Biosynthesis of the terpene phenalinolactone in Streptomyces sp. Tü6071: analysis of the gene cluster and generation of derivatives

Phenalinolactones are terpene glycosides with antibacterial activity. A striking structural feature is a highly oxidized gamma-butyrolactone of elusive biosynthetic origin. To investigate the genetic basis of the phenalinolactones biosynthesis, we cloned and sequenced the corresponding gene cluster from the producer strain Streptomyces sp. Tü6071. Spanning a 42 kbp region, 35 candidate genes could be assigned to putatively encode biosynthetic, regulatory, and resistance-conferring functions. Targeted gene inactivations were carried out to specifically manipulate the phenalinolactones pathway. The inactivation of a sugar methyltransferase gene and a cytochrome P450 monoxygenase gene led to the production of modified phenalinolactone derivatives. The inactivation of a Fe(II)/alpha-ketoglutarate-dependent dioxygenase gene disrupted the biosynthetic pathway within gamma-butyrolactone formation. The structure elucidation of the accumulating intermediate indicated that pyruvate is the biosynthetic precursor of the gamma butyrolactone moiety.     

Biosynthesis of the orthosomycin antibiotic avilamycin A: deductions from the molecular analysis of the avi biosynthetic gene cluster of Streptomyces viridochromogenes Tü57 and production of new antibiotics.

Background: Streptomyces viridochromogenes Tü57 is the producer of avilamycin A. The antibiotic consists of a heptasaccharide side chain and a polyketide-derived dichloroisoeverninic acid as aglycone. Molecular cloning and characterization of the genes governing the avilamycin A biosynthesis is of major interest as this information might set the direction for the development of new antimicrobial agents.Results: A 60-kb section of the S. viridochromogenes Tü57 chromosome containing genes involved in avilamycin biosynthesis was sequenced. Analysis of the DNA sequence revealed 54 open reading frames. Based on the putative function of the gene products a model for avilamycin biosynthesis is proposed. Inactivation of aviG4 and aviH, encoding a methyltransferase and a halogenase, respectively, prevented the mutant strains from producing the complete dichloroisoeverninic acid moiety resulting in the accumulation of new antibiotics named gavibamycins.Conclusions: The avilamycin A biosynthetic gene cluster represents an interesting system to study the formation and attachment of unusual deoxysugars. Several enzymes putatively responsible for specific steps of this pathway could be assigned. Two genes encoding enzymes involved in post-PKS tailoring reactions were deleted allowing the production of new analogues of avilamycin A.     

Biosynthesis of the angiogenesis inhibitor borrelidin by Streptomyces parvulus Tü4055: cluster analysis and assignment of functions

The biosynthetic gene cluster for the angiogenesis inhibitor borrelidin has been cloned from Streptomyces parvulus Tü4055. Sequence analysis indicates that the macrolide ring of borrelidin is formed by a modular polyketide synthase (PKS) (borA1-A6), a result that was confirmed by disruption of borA3. The borrelidin PKS is striking because only seven rather than the nine modules expected for a nonaketide product are encoded by borA1-A6. The starter unit of the PKS has been verified as trans-cyclopentane-1,2-dicarboxylic acid (trans-1,2-CPDA), and the genes involved in its biosynthesis identified. Other genes responsible for biosynthesis of the nitrile moiety, regulation, and self-resistance were also identified.     

Studies on the biosynthesis of arteannuin——Ⅳ.The biosynthesis of arteanniuin and arteannuin B by the leaf homogenate of Artemisia annua L.

Arteannuic acid and(-)11R,13-d ydroarteannuic acid are the key intermediates in thebiosynthesis of arteannuin by the leaf homogenate of Artemisia annua L.,and arteannuic acid andepoxyarteannuic acid are the intermediates of arteannuin B.and epoxyarteannuic acid can not betransformed into arteannuin by the homogenate.     

A new approach to the spectral analysis of liquid membrane oscillators by Gábor transformation

Liquid membrane oscillators very frequently have an irregular oscillatory behavior. Fourier transformation cannot be used for these nonstationary oscillations to establish their power spectra. This important point seems to be overlooked in the field of chemical oscillators. A new approach is presented here based on Gábor transformation allowing one to obtain power spectra of any kind of oscillations that can be met experimentally. The proposed Gábor analysis is applied to a liquid membrane oscillator containing a cationic surfactant. It was found that the power spectra are strongly influenced by the presence of various added substances.     

Stereochemical control in microbial reduction 4. Effect of cultivation conditions on the reduction of β-keto esters by methylotrophic yeasts.

When β-keto esters are reduced by methanol grown yeast, drastic shift of the enantiomer excess of the products toward D-isomer formation is caused compared with those by glucose grown cells.     

