The biosynthesis of the enediyne moiety of the antitumor natural product calicheamicin involves an iterative polyketide synthase (CalE8) and other ancillary enzymes. In the proposed mechanism for the early stage of 10-membered enediyne biosynthesis, CalE8 produces a carbonyl-conjugated polyene with the assistance of a putative thioesterase (CalE7). We have determined the x-ray crystal structure of CalE7 and found that the subunit adopts a hotdog fold with an elongated and kinked substrate-binding channel embedded between two subunits. The 1.75-Å crystal structure revealed that CalE7 does not contain a critical catalytic residue (Glu or Asp) conserved in other hotdog fold thioesterases. Based on biochemical and site-directed mutagenesis studies, we proposed a catalytic mechanism in which the conserved Arg 37 plays a crucial role in the hydrolysis of the thioester bond, and that Tyr 29 and a hydrogen-bonded water network assist the decarboxylation of the ␤-ketocarboxylic acid intermediate. Moreover, computational docking suggested that the substrate-binding channel binds a polyene substrate that contains a single cis double bond at the C4/C5 position, raising the possibility that the C4‫؍‬C5 double bond in the enediyne moiety could be generated by the iterative polyketide synthase. Together, the results revealed a hotdog fold thioesterase distinct from the common type I and type II thioesterases associated with polyketide biosynthesis and provided interesting insight into the enediyne biosynthetic mechanism.Enediyne natural products represent a family of structurally unique secondary metabolites with potent antitumor and antibiotic activities. Based on the structure of the bicyclic enediyne core, enediyne natural products are categorized into two groups with either a 9-or 10-membered enediyne moiety (1, 2). The antitumor activity of enediyne natural products derives from their capacity to induce chromosomal DNA cleavage through an oxidative radical mechanism (3). The biosynthetic mechanism for the enediyne moiety has been, however, elusive despite clues gleaned from early isotope-feeding experiments (4, 5). Pioneering genetic studies of the biosynthesis of calicheamicin and C-1027 from two research groups yielded major insights into the biosynthetic pathways, suggesting that an iterative polyketide synthase (PKS) 5 plays a central role in the assembly of both the 9-and 10-membered enediyne moieties (6, 7). The gene clusters also contain open reading frames encoding hypothetical proteins for the downstream processing of the PKS product. The involvement of similar genes in enediyne biosynthesis was later confirmed for neocarzinostatin, maduropeptin, dynemicin, and several putative enediyne natural products in soil and marine microorganisms (8 -11). Recently, based on the study on the 9-membered enediynecontaining C-1027, Shen and coworkers found that the iterative PKS (SgcE) and the putative thioesterase (SgcE10) generated a conjugated polyene (1,3,5,7,9,11,13-pentadecaheptaene) through an ACP-tethered 3-hydroxy-4...

Multidrug-resistant Escherichia coli infections are a growing public health concern. This study analyzed the possibility of contamination of commercial poultry meat (broiler and free-range) with pathogenic and or multi-resistant E. coli in retail chain poultry meat markets in India. We analyzed 168 E. coli isolates from broiler and free-range retail poultry (meat/ceca) sampled over a wide geographical area, for their antimicrobial sensitivity, phylogenetic groupings, virulence determinants, extended-spectrum-β-lactamase (ESBL) genotypes, fingerprinting by Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR and genetic relatedness to human pathogenic E. coli using whole genome sequencing (WGS). The prevalence rates of ESBL producing E. coli among broiler chicken were: meat 46%; ceca 40%. Whereas, those for free range chicken were: meat 15%; ceca 30%. E. coli from broiler and free-range chicken exhibited varied prevalence rates for multi-drug resistance (meat 68%; ceca 64% and meat 8%; ceca 26%, respectively) and extraintestinal pathogenic E. coli (ExPEC) contamination (5 and 0%, respectively). WGS analysis confirmed two globally emergent human pathogenic lineages of E. coli, namely the ST131 (H30-Rx subclone) and ST117 among our poultry E. coli isolates. These results suggest that commercial poultry meat is not only an indirect public health risk by being a possible carrier of non-pathogenic multi-drug resistant (MDR)-E. coli, but could as well be the carrier of human E. coli pathotypes. Further, the free-range chicken appears to carry low risk of contamination with antimicrobial resistant and extraintestinal pathogenic E. coli (ExPEC). Overall, these observations reinforce the understanding that poultry meat in the retail chain could possibly be contaminated by MDR and/or pathogenic E. coli.

Mammalian Pellino isoforms are phosphorylated by IRAK (interleukin receptor associated kinase) 1/IRAK4 in vitro, converting them into active E3 ubiquitin ligases. In the present paper we report a striking enhancement in both transcription of the gene encoding Pellino 1 and Pellino 1 protein expression when murine BMDMs (bone-marrow-derived macrophages) are stimulated with LPS (lipopolysaccharide) or poly(I:C). This induction occurs via a TRIF [TIR (Toll/interleukin-1 receptor)-domain-containing adaptor-inducing interferon-β]-dependent IRAK-independent pathway and is prevented by inhibition of the IKK [IκB (inhibitor of nuclear factor κB) kinase]-related protein kinases, TBK1 {TANK [TRAF (tumour-necrosis-factor-receptor-associated factor)-associated nuclear factor κB activator]-binding kinase 1} and IKKε. Pellino 1 is not induced in IRF3 (interferon regulatory factor 3)-/- BMDMs, and its induction is only reduced slightly in type 1 interferon receptor-/- BMDMs, identifying Pellino 1 as a new IRF3-dependent gene. We also identify Pellino 1 in a two-hybrid screen using IKKε as bait, and show that IKKε/TBK1 activate Pellino 1 in vitro by phosphorylating Ser76, Thr288 and Ser293. Moreover, we show that the E3 ligase activity of endogenous Pellino 1 is activated in LPS- or poly(I:C)-stimulated macrophages. This occurs more rapidly than the increase in Pellino 1 mRNA and protein expression, is prevented by the inhibition of IKKε/TBK1 and is reversed by phosphatase treatment. Thus IKKε/TBK1 mediate the activation of Pellino 1's E3 ligase activity, as well as inducing the transcription of its gene and protein expression in response to TLR3 and TLR4 agonists.

