Antibiotic Y: biosynthesis by Fusarium avenaceum (Corda ex Fries) Sacc., isolation, and some physicochemical and biological properties

A compound very similar to the mycotoxin citrinin was observed on thin-layer chromatographic plates during the screening analysis of grain extracts. This compound was produced by 22 of the tested Fusarium avenaceum (Corda ex Fries) Sacc. strains isolated from wheat, triticale, barley, corn, and potatoes. A chemical test confirmed the presence of an unknown compound, which was given the preliminary name of antibiotic Y (indicating yellow fluorescence). The following properties of the new metabolite are described: spectroscopic (UV, infrared, proton nuclear magnetic resonance, fluorescence, and mass spectrometry), phytotoxic, antibiotic (inhibitory effect of bacterial growth), and toxic (toxicity to Artemia salina, chicken embryos, and mouse fibroblasts). Elemental analysis of the compound showed that it had the general formula C15H10O8, in agreement with the mass spectrometric finding that the molecular ion had a molecular weight of 318. The structure of the compound is presently under study.     

Detection and characterization of the Gloeosporium gloeosporioides growth inhibitory compound iturin A from Bacillus subtilis strain KS03

The Bacillus subtilis strain KS03 was isolated, and identified as a biological control agent that inhibits the anthracnose disease fungus Gloeosporium gloeosporioides. The antifungal compound was purified from its culture broth through butanol extraction, diethylaminoethyl (DEAE) Sepharose CL-6B chromatography, and preparative thin layer chromatography. Tandem mass spectrometric analyses (MS/MS), with matrix-assisted laser desorption ionization (MALDI) time-of-fight/time-of-flight (TOF/TOF) mass spectrometry, showed that the antifungal compound was iturin A, a cyclic lipopeptide antibiotic. The major compound, with a molecular mass of 1042 Da, was identified as iturin A(2).     

Filipin fluorescence quenching by spin-labeled probes: studies in aqueous solution and in a membrane model system.

A detailed photophysical study of the fluorescence quenching (transient and steady state) of the macrolide antibiotic filipin by nitroxide-substituted fatty acids and a cholesterol derivative was carried out, aimed at determining its transverse position in a model system of membranes (multilamellar vesicles of dipalmitoylphosphatidylcholine). Filipin partitions efficiently into membranes (Kp = (5.0 +/- 1.0).10(3), 20 degrees C) and it was concluded that the antibiotic is buried in the membrane, away from the lipid-water interface. In addition, information on the organization of the quenchers was also obtained. The 5-nitroxide derivative of the fatty acid is essentially randomly distributed, while the 16-nitroxide is aggregated at concentrations higher than approximately 5% molar. For the cholesterol compound the results point to a phase separation at concentrations higher than 3% molar (below this limit concentration filipin associates with the derivatized sterol with KA = 20 M-1, assuming a 1:1 interaction). We propose that this phase separation and the aggregation state of filipin in the aqueous solution may be key processes in the antibiotic mode of action. A systematic and general approach to fluorescence quenching data analysis in complex (e.g., biochemical) systems is also presented.     

Identification of glutathionyl-3-hydroxykynurenine glucoside as a novel fluorophore associated with aging of the human lens.

A novel fluorophore was isolated from human lenses using high performance liquid chromatography (HPLC). The new fluorophore was well separated from 3-hydroxykynurenine glucoside (3-OHKG) and its deaminated isoform, 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-glucoside, which are known UV filter compounds. The new compound exhibited UV absorbance maxima at 260 and 365 nm, was fluorescent (Ex(360 nm)/Em(500 nm)), and increased in concentration with age. Further analysis of the purified compound by microbore HPLC with in-line electrospray ionization mass spectrometry revealed a molecular mass of 676 Da. This mass corresponds to that of an adduct of GSH with a deaminated form of 3-OHKG. This adduct was synthesized using 3-OHKG and GSH as starting materials. The synthetic glutathionyl-3-hydroxykynurenine glucoside (GSH-3-OHKG) adduct had the same HPLC elution time, thin-layer chromatography R(F) value, UV absorbance maxima, fluorescence characteristics, and mass spectrum as the lens-derived fluorophore. Furthermore, the (1)H and (13)C NMR spectra of the synthetic adduct were entirely consistent with the proposed structure of GSH-3-OHKG. These data indicate that GSH-3-OHKG is present as a novel fluorophore in aged human lenses. The GSH-3-OHKG adduct was found to be less reactive with beta-glucosidase compared with 3-OHKG, and this could be due to a folded conformation of the adduct that was suggested by molecular modeling.     

