Phospholipase A2 as a target protein for nonsteroidal anti-inflammatory drugs (NSAIDS): crystal structure of the complex formed between phospholipase A2 and oxyphenbutazone at 1.6 A resolution

Phospholipase A(2) (PLA(2); EC 3.1.1.4) is a key enzyme involved in the production of proinflammatory mediators known as eicosanoids. The binding of the substrate to PLA(2) occurs through a well-formed hydrophobic channel. To determine the viability of PLA(2) as a target molecule for the structure-based drug design against inflammation, arthritis, and rheumatism, the crystal structure of the complex of PLA(2) with a known anti-inflammatory compound oxyphenbutazone (OPB), which has been determined at 1.6 A resolution. The structure has been refined to an R factor of 0.209. The structure contains 1 molecule each of PLA(2) and OPB with 2 sulfate ions and 111 water molecules. The binding studies using surface plasmon resonance show that OPB binds to PLA(2) with a dissociation constant of 6.4 x 10(-8) M. The structure determination has revealed the presence of an OPB molecule at the binding site of PLA(2). It fits well in the binding region, thus displaying a high level of complementarity. The structure also indicates that OPB works as a competitive inhibitor. A large number of hydrophobic interactions between the enzyme and the OPB molecule have been observed. The hydrophobic interactions involving residues Tyr(52) and Lys(69) with OPB are particularly noteworthy. Other residues of the hydrophobic channel such as Leu(3), Phe(5), Met(8), Ile(9), and Ala(18) are also interacting extensively with the inhibitor. The crystal structure clearly reveals that the binding of OPB to PLA(2) is specific in nature and possibly suggests that the basis of its anti-inflammatory effects may be due to its binding to PLA(2) as well.     

Influence of Physicochemical Parameters on the Abundance of Coliform Bacteria in an Industrial Site of the Hooghly River, India

Industrial effluents from jute, paper, pulp mills and sewage from households are regularly discharged into the Hooghly River. It generates a potential risk for both humans and animals of the area concerned. In the present study, water quality of the Hooghly River passing by the site of a growing township (Naihati, North 24 Parganas, West Bengal, India) was assessed throughout the year 2010 on the basis of the data collected on the physicochemical and microbiological parameters. The water samples collected on each month revealed the presence of higher amount of coliform bacteria, Streptococcus sp. and Escherichia coli, than the standard limit. Different physicochemical parameters like chemical oxygen demand, biological oxygen demand, dissolved oxygen (DO), total suspended solids, total dissolved solids (TDS), total hardness, alkalinity, chlorinity, nitrate and nitrite of the water at the sampling sites were found to be considerably higher than the levels standardized by WHO (2006). It was found that the relative abundance of Streptococcus and E. coli was influenced by two independent variables (water quality parameters), namely, DO and TDS. The abundance of coliform bacteria in the water sample warrants the adoption of proper measures to reduce the pollution level at the point source on way of scientific disposal of industrial effluents.     

Nod factor-independent ‘crack-entry’ symbiosis in dalbergoid legume Arachis hypogaea

Dalbergoids are typified by crack-entry symbiosis which is evidenced to be Nod Factor (NF)-independent in several Aeschynomene legumes. Natural symbionts of the dalbergoid legume Arachis hypogaea are always NF-producing, prompting us to check whether symbiosis in this legume could also be NF-independent. For this, we followed the symbiosis with two NF-containing bradyrhizobial strains – SEMIA6144, a natural symbiont of Arachis and ORS285, a versatile nodulator of Aeschynomene legumes, along with their corresponding nodulation (nod) mutants. Additionally, we investigated NF-deficient bradyrhizobia like BTAi1, a natural symbiont of Aeschynomene indica and the WBOS strains that were natural endophytes of Oryza sativa, collected from an Arachis-Oryza intercropped field. While SEMIA6144ΔnodC was non-nodulating, both ORS285 and ORS285ΔnodB could induce functional nodulation, although with lower efficiency than SEMIA6144. On the other hand, all the NF-deficient strains – BTAi1, WBOS2 and WBOS4 showed comparable nodulation with ORS285 indicating Arachis to harbour an NF-independent mechanism of symbiosis. Intriguingly, symbiosis in Arachis, irrespective of whether it was NF-dependent or independent, was always associated with the curling or branching of the rosette root hairs at the lateral root bases. Thus, despite being predominantly described as an NF-dependent legume, Arachis does retain a vestigial, less-efficient form of NF-independent symbiosis.     

Meta analysis of bioactive compounds,miRNA, siRNA and cell death regulators as sensitizers to doxorubicin induced chemoresistance

Cancer has presented to be the most challenging disease, contributing to one in six mortalities worldwide. The current treatment regimen involves multiple rounds of chemotherapy administration, alone or in combination. The treatment has adverse effects including cardiomyopathy, hepatotoxicity, and nephrotoxicity. In addition, the development of resistance to chemo has been attributed to cancer relapse and low patient overall survivability. Multiple drug resistance development may be through numerous factors such as up-regulation of drug transporters, drug inactivation, alteration of drug targets and drug degradation. Doxorubicin is a widely used first line chemotherapeutic drug for a myriad of cancers. It has multiple intracellular targets, DNA intercalation, adduct formation, topoisomerase inhibition, iron chelation, reactive oxygen species generation and promotes immune mediated clearance of the tumor. Agents that can sensitize the resistant cancer cells to the chemotherapeutic drug are currently the focus to improve the clinical efficiency of cancer therapy. This review summarizes the recent 10-year research on the use of natural phytochemicals, inhibitors of apoptosis and autophagy, miRNAs, siRNAs and nanoformulations being investigated for doxorubicin chemosensitization.

