ATM regulates cell fate choice upon p53 activation by modulating mitochondrial turnover and ROS levels

Despite extensive study, the mechanisms of cell fate choice upon p53 activation remain poorly understood. Using genome-wide shRNA screening, we recently identified the ATM kinase as synthetic lethal with Nutlin-3, an MDM2 inhibitor that leads to non-genotoxic p53 activation. Here, we demonstrate that while this synthetic lethal interaction relies upon components of both the intrinsic and extrinsic apoptotic pathways (e.g., BAX and BID), it is not due to significant ATM effects on the expression of p53 target genes. Instead, loss of ATM activity results in increased mitochondria and reactive oxygen species that drive apoptosis. Finally, we provide evidence that pharmacologic inhibition of ATM blocks autophagy in direct opposition to p53, which activates this process, and that inhibition of autophagy is sufficient to elicit an apoptotic response when combined with Nutlin-3.     

Controlled Porosity Solubility Modulated Osmotic Pump Tablets of Gliclazide

A system that can deliver drug at a controlled rate is very important for the treatment of various chronic diseases such as diabetes, asthma, and heart disease. Poorly water-soluble drug with pH-dependent solubility such as gliclazide (GLZ) offers challenges in the controlled-release formulation because of low dissolution rate and poor bioavailability. Solid dispersion (SD) of GLZ consisted of hydroxypropyl cellulose (HPC-SSL) as a polymeric solubilizer was manufactured by hot melt extrusion (HME) technology. Then, controlled porosity osmotic pump (CPOP) tablet of gliclazide was designed to deliver drug in a controlled manner up to 16 h. The developed formulation was optimized for type and level of pore former and coating weight gain. The optimized formulation was found to exhibit zero order kinetics independent of pH and agitation speed but depends on osmotic pressure of dissolution media indicated that mechanism of drug release was osmotic pressure. The in vivo performance prediction of developed formulation using convolution approach revealed that the developed formulation was superior to the existing marketed extended-release formulation in terms of attaining steady state plasma levels and indicated adequate exposure in translating hypoglycemic response. The prototype solubilization method combined with controlled porosity osmotic pump based technique could provide a unique way to increase dissolution rate and bioavailability of many poorly water-soluble, narrow therapeutic index drugs used in diabetes, cardiovascular diseases, etc.KEY WORDS: convolution approach, gliclazide, hot melt extrusion (HME), hydroxypropyl cellulose, solid dispersion     

Azadirachta indica and Ocimum sanctum leaf extracts alleviate arsenic toxicity by reducing arsenic uptake and improving antioxidant system in rice seedlings

In the present study the potentials of aqueous extracts of the two plants, neem (Azadirachta indica) and Tulsi (Ocimum sanctum) were examined in alleviating arsenic toxicity in rice (Oryza sativa L.) plants grown in hydroponics. Seedlings of rice grown for 8 days in nutrient solution containing 50 μM sodium arsenite showed decline in growth, reduced biomass, altered membrane permeability and increased production of superoxide anion (O2·−), H2O2 and hydroxyl radicals (·OH). Increased lipid peroxidation marked by elevated TBARS (thiobarbituric acid reactive substances) level, increased protein carbonylation, alterated levels of ascorbate, glutathione and increased activities of enzymes SOD (superoxide dismutase), CAT (catalase), APX (ascorbate peroxidase) and GPX (glutathione peroxidase) were noted in the seedlings on As treatment. Exogenously added leaf aqueous extracts of Azadirachta indica (0.75 mg mL−1, w/v) and Ocimum sanctum (0.87 mg mL−1, w/v) in the growth medium considerably alleviated As toxicity effects in the seedlings, marked by reduced As uptake, restoration of membrane integrity, reduced production of ROS, lowering oxidative damage and restoring the levels of ascorbate, glutathione and activity levels of antioxidative enzymes. Arsenic uptake in the seedlings declined by 72.5% in roots and 72.8% in shoots, when A. indica extract was present in the As treatment medium whereas with O. sanctum extract, the uptake declined by 67.2% in roots and 70.01% in shoots. Results suggest that both A. indica and O. sanctum aqueous extracts have potentials to alleviate arsenic toxicity in rice plants and that A. indica can serve as better As toxicity alleviator compared to O. sanctum.     

