A novel lipid compound, epolactaene, induces apoptosis: its action is modulated by its side chain structure

A novel lipid compound, epolactaene, was isolated from the culture supernatant of Penicillium sp. 1689-P and it has already been reported that it induced neurite outgrowth in a human neuroblastoma cell line. In this study, we first investigated the effects of epolactaene on a human leukemia B-cell line, BALL-1 cells, and clarified that epolactaene induces apoptosis in BALL-1 cells in a dose- and time-dependent manner. Furthermore, we focused on the side chain structure of epolactaene, and chemically synthesized epolactaene derivatives. One derivative, which has a straight long alkyl chain as its side chain, induced apoptosis more effectively than epolactaene. On the other hand, other derivatives with a short alkyl side chain had weaker apoptosis-inducing actions. A good correlation was found between the apoptosis-inducing action of these compounds and their octanol/water partition coefficients (log P). These results suggested that the apoptosis-inducing activities of epolactaene and its derivatives were related to the hydrophobicity of these compounds; so that side chain structure of epolactaene is very important for its apoptosis-inducing activities. These apoptosis-inducing actions of epolactaene and its derivatives were also observed in various blood tumor cell lines and normal lymphocytes.     

Microbiological transformation of mycophenolic acid

In our microbial screening program, we have isolated a fungal strain which produced mycophenolic acid (MPA). This compound is a selective inhibitor of guanine synthesis and, therefore, it has antibacterial, antiviral, antitumor and selective immunosuppressive activities, too. This last effect was utilised by Roche-Syntex to develop a derivative of MPA to the immunosuppressive drug CellCept®.

In order to obtain novel derivatives of MPA with an enhanced activity, we applied bioconversion of MPA with various microorganisms. TLC with densitometric evaluation and HPLC methods were developed for measurement of MPA derivatives. In the course of the bioconversion of MPA by using various types of microorganisms amidation of the carboxyl group, hydroxylation of the C₄-methyl group and formation of glycoside derivatives from the hydroxyl group located on C₇ were observed as the most frequently occurring transformations. The structures of bioconversion products were determined by UV, IR, ¹H NMR, ¹³C NMR and mass spectroscopic methods.

The taxonomic features of cultures of the species applied in the bioconversion were also determined.     

Interaction of hydroxycinnamic acid derivatives with the Cl3COO radical: a pulse radiolysis study

The electron transfer reactions between the trichloromethylperoxyl radical (Cl₃COO^·) and hydroxycinnamic acid derivatives, including chlorogenic acid, sinapic acid, caffeic acid, ferulic acid and 3,4-(methylenedioxy)cinnamic acid, have been studied by pulse radiolysis. The hydroxycinnamic acid derivatives, especially sinapic acid, are identified as good antioxidants for reduction of Cl₃COO^· via electron transfer reactions. From buildup kinetic analysis of phenoxyl radical, the rate constant for reaction of Cl₃COO^· with sinapic acid has been determined to be 8.2 × 10⁷ dm³ mol^-1 s^-1, while the rate constants of electron transfer from other hydroxycinnamic acid derivatives to Cl₃COO^· were obtained to be about 2 × 10⁷dm³ mol^-1 s^-1. The reaction of 3,4-(methylenedioxy)cinnamic acid with Cl₃COO^· was investigated as an evidence for the electron transfer mechanism.     

Enzymes and pathways of polyamine breakdown in microorganisms

Abstract The information currently available on the breakdown of spermidine and putrescine by microorganisms is reviewed. Two major metabolic routes have been described, one for the free bases via δ¹-pyrroline (4-aminobutyraldehyde), the other via N -acetyl derivatives. In both pathways oxidases or aminotransferases are the key enzymes in removing the nitrogen atoms. The two routes converge at 4-aminobutyrate, which is then metabolized via succinate. The degradation of putrescine in Escherichia coli has been well characterized at both genetic and biochemical levels, but for other bacteria much less information is available. The C₃ moiety of spermidine is broken down via β-alanine, but the metabolism of this compound and its precursors is poorly understood. In yeasts, a catabolic route for spermidine and putrescine via N -acetyl derivatives has been described in Candida boidinii , and the evidence for its occurrence in other species is reviewed. Except for the terminal step of this pathway, the same group of enzymes can metabolize both the C₃ and C₄ moieties of spermidine. It is likely that other routes of polyamine catabolism also exist in both bacteria and yeasts.     