     

Conformational stability and thermodynamic characterization of homotetrameric Plasmodium falciparum beta-ketoacyl-ACP reductase

The conformational stability of the homotetrameric Plasmodium falciparum beta-ketoacyl-ACP reductase (FabG) was determined by guanidinium chloride-induced isothermal and thermal denaturation. The reversible unfolding transitions were monitored by intrinsic fluorescence, circular dichroism (CD) spectroscopy and by measuring the enzyme activity of FabG. The denaturation profiles were analyzed to obtain the thermodynamic parameters associated with unfolding of the protein. The data confirm the simple A(4) <--> 4A model of unfolding, based on the corroboration of CD data by fluorescence transition and similar Delta G estimation for denaturation curves obtained at four different concentration of the FabG. Denaturation is well described by the linear extrapolation model for denaturant-protein interactions. In addition, the conformational stability (Delta G(s)) as well as the Delta C(p) for the protein unfolding is quite high, 22.68 kcal/mole and 5.83 kcal/(mole K), respectively, which may be a reflection of the relatively large size of the tetrameric molecule (Mr 120, 000) and a large buried hydrophobic core in the folded protein. This study provides a prototype for determining conformational stability of other members of the short-chain alcohol dehydrogenase/reductase superfamily of proteins to which PfFabG belongs.     

Unfolding energetics and stability of banana lectin

The unfolding pathway of banana lectin from Musa paradisiaca was determined by isothermal denaturation induced by the chaotrope GdnCl. The unfolding was found to be a reversible process. The data obtained by isothermal denaturation provided information on conformational stability of banana lectin. The high values of DeltaG of unfolding at various temperatures indicated the strength of intersubunit interactions. It was found that banana lectin is a very stable and denatures at high chaotrope concentrations only. The basis of the stability may be attributed to strong hydrogen bonds of the order 2.5-3.1 A at the dimeric interface along with the presence of water bridges. This is perhaps very unique example in proteins where subunit association is not a consequence of the predominance of hydrophobic interactions.     

Kinetic and Structural Studies on the Interactions of Heparin and Proteins of Human Seminal Plasma using Surface Plasmon Resonance

Heparin is naturally occurring polysaccharides which interacts with seminal plasma proteins and regulate multiple steps in fertilization process. Qualitative and quantitative information regarding the affinity for heparin-seminal plasma proteins interactions is not generally well documented and there are no reports of a comprehensive analysis of these interactions in human seminal plasma. Such information should improve our understanding of how GAGs especially heparin present in the reproductive tract regulate fertilization. In this study, we use SPR to study interactions of heparin with various seminal plasma heparin-binding proteins (HBPs). HBPs like lactoferrin (LF), fibronectin fragment (FNIII), semenogelinI (SGI) and prostate specific antigen (PSA) all bind heparin with different binding kinetics and affinities. Kinetic data suggests that FNIII binds heparin with a high affinity (KD=3.2 nM), while PSA binds heparin with a micromolar affinity (KD=11.1 μM). Preincubation of SGI with heparin inhibits the binding of SGI to immobilized PSA in a dosedependent manner, while FNIII incubated with heparin binds with an increased affinity to PSA. Solution-competition studies show that the minimum size of a heparin oligosaccharide capable of binding with PSA is greater than a tetrasaccharide, with LF and SGI is larger than a hexasaccharide and for FNIII is larger than an octasaccharide.     

Overview on current status of biotechnological interventions on yellow stem borer Scirpophaga incertulas (Lepidoptera: Crambidae) resistance in rice

Yellow stem borer (YSB), Scirpophaga incertulas (Lepidoptera: Crambidae), a monophagous pest of paddy is considered as most important pest of rain fed low land and flood prone rice eco-systems. Breeding of yellow stem borer resistance in rice is difficult owing to the complex genetics of the trait, inherent difficulties in screening and poor understanding of the genetics of resistance. On the other hand, a good level of resistance against the widespread yellow stem borer has been rare in the rice germplasm. Resistance to insects has been demonstrated in transgenic plants expressing genes for δ-endotoxins from Bacillus thuringiensis (Bt), protease inhibitors, enzymes and plant lectins. The performance of insect resistant GM rice in trials in China has been quite impressive. The present review is an attempt to assess the current state of development in biotechnological intervention for yellow stem borer resistance in rice.     

SD-8, a novel therapeutic agent active against multidrug-resistant Gram positive cocci

Anti-bacterial drug resistance is one of the most critical concerns among the scientist worldwide. The novel antimicrobial decapeptide SD-8 is designed and its minimal inhibitory concentration and therapeutic index (TI) was found in the range of 1–8 μg/ml and 45–360, respectively, against major group of Gram positive pathogens (GPP). The peptide was also found to be least hemolytic at a concentration of 180 μg/ml, i.e., nearly 77 times higher than its average effective concentration. The kinetics assay showed that the killing time is 120 min for methicillin-sensitive Staphylococcus aureus (MSSA) and 90 min for methicillin-resistant S. aureus (MRSA). Membrane permeabilization is the cause of peptide antimicrobial activity as shown by the transmission electron microscopy studies. The peptide showed the anti-inflammatory property by inhibiting COX-2 with a K _D and K _i values of 2.36 × 10⁻⁹ and 4.8 × 10⁻⁸ M, respectively. The peptide was also found to be effective in vivo as derived from histopathological observations in a Staphylococcal skin infection rat model with MRSA as causative organism.