A switch‐off fluorescence probe towards Pb(II) and cu(II) ions based on a calix[4]pyrrole bearing amino‐quinoline group

A new fluorescence receptor calix[4]pyrrole‐N‐(quinoline‐8‐yl) acetamide (CAMQ) containing a pyrrolic ring connected via the meso‐position was synthesized, purified and characterized by elemental analysis, NMR and mass spectroscopy. This compound was examined for its fluorescence properties towards different metal ions e.g. Ag(I), Hg(II), Co(II), Ca(II), Ni(II), Zn(II), Cr(II), Ba(II), Fe(II), Cu(II), Pb(II)and Mg(II) ions by spectrophotometry and spectrofluorometry. It was concluded that the compound (CAMQ) possessed significantly enhanced selectivity for Pb(II) and Cu(II) ions in dimethyl sulfoxide (DMSO) even at very low concentrations (1 μM). It exhibit ‘turn‐on’ fluorescence when exposed to Pb(II) and Cu(II) and did so in preference to other metal ions. The binding constants, stoichiometry and quantum yields have been determined. The quenching mechanism was assessed using the Stern–Volmer equation and was also discussed.     

蜡梅种子抑菌成分研究

在抑菌活性追踪指导下,采用硅胶柱层析的方法。从蜡梅种子中分离得到一活性化合物A,经质谱、核磁共振波谱等技术鉴定为d－洋蜡梅碱。经杀虫活性和抑菌活性测定,该化合物对粘虫的幼虫无毒杀活性;对西瓜枯萎病菌、玉米小斑病菌、玉米大斑病菌、番茄早疫病菌均有显著的抑菌活性。其抑制中浓（EC50）分别为：3878．8、29．3、103．1、328．3mg／L,但对油菜菌核病菌无抑制活性。     

枯草芽孢杆菌JA产生的脂肽类抗生素－iturin A的纯化 及电喷雾质谱鉴定

枯草芽孢杆菌JA产生的抗生素对植物病原真菌具有广谱抗性,明确抗生素的种类是进一步研究的基础.用6mol/L盐酸沉淀JA菌株的去菌体培养基,再用甲醇抽提获得抗生素的粗提物.利用反相HPLC系统,将粗提物过Diamonsil C18柱,收集有抗小麦赤霉病等病原真菌活性的化合物1、2.运用电喷雾质谱法(ESI/MS)测得其分子量分别为1042.4D和1056.5D.再利用碰撞诱导解离(CID)技术获得化合物的典型结构特征离子碎片,结果表明分子量为1042.4D的化合物一级结构为Pro-Asn-Tyr-βAA-Asn-Tyr-Asn-Gln(βAA为14个碳原子的氨基脂肪酸),属于脂iturin A.化合物1、2为相差一个亚甲基(-CH2)的iturin A同系物.研究结果提供了一种从枯草芽孢杆菌发酵液中快速分离纯化和鉴定脂肽类抗生素iturin A的新方法.     

Chlorxanthomycin,a fluorescent,chlorinated, pentacyclic pyrene from a Bacillus sp

A gram-positive Bacillus sp. that fluoresces yellow under long-wavelength UV light on several common culture media was isolated from soil samples. On the basis of carbon source utilization studies, fatty acid methyl ester analysis, and 16S ribosomal DNA analysis, this bacterium was most similar to Bacillus megaterium. Chemical extraction yielded a yellow-orange fluorescent pigment, which was characterized by X-ray crystallography, mass spectrometry, and nuclear magnetic resonance spectroscopy. The fluorescent compound, chlorxanthomycin, is a pentacyclic, chlorinated molecule with the molecular formula C22H15O6Cl and a molecular weight of 409.7865. Chlorxanthomycin appears to be located in the cytoplasm, does not diffuse out of the cells into the culture medium, and has selective antibiotic activity.     

Gold(III) compounds of 1,4,7-triazacyclononane showing high cytotoxicity against A-549 and HCT-116 tumor cell lines

Two gold(III) compounds [Au(TACN)Cl(2)]Cl (1) and [Au(TACN)Cl(2)][AuCl(4)] (2) (where TACN=1,4,7-triazacyclononane), have been synthesized and characterized by electrospray ionization mass spectrometry (ESI-MS), (1)H NMR spectroscopy and elemental analyses. The structure of compound 2 was determined by X-ray crystallography, in which TACN coordinates to the gold(III) center in a bidentate mode and the unbound amine group forms a very short intramolecular Au-H(-N) contact (1.91A). Biological activity data showed that compound 1 is more cytotoxic than cisplatin against A-549 and HCT-116 tumor cell lines. The interactions of compound 1 with CT-DNA were studied by UV-Vis, fluorescence and CD spectroscopy, which suggests that compound 1 can induce the distortion of DNA double helix.     

