Functional assignment to JEV proteins using SVM

Identification of different protein functions facilitates a mechanistic understanding of Japanese encephalitis virus (JEV) infection and opens novel means for drug development. Support vector machines (SVM), useful for predicting the functional class of distantly related proteins, is employed to ascribe a possible functional class to Japanese encephalitis virus protein. Our study from SVMProt and available JE virus sequences suggests that structural and nonstructural proteins of JEV genome possibly belong to diverse protein functions, are expected to occur in the life cycle of JE virus. Protein functions common to both structural and non-structural proteins are iron-binding, metal-binding, lipid-binding, copper-binding, transmembrane, outer membrane, channels/Pores - Pore-forming toxins (proteins and peptides) group of proteins. Non-structural proteins perform functions like actin binding, zinc-binding, calcium-binding, hydrolases, Carbon-Oxygen Lyases, P-type ATPase, proteins belonging to major facilitator family (MFS), secreting main terminal branch (MTB) family, phosphotransfer-driven group translocators and ATP-binding cassette (ABC) family group of proteins. Whereas structural proteins besides belonging to same structural group of proteins (capsid, structural, envelope), they also perform functions like nuclear receptor, antibiotic resistance, RNA-binding, DNA-binding, magnesium-binding, isomerase (intra-molecular), oxidoreductase and participate in