In silico studies of urease inhibitors to explore ligand-enzyme interactions

In continuation of our previous study on the urease inhibition by a number of chalcones, 2,3-dihydro-1,5-benzothiazepines and 2,3,4,5-tetrahydro-1,5-benzothiazepines, FlexX docking has been exploited to get a deeper insight into the mechanism of their inhibitory action. A comparison of the IC₅₀ values of the active compounds reveals that, of the three classes of compounds studied, 2,3-dihydro-1,5-benzothiazepines were the most potent urease inhibitors. An in silico examination of these compounds showed that the activity is related to the interaction of ligand with the nickel metallocentre, its interaction with two amino acid residues, Asp224 and Cys322, in addition to the orientation of rings A and B in the catalytic core of the enzyme. The most active compound 2,3-dihydro-1,5-benzothiazepine (4) anchor tightly through a network of interactions with Ni701 and Ni702. This includes a number of hydrogen bonds and hydrophobic contacts with the amino acid residues in its vicinity. For their reduced analogs, the difference in the activity of different diastereomers has been observed to be configuration-dependent. This may be ascribed mainly to the difference in the orientation of ring B of the two stereoisomers and the extent of their interaction with Asp224 and Cys322 present in the catalytic core of the enzyme.     

Opposite association of two PPARG variants with cancer: overrepresentation of H449H in endometrial carcinoma cases and underrepresentation of P12A in renal cell carcinoma cases

Peroxisome proliferator activated receptor gamma (PPARgamma) is a nuclear hormone receptor that has been shown to regulate differentiation and cell growth. Studies of the differentiative effects of PPARgamma agonists on several cancer cell lines led to the hypothesis that dysfunction of PPARgamma contributes to tumorigenesis. These functional observations were strengthened by genetic evidence: somatic loss-of-function mutations in PPARG, encoding PPARgamma, in sporadic colorectal carcinomas and somatic translocation of PAX8 and PPARG in follicular thyroid carcinoma. Recently overrepresentation of the H449H variant was found in a cohort of American patients with glioblastoma multiforme. The glioblastoma multiforme data suggest that PPARG contributes common, low-penetrance alleles for cancer susceptibility. To test this hypothesis in a broader range of cancers we examined a series of carcinomas of the cervix, endometrium, ovary, prostate, and kidney for germline sequence variation in PPARG. In addition to the two common sequence variants, P12A and H449H, there were five other sequence variants. P12A alleles were underrepresented in renal cell carcinoma patients compared to country-of-origin race-matched controls (3.75% vs. 12.1%, P<0.04). In contrast, the H449H variant was overrepresented in individuals with endometrial carcinoma compared to controls (14.4% vs. 6.25%, P<0.02). These observations lend genetic evidence consistent with our hypothesis that PPARG serves as a common, low-penetrance susceptibility gene for cancers of several types, especially those epidemiologically associated with obesity and fat intake.     

Metabolomic approaches for the identification of flavonoids associated with weed suppression in selected Hardseeded annual pasture legumes

Plant and Soil - Weed suppressive potential of annual pasture legumes has been previously described, the mechanism of interference with weeds has not been clearly elucidated. We, therefore, aimed...     

Rapid protein production using CHO stable transfection pools

During early preclinical development of therapeutic proteins, representative materials are often required for process development, such as for pharmacokinetic/pharmacodynamic studies in animals, formulation design, and analytical assay development. To rapidly generate large amounts of representative materials, transient transfection is commonly used. Because of the typical low yields with transient transfection, especially in CHO cells, here we describe an alternative strategy using stable transfection pool technology. Using stable transfection pools, gram quantities of monoclonal antibody (Mab) can be generated within 2 months post‐transfection. Expression levels for monoclonal antibodies can be achieved ranging from 100 mg/L to over 1000 mg/L. This methodology was successfully scaled up to a 200 L scale using disposable bioreactor technology for ease of rapid implementation. When fluorescence‐activated cell sorting was implemented to enrich the transfection pools for high producers, the productivity could be improved by about three‐fold. We also found that an optimal production time window exists to achieve the highest yield because the transfection pools were not stable and productivity generally decreased over length in culture. The introduction of Universal chromatin‐opening elements elements into the expression vectors led to significant productivity improvement. The glycan distribution of the Mab product generated from the stable transfection pools was comparable to that from the clonal stable cell lines. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Biochemical studies on malathion resistance,inheritance and association of carboxylesterase activity in brown planthopper,Nilaparvata lugens complex in Peninsular Malaysia

