Kinetic and expression profiling of cytosolic pyruvate kinase enzyme during seed development of Indian mustard (Brassica juncea)

In the recent years tremendous progress has been achieved in deconstructing the oil biosynthetic pathways, majority of which is in Arabidopsis. Glycolysis is fundamental to this process as it is the cardinal supplier of precursors for fatty acid metabolism. Recent reports suggest that modification and expression of pyruvate kinase (PK), a crucial regulatory enzyme involved in glycolysis is one of the plausible ways to alter seed oil content in plants. In the present study we evaluated the kinetic behavior and expression profiling of pyruvate kinase, associated with seed development in a major oilseed crop B. juncea. Developmental profiling of the enzyme showed that enzyme activity was highest during middle stage (35 DAF) of seed development which is strongly corroborated by the expression profiling of the enzyme using RT-PCR approach. Oil accumulation pattern also correlated with the enzyme expression study. Comparative activity profiling from different tissues showed seedlings to have elevated activity than other tissues. For kinetic characterization, the enzyme was partially purified by 12.3 fold using DEAE-Sephadex column and showed a narrow pH optimum of 7.0. In presence of saturated substrate concentration, the enzyme exhibited hyperbolic kinetics for both ADP and PEP with Km (Michaelis constant value) for PEP and ADP was found to be 178.5 and 96.45 μM respectively. ATP and citrate are the most significant allosteric effectors of the partially purified PK. Study on isozymes of PK resulted in a single band.     

Green tea extract affects porcine ovarian cell apoptosis

Green tea is a commonly used beverage and green tea extract is a common dietary herbal supplement manufactured into different over-the-counter products. The aim of this in vitro study was to examine the steroid hormone secretion (progesterone and 17-β estradiol), proliferation and apoptosis of porcine ovarian granulosa cells after addition of green tea extract. Granulosa cells were incubated with green tea extract at five doses (0.1, 1, 10, 100 and 200?μg/ml) and the release of hormones by granulosa cells was assessed by EIA after 24?h exposure. The presence of proliferation and apoptotic markers was assessed by immunocytochemistry. Secretion of steroid hormones was not affected by green tea extract at all the doses in comparison to control. Also, markers of proliferation (PCNA and cyclin B1) were not affected by green tea extract. However, the highest dose (200?μg/ml) of green tea extract used in this study increased the accumulation of apoptotic markers caspase-3 and p53 in granulosa cells. In conclusion, our results indicate the impact of green tea extract at the highest dose used in this study on ovarian apoptosis through pathway that includes activation of caspase-3 and p53. Potential stimulation of these intracellular regulators could induce the process of apoptosis in ovarian cells.     

A strategy for theoretical binding constant,Ki, calculations for neuraminidase aromatic inhibitors designed on the basis of the active site structure of influenza virus neuraminidase

Neuraminidase (NA) is one of the two major surface antigens of influenza virus. It plays an indispensable role in the release and spread of progeny virus particles during infection. NA inhibitors reduce virus infection in animals. To improve the clinical efficacy of NA inhibitors, we have begun the design of non-carbohydrate inhibitors based on the active site structure of NA. The approach is an iterative process of ligand modeling and electrostatic calculations followed by chemical synthesis of compounds, biological testing, and NA-inhibitor complex structure determination by X-ray crystallography. A strategy has been developed to calculate K_i for newly designed inhibitors. The calculations using the DelPhi program were performed for carbohydrate inhibitors and three preliminary benzoic acid inhibitors of neuraminidase (BANA) that have been synthesized and shown to bind to the active site of NA in the crystal structure. The calculated K_is of these inhibitors have an enlightening agreement with their in vitro biological activities. This demonstrates that the calculations produce informative results on the affinity of modeled inhibitors. GRID maps were also calculated and several pockets were identified for accepting possible new ligands. The calculated K_is for newly designed ligands suggest that these potential compounds will have high inhibitory activities. © 1995 Wiley-Liss, Inc.     

Biological clocks help reduce the physiological conflicts in avian migrants

We present evidence from experiments on overwintering populations of two Palearctic-Indian latitudinal migratory birds, the black-headed bunting (Emberiza melanocephala) and the red-headed bunting (E. bruniceps), that the bird’s clock in interaction with day length regulates seasonal rhythms of migration and reproduction such that physiological conflict between them is reduced. Initiation and termination of the body mass and testicular cycles are separately regulated photoperiodic events. For example, under stimulatory photoperiods the response curve of body mass does not overlap with that of the testicular growth. A response-specific photoperiodism is adaptive, since gain in body mass, critical to spring migration, precedes gonadal recrudescence. Finally, migration as indicated by the night-time migratory restlessness under experimental situations (e.g., intense locomotion under caged condition-called Zugunruhe) appears to be regulated by a separate circadian oscillator.     

Holistic process development to mitigate proteolysis of a subunit rotavirus vaccine candidate produced in Pichia pastoris by means of an acid pH pulse during fed-batch fermentation

To meet the challenges of global health, vaccine design and development must be reconsidered to achieve cost of goods as low as 15¢ per dose. A new recombinant protein-based rotavirus vaccine candidate derived from non-replicative viral subunits fused to a P2 tetanus toxoid CD4(+) T cell epitope is currently under clinical development. We have sought to simplify the existing manufacturing process to meet these aims. To this end, we have taken a holistic process development approach to reduce process complexity and costs while producing a product with the required characteristics. We have changed expression system from Escherichia coli to Pichia pastoris, to produce a secreted product, thereby reducing the number of purification steps. However, the presence of proteases poses challenges to product quality. To understand the effect of fermentation parameters on product quality small-scale fermentations were carried out. Media pH and fermentation duration had the greatest impact on the proportion of full-length product. A novel acidic pH pulse strategy was used to minimize proteolysis, and this combined with an early harvest time significantly increased the proportion of full-length material (60–75%). An improved downstream process using a combination of CIEX and AIEX to further reduce proteases, resulted in maintaining product quality (95% yield).