Mutagenicities of nitrosated carboline derivatives

Food-borne amines have been considered as the potential precursors of endogenous carcinogenic N-nitroso compounds in humans. A compound which yields a direct mutagen after nitrite treatment was isolated from soy sauce and was identified as 1-methyl-1,2,3,4-tetrahydro-2-carboline-3-carboxylic acid (MTCA) (Wakabayashi, et al., 1983). The mutagenicities of other carboline derivatives such as harman, norharman, harmaline, harmalol, harmine, and harmol were studied. Like MTCA, the nitrosated carboline derivatives showed higher mutagenic activity as compared to their corresponding parent compounds. The demethylated analogue of MTCA, 1,2,3,4-tetrahydro-2-carboline-3-carboxylic acid was synthesized and its nitrosated products were shown to be mutagenic to Salmonella typhimurium TA 100 and TA 98. The potent mutagen Trp-P-2 is a typical 3-carboline derivative. The mutagenicity of Trp-P-2 was suppressed remarkably after nitrosation. Several 3-carboline derivatives also showed the similar property. Nitrosation of MTCA gave several derivatives which were isolated and showed direct mutagenicity to Salmonella typhimurium TA 98. Further characterization of these new carboline derivatives is in progress.     

CHEMICAL STRUCTURAL REQUIREMENTS FOR INDUCTION OF OOCYTE MATURATION AND SPAWNING IN STAREISHES

Effects of various adenine derivatives on oocyte maturation and spawning were studied in the starfishes, Marthasterias glacialis, Astropecten aurantiacus, Patiria miniata, Asterina pectinifera and Asterias forbesi . 1-Methyladenine and 1-ethyladenine were very effective in inducing oocyte maturation and spawning, whereas the following related compounds had no effect: adenine, 3-methyladenine, 7-methyl-adenine, 9-methyladenine, 1-methylguanine, 1-methylhypoxanthine, 6-methylpurine, N₆-methyladenine, N₆-
dimethyladenine, N₆-benzyladenine, N₆-furfuryladenine(kinetin), adenosine, 5' -adenylic acid, adenosine 3',5'-cyclic monophosphate, adenosine triphos-phate, inosine, 5'-inocinic acid, guanine, guanosine, 5'-guanylic acid, hypoxanthine, xanthine, xanthosine, 3-methylcytidine and 5-methylcytosine. 1-Methyladenosine induced oocyte maturation and spawning when isolated ovarian fragments were used as assay material; however, it had little effect in inducing maturation of isolated oocytes. Therefore, this compound seems to active only after its decomposition to 1-methyladenine and ribose. The chemical structure responsible for inducing oocyte maturation and spawning in starfishes is proposed: a short alkyl radical such as methyl or ethyl at N₁ site and an imino radical at C₆ site of the purine nucleus.     

Identification of an Acidic Dipeptide, β-Aspartylglycine, in the CNS of Aplysia

Abstract: A novel dipeptide, (β-aspartylglycine (β-DG), has been isolated from tissues of the marine gastropod mollusc Aplysia californica. This compound was detected only in Aplysia and not in other molluscs, such as Helix or Mercenaria , or in lobster or frog. Among the Aplysia tissues, the highest levels of β-DG were in nervous tissue and in the reproductive tract. β-DG was assayed by HPLC as the o -phthaldialdehyde derivative and found to be present in all individual, identified neurons at a concentration of approximately 40 pmol/μg protein. The peptide was identified as β-DG by gas chromatography-mass spectrometry (GCMS) using trimethylsilyl derivatives prepared before and after acid hydrolysis. It was further characterized as the β-isomer by TLC, including R_f, atypical blue-gray color with ninhydrin, and a violet color with Cu²⁺-ninhydrin. A fractionation scheme is described whereby acid-soluble tissue constituents can be divided into acidic, neutral, and basic components using mini ion-exchange columns. This partial purification prior to TLC analysis was necessary to remove compounds that interfered with the isolation of β-DG.     