Detection and characterization of cerein 7, a new bacteriocin produced by Bacillus cereus with a broad spectrum of activity

A bacteriocin-producing strain of Bacillus cereus was identified and isolated from a soil sample. The bacteriocin could be purified by a two-step procedure: ammonium sulfate precipitation of culture supernatants followed by a butanol extraction step, the antibiotic was recovered from the organic phase. The peptidic nature of the bacteriocin was proven by its sensitivity to proteolytic enzymes; its molecular mass, determined by mass spectrometry, was 3940 Da; and its amino-terminal sequence (GWGDVL) is unique in the databases. The compound was active against most Gram-positive but not Gram-negative bacteria. This is to our knowledge the first bacteriocin with these characteristics reported to be produced by B. cereus.     

Design and synthesis of ICT-based fluorescent probe for high-sensitivity protein detection and application to rapid protein staining for SDS-PAGE

A novel fluorescent molecular probe possessing styryl, sulfonyl, and cyanopyranyl moieties that was termed compound 1 was designed and synthesized to detect proteins through noncovalent bonding. Compound 1 did not produce fluorescence emission in the absence of proteins. However, its fluorescence spectrum showed a dramatic increase in the fluorescence intensity and strong orange emission after the addition of BSA. These changes were caused by intramolecular charge transfer (ICT). The fluorescence intensities of compound 1 were plotted as a function of the protein concentrations. A good linear relationship was observed up to a protein concentration of 325 mug/mL, and the detection limit was 70 ng/mL under the given assay conditions; this detection limit was higher than that of previously reported compounds. To demonstrate the application of compound 1, proteins in an SDS-PAGE gel were stained with compound 1 and were successfully imaged with a higher sensitivity and shorter staining operation time as compared to those of the silver staining method and SYPRO Ruby staining method. Thus, easy and high-sensitivity protein detection can be performed with the fluorescent probe, and this probe is ideally suited to proteomic applications.     

New antibiotic no. 792 formed by Actinomyces bottropensis

An actinomycetes strain 792 producing a new antibiotic was isolated under the programme of antitumor antibiotic screening. By its morphological and cultural properties strain 792 was classified as belonging to species Actinomyces bottropensis. Antibiotic 792 was recovered from the culture fluid of the strain by the extraction method in the form of a crystalline orange substances. lambda max 235, 305, 410 nm (E 1% 1cm 705, 105, 168), m. p. 232-255 degrees (dec), molecular weight 340, C 67 per cent, H 4.8 per cent, no nitrogen, sulphur or halogens. The antibiotic was inactivated in alkaline solutions forming a hardly soluble compound crystallizing in the form of red needles, lambda max 256, 485 nm (E 1% 1ct 800, 195), m. p. 202-204 degrees (dec), molecular weight 320, C 69.5 per cent, H 4.7 per cent. Antibiotic 792 had antitumor and antimicrobial activity.     

Determination of cell mass and polymyxin using multi-wavelength fluorescence

Multi-wavelength fluorescence was applied for on-line monitoring of cell mass and the antibiotic polymyxin B in Bacillus polymyxa cultivations. By varying the phosphate and nitrogen content of the medium different polymyxin-cell mass ratios could be obtained. Using this strategy, it was possible to investigate if multi-wavelength fluorescence is able to give independent prediction of the two parameters. Partial least square (PLS) regression was applied to establish mathematical relationships between off-line determined cell mass and polymyxin concentrations and on-line collected fluorescence data. For polymyxin one universal PLS model, with a correlation of 0.95 and a root mean square error of cross validation (RMSECV) of 35 mgl(-1), could be constructed. However, correlation between fluorescence and cell mass dry weight could not be established including data from all three types of cultivations. For data from each type of cultivation, separate models with high correlation and low RMSECV values were built. A large variation in cellular composition as a result of the different levels of nitrogen and phosphorus in the cultivations was the probable reason to the necessity of building three models. The results of the present investigation indicate that production of polymyxin is concomitantly regulated by phosphate and nitrogen as the highest polymyxin yield on cell mass, 0.17+/-0.01 gg(-1), was reached in the cultivations where both nitrogen and phosphate concentrations were kept low.     