BackgroundAbelmoschus moschatus Medik. leaves and seeds are considered as valuable traditional medicine. The aromatic seeds of this plant are aphrodisiac, ophthalmic, cardio tonic, antispasmodic and used in the treatment of intestinal complaints and check queasiness. To give a scientific basis for traditional usage of this medicinal plant, the seed and leaf extracts were evaluated for their antioxidant, free radical scavenging, antimicrobial and antiproliferative activities.MethodsIn this study, antioxidant, antimicrobial and antiproliferative activities of A. moschatus extracts were evaluated in a series of in vitro assay involving free radicals, reactive oxygen species and their IC50 values were also determined. The antioxidant activities of the seed and leaf extracts of A. moschatus were determined by total antioxidant, DPPH, and ferrous reducing antioxidant property (FRAP) methods. In addition, the antiproliferative activity was also evaluated using colorectal adenocarcinoma and retinoblastoma human cancer cell lines. Moreover, six bacterial reference strains, two gram-positive (Bacillus subtilis and Staphylococcus aureus), four gram-negative (Escherichia coli, Pseudomonas aeruginosa, Proteus vulgaris and Salmonella enterica paratyphi) and one fungal strain (Candida albicans) were used to evaluate its antimicrobial activity.ResultsThe results from this study showed that the antioxidant activities of A. moschatus as determined by the total phenol, flavonoids, total antioxidant and FRAP methods were higher in leaf than that of the seed extracts. On the other hand, the aqueous overnight seed extract (AMS-I) has shown significant radical scavenging activity as in 1, 1- Diphenyl-2-picrylhydrazyl (DPPH), hydrogen peroxide, hydroxyl radical, superoxide and lipid peroxidation as compared to other seed and leaf extracts. The AMS-I and AML-IV have shown activity against six and seven microorganisms respectively. Simulteneously, AMS-IV and AML-IV have demonstrated potential antiproliferative activity against two human cell lines - Colorectal adenocarcinoma (COLO-205) and retinoblastoma (Y79).ConclusionThe seed and leaf extracts of A. moschatus possess significant antioxidant activity and could serve as free radical inhibitors or scavenger, or substitute, probably as primary antioxidants. The plant possesses moderate antibacterial activity against bacterial strains used in this study. Hydroalcoholic seed and leaf extracts also exhibited antiproliferative activity against two human cancer cell lines. A. moschatus may therefore, be a good candidate for functional foods as well as pharmaceutics.

BackgroundThe use of traditional medicine at the primary health care level is widespread and plant-based treatments are being recommended for curing various diseases by traditional medical practitioners all over the world. The phytochemicals present in the fruits, vegetables and medicinal plants are getting attention day-by-day for their active role in the prevention of several human diseases. Abrus precatorius is a widely distributed tropical medicinal plant with several therapeutic properties. Therefore in the present study, A. precatorius leaf extracts were examined for their antioxidant and cytotoxic properties in vitro in order to discover resources for new lead structures or to improve the traditional medicine.MethodsIn this study, antioxidant and antiproliferative properties of the different leaf extracts (hexane, ethyl acetate, ethanol and water) from A. precatorius were investigated along with the quantification of the polyphenol and flavonoid contents. The ability of deactivating free radicals was extensively investigated with in vitro biochemical methods like DPPH•, •OH, NO, SO2- scavenging assays and inhibition capability of Fe(II)-induced lipid peroxidation. Furthermore, antiproliferative activities using different human cancer cell lines and primary cell line was carried out by MTT method.ResultsTotal phenolic content and total flavonoid content of the extracts were found in the range of 1.65 ± 0.22 to 25.48 ± 0.62 GAE mg/g dw and 6.20 ± 0.41 to 17.16 ± 1.04 QE mg/g dw respectively. The experimental results further revealed that A. precatorius extracts showed strong antiradical properties, capable to chelate Fe2+ and possess good inhibition ability of lipid peroxidation. In addition, as a first step towards the identification of phytoconstituents endowed with potent chemopreventive activities, we evaluated the inhibitory effects of A. precatorius extracts on the proliferation of four different human tumour cell lines such as human colon adenocarcinoma cells (Colo-205), human retinoblastoma cancer cells (Y79), human hepatocellular carcinoma cells (HepG2) and Leukemia cells (SupT1). Ethanol extract (APA) and ethyl acetate extract (APE) of A. precatorius had apparent capabilities of inhibiting the survival of tested human cancer cell lines. Moreover, it was observed that the A. precatorius extracts did not inhibit the growth of mice peritoneal macrophages, thus confirming that plants extracts are selective against the cancer cell lines.ConclusionThis work provides a scientific support for the high antioxidant and antiproliferative activity of this plant and thus it may find potential applications in the treatment of the diseases caused by ROS. Further studies are needed to confirm in vivo anti-tumorgenicity and subsequent chemical characterization of the active molecule(s).