Alpha-synuclein overexpression and aggregation exacerbates impairment of mitochondrial functions by augmenting oxidative stress in human neuroblastoma cells

Overexpression of alpha-synuclein and oxidative stress has been implicated in the neuronal cell death in Parkinson's disease. Alpha-synuclein associates with mitochondria and excessive accumulation of alpha-synuclein causes impairment of mitochondrial functions. However, the mechanism of mitochondrial impairment caused by alpha-synuclein is not fully understood. We recently reported that alpha-synuclein associates with mitochondria and that overexpression of alpha-synuclein causes nitration of mitochondrial proteins and release of cytochrome c from the mitochondria [Parihar M.S., Parihar A., Fujita M., Hashimoto M., Ghafourifar P. Mitochondrial association of alpha-synuclein causes oxidative stress. Cell Mol Life Sci. 2008a;65:1272–1284]. The present study shows that overexpression of alpha-synuclein A53T or A30P mutants or wild-type in human neuroblastoma cells augmented aggregation of alpha-synuclein. Immunoblotting and immuno-gold electron transmission microscopy show localization of alpha-synuclein aggregates within the mitochondria of overexpressing cells. Overexpressing cells show increased mitochondrial reactive oxygen species, increased protein tyrosine nitration, decreased mitochondrial transmembrane potential, and hampered cellular respiration. These findings suggest an important role for mitochondria in cellular responses to alpha-synuclein.     

Characterization of plant-growth promoting diazotrophic bacteria isolated from field grown Chinese cabbage under different fertilization conditions

Diazotrophic bacteria isolated from the rhizosphere of Chinese cabbage were assessed for other plant growth promoting characteristics viz., production of IAA, ethylene, ACC deaminase, phosphate solubilization, and gnotobiotic root elongation. Their effect on inoculation to Chinese cabbage was also observed under growth chamber conditions. A total of 19 strains that showed higher nitrogenase activity identified by 16S rRNA gene sequence analysis were found to be the members of the genera Pseudomonas and Agrobacterium belonging to α- and γ-Proteobacteria groups. These strains were also efficient in producing IAA and ACC deaminase though they produced low levels of ethylene and no phosphate solubilization. In addition, inoculation of selected diazotrophic bacterial strains significantly increased seedling length, dry weight, and total nitrogen when compared to uninoculated control. The colonization of crop plants by diazotrophic bacteria can be affected by many biotic and abiotic factors, and further studies are oriented towards investigating the factors that could influence the establishment of a selected bacterial community.     

Development and Evaluation of Sustained Release Gastroretentive Minimatrices for Effective Treatment of H. pylori Infection

In the present work, sustained release gastroretentive minimatrices of amoxicillin have been designed and optimized using central composite design. Effect of amount of xanthan gum, rate controlling polymers (HPMC K100M CR/PEO coagulant (1:1)), carbopol 974P, and gas generating couple (sodium bicarbonate/citric acid (3:1)) was studied on dependent (response) variables, i.e., buoyancy lag time, drug release at 1 h, time required for 95% drug release, swelling index, and bioadhesive strength. Minimatrices were prepared by non aqueous granulation method using solution of PVP K30 in isopropyl alcohol. All the formulations were found to contain 99.2% to 100.9% of amoxicillin per minimatrix. Optimum formulation (Formulation number AGT09) containing high level of the independent variables was having buoyancy lag time of 7 min and drug release at 1 h was 32.5%. It required 9.39 h for 95% drug release while swelling index and bioadhesive strength were 341 and 17.9 dyn/cm², respectively. This formulation was said to be optimum because it has minimum buoyancy lag time, requires maximum time for 95% drug release, and has higher bioadhesive capabilities. In vitro results of an optimized formulation indicate its sustained drug release and gastric retention capability, which may be very useful for effective treatment of H. pylori infection.     

5-Nitrofuran-2-yl derivatives: Synthesis and inhibitory activities against growing and dormant mycobacterium species

Eighteen 5-nitrofuran-2-yl derivatives were prepared by reacting 5-nitro-2-furfural with various (sub)phenyl/pyridyl thiosemicarbazide using microwave irradiation. The compounds were tested for their in vitro activity against tubercular and various non-tubercular mycobacterium species in log-phase and 6-week-starved cultures. Compound N-(3,5-dibromopyridin-2-yl)-2-((5-nitrofuran-2-yl)methylene)hydrazinecarbothioamide (4r) was found to be the most potent compound (MIC: 0.22 μM) and was 3 times more active than standard isoniazid (INH) and equally active as rifampicin (RIF) in log-phase culture of Mycobacterium tuberculosis H37Rv. In starved M. tuberculosis H37Rv, 4r inhibited with MIC of 13.9 μM and was found to be 50 times more active than INH and slightly more active than RIF.