Small Molecule Inhibitors of Phospholipase C from a Novel High-throughput Screen

Phospholipase C (PLC) isozymes are important signaling molecules, but few small molecule modulators are available to pharmacologically regulate their function. With the goal of developing a general approach for identification of novel PLC inhibitors, we developed a high-throughput assay based on the fluorogenic substrate reporter WH-15. The assay is highly sensitive and reproducible: screening a chemical library of 6280 compounds identified three novel PLC inhibitors that exhibited potent activities in two separate assay formats with purified PLC isozymes in vitro. Two of the three inhibitors also inhibited G protein-coupled receptor-stimulated PLC activity in intact cell systems. These results demonstrate the power of the high-throughput assay for screening large collections of small molecules to identify novel PLC modulators. Potent and selective modulators of PLCs will ultimately be useful for dissecting the roles of PLCs in cellular processes, as well as provide lead compounds for the development of drugs to treat diseases arising from aberrant phospholipase activity.     

Molecular Mechanism by Which a Potent Hepatitis C Virus NS3-NS4A Protease Inhibitor Overcomes Emergence of Resistance

Although optimizing the resistance profile of an inhibitor can be challenging, it is potentially important for improving the long term effectiveness of antiviral therapy. This work describes our rational approach toward the identification of a macrocyclic acylsulfonamide that is a potent inhibitor of the NS3-NS4A proteases of all hepatitis C virus genotypes and of a panel of genotype 1-resistant variants. The enhanced potency of this compound versus variants D168V and R155K facilitated x-ray determination of the inhibitor-variant complexes. In turn, these structural studies revealed a complex molecular basis of resistance and rationalized how such compounds are able to circumvent these mechanisms.     

Fatty acid-induced angiogenesis in first trimester placental trophoblast cells: Possible roles of cellular fatty acid-binding proteins

Angiogenesis is involved in the growth of new blood vessels from the existing one. Consequently, angiogenesis plays an indispensable role in tissue growth and repair including early placentation processes. Besides angiogenic growth factors (vascular endothelial growth factor (VEGF), angiopoietin-like 4 (ANGPTL4), placental growth factor (PlGF), platelet derived growth factor (PDGF), fibroblast growth factors (FGF)), dietary fatty acids (c>16) also directly or indirectly modulate angiogenic processes in tumors and other cell systems. Usually n − 3 fatty acids inhibit whereas n − 6 fatty acids stimulate angiogenesis in tumors and other cells. Contrary to this, docosahexaenoic acid, 22:6n − 3 (DHA) and other fatty acids including conjugated linoleic acid stimulate angiogenesis in placental first trimester cells. In addition to the stimulation of expression of major angiogenic factors such as VEGF and ANGPTL4, fatty acids also stimulate expression of intracellular fatty acid-binding proteins (FABPs) FABP-4 and FABP-3 those are known to directly modulate angiogenesis. Emerging data indicate that FABPs may be involved in the angiogenesis process. This paper reviews the fatty acid mediated angiogenesis process and the involvement of their binding proteins in these processes.     

Role of Microorganisms in Emission of Nitrous Oxide and Methane in Pulse Cultivated Soil Under Laboratory Incubation Condition

Soil from a pulse cultivated farmers land of Odisha, India, have been subjected to incubation studies for 40 consecutive days, to establish the impact of various nitrogenous fertilizers and water filled pore space (WFPS) on green house gas emission (N₂O & CH₄). C₂H₂ inhibition technique was followed to have a comprehensive understanding about the individual contribution of nitrifiers and denitrifiers towards the emission of N₂O. Nevertheless, low concentration of C₂H₂ (5 ml: flow rate 0.1 kg/cm²) is hypothesized to partially impede the metabolic pathways of denitrifying bacterial population, thus reducing the overall N₂O emission rate. Different soil parameters of the experimental soil such as moisture, total organic carbon, ammonium content and nitrate–nitrogen contents were measured at regular intervals. Application of external N-sources under different WFPS conditions revealed the diverse role played by the indigenous soil microorganism towards green house gas emission. Isolation of heterotrophic microorganisms (Pseudomonas) from the soil samples, further supported the fact that denitrification might be prevailing during specific conditions thus contributing to N₂O emission. Statistical analysis showed that WFPS was the most influential parameter affecting N₂O formation in soil in absence of an inhibitor like C₂H₂.     

Vom Phänotyp zum Genotyp

Forward Genomics – a comparative genomics approach to link phenotype to genotype Despite availability of several sequenced genomes, we know very little about the specific changes in the DNA that underlie phenotypic differences between species. The main reason is that species differ by both numerous genomic and phenotypic changes. A new comparative genomics method addresses this question by for phenotypes with independent evolutionary losses by searching for genomic regions that exhibit an elevated number of mutations in exactly these phenotype‐loss species. The near future sequencing of thousands of novel genomes will make it possible to use comparative genomics to systematically search for such DNA changes that are associated with phenotypic differences.     

Nontuberculous mycobacteria in respiratory samples from patients with pulmonary tuberculosis in the state of Rond?nia, Brazil

The main cause of pulmonary tuberculosis (TB) is infection with Mycobacterium tuberculosis (MTB). We aimed to evaluate the contribution of nontuberculous mycobacteria (NTM) to pulmonary disease in patients from the state of Rondônia using respiratory samples and epidemiological data from TB cases. Mycobacterium isolates were identified using a combination of conventional tests, polymerase chain reaction-based restriction enzyme analysis of hsp65 gene and hsp65 gene sequencing. Among the 1,812 cases suspected of having pulmonary TB, 444 yielded bacterial cultures, including 369 cases positive for MTB and 75 cases positive for NTM. Within the latter group, 14 species were identified as Mycobacterium abscessus, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium intracellulare, Mycobacterium gilvum, Mycobacterium gordonae, Mycobacterium asiaticum, Mycobacterium tusciae, Mycobacterium porcinum, Mycobacterium novocastrense, Mycobacterium simiae, Mycobacterium szulgai, Mycobacterium phlei and Mycobacterium holsaticum and 13 isolates could not be identified at the species level. The majority of NTM cases were observed in Porto Velho and the relative frequency of NTM compared with MTB was highest in Ji-Paraná. In approximately half of the TB subjects with NTM, a second sample containing NTM was obtained, confirming this as the disease-causing agent. The most frequently observed NTM species were M. abscessus and M. avium and because the former species is resistant to many antibiotics and displays unsatisfactory cure rates, the implementation of rapid identification of mycobacterium species is of considerable importance.