Evaluation of the Synthetic Potential of an AHBA Knockout Mutant of the Rifamycin Producer Amycolatopsis mediterranei

Supplementing an AHBA(?) mutant strain of Amycolatopsis mediterranei, the rifamycin producer, with a series of benzoic acid derivatives yielded new tetraketides containing different phenyl groups. These mutasynthetic studies revealed unique reductive properties of A. mediterranei towards nitro‐ and azidoarenes, leading to the corresponding anilines. In selected cases, the yields of mutaproducts (fermentation products isolated after feeding bacteria with chemically prepared analogs of natural building blocks) obtained are in a range (up to 118 mg L^?1) that renders them useful as chiral building blocks for further synthetic endeavors. The configuration of the stereogenic centers at C6 and C7 was determined to be 6R,7S for one representative tetraketide. Importantly, processing beyond the tetraketide stage is not always blocked when the formation of the bicyclic naphthalene precursor cannot occur. This was proven by formation of a bromo undecaketide, an observation that has implications regarding the evolutionary development of rifamycin biosynthesis.     

New antituberculosis drugs targeting the respiratory chain

With the emergence of multidrug-resistant tuberculosis and extensive drug-resistant tuberculosis strains,there is an urgent need to develop novel drugs for the treatment of tuberculosis.The respiratory chain is a promising target for the development of newantimycobacterial agents,and a growing number of compounds have been reported and some have entered clinical trials.In this review,we summarize the main features and the electron transfer process of the mycobacterial respiratory chain,and the recent progress in the search for new small molecule inhibitors to rgeting the three main potential targets in the respiratory chain of Mycrobacterium tuberculosis.Our emphasis is on the optimization strategy of QcrB inhibitors and the challenges of developing QcrB inhibitors as antituberculosis drugs due to the alternate bd-type oxidase oxidative compensation pathway are discussed.     

Hyaluronic Acid: Its Role in Voice

The extracellular matrix (ECM), once regarded simply as a structural scaffold, is now recognized as an important modulator of cellular behavior and function. One component that plays a prominent role in this process is hyaluronic acid (HA)--a molecule found in many different tissues. Research into the roles of HA indicates that it plays a key role in tissue viscosity, shock absorption, and space filling. Specifically, research into the role of HA in laryngology indicates that it has profound effects on the structure and viscosity of vocal folds. This article provides an introduction to the structure and biological functions of HA and its importance in voice. In addition, an overview of the pharmaceutical applications of HA is discussed.     

水/有机溶剂双相体系中色氨酸合成酶酶法合成L-色氨酸

在水/有机溶剂双相体系中,利用重组大肠杆菌DM206 [pET28a-trpBA/BL21 (DE3)]色氨酸合成酶催化L-丝氨酸和吲哚合成了L-色氨酸.考察了有机溶剂、有机相乙酸乙酯体积分数、反应温度、水相pH、底物L-丝氨酸与吲哚摩尔比、底物L-丝氨酸浓度、表面活性剂和酶添加量对色氨酸合成酶酶活的影响.结果表明,酶...     

Biosynthesis of the Apoptolidins in Nocardiopsis sp. FU 40

The apoptolidins are 20/21-membered macrolides produced by Nocardiopsis sp. FU40. Several members of this family are potent and remarkably selective inducers of apoptosis in cancer cell lines, likely via a distinct mitochondria associated target. To investigate the biosynthesis of this natural product, the complete genome of the apoptolidin producer Nocardiopsis sp. FU40 was sequenced and a 116 kb region was identified containing a putative apoptolidin biosynthetic gene cluster. The apoptolidin gene cluster comprises a type I polyketide synthase, with 13 homologating modules, apparently initiated in an unprecedented fashion via transfer from a methoxymalonyl-acyl carrier protein loading module. Spanning approximately 39 open reading frames, the gene cluster was cloned into a series of overlapping cosmids and functionally validated by targeted gene disruption experiments in the producing organism. Disruption of putative PKS and P₄₅₀ genes delineated the roles of these genes in apoptolidin biosynthesis and chemical complementation studies demonstrated intact biosynthesis peripheral to the disrupted genes. This work provides insight into details of the biosynthesis of this biologically significant natural product and provides a basis for future mutasynthetic methods for the generation of non-natural apoptolidins.     