有害赤潮生物球形棕囊藻对卤虫的毒性研究

利用1997年10月和1998年6月分别在广东汕头饶平海域和香港海域分离到的球形棕囊藻,即球形棕囊藻香港株(Phaeocystis globosastrain HK)和球形棕囊藻汕头株(Phaeocystis globosastrain ST),在实验室条件下研究其对四日龄卤虫(Artemia sinica)的急性毒性。结果表明,对数生长期的HK株藻液24h对卤虫LC₅₀约为2.9×10⁵cell·mL^-1。对数期的ST株对卤虫的毒性难以达到半致死浓度,衰亡期的ST株24h对卤虫LC₅₀约为9.89×10⁶cell·mL^-1。在上述浓度下两者半致死时间分别为20.91h和26.62h,而对数生长期的HK株和衰亡期的ST株的培养物过滤液24h对卤虫LC₅₀则分别为7.1×10⁵cell·mL^-1和1.457×10⁷cell·mL^-1,且在该浓度下的半致死时间分别为26.56h和28.02h。实验表明,HK株藻液和滤液的毒性均大于ST株,且藻液的毒性均大于滤液。     

Ethylene is required for elicitin-induced oxidative burst but not for cell death induction in tobacco cell suspension cultures

The signal compound ethylene and its relationships with oxidative burst and cell death were analyzed in cultured tobacco cells treated with the proteinaceous elicitor quercinin. Quercinin belongs to the protein family of elicitins and was isolated from the soil-born oak pathogen Phytophthora quercina. It was shown to induce a dose-dependent oxidative burst in tobacco cell culture in concentrations from 0.05 to 0.5 nM, and subsequently, cell death. The characteristics of quercinin-induced cell death included both membrane damage and DNA fragmentation in tobacco cell culture.

At higher quercinin concentrations (2 nM), H₂O₂ formation and ethylene biosynthesis were inhibited. Ethylene at low concentrations proved to be necessary for induction and maintenance of H₂O₂ production in tobacco cells treated with quercinin. It was demonstrated that external addition of inhibitors of ethylene biosynthesis such as -amino-oxy-acetic acid (AOA) and CoCl₂ also decreased or even inhibited the quercinin-induced oxidative burst, but did not influence cell death induction. These results demonstrate evidence for a requirement of the plant hormone ethylene for the onset of the quercinin-induced oxidative burst.     

The chemistry of flavins and flavoproteins III. The reaction of dihydrolipoic acid with flavins

1. The reaction between dihydrolipoic acid and a number of flavin derivatives has been found to be of first order in both reactants.

2. The rates are strongly pH dependent and are of first order in OH- concentration as well as being generally base catalysed.

3. The polarographic half-wave potentials of flavin derivatives and their reactivities towards NADH correlate reasonably well with their reactivities towards dihydrolipoic acid.

4. On the basis of these observations and on some electron spin resonance evidence a two-step mechanism is suggested for the reaction, the first being a fast dissociation of one of the -SH groups of dihydrolipoic acid into its anion followed by a second-order reaction between this anion and the flavin, probably by a two-electron transfer.

5. The pK_a of the -SH-groups of the acid has been measured spectrophotometrically, and the value derived (10.7) is in good agreement with the value arrived at from the kinetic studies.     

Investigation on the Toxin Production by Several Blast Fungus Strains and Isolation of Tenuazonic Acid as a Novel Toxin

A survey of the toxin production by several strains of blast fungus was carried out. Among the strains tested only one strain, C₁(THU 69-03) produced α-picolinic acid with good yield and other strains produced neither α-picolinic acid nor piricularin. On the other hand, tenuazonic acid and its Fe-cbelate compound were isolated as novel blast fungus toxins from the surface culture brothes of these strains, but they could not be isolated from the shaking culture brothes. The phytotoxic effect of tenuazonic acid on rice plants in regard to varietal difference was also studied.     

Efficient enzymatic preparation of hydroxycinnamates in ionic liquids enhances their antioxidant effect on lipoproteins oxidative modification

Biocatalytic lipophilization of hydroxycinnamic acids was performed in several BF₄^? and PF₆^? imidazolium ionic liquids using immobilized lipases. The influence of various reaction parameters on the performance of the biocatalytic process was pointed out, using as model reaction the esterification of ferulic acid. The biocatalytic lipophilization strongly depended on the ion composition of ionic liquids used. Conversions and initial reaction rates were significantly higher in PF₆^? as compared with BF₄^? ionic liquids and commonly used organic solvents. The high enzyme stability and the relative solubility of substrate versus product in PF₆^? ionic liquids can account for the improved synthesis of lipophilic ferulates. These lipophilic derivatives, when used at a concentration of up to 400-fold lower than the parental compound, efficiently inhibited the oxidation of isolated LDL, HDL and total serum in vitro. Moreover, it has been shown for the first time that the lipophilic ferulates improve the antioxidant efficiency of the HDL_3c towards LDL in vitro oxidation.     