Studies towards the synthesis of superstolide A. Synthesis and stereochemical assignment of the C(21)C(26) fragment of superstolide A

An asymmetric synthesis of the C(21)C(26) fragment of superstolide A is described. A fragment, corresponding to a reductive ozonolysis product of superstolide, was also prepared. Comparison of spectroscopic and optical properties of the corresponding fragment obtained by degradation of natural superstolide A allowed the confirmation of the stereochemistry of the natural product.     

A new compound with cytotoxic activities from the leaves of Panax ginseng C.A. Meyer

A new compound, 3,6,20（S）-trihydroxy- 12,23-epoxydammar-24-ene,6,20-di-O-β-D-glucopyranoside （1）, was isolated from the leaves of Panax ginseng C.A. Meyer, whose structural elucidation was carried out by means of spectral analysis （including IR, HR- FAB-MS and NMR）. This compound showed the moderate cytotoxic activities against U937 and HeLa cells by using the MTT method.     

Doping control analysis of seven bioactive peptides in horse plasma by liquid chromatography–mass spectrometry

In recent years, there has been an ongoing focus for both human and equine doping control laboratories on developing detection methods to control the misuse of peptide therapeutics. Immunoaffinity purification is a common extraction method to isolate peptides from biological matrices and obtain sufficient detectability in subsequent instrumental analysis. However, monoclonal or polyclonal antibodies for immunoaffinity purification may not be commercially available, and even if available, such antibodies are usually very costly. In our study, a simple mixed-mode anion exchange solid-phase extraction cartridge was employed for the extraction of seven target peptides (GHRP-1, GHRP-2, GHRP-6, ipamorelin, hexarelin, CJC-1295, and N-acetylated LKKTETQ (active ingredient of TB-500)) and their in vitro metabolites from horse plasma. The final extract was subject to ultra-high-performance liquid chromatographic separation and analysed with a hybrid high-resolution mass spectrometer. The limits of detection for all seven peptides were estimated to be less than 50 pg/mL. Method validation was performed with respect to specificity, precision, and recovery. The applicability of this multi-analyte method was demonstrated by the detection of N-acetylated LKKTETQ and its metabolite N-acetylated LK from plasma samples obtained after subcutaneous administration of TB-500 (10 mg N-acetylated LKKTETQ) to two thoroughbred geldings. This method could easily be modified to cover more bioactive peptides, such as dermorphin, β-casomorphin, and desmopressin. With the use of high-resolution mass spectrometry, the full-scan data acquired can also be re-processed retrospectively to search for peptides and their metabolites that have not been targeted at the time of analysis. To our knowledge, this is the first identification of in vitro metabolites of all the studied peptides other than TB-500 in horses.

Quantitative analysis of urinary phospholipids found in patients with breast cancer by nanoflow liquid chromatography–tandem mass spectrometry: II. Negative ion mode analysis of four phospholipid classes

Analysis was performed on four different categories of phospholipids (phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), and phosphatidic acid (PA)) from urine in patients with breast cancer. This quantitative analysis was conducted using nanoflow liquid chromatography–electrospray ionization–tandem mass spectrometry (nLC-ESI-MS-MS). This study shows the profiling of the phospholipids (PLs) that can be identified by the negative ion mode of MS. A previous study (Kim et al. Anal. Bioanal. Chem. 393:1649, 21) focused on only two PL classes: phosphatidylcholines (PCs) and phosphatidylethanolamines (PEs) and were identified by positive ion mode. PLs were extracted by lyophilization of 1 mL of urine from both healthy normal females and breast cancer patients before and after surgery. Separation of PLs was performed by nLC followed by structural identification of PLs using data-dependent collision-induced dissociation. A total of 34 urinary PL molecules (12 PSs, 12 PIs, four PGs, and six PAs) were quantitatively examined. Among the four PL categories examined in this study, most PL classes showed an increase in the total amounts in the cancer patients, yet PIs exhibited some decreases. The present study suggests that the lipid composition found in the urine of breast cancer patients can be utilized for the possible development of disease markers, when the analysis is performed with negative ion mode of nLC-ESI-MS-MS.      

Taylor dispersion analysis discloses the impairment of Aβ peptide aggregation by the presence of a fluorescent tag

The use of fluorescently tagged amyloid peptides, implicated in Alzheimer's disease, to study their aggregation at low concentrations is a common method; however, the fluorescent tag should not introduce a bias in the aggregation process. In this work, native amyloid peptides Aβ(1–40) and Aβ(1–42) and fluorescein-5-isothiocyanate (FITC), tagged ones, were studied using Taylor dispersion analysis coupled with a simultaneous UV and light-emitting diode-induced fluorescence detection, to unravel the effect of FITC on the aggregation process. For that, a total concentration of 100 µM of peptides consisting of a mixture of native and tagged ones (up to 10% in moles) was applied. Results demonstrated that FITC had a strong inhibition effect upon the aggregation behaviour of Aβ(1–42), whereas for Aβ(1–40), only a retardation in kinetics was observed. It was also shown that when mixed solutions of Aβ(1–40) and Aβ(1–42) are used, the Aβ(1–42) alloform was the leading peptide in the aggregation process, and when the latter was tagged, the aggregation kinetics decreased but the lifetime of potentially toxic oligomers was drastically increased. These results confirmed that the hydrophilicity of the N-terminus part of the peptide plays a major role in the aggregation process.     