Single- and double-chained truncated jaspine B analogues: asymmetric synthesis, biological evaluation and theoretical study of an unexpected 5-endo-dig process

An optimized synthesis of jaspine B analogues bearing an n-octyl or a p-fluorophenethyl lipophilic appendage was developed. Key to the approach was the use of acetylenic nucleophiles for the stereocontrolled introduction of the side chain and the implementation of a novel cyclization procedure to build the tetrahydrofuran ring. Three N-substituted amine or amide derivatives were also accessed. The biological activity of these four jaspine B analogues was shown to strongly depend on the nature of both the N-substituent and the aliphatic moiety connected to the tetrahydrofuran ring. Gratifyingly, the truncated jaspine B derivative proved to be a pro-apoptotic inhibitor of the conversion of ceramide into sphingomyelin. Finally, the efficient formation of a fused bis-furan derivative according to a 5-endo-dig process was observed under saponification conditions. On the basis of a theoretical study, a mechanistic pathway was delineated highlighting the Lewis acidity of the K⁺ ion as the driving force for this transformation in a strongly alkaline medium.     

A facile electrophoretic technique to monitor phosphoenolpyruvate-dependent kinases

Phosphoenolpyruvate (PEP)-dependent kinases are central to numerous metabolic processes and mediate the production of adenosine triphosphate (ATP) by substrate-level phosphorylation (SLP). While pyruvate kinase (PK, EC: 2.7.1.40), the final enzyme of the glycolytic pathway is critical in the anaerobic synthesis of ATP from ADP, pyruvate phosphate dikinase (PPDK, EC: 2.7.9.1), and phosphoenolpyruvate synthase (PEPS, EC: 2.7.9.2) help generate ATP from AMP coupled to PEP as a substrate. Here we demonstrate an inexpensive and effective electrophoretic technology to determine the activities of these enzymes by blue-native polyacrylamide gel electrophoresis (BN-PAGE). The generation of pyruvate is linked to exogenous lactate dehydrogenase (LDH), and the oxidation of reduced nicotinamide adenine dinucleotide (NADH) coupled to 2,6-dichloroindophenol (DCIP) and iodonitrotetrazolium chloride (INT) results in a formazan precipitate which is easily quantifiable. The selectivity of the enzymes is ensured by including either AMP or ADP and pyrophosphate (PP(i) ) or inorganic phosphate (P(i) ). Activity bands were readily obtained after incubation in the respective reaction mixtures for 20-30 min. Cell-free extract concentrations as low as 20 μg protein equivalent yielded activity bands and substrate levels were manipulated to optimize sensitivity of this analytical technique. High-pressure liquid chromatography (HPLC), two-dimensional (2-D) SDS-PAGE (where SDS is sodium dodecyl sulfate), and immunoblot studies of the excised activity band help further characterize these PEP-dependent kinases. Furthermore, these enzymes were readily identified on the same gel by incubating it sequentially in the respective reaction mixtures. This technique provides a facile method to elucidate these kinases in biological systems.     

The Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein, a toll-like receptor 4 agonist, enhances dendritic cell-based cancer vaccine potency

In this study, we showed the direct interaction between Mycobacterium avium subsp. paratuberculosis fibronectin attachment protein (FAP) and toll-like receptor4 (TLR4) via co-localization and binding by using confocal microscopy and co-immunoprecipitation assays. FAP triggered the expression of pro- and antiinflammatory cytokines in a TLR4-dependent manner. In addition, FAP-induced cytokine expression in bone marrow-derived dendritic cells (BMDCs) was modulated in part by glycogen synthase kinase-3 (GSK-3). FAP-induced expression of CD80, CD86, major histocompatibility complex (MHC) class I, and MHC class II in TLR4(+/+) BMDCs was not observed in TLR4(-/-) BMDCs. Furthermore, FAP induced DC-mediated CD8(+) T cell proliferation and cytotoxic T lymphocyte (CTL) activity, and suppressed tumor growth with DC-based tumor vaccination in EG7 thymoma murine model. Taken together, these results indicate that the TLR4 agonist, FAP, a potential immunoadjuvant for DC-based cancer vaccination, improves the DC-based immune response via the TLR4 signaling pathway.     

New Fluorometric Method for Continuous Measurement of ß-Cyanoalanine Synthase Activity

Abstract

A new method for the determination of injectase has been described. Assay conditions have been established which permit the assay of injectase over the range from 10^?3 to 0. 1 unit. The method is simple, rapid, sensitive, and reliable.