Chlorogenic acid in a Nicotiana plumbaginifolia cell suspension

A phenylpropanoid compound has been characterized in a Nicotiana plumbaginifolia cell suspension. This compound has been isolated and purified by semi-preparative reverse phase-high performance liquid chromatography. Its structure has been identified by NMR spectroscopy as 5-O-caffeoylquinic acid, which is chlorogenic acid (CA). The influence of culture conditions on the accumulation of this metabolite by N. plumbaginifolia cell suspensions has been studied. Darkness strongly inhibits the CA accumulation. Moreover, it has been shown that feeding experiments with caffeic acid had a deleterious effect upon the CA content. This one was not influenced by a supplementation with quinic acid.     

Bilirubin conjugates of human bile. Nuclear-magnetic-resonance, infrared and optical spectra of model compounds

N.m.r., i.r. and optical spectra of model compounds were recorded. These were to help in elucidating the structures of the phenylazo derivatives of bilirubin conjugates isolated from human bile. Model compounds included commercial and human bile bilirubin, mesobilirubin, bilirubin dimethyl ester, dimethoxybilirubin dimethyl ester and the corresponding phenylazo derivatives. The phenylazo derivative of vinylneoxanthobilirubinic acid was also investigated. All compounds were of the type IXα, and no other isomer could be detected with the spectroscopic methods employed. The compounds crystallize as the lactams, except for dimethoxybilirubin dimethyl ester and its phenylazo derivative, which are held in the lactim ether configuration. With all other compounds no tautomeric forms other than the lactams could be detected, although small proportions of bilirubin must exist as the lactim. Bilirubin does not form a betaine, a structure that has been proposed by von Dobeneck & Brunner (1965) to explain the bathochromic shift of its optical spectrum as compared with the expected position of the absorption maximum at 420nm. However, this shift to 453nm can be explained on the basis of internal hydrogen bonds occurring between the carboxylic protons and the pyrrole rings of bilirubin, as proposed by Fog & Jellum (1963), and new evidence for such a bonding has been accumulated. The bilirubin sulphate described by Watson (1958), which is formed by treatment of bilirubin with concentrated sulphuric acid and acetic anhydride, was also investigated. The main product of this reaction was isolated as its phenylazo derivative, and was shown to be 3,18-di(ethylidene sulphate)-2,7,13,17-tetramethylbiladiene-ac-8,12-dipropionic acid. The reaction leading to this compound is an addition of sulphuric acid to the vinyl side chains of bilirubin according to Markownikoff's rule.     

Simultaneous determination of volatile and non-volatile acidic fermentation products of anaerobes by capillary gas chromatography

A method previously used for the analysis of organic acids in silage has been applied to the detection and quantification of acidic fermentation products (C₁ to C₆ volatile fatty acids, lactic and succinic acid) of rumen bacteria and anaerobic fungi grown in pure culture. The acids were converted to tertiary butyldimethylsilyl derivatives prior to separation on a 30 m DB1 capillary gas chromatographic column. The quantitative recoveries of formic and succinic acids were found to be comparable to the recoveries of other acids reported in the original study. The quantitative recovery of lactic acid was found to be dependent on storage of the samples at ambient temperature for at least 24 h following derivatization. The simultaneous determination of a wide range of volatile and non-volatile acidic products is an important feature of this method.     

The structure of the glucuronide of sulphadimethoxine formed in man

1. The major metabolite of 2,4-dimethoxy-6-sulphanilamidopyrimidine (sulphadimethoxine) in urine in man is a non-reducing glucuronide, which has been isolated and characterized as its S-benzylthiouronium salt. 2. The same compound was made synthetically by standard methods from sodium sulphadimethoxine and methyl 2,3,4-tri-O-acetyl-1-bromoglucuronate. 3. On hydrolysis with acid, the glucuronide yielded sulphanilic acid, glucuronic acid and barbituric acid, and with beta-glucuronidase it slowly yielded sulphadimethoxine and glucuronic acid. 4. Evidence based on infrared spectra and other data showed that the urinary and synthetic glucuronide was 1-deoxy-1-[N(1)'-(2',4'-dimethoxypyrimidin-6' -yl)sulphanilamido-beta-d-glucosid]uronic acid or sulphadimethoxine N(1)-glucuronide. 5. N(1)-Methyl- and N(ring)-methyl derivatives of sulphadimethoxine and 4-methoxy-6-sulphanilamidopyrimidine were prepared and their infrared and ultraviolet spectra determined for comparison.     

