Effect of PVA on the gel temperature of MC and release kinetics of KT from MC based ophthalmic formulations

The effect of molecular weight of poly(vinyl alcohol) (PVA) and sodium chloride on the gelation temperature of methylcellulose (MC) was studied with the objective to develop a MC based formulation for sustained delivery of ketorolac tromethamine a model ophthalmic drug. Pure MC showed sol-gel transition at 61.2 °C. In order to reduce the gelation temperature of MC and to increase the drug release time, PVA was used. Different techniques such as test tube tilting method, UV-vis spectroscopy, viscometry and rheometry were used to measure gelation temperature of all the binary combinations of MC and PVA. It was observed that the gelation temperature of MC was reduced with the addition of 4% PVA and also the extent of reduction of the gelation temperature of MC was dependent on the molecular weight of PVA. The strong interactions between MC and PVA molecules were established using Fourier transform infrared spectroscopy. To study the in vitro drug release properties of the MC-PVA binary combinations, 6% sodium chloride was used to reduce the gelation temperature further up to physiological temperature. It was observed that the drug release time increased from 5 to 8h with the increase of molecular weight of PVA from 9×10(3) to 1.3×10(5) and this was due to the higher viscosity, better gel strength and greater interactions between the drug and PVA molecules in case of PVA (1.3×10(5)) compared to PVA (9×10(3)). In order to have an idea about the nature of interactions between the functional moieties of the drug and the polymer unit of PVA, a theoretical study was carried out.     

In vitro protein folding by E. coli ribosome: unfolded protein splitting 70S to interact with 50S subunit

Folding of unfolded protein on Escherichia coli 70S ribosome is accompanied by rapid dissociation of the ribosome into 50S and 30S subunits. The dissociation rate of 70S ribosome with unfolded protein is much faster than that caused by combined effect of translation and polypeptide release factors known to be involved in the dissociation of ribosome into subunits. The protein then reaches a “folding competent” state on 50S and is released to take up native conformation by itself. Release before attaining the folding competent state or prevention of release by cross-linking it with ribosome, would not allow the protein to get back to its native conformation.     

Genetic interaction ofDrosophila homologue ofabelson (abl) proto-oncogene (D-abl) andlethal(2)giant larvae (lgl) tumour suppressor gene during embryonic development

TheDrosophila homologue (D-abl) of the mammalianabelson proto-oncogene (c-abl) encodes a cytoplasmic protein tyrosine kinase which localizes to axons of the developing embryonic central nervous system (CNS) and has been shown to be required for redundant functions during axonogenesis. These redundant functions become indispensable when other components of the redundant pathway, such as those encoded bydisabled (dab), fasciclin I (fas I) orfailed axon connection (fax) are removed fromabl mutants. Second-site mutations can thus uncover redundant aspects ofabl-mediated axonogenesis. We used this strategy, and present evidence to suggest a redundant function of the cytoskeletal protein encoded by thelethal(2)giant larvae (lgl) tumour suppressor gene during embryonic axonogenesis. Simultaneous mutation inlgl andabl shifts lethality of the mutations to late embryogenesis while mutation in only one of these genes permits development up to late larval/pupal or pharate adult stages. Thelgl ^-;abl ^- embryos show defective development of the CNS, characterized by loss of axonal commissures and longitudinal axonal tracts. Lethality of the double mutation is aggravated or suppressed bydisabled (dab) orenabled (ena) mutations, which act, respectively, as dominant enhancers or suppressors ofabl. The redundant function oflgl tumour suppressor gene during axonogenesis therefore appears to involve aspects of D-abl-mediated signalling.     