Abstract Two sympatric populations of brown planthopper (BPH), one from rice and the other from Leersia hexandra were collected from each of five locations in Malaysia. All the tested malathion-resistant individuals of the rice BPH population and F₁ generation (cross between malathion-resistant [usually caught on rice] and malathion-susceptible [usually caught on Leersia]) showed high esterase activity, while all malathion-susceptible individuals on L. hexandra showed low esterase activity. In the F₂ generation, all the individuals tested against malathion were approximately 75% resistant and 25% susceptible and the inheritance pattern of esterase activity (high and low esterase activity) segregated in the same manner to a 3: 1 ratio. This confirms that resistance to malathion is mono-factorial and inheritance pattern of esterase activity is also linked to malathion resistance. Carboxylesterase or total esterase activity in BPH is inherited in a simple Mendelian fashion that is encoded by a single dominant gene. For the total esterase assay, average esterase activity levels in the rice-infesting population ranged from 17.64 to 19.37 nmoles 1-napthol/mg protein while that in the Leersia-infesting population ranged from 5.29 to 6.11 nmoles 1-napthol/mg protein. In terms of esterase activity, the two sympatric Nilaparvata lugens populations separated into two distinct groups. Results based on the tube color intensity test showed 96% and 98% resistant and susceptible individuals were present in the rice- and Leersia-infesting populations, respectively. In a filter paper test, the rice-infesting population had 94% with high esterase activity while the Leersia-infesting population had 96% with low esterase activity.     

Oligomerization of the human thyrotropin receptor: fluorescent protein-tagged hTSHR reveals post-translational complexes

To examine thyrotropin (TSH) receptor homophilic interactions we fused the human TSH receptor (hTSHR) carboxyl terminus to green fluorescent protein (GFP) and the corresponding chimeric cDNA was expressed in Chinese hamster ovary cells. Fluorescent TSH receptors on the plasma membrane were functional as assessed by TSH-induced cAMP synthesis. The binding of TSH, as well as TSHR autoantibodies, induced time- and dose-dependent receptor capping. Fluorescence resonance energy transfer between receptors differentially tagged with GFP variants (RFP and YFP) provided evidence for the close proximity of individual receptor molecules. This was consistent with previous studies demonstrating the presence of TSHR dimers and oligomers in thyroid tissue. Co-immunoprecipitation of GFP-tagged and Myc-tagged receptor complexes was performed using doubly transfected cells with Myc antibody. Western blotting of the immunoprecipitated complex revealed the absence of noncleaved TSH holoreceptors. This further suggested that cleavage of the holoreceptor into its two-subunit structure, comprising disulfide-linked TSHR-alpha and TSHR-beta subunits, was required for the formation of TSHR dimers and higher order complexes.     

In Silico Analysis of Acinetobacter baumannii Phospholipase D as a Subunit Vaccine Candidate

The rate of human health care-associated infections caused by Acinetobacter baumannii has increased significantly in recent years for its remarkable resistance to desiccation and most antibiotics. Phospholipases, capable of destroying a phospholipid substrate, are heterologous group of enzymes which are believed to be the bacterial virulence determinants. There is a need for in silico studies to identify potential vaccine candidates. A. baumannii phospholipase D (PLD) role has been proved in increasing organism’s resistance to human serum, destruction of host epithelial cell and pathogenesis in murine model. In this in silico study high potentials of A. baumannii PLD in elicitation of humoral and cellular immunities were elucidated. Thermal stability, long half-life, non-similarity to human and gut flora proteome and non-allergenicity were in a list of A. baumannii PLD positive properties. Potential epitopic sequences were also identified that could be used as peptide vaccines against A. baumannii and various other human bacterial pathogens.