Bulky adducts detected by P-postlabeling in DNA modified by oxidative damage in vitro. Comparison with rat lung I-compounds

Oxygen free radicals, such as the hydroxyl radical generated by interaction of Fe²⁺ and H₂O₂ (Fenton reaction), are produced in mammalian cells as a result of aerobic metabolism and under various pathological conditions and are known to elicit mutations and potentially other adverse effects by reacting with DNA bases. Several products thus formed have recently been characterized as hydroxylated derivatives of cytosine, thymine, adenine, and guanine and imidazole-ring-opened derivatives of adenine and guanine in DNA. As shown herein by ³²P-postlabeling, incubation of DNA under Fenton reaction conditions led to additional products which, by virtue of resistance to nuclease P1 catalyzed 3′-dephosphorylation and chromatographic behavior, appeared to be bulky adducts rather than small polar, hydroxylated or ring-opened nucleotide derivatives. Two major and five minor DNA derivatives were measured after ³²P-postlabeling and TLC mapping of DNA oxidized in vitro under conditions known to lead to formation of reactive oxygen species. Amounts of products formed depended on Fe²⁺ and H₂O₂ concentrations and increased in the presence of -ascorbic acid. One of the two major products was also detected in lung DNA of rats where its amount increased with animal age. Thus, at least one I-compound appeared to have its origin in the interaction of DNA with reactive oxygen species.     

Isolation of 9-hydroxy-δ-tetradecalactone from lipid A of Pseudomonas diminuta and Pseudomonas vesicularis

Abstract A lipid component was isolated from the fatty acid fraction of acid hydrolysates of lipid A derived from Pseudomonas diminuta JCM 2788 and Pseudomonas vesicularis JCM 1477 lipopolysaccharide. By structural analysis of the lipid and its trimethylsilyl and acetyl derivatives by thin-layer chromatography, gas chromatography-mass spectrometry, mass spectrometry, infrared spectrometry and ¹³C-NMR, it was identified as 9-hydroxy-δ-tetradecalactone.     

A chemotaxonomic survey of kaurene derivatives in the genus Alepidea (Apiaceae)

Lipophilic extracts from the roots of 16 species of Alepidea were studied by GC-MS for the presence of kaurenoic acids, dehydrokaurenoic acids, kaurenic lactones, hydroxykaurenoic acids and other kaurene derivatives. Various mixtures of these compounds are present in yields of up to 11.8% dry wt. in the rhizomes and roots. Two different isomers of kaurenoic acid occur in Alepidea, the one as a major compound in all the species, the other as a major compound only in some of them. The first isomer is clearly a useful chemical marker for the genus Alepidea, but the apparent chemical dichotomy is not obviously correlated with morphological patterns. Alepidea amatymbica, however, is part of a morphologically isolated group within the genus and also differs from other species in its chemical complexity and in the unique presence of kaurenoic lactones.     

Activity of dehydroabietic acid derivatives against wood contaminant fungi

The antifungal activity of 10 dehydroabietic acid derivatives with different configuration in A and B rings (cis/trans A/B junction) and different substituents and/or functionalities was evaluated in bioassays in vitro and in situ (pine wood blocks).

The test compounds dissolved in acetone were assayed at several concentrations w/w (test compound/culture medium) against the fungi. The Relative Inhibition (RI) was determined by measuring the radial growth of colonies of the fungi treated with the test compounds by comparison with those of control cultures; the results are expressed as EC₅₀.

The results of bioassays in vitro have shown that hydroxyl and aldehyde functions are required for antifungal activity in this group of compounds and deisopropylation can increase the activity. Our assay of antifungal activity in situ (in pine wood blocks) provides a means to investigate the preservative activities of these antifungal compounds under actual conditions of use.

The dehydroabietic acid derivative cis-deisopropyldehydroabietanol (10) inhibited the growth of several of the fungi tested, in vitro and in situ.

The results obtained in situ with the test compound (10) at 6% and 8% were not significantly different from the reference products and a good level of protection of the wood against the organisms tested was achieved.

The results in wood bioassays present new possibilities in the search for natural new compounds in the wood protection, as an alternative to conventional fungicides.