Conformation and reactivity changes induced by N-methylkirromycin (aurodox) in elongation factor Tu

Kirromycin and related antibiotics inhibit protein synthesis in bacteria by acting on elongation factor Tu (EF-Tu). We have studied the effects of N-methylkirromycin (aurodox) on some molecular properties of this protein. The binding of the antibiotic causes a dramatic variation in the protein fluorescence emission spectrum with the appearance of a new maximum at around 340 nm. Addition of aurodox to trypsinized EF-Tu resulted in an emission spectrum similar to that of the denatured intact factor. Fluorescence lifetime analysis performed by a multifrequency phase fluorometer indicated that the fluorescence emission of the factor is heterogeneous with the major component having a lifetime near 4.8 ns in the absence and 6.6 ns in the presence of the antibiotic. These results were interpreted in terms of an antibiotic-induced environmental modification of the unique tryptophan residue of the protein leading to an increase in its quantum yield. However, aurodox did not modify the solvent exposure of this residue, as judged by fluorescence quenching experiments. Moreover, 1-anilino-8-naphthalenesulfonate (ANS) binding studies, as well as analysis of the protein reactivity toward the sulfhydryl group reagent 5,5'-dithiobis(2-nitrobenzoate) (DTNB), showed that, in the presence of aurodox, the behavior of the EF-Tu-GDP complex nears that of EF-Tu.GTP. These results strongly support the hypothesis that aurodox not only confers a "GTP-like" conformation to the EF-Tu.GDP complex but also produces a less stable folding of the protein around the tryptophan residue that may contribute to the multiple functional effects of this antibiotic.     

Antibiotic transport in resistant bacteria: synchrotron UV fluorescence microscopy to determine antibiotic accumulation with single cell resolution

A molecular definition of the mechanism conferring bacterial multidrug resistance is clinically crucial and today methods for quantitative determination of the uptake of antimicrobial agents with single cell resolution are missing. Using the naturally occurring fluorescence of antibacterial agents after deep ultraviolet (DUV) excitation, we developed a method to non-invasively monitor the quinolones uptake in single bacteria. Our approach is based on a DUV fluorescence microscope coupled to a synchrotron beamline providing tuneable excitation from 200 to 600 nm. A full spectrum was acquired at each pixel of the image, to study the DUV excited fluorescence emitted from quinolones within single bacteria. Measuring spectra allowed us to separate the antibiotic fluorescence from the autofluorescence contribution. By performing spectroscopic analysis, the quantification of the antibiotic signal was possible. To our knowledge, this is the first time that the intracellular accumulation of a clinical antibiotic could be determined and discussed in relation with the level of drug susceptibility for a multiresistant strain. This method is especially important to follow the behavior of quinolone molecules at individual cell level, to quantify the intracellular concentration of the antibiotic and develop new strategies to combat the dissemination of MDR-bacteria. In addition, this original approach also indicates the heterogeneity of bacterial population when the same strain is under environmental stress like antibiotic attack.     

A study of the structure and dynamics of complexes between polymyxin B and phosphatidylglycerol in monolayers by fluorescence

Interactions between the antibiotic polymyxin B and monolayers of dipalmitoylglycerophosphoglycerol have been reinvestigated through a study of the structure and dynamics of the complexes by means of an interface fluorimeter of our fabrication. A fluorescence technique has been developed where the use of linearly polarized incident beams gives the simultaneous determination of the orientation and the lateral diffusion rate of a fluorescent probe inserted in the film. The present investigation was carried out with 12-(9-anthroyloxy)-stearic acid, a fluorescent compound which forms non-fluorescent photodimers upon illumination. Orientation of the probe was studied by computing the ratio of the two dimerization constants KD and the ratio of the fluorescence intensities obtained with crossed linearly polarized incident lights. The lateral diffusion rate of the probe was obtained by measuring fluorescence recovery after photobleaching (photodimerization) of the probe. Control experiments, carried out with dimyristoylglycerophosphocholine, a lipid which does not interact with polymyxin B, show that the antibiotic does not significantly modify the behaviour of the probe. Both in terms of orientation and dynamics, with respect to dipalmitoylglycerophosphoglycerol, when the antibiotic is present in the subphase (1 microM, saturating conditions), data indicate that the lipid remains in a liquid-expanded state. This is true even at a high surface pressure (pi approximately equal to 37 mN X m-1), above the apparent 'transition' which can be observed at 30-35 mN X m-1 on its compression isotherm. Computation of the contribution of polymyxin B to the film expansion to the conclusion that this 'transition' would be a structural transition between two models of interaction: one, below the 'transition', where the polypeptide ring penetrates between the film-forming lipid molecules and another one, above the 'transition', were the antibiotic is adsorbed at the lipid-water interface with only its hydrocarbon chain penetrating the film.     

