Molecular cloning and characterization of pentachlorophenol-degrading monooxygenase genes of Pseudomonas sp. from the chemostat.

Pseudomonas sp. strain IST 103 (PCP103) capable of utilizing pentachlorophenol (PCP) was determined by utilization of a carbon source and release of the hydroxylating enzyme PCP-4 monooxygenase. The metabolites were extracted from the culture medium and analyzed by high-performance liquid chromatography. The enzyme purified to apparent homogeneity from an extract of PCP-grown cells indicated that a fraction of DEAE-cellulose ion exchange chromatography of molecular size of 30,000 kDa determined by gel filtration chromatography and SDS-polyacrylamide gel electrophoresis was responsible for dechlorination of PCP. The plasmid isolated from the bacterium was subjected to Shotgun cloning by restriction digestion by BamHI, HindIII, and SalI, ligated to pUC19 vector, and transformed into Escherichia coli XLBlue1alpha. The recombinant clones having higher potentiality to degrade PCP were selected by utilization of a carbon source and release of intermediary metabolites during degradation of PCP as the sole source of carbon and energy. The recombinant clones, which contained an insert of 3.0 kb of SalI and HindIII sites, were sequenced and compared with gene sequences deposited in GenBank by BLAST search; this indicated homology with the thdf gene of monooxygenase of thiophene and furan. Southern blot analysis performed by developing gene probes indicated the presence of the PCP monooxygenase gene in plasmids of the bacterium.     

Antiviral potential of 4-hydroxypanduratin A,secondary metabolite of Fingerroot,Boesenbergia pandurata (Schult.), towards Japanese Encephalitis virus NS2B/NS3 protease

4-hydroxypanduratin A is a secondary metabolite of Boesenbergia pandurata Schult. (Fingerroot) plant with various pharmacological activities such as neuroprotective, potent antioxidant, antibacterial and antifungal. Flaviviral NS2B/NS3 protease activity is essential for polyprotein processing and viral replication for Japanese Encephalitis Virus (JEV), a major cause of Acute Encephaltis in Asia. Inhibition of formation of this complex by arresting the binding of NS2B with NS3 would reduce the enzyme''s activity to meager proportions and hence would prevent further viral proliferation. The automated 3D structure of NS2B protein of the JEV GP78 was predicted based on the sequence-to-structure-to-function paradigm using I-TASSER and the function of NS2B protein was inferred by matching to other known proteins. The stereochemical quality of predicted structure was checked by PROCHECK. The antiviral activity of 4-hydroxypanduratin A against NS2B protein as a potential drug has been elucidated in this paper. Docking simulation analysis showed 4-hydroxypanduratin A as potential inhibitor of NS2B protein/cofactor which is necessary for NS3 protease activity. 220 derivatives of 4-hydroxypanduratin A were virtually screened with rigid criteria of Lipinski''s rule of 5 using Autodock4.2. 4-hydroxypanduratin A was found interacting with target hydrophilic domain in NS2B protein by two Hbonds (Gly80 and Asp81) with active residues, several hydrophobic interactions, Log P value of 5.6, inhibition constant (Ki) of 51.07nM and lowest binding energy of -9.95Kcal/Mol. Hence, 4-hydroxypanduratin A targeted to Site 2 will have sufficient profound effect to inhibit protease activity to abrogate viral replication. It could be a promising potential drug candidate for JEV infections using NS2B Site 2 as a Drug target.     

Interaction pattern for the complex of B-DNA Fullerene compounds with a set of known replication proteins using docking study

Fullerenes have attracted considerable attention due to their unique chemical structure and potential applications which has opened wide venues for possible human exposure to various fullerene types. Therefore, in depth knowledge of how fullerene may interfere with various cellular processes becomes quite imperative. The present study was designed to investigate how the presence of fullerene affect the binding of DNA with different enzymes involved in replication process. Different fullerenes were first docked with DNA and then binding scores of different enzymes was analyzed with fullerene docked DNA. C30, C40 & C50 once docked with DNA, reduced the binding score of primase, whereas no significant change in the binding score was observed with the helicase, ssb protein, dna pol δ, dna pol ε, ligase, DNA clamp, and topoisomerases. On the contrast, the binding score of RPA14 decreases in fluctuating manner while interacting with increasing molecular weight of fullerene bound single-stranded DNA complex. The study revealed the affect of fullerene family interacting with DNA on the binding pattern of enzymes involved in replication process. Study suggests that the presence of most of fullerenes may not affect the activity of these enzymes necessary for replication process whereas C30, C40 & C50 may disrupt the activity of primase, (strating point for DNA polymerase) its docking score decreases from 13820 to 10702.     

