An efficient method for the purification and characterization of nematicidal azadirachtins A,B, and H,using MPLC and ESIMS

Azadirachtin A enriched concentrate containing 60% active ingredient (a.i.) was prepared from the methanolic extract of the de-fatted neem (Azadirachta indica A. Juss) seed kernels. Azadirachtins A, B, and H, the three major bioactive constituents of neem seed kernel, were purified from this methanolic concentrate by employing reverse phase medium-pressure liquid chromatography (MPLC), using methanol-water solvent system as an eluant. The three pure azadirachtin congeners thus obtained were characterized by their unique mass spectral fragmentation, using electrospray probe in positive ion mode (ESI). All three azadirachtins exhibited nematicidal and antifungal activities. Azadirachtin B was the most effective against the reniform nematode Rotylenchulus reniformis (EC(50) 96.6 ppm), followed by Azadirachtin A (119.1 ppm) and H (141.2 ppm). At 200-ppm concentration, the test compounds caused 50-65% mortality of Caenorhabditis elegans nematode. Azadirachtin H showed the highest activity against the phytophagous fungi Rhizoctonia solani (EC(50) 63.7 ppm) and Sclerotium rolfsii (EC(50) 43.9 ppm), followed by B and A. The isolation of pure azadirachtins A, B, and H directly by MPLC purification from its concentrate and their characterization by ESIMS are unique and less time-consuming.     

Evolution of Marek's disease -- a paradigm for incessant race between the pathogen and the host

Marek's disease (MD) is a highly contagious lymphoproliferative disease of poultry caused by the oncogenic herpesvirus designated Marek's disease virus (MDV). MD has a worldwide distribution and is thought to cause an annual loss over 1 bn US dollars to the poultry industry. Originally described as a paralytic disease, today MD is mostly manifested as an acute disease with tumours in multiple visceral organs. MD is controlled essentially by the widespread use of live vaccines administered either in ovo into 18-day-old embryos or into chicks immediately after they hatch. In spite of the success of the vaccines in reducing the losses from the disease in the last 30 years, MDV strains have shown continuous evolution in virulence acquiring the ability to overcome the immune responses induced by the vaccines. During this period, different generations of MD vaccines have been introduced to protect birds from the increasingly virulent MDV strains. However, the virus has countered each new vaccine with ever more virulent strains. This continuous race between the virus and the host is making the control of this poultry health problem a major challenge for the future.     

Structure and mechanism of the lantibiotic cyclase involved in nisin biosynthesis

Nisin is a posttranslationally modified antimicrobial peptide that is widely used as a food preservative. It contains five cyclic thioethers of varying sizes that are installed by a single enzyme, NisC. Reported here are the in vitro reconstitution of the cyclization process and the x-ray crystal structure of the NisC enzyme. The structure reveals similarities in fold and substrate activation with mammalian farnesyl transferases, suggesting that human homologs of NisC posttranslationally modify a cysteine of a protein substrate.     

Dietary anthocyanin-rich tart cherry extract inhibits intestinal tumorigenesis in APC(Min) mice fed suboptimal levels of sulindac

A promising approach for cancer chemoprevention might be a combination therapy utilizing dietary phytochemicals and anticarcinogenic pharmaceuticals at a suboptimal dosage to minimize any potential adverse side effects. To test this hypothesis, various dosages of anthocyanin-rich tart cherry extract were fed in combination with suboptimal levels of the nonsteroidal anti-inflammatory drug sulindac to APCMin mice for 19 weeks. By the end of the feeding period, fewer mice that were fed the anthocyanin-rich extract in combination with sulindac lost more than 10% of body weight than mice fed sulindac alone. Mice that were fed anthocyanin-rich extract (at any dose) in combination with sulindac had fewer tumors and a smaller total tumor burden (total tumor area per mouse) in the small intestine when compared to mice fed sulindac alone. These results suggest that a dietary combination of tart cherry anthocyanins and sulindac is more protective against colon cancer than sulindac alone.     

Diagnosis of paratuberculosis in goats by cell mediated immune response,conventional and molecular diagnostic techniques

In the present study efficacy of single intradermal Johnin test, acid fast staining of faecal smear and IS 900 faecal polymerase chain reaction tests was evaluated in 200 goats for detection of Mycobacterium avium subsp paratuberculosis. Two hundred goats comprising 150 goats from an organised farm in Trichur district and 50 goats reared under field condition at farmers premise from Malappuram district of Kerala state formed the study population. Faecal smear from all the 200 goats was stained by Ziehl–Neelsen acid fast stain and faecal polymerase chain reaction (PCR) specific for M. avium subsp paratuberculosis (MAP); IS 900 was performed on all samples. All the animals were subjected to single intradermal Johnin test. Out of 200 goats screened for paratuberculosis, six goats (3%), 11 goats (5.5%) and 42 goats (21%) were found positive by Ziehl–Neelsen acid fast staining of faecal smear, single intradermal Johnin test and IS 900 PCR respectively. Results of the present study indicate that amplification of IS 900 insertion element was the most specific and sensitive diagnostic detection method. Single intradermal Johnin test and Ziehl–Neelsen acid fast staining did not show any significant difference.