A mixed-alpha,beta miniprotein stereochemically reprogrammed to high-binding affinity for acetylcholine

The protein-structure space is limited to L configuration in the asymmetric alpha-amino acid structures; the function space, on other hand, seems limitless because of the chemical diversity in the amino acid side chain structures. The chemical diversity in side chain structure may be multiplied beneficially with the stereochemical diversity in main chain structure; thus, de novo protein design may be explored for customizing molecular structures stereochemically and molecular functions chemically. Illustrating de novo design in the structure space of L and D alphabet, canonical all-beta folds of poly-L structure were reprogrammed as bracelet, boat, and canoe-shaped molecules-the "boat" as a receptor-like pocket and the "canoe" as a metal-ion receptor-simply by mutating specific L-amino acid residues to the corresponding D stereochemical structure. Demonstrating customization of molecular shape stereochemically and function chemically, a 15-residue mixed-alpha, beta miniprotein of canonical poly-L structure is now reprogrammed stereochemically as a cup-shaped receptor for acetylcholine via cation-pi interaction with a triad of aromatic side chains placed in mimicry of the acetylcholine-receptor sites both natural and artificial. Evidence from CD, fluorescence, NMR, DSC, ITC, MD, and molecular-docking studies is presented to show that a rationally designed 15-residue mixed-L, D peptide is a cooperatively ordered molecular fold in the stereochemically specified molecular morphology, submicromolar in affinity of acetylcholine and thus an acetylcholine receptor exceptionally small and simple. .     

Synthesis and anticonvulsant activity of sulfonamide derivatives-hydrophobic domain

A series of sulphonamide derivatives (1-11) were synthesized in good yield and evaluated for their possible anticonvulsant activity and neurotoxic study. The structures of the synthesized compounds were confirmed on the basis of their spectral data and elemental analysis. Majority of the compounds were active in MES and scPTZ tests. All the compounds were less toxic than the standard drug phenytoin.     

Spoligotyping of Mycobacterium tuberculosis DNA from Archival Ziehl-Neelsen-stained sputum smears

Spoligotyping was applied to old (5-11 years) Ziehl-Neelsen (ZN)-stained smears for strain identification and differentiation and to predict the utility of the technique in epidemiological studies. Among 57 DNA samples extracted from ZN slides lying stored at room temperature, 93% (53) amplification was achieved for mpt64 gene. Spoligopatterns were generated from 77.7% (41/53) DNA samples, whereas negative controls did not yield any spoligopatterns. All slides with 2+ (n=20) and 3+ (n=13) positivity while 42% (11/26) of slides with low positivity (< or 1+) showed a good signal and a reproducible pattern. This technique may have application in identification of spoligotypes in control programme implemented areas remote from research laboratory and would also increase our knowledge about the clonal structure of Mycobacterium tuberculosis in the population, when applied to old samples in different locations.     

Simultaneous identification of 2-carboxy-tetrahydrocannabinol, tetrahydrocannabinol, cannabinol and cannabidiol in oral fluid

Tetrahydrocannabinol (THC) is an important psychoactive ingredient in marijuana, which is the most widely used illegal recreational drug in the USA. Since it is generally smoked, the constituents of the plant material, as well as THC may be present in oral fluid specimens collected for the purposes of drug testing. We present an analytical procedure for the simultaneous determination of the pyrolytic precursor Delta(9)-tetrahydrocannabinolic acid A, tetrahydrocannabinol, cannabinol and cannabidiol in human oral fluid specimens using gas chromatography mass spectrometry (GC/MS). Solid phase extraction and GC/MS/EI with selected ion monitoring were used, and the linearity of the method ranged from 0-16 ng/mL of neat oral fluid. The recovery of the cannabinoids from the collection pad into the transportation buffer was greater than 70% for all cannabinoids tested at 4 ng/mL, and the intra- and inter-day precision was less than 10.3 and 15.2% for all analytes. The stability of the drugs in oral fluid and of the extracted derivatives was investigated. The procedure was applied to oral fluid specimens taken from habitual marijuana smokers. We have previously reported the presence of the metabolite 11-nor-Delta(9)-tetra-hydrocannabinol-9-carboxylic acid in oral fluid, but this is the first report of the plant constituent 2-carboxy-THC being detected in saliva.     

Determination of meperidine, tramadol and oxycodone in human oral fluid using solid phase extraction and gas chromatography-mass spectrometry

Analytical procedures for the determination of meperidine, tramadol and oxycodone in oral fluid have been developed and validated using gas chromatography-mass spectrometry (GC/MS) following initial screening with enzyme linked immunosorbent assay (ELISA). The oral fluid samples were collected using the Quantisal device, and any drugs present were quantified using mixed mode solid-phase extraction and electron impact GC/MS. For confirmation, three ions were monitored and two ion ratios determined, which were within 20% of those of the known calibration standards. The limits of quantitation were 10 ng/mL; the intra-day precision of the assays (n=5) was 2.33%, 1.00% and 7.61%; inter-day precision 2.48%, 2.44% and 5.8% (n=10) for meperidine, tramadol and oxycodone, respectively. The percentage recovery of the drugs from the collection pads was 86.7%, 87.7% and 96.6%, respectively (n=6). The methods were applied to specimens obtained during research studies in the USA.     

Berberine enhances posttranslational protein stability of p21/cip1 in breast cancer cells via down-regulation of Akt

Berberine has shown anticancer properties and has potential for a chemopreventive and/or chemotherapeutic agent for breast cancer. Berberine showed cytotoxicity to breast cancer cells, with an increase in the levels of p21/cip1 and p27/kip1, cyclin-dependent kinase inhibitors (CDKI), but mechanisms involved in up-regulating these molecules are largely unknown. Herein, we studied the key regulatory mechanisms involved in berberine-mediated up-regulation of p21/cip1 and p27/kip1. Berberine treatment for 24 and 48 h decreased the number of cells by 44–84% (P?<?0.0001) and 38–78% (P?<?0.0001), and increased cell death by 12–17% (P?<?0.005) and 38–78% (P?<?0.0001) in MCF-7 and MDA-MB-231 cells, respectively. Cells were arrested in G1 phase by berberine which was accompanied with up-regulation of mRNA and protein level of both p21/cip1 and p27/kip1. Berberine decreased the expression of protein levels of cyclin D1, cyclin E, CDK2, CDK4, and CDK6 to cause G1 phase arrest. Berberine caused nuclear localization of p21/cip1 in both the cell lines. Our data for the first time showed that the post-translational stability of both the proteins was strongly increased by berberine as examined by cycloheximide chase assay. Inhibition of Akt was associated with berberine-mediated up-regulation of p21/cip1 and also led to a decrease in cell viability accompanied with significant G1 phase cell cycle arrest. Our study revealed that berberine not only up-regulates mRNA and protein levels of p21/cip1 and p27/kip1 but also increases their nuclear localization and post-translational protein stability. Further, Akt inhibition was found to mediate berberine-mediated up-regulation of p21/cip1 but not the p27/kip1.