihydroxy-2-methoxy-benzoic acid. The structure of this compound was determined by 1D and 2D NMR and MS data analysis. The new compound showed moderate antioxidant activity in the assay and the auto-oxidation of β-carotene in a linolenic acid suspension method.     

A new prostaglandin E1 analogue (CL-115,574) II. Effects on gastric acid secretion in the dog

The efficacy of CL-115,574, a prostaglandin E₁ analogue, as an acid antisecretory agent was evaluated in dogs. CL-115,574 inhibited acid secretion maximally at an oral dose of 20 μg/kg causing 100% inhibition of acid secretion up to one hour after administration, with significant inhibition of secretion (30%) still present nearly four hours after drug administration. The wide disparity between the maximally effective antisecretory dose 20 μg/kg and the dose at which reproducible side effects occurred (1 mg/kg) suggests that this compound may be developed as an antisecretory compound for use in man.     

Evaluation in Drosophila melanogaster of the mutagenic potential of furfural in the mei-9a test for chromosome loss in germ-line cells and the wing spot test for mutational activity in somatic cells.

The mutagenic potential of furfural was evaluated by means of the chromosome loss test in germ cells and the wing spot test in somatic cells of Drosophila melanogaster. The chromosome loss test was carried out employing repair-proficient as well as repair-deficient females. Males carried the compound Y chromosome, BSYy+. Two routes of administration were used: injection and feeding of adult males. Genetic damage was demonstrable after matings of treated males with females carrying the excision repair-deficient mutant mei-9a. The somatic mutation and recombination test was carried out treating 72-h transheterozygous mwh+/+flr3 larvae. Acute treatment of larvae was chosen as the method of exposure. Evidence indicates that furfural induces somatic damage as measured in the wing spot test.     

Tetrazine-Containing Amino Acid for Peptide Modification and Live Cell Labeling

A novel amino acid derivative 3-(4-(1, 2, 4, 5-tetrazine-3-yl) phenyl)-2-aminopropanoic acid was synthesized in this study. The compound possessed better water-solubility and was synthesized more easily compared with the well-known and commercially available 3-(p-benzylamino)-1, 2, 4, 5-tetrazine. Tetrazine-containing amino acid showed excellent stability in biological media and might be used for cancer cell labeling. Moreover, the compound remained relatively stable in 50% TFA/DCM with little decomposition after prolonged exposure at room temperature. The compound could be utilized as phenylalanine or tyrosine analogue in peptide modification, and the tetrazine-containing peptide demonstrated more significant biological activity than that of the parent peptide. The combination of tetrazine group and amino acid offered broad development prospects of the bioorthogonal labeling and peptide synthesis.     

A specific assay for subnanogram concentrations of reserpine in human plasma

A sensitive analytical procedure was developed for the measurement of reserpine in human plasma. The method is based on thin layer chromatography followed by development of a fluorophor with acetic acid vapor. The thin layer plates were scanned with a Vitatron “flying spot” densitometer. Use of an internal standard minimized errors in quantitative spotting. Plasma levels were measured in healthy subjects after single oral and intramuscular doses of 1 mg of reserpine.     

A new PGE1 analogue (CL115,574) III. Effects on gastric acid and mucus secretion in man

CL115,574, an analogue of PGE₁, is a potent inhibitor of gastric acid secretion in animals. The effects of this compound on gastric acid and mucus secretion were studied in 8 male volunteers. The compound was well tolerated, and its maximally effective antisecretory dose (750 μg) inhibited pentagastrin stimulated acid secretion by approximately 40% over a 2-hour period, with stimulation beginning one hour after the drug was orally administered. CL115,574 proved to have a significant and sustained effect upon the stimulation of mucus secretion into gastric juice. Considering the possible role that mucuse may play in mucosal cytoprotection, CL115,574 because of its antisecretory and mucogenic actions may prove to be an important clinical anti-ulcer compound.