Echinicola shivajiensis sp. nov., a novel bacterium of the family “Cyclobacteriaceae” isolated from brackish water pond

Strain AK12^T, an orange pigmented Gram-negative, rod shaped, non-motile bacterium, was isolated from a mud sample collected from a brackish water pond at Rampur of West Bengal, India. The strain was positive for oxidase, catalase and phosphatase. The predominant fatty acids were iso-C_15:0 (42.7%), iso-C_17:0 3OH (13.2%), C_16:1ω7c/C_16:1ω6c (summed feature 3) (8.0%), iso-C_17:1 I/anteiso-C_17:1 B (summed feature 4) (6.1%) and iso-C_17:1ω9c/C_16:0 10-methyl (summed feature 9) (9.4%). Strain AK12^T contained MK-7 as the major respiratory quinone and phosphatidylethanolamine, one unidentified aminophospholipid and six unidentified lipids as the polar lipids. The G + C content of DNA of the strain AK12^T was 46.2 mol%. The 16S rRNA gene sequence analysis indicated that strain AK12^T was member of the genus Echinicola and closely related to Echinicola vietnamensis, Echinicola pacifica and Echinicola jeungdonensis with pair-wise sequence similarity of 96.8, 96.3 and 96.0% respectively. Phylogenetic analyses indicated that the strain AK12^T clustered with E. vietnamensis and together with E. pacifica and E. jeungdonensis with a phylogenetic distance of 5.1, 6.3 and 6.6% (94.9, 93.7 and 93.4% similarity) respectively. Based on data from the current polyphasic study, strain AK12^T is proposed as a novel species of the genus Echinicola, for which the name Echinicola shivajiensis sp. nov. is proposed. The type strain of E. shivajiensis is AK12^T (= MTCC 11083^T = JCM 17847^T).     

IL-15 activated human peripheral blood dendritic cell kill allogeneic and xenogeneic endothelial cells via apoptosis

IL-15 is a pleotropic cytokine, which plays an important role in natural killer (NK) cell activity, T cell proliferation, and T cell cytotoxic activity. Dendritic cells (DCs) are the major antigen presenting cells in the immune system and presumed to play an important role in immune recognition of allo and xenotransplantation. We showed that IL-15 activated human peripheral blood DC is cytotoxic to human and porcine aortic endothelial cells. Unlike DCs, CD14+ monocytes show no cytotoxicity against the endothelial cells. This cytotoxic potential of IL-15 activated DC against endothelial cells is dose dependent and increases significantly upon treatment of endothelial cells with inflammatory cytokines like TNF-α or IFN-γ. The cytotoxic potential of IL-15 activated DC is associated with apoptosis of endothelial cells, as indicated by the increased Annexin V staining, caspase activation and loss of mitochondrial membrane potential. Further it was observed that DC mediated cytotoxicity against endothelial cell is mediated via granzyme B possibly secreted by the activated DCs.     