Nucleotide-induced conformational change in the catalytic subunit of the phosphate-specific transporter from M. tuberculosis: implications for the ATPase structure

The nucleotide binding subunit of the phosphate-specific transporter (PstB) from Mycobacterium tuberculosis is a member of the ABC family of permeases, which provides energy for transport through ATP hydrolysis. We utilized the intrinsic fluorescence of the single tryptophan containing protein to study the structural and conformational changes that occur upon nucleotide binding. ATP binding appeared to lead to a conformation in which the tryptophan residue had a higher degree of solvent exposure and fluorescence quenching. Substantial alteration in the proteolysis profile of PstB owing to nucleotide binding was used to decipher conformational change in the protein. In limited proteolysis experiments, we found that ATP or its nonhydrolyzable analog provided significant protection of the native protein, indicating that the effect of nucleotide on PstB conformation is directly associated with nucleotide binding. Taken together, these results indicate that nucleotide binding to PstB is accompanied by a global conformational change of the protein, which involves the helical domain from Arg137 to Trp150. Results reported here provide evidence that the putative movement of the alpha-helical sub-domain relative to the core sub-domain, until now only inferred from X-ray structures and modeling, is indeed a physiological phenomenon and is nucleotide dependent.     

Expression of Mammalian Antiviral Enzymes from the 2–5A System in Transgenic Plants

The 2–5A system is an interferon-regulated antiviral RNA decay pathway present in cells of higher vertebrates. Two enzymes are essential, a 2–5A synthetase which produces 5′-phosphorylated, 2′,5′-linked oligoadenylates (2–5A) in response to doublestranded RNA, and the 2–5A-dependent RNase L. To determine if these human proteins would be functional in plants, we expressed the human cDNAs for a 2–5A synthetase and RNase L in separate tobacco plants. Both proteins were enzymatically active in extracts of transgenic plants while such activities were not detected in the control plants. Furthermore, activation by 2–5A of RNase L in the transgenic plant leaves was shown to cause degradation of ribosomal RNA. The requirement for both the synthetase and RNase L for antiviral activity was underscored by the observations that expression of human RNase L alone or 2–5A-synthetase alone was insufficient to protect plants against either tobacco etch virus or tobacco mosaic virus.     

Chaotropic effects on 2,4,6-trinitrotoluene uptake by wheat (Triticum aestivum)

Previous research in our laboratory investigated the effectiveness of a common agrochemical, urea used as a chaotropic agent to facilitate 2,4,6-trinitrotoluene (TNT) removal by vetiver grass (Vetiveria zizanioides L.). Chaotropic agents disrupt water structure, increasing solubilization of hydrophobic compounds (TNT), and enhancing plant TNT uptake. Our findings showed that urea significantly enhanced TNT uptake kinetics by vetiver. We hypothesized that the beneficial effect of urea on the overall TNT uptake by vetiver grass was not plant-specific. We explored this hypothesis by testing the ability of wheat (Triticum aestivum L.) in removing TNT from aqueous media in the presence of urea. Results showed that untreated (no urea) wheat exhibited a slow, kinetically limited TNT uptake that was nearly half of the urea-treated wheat TNT capacity (250 mg kg⁻¹). Chaotropic effects of urea were illustrated by the significant (P < 0.001) increase in the TNT second-order reaction rate constants over those of the untreated (no urea) controls. Plant TNT speciation showed that TNT and several of its metabolites were detected in both root and shoot compartments of the plant, allowing for 110 and 36% recovery for the untreated and 0.1% urea treated plants. The lower % recovery of the urea-treated plants was attributed to a number of unknown polar TNT metabolites. Responsible Editor: Hans Lambers.     

Force-Induced Dynamical Properties of Multiple Cytoskeletal Filaments Are Distinct from that of Single Filaments

How cytoskeletal filaments collectively undergo growth and shrinkage is an intriguing question. Collective properties of multiple bio-filaments (actin or microtubules) undergoing hydrolysis have not been studied extensively earlier within simple theoretical frameworks. In this paper, we study the collective dynamical properties of multiple filaments under force, and demonstrate the distinct properties of a multi-filament system in comparison to a single filament. Comparing stochastic simulation results with recent experimental data, we show that multi-filament collective catastrophes are slower than catastrophes of single filaments. Our study also shows further distinctions as follows: (i) force-dependence of the cap-size distribution of multiple filaments are quantitatively different from that of single filaments, (ii) the diffusion constant associated with the system length fluctuations is distinct for multiple filaments, and (iii) switching dynamics of multiple filaments between capped and uncapped states and the fluctuations therein are also distinct. We build a unified picture by establishing interconnections among all these collective phenomena. Additionally, we show that the collapse times during catastrophes can be sharp indicators of collective stall forces exceeding the additive contributions of single filaments.