Isolation and characterization of a novel nonheme chloroperoxidase

Chloroperoxidase, purified from the fermentation of Curvularia inaequalis, had a molecular weight of approximately 240,000 and was composed of 4 subunits of identical molecular weight (Mr 66,000). The enzyme was specific for I-, Br- and Cl-, and inactive toward F-. The optimum pH of the enzyme was centered around 5.0. X-ray fluorescence revealed that the enzyme contained 2.2 atoms of zinc and 0.7 atom of Fe per molecule of protein. The enzyme had no heme-like compound as a prosthetic group, making it the first nonheme chloroperoxidase to be reported. Under oxidative conditions that rapidly inactivated other haloperoxidases, this enzyme was remarkably stable.     

Structure and mechanism of formation of human lens fluorophore LM-1. Relationship to vesperlysine A and the advanced Maillard reaction in aging, diabetes, and cataractogenesis.

Human lens crystallins become progressively yellow-brown pigmented with age. Both fluorescent and non-fluorescent protein adducts and cross-links are formed, many of which result from the advanced Maillard reaction. One of them, LM-1, is a blue fluorophore that was earlier tentatively identified as a cross-link involving lysine residues (1). A two-step chromatographic system was used to unequivocally identify and quantitatively prepare a synthetic fluorescent cross-link with lysine residues that had identical UV, fluorescent, and chromatographic properties with both acetylated and non-acetylated LM-1. Proton, (13)C NMR, and molecular mass of the synthetic compound were identical with vesperlysine A, a fluorescent cross-link discovered by Nakamura et al. (2). The fragmentation patterns of vesperlysine A and LM-1 were identical as determined by NMR/mass spectrometry. Lenticular levels of vesperlysine A increase curvilinearly with age and reach 20 pmol/mg at 90 years. Levels correlate with degree of lens crystallin pigmentation and fluorescence and are increased in diabetes, in contrast to N(epsilon)-(carboxymethyl)lysine and pentosidine. Ascorbate, D-pentoses, and D-threose, but neither D-glucose under oxidative conditions, DL-glyceraldehyde, methylglyoxal, glyoxal, nor glycolaldehyde, are precursors. However, addition of C-2 compounds greatly catalyzes vesperlysine A formation from ribose. Thus, vesperlysine A/LM-1 is a novel product of the advanced Maillard reaction in vivo and a specific marker of a diabetic process in the lens that is different from glyco- and lipoxidation.     

Identification of 1-lysophosphatidylethanolamine (C(16:1)) as an antimicrobial compound in the housefly, Musca domestica

We observed that a methanolic whole body extract of uninfected last instar larvae of the housefly, Musca domestica, displayed antifungal and antibacterial activity. We have further purified this extract to a single active fraction using reversed phase high performance liquid chromatography. The pure fraction inhibited growth of the Gram-positive bacteria Bacillus thuringiensis and the yeast Saccharomyces cerevisiae, but not the Gram-negative bacteria Escherichia coli. The active compound was determined to have a molecular mass of 451.2 Da. Further analysis by nuclear magnetic resonance identified the substance as mono-unsaturated 1-lysophosphatidylethanolamine (C(16:1)) (1-LPE). The structurally different and more common 2-LPE have been described as mediators of the antimicrobial activity of rimenophenazine antibiotic agents (Van Rensburg et al., 1992). Our results suggest that the isolated 1-LPE displays a higher activity in comparison, possibly based on structure-specific differences in activity.     

Isolation and identification of N-mercapto-4-formylcarbostyril, an antibiotic produced by Pseudomonas fluorescens.

Pseudomonas fluorescens strain G308 isolated from barley leaves produces a novel antibiotic substance that was purified by preparative TLC and HPLC and identified as N-mercapto-4-formylcarbostyril (Cbs) by LC/DAD, IR, LC-ES(+)/MS, LC-ES(-)/MS, GC-EI/MS, LC-HRES(+)/MS, mass isotope ratios analysis, 1H NMR and 13C NMR analysis. The purified new antibiotic compound is effective against many phytopathogenic fungi in vitro. The compound inhibited at 25 ppm spore germination and germ tube growth of the following fungi; Fusarium oxysporum f. sp. lycopersici, Fusarium culmorum, Cladosporium cucumerinum and Colletotrichum lagenarium. At concentrations up to 125 ppm, the compound did not interfere with release of zoospores from sporangia and germination of encysted zoospores of Phytophthora infestans.