Antioxidant activity of allicin,an active principle in garlic

Garlic has been claimed to be effective against diseases, in the pathophysiology of which oxygen free radicals (OFRs) have been implicated. Effectiveness of garlic could be due to its ability to scavenge OFRs. However, its antioxidant activity is not known. We investigated the ability of allicin (active ingredient of garlic) contained in the commercial preparation Garlicin to scavenge hydroxyl radicals (·OH) using high pressure liquid chromatographic (HPLC) method. ·OH was generated by photolysis of H₂O₂ (1.25–10 moles/ml) with ultraviolet light and was trapped with salicylic acid which is hydroxylated to produce ·OH adduct products 2,3- and 2,5-dihydroxybenzoic acid (DHBA). H₂O₂ produced a concentration-dependent ·OH as estimated by ·OH adduct products 2,3-DHBA and 2,5-DHBA. Allicin equivalent in Garlicin (1.8, 3.6, 7.2, 14.4, 21.6, 28.8 and 36 g) produced concentration-dependent decreases in the formation of 2,3-DHBA and 2,5-DHBA. The inhibition of formation of 2,3-DHBA and 2,5-DHBA with 1.8 g/ml was 32.36% and 43.2% respectively while with 36.0 g/ml the inhibition was approximately 94.0% and 90.0% respectively. The decrease in ·OH adduct products was due to scavenging of ·OH and not by scavenging of formed ·OH adduct products. Allicin prevented the lipid peroxidation of liver homogenate in a concentration-dependent manner. These results suggest that allicin scavenges ·OH and Garlicin has antioxidant activity.     

Oxidative stress as a mechanism of diabetes in diabetic BB prone rats: Effect of secoisolariciresinol diglucoside (SDG)

Secoisolariciresinol diglucoside (SDG) isolated from flaxseed has antioxidant activity and has been shown to prevent hypercholesterolemic atherosclerosis. An investigation was made of the effects of SDG on the development of diabetes in diabetic prone BioBreeding rats (BBdp rats), a model of human type I diabetes [insulin dependent diabetes mellitus (IDDM)] to determine if this type of diabetes is due to oxidative stress and if SDG can prevent the incidence of diabetes. The rats were divided into three groups: Group I, BioBreeding normal rats (BBn rats) (n = 10); group II, BBdp untreated (n = 11); and group III, BBdp treated with SDG 22 mg/kg body wt, orally) (n = 14). Oxidative stress was determined by measuring lipid peroxidation product malondialdehyde (MDA) an index of level of reactive oxygen species in blood and pancreas; and pancreatic chemiluminescence (Pancreatic-CL), a measure of antioxidant reserve. Incidence of diabetes was 72.7% in untreated and 21.4% in SDG-treated group as determined by glycosuria and hyperglycemia. SDG prevented the development of diabetes by approximately 71%. Development of diabetes was associated with an increase in serum and pancreatic MDA and a decrease in antioxidant reserve. Prevention in development of diabetes by SDG was associated with a decrease in serum and pancreatic-MDA and an increase in antioxidant reserve. These results suggest that IDDM is mediated through oxidative stress and that SDG prevents the development of diabetes.     

Plant diversity of the Kangchenjunga Landscape,Eastern Himalayas

The Kangchenjunga Landscape (KL) in the Eastern Himalayas is a transboundary complex shared by Bhutan, India, and Nepal. It forms a part of the ‘Himalayan Biodiversity Hotspot’ and is one of the biologically richest landscapes in the Eastern Himalayas. In this paper, we use secondary information to review and consolidate the knowledge on the flora of the KL. We reviewed 215 journal articles, analysed the history of publications on the flora of the KL, their publication pattern in terms of temporal and spatial distribution and key research areas. Our review shows that the landscape has a long history of botanical research that dates back to the 1840s and progressed remarkably after the 1980s. Most of the studies have been carried out in India, followed by Nepal and Bhutan. The majority of these have been vegetation surveys, followed by research on ethnobotanical aspects and Non-Timber Forest Products (NTFPs). This paper describes the forest types and characteristic species of the KL and details the species richness, diversity and dominant families of seed plants. A total of 5198 species of seed plants belonging to 1548 genera and 216 families have been recorded from the landscape, including 3860 dicots, 1315 monocots and 23 gymnosperms. Among families, Orchidaceae is the most diversely represented family in terms of species richness. This paper also draws attention to the threatened and endemic flora of the KL, including 44 species that are threatened at national and global level and 182 species that are endemic. Finally, the paper reviews the major challenges facing the KL, the conservation efforts and practices that are currently in place and recommends systematic and comprehensive floral surveys, particularly long-term data collection and monitoring and transboundary collaboration, to address the existing knowledge gaps on floral diversity of the KL.