DMMIC derivatization-assisted liquid chromatography-mass spectrometry method for metabolite profiling of the glutathione anabolic pathway in esophageal cancer tissues and cells.

In this work, a new pyrylium derivatization-assisted liquid chromatography-mass spectrometry (LC-MS) method was developed for metabolite profiling of the glutathione anabolic pathway (GAP) in cancer tissues and cells. The pyrylium salt of 6,7-dimethoxy-3-methyl isochromenylium tetrafluoroborate (DMMIC) was used to label the amino group of metabolites, and a reductant of dithiothreitol (DTT) was employed to stabilize the thiol group. By combining DMMIC derivatization with LC-MS, it was feasible to quantify the 13 main metabolites on the GAP in complex biological samples, which had good linearity (R2 = 0.9981-0.9999), precision (interday precision of 1.6%-19.0% and intraday precision of 1.4%-19.8%) and accuracy (83.4%-115.7%). Moreover, the recovery assessments in tissues (82.5%-107.3%) and in cells (98.1%-118.9%) with GSH-13C2, 15N, and Cys-15N demonstrated the reliability of the method in detecting tissues and cells. Following a methodological evaluation, the method was applied successfully to investigate difference in the GAP between the carcinoma and para-carcinoma tissues of esophageal squamous cell carcinoma (ESCC) and the effect of p-hydroxycinnamaldehyde (CMSP) on the GAP in KYSE-150 esophageal cancer cells. The results demonstrate that the developed method provides a promising new tool to elucidate the roles of GAP in physiological and pathological processes, which can contribute to research on drugs and diseases.

Characterization of AvaR1, an autoregulator receptor that negatively controls avermectins production in a high avermectin-producing strain.

Many γ-butyrolactone-autoregulator receptors control the production of secondary metabolites in Streptomyces spp. Hence, AvaR1, an autoregulator receptor protein in Streptomyces avermitilis, was characterized as a negative regulator of avermectin (Ave) production. Deletion of AvaR1 in a high-producing strain increased production of Ave B1a approx. 1.75 times (~700 µg/ml) compared with the parent strain. Semi-quantitative RT-PCR and electrophoretic mobility shift assays revealed that AvaR1 regulates the biosynthesis of Ave but not through the aveR pathway-specific regulatory gene. A special signaling molecule, avenolide, increased production of Ave. This study has refined our understanding of how avenolide regulates the production of Aves which is promising for developing new methods to improve the production of antibiotics in industrial strains.

Characterization of the saframycin A gene cluster from Streptomyces lavendulae NRRL 11002 revealing a nonribosomal peptide synthetase system for assembling the unusual tetrapeptidyl skeleton in an iterative manner.

Saframycin A (SFM-A), produced by Streptomyces lavendulae NRRL 11002, belongs to the tetrahydroisoquinoline family of antibiotics, and its core is structurally similar to the core of ecteinascidin 743, which is a highly potent antitumor drug isolated from a marine tunicate. In this study, the biosynthetic gene cluster for SFM-A was cloned and localized to a 62-kb contiguous DNA region. Sequence analysis revealed 30 genes that constitute the SFM-A gene cluster, encoding an unusual nonribosomal peptide synthetase (NRPS) system and tailoring enzymes and regulatory and resistance proteins. The results of substrate prediction and in vitro characterization of the adenylation specificities of this NRPS system support the hypothesis that the last module acts in an iterative manner to form a tetrapeptidyl intermediate and that the colinearity rule does not apply. Although this mechanism is different from those proposed for the SFM-A analogs SFM-Mx1 and safracin B (SAC-B), based on the high similarity of these systems, it is likely they share a common mechanism of biosynthesis as we describe here. Construction of the biosynthetic pathway of SFM-Y3, an aminated SFM-A, was achieved in the SAC-B producer (Pseudomonas fluorescens). These findings not only shed new insight on tetrahydroisoquinoline biosynthesis but also demonstrate the feasibility of engineering microorganisms to generate structurally more complex and biologically more active analogs by combinatorial biosynthesis.

Genetic characterization of the chlorothricin gene cluster as a model for spirotetronate antibiotic biosynthesis.

The biosynthetic gene cluster for chlorothricin (CHL) was localized to a 122 kb contiguous DNA from Streptomyces antibioticus DSM 40725, and its involvement in CHL biosynthesis was confirmed by gene inactivation and complementation. Bioinformatic analysis of the sequenced 111.989 kb DNA region revealed 42 open reading frames, 35 of which were defined to constitute the CHL gene cluster. An assembly model for CHL biosynthesis from D-olivose, 2-methoxy-5-chloro-6-methylsalicyclic acid, and chlorothricolide building blocks was proposed. This work represents cloning of a gene cluster for spirotetronate antibiotic biosynthesis and sets the stage to investigate the unusual macrolide biosynthesis including tandem Diels-Alder cyclizations, Baeyer-Villiger oxidation, and incorporation of an enoylpyruvate unit.

Cloning and characterization of a bacterial iterative type I polyketide synthase gene encoding the 6-methylsalicyclic acid synthase.

Unusual polyketide synthases (PKSs), that are structurally type I but act in an iterative manner for aromatic polyketide biosynthesis, are a new family found in bacteria. Here we report the cloning of the iterative type I PKS gene chlB1 from the chlorothricin (CHL) producer Streptomyces antibioticus DSM 40725 by a rapid PCR approach, and characterization of the function of the gene product as a 6-methylsalicyclic acid synthase (6-MSAS). Sequence analysis of various iterative type I PKSs suggests that the resulting aromatic or aliphatic structure of the products might be intrinsically determined by a catalytic feature of the paired KR-DH domains in the control of the double bond geometry. The finding of ChlB1 as a 6-MSAS not only enriches the current knowledge of aromatic polyketide biosynthesis in bacteria, but will also contribute to the generation of novel polyketide analogs via combinatorial biosynthesis with engineered PKSs.

[Study on the effect of H2O2 against Acanthamoeba in vitro].

OBJECTIVE: To detect the effect of H2O2 on Acanthamoeba spp.. METHODS: By Wright's stain, quantitative culture, MTT assay and lactate dehydrogenase(LDH) assessment, the influence of H2O2 on the morphological feature, proliferation speed and the survival rate of Acanthamoeba was tested. RESULTS: At low concentration of 0.125%, H2O2 can force the Acanthamoeba trophozoites into cysts irreversibly, and inhibit its proliferation. 1% H2O2 can directly destroy Acanthamoeba trophozoites. CONCLUSION: H2O2 is effective in destroying Acanthamoeba. It is possible to be used as an ideal reagent for the prevention of Acanthamoeba keratitis.