A new treatment by dispersive liquid–liquid microextraction for the determination of parabens in human serum samples

Alkyl esters of p-hydroxybenzoic acid (parabens) are a family of compounds that have been in use since the 1920s as preservatives in cosmetic formulations, with one of the lowest rates of skin problems reported in dermatological patients. However, in the last few years, many scientific publications have demonstrated that parabens are weak endocrine disruptors, meaning that they can interfere with the function of endogenous hormones, increasing the risk of breast cancer. In the present work, a new sample treatment method is introduced based on dispersive liquid–liquid microextraction for the extraction of the most commonly used parabens (methyl-, ethyl-, propyl-, and butylparaben) from human serum samples followed by separation and quantification using ultrahigh performance liquid chromatography–tandem mass spectrometry. The method involves an enzymatic treatment to quantify the total content of parabens. The extraction parameters (solvent and disperser solvent, extractant and dispersant volume, pH of the sample, salt addition, and extraction time) were accurately optimized using multivariate optimization strategies. Ethylparaben ring ¹³C₆-labeled was used as surrogate. Limits of quantification ranging from 0.2 to 0.7 ng mL^?1 and an interday variability (evaluated as relative standard deviations) from 3.8 to 11.9 % were obtained. The method was validated using matrix-matched calibration standard and a spike recovery assay. Recovery rates for spiked samples ranged from 96 to 106 %, and a good linearity up to concentrations of 100 ng mL^?1 was obtained. The method was satisfactorily applied for the determination of target compounds in human serum samples.     

A new sesterterpenoid from the fruits of Phellodendron chinense var.glabriusculum Schneid

A new sesterterpenoid phellogine was isolated from the fruits of Phellodendron chinense var. glabriusculum Schneid. Its structure was elucidated on the basis of spectral analysis.     

Temperature and pressure dependence of the mode Grüneisen parameters close to the melting point in hexagonal ice

We reexamine the Pippard relations in this study by relating the specific heat CP to the Raman frequency shifts 1/nu (partial differentialnu/partial differentialT) P and the thermal expansivity alphap to the 1/nu (partial differentialnu/partial differentialP) T, when the mode Grüneisen parameter depends on the temperature and pressure close to the melting point in hexagonal ice. From linear relations between them, the values of the slope dP/dT are deduced in this crystal. Our slope values calculated here do not change significantly compared to those obtained when the mode Grüneisen parameter is taken as a constant close to the melting point in hexagonal ice.     

A new route of the reaction of EtAlCl2 with α-olefins catalyzed by Ti complexes

A new method for the synthesis of dialkyl(ethyl)alanes by the reaction of EtAlCl₂ with -olefins in the presence of Mg and a catalytic amount of Cp₂TiCl₂ (Ti(OPrⁱ)₄, Ti(OBuⁿ)₄) in THF was developed.     

Alkaloids of the Monolian flora. IV. Turcosine — A new alkaloid from Aconitum turczaninowi

As the result of a further study of the total alkaloids from the epigeal part ofAconitum turczaninowi we have isolated beiwutine and delcaroline and also a new alkaloid, which has been called turcosine. The structure of turcosine as 6,16-dimethoxy-4-methoxymethyl-1,7,8,10,14-pentahydroxy-N-ethylaconitane has been shown from a study of the IR, mass, PMR, and¹³C NMR spectra of the alkaloid and of the triacetate obtained from it.Institute of Chemistry, Mongolian People's Republic Academy of Sciences. Institute of the Chemistry of Plant Substances, Uzbekistan Republic Academy of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedeninii, No. 1, pp. 60–62, January–February, 1993.     

Alkaloids of the Mongolian flora. III. Altaconitine — A new alkaloid from Aconitum altaicum

Aconitine, mesaconitine, and a new alkaloid, which has been called altaconitine, have been isolated from the total alkaloids of the epigeal part ofAconitum altaicum. Altaconitine has the structure of 8-acetoxy-14-benzoyloxy-2,3,13,-15-tetrahydroxy-1,6, 16-trimethoxy-4-methoxymethy-N-ethylaconitan, which was established on the basis of a study of IR, PMR,¹³C NMR, and mass spectra and by the x-ray structural method.Institute of Chemistry, Mongolian Peoples' Republic Academy of Sciences, Ulan-Bator. Institute of the Chemistry of Plant Substances of the Uzbekistan Republic of Sciences, Tashkent. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 47–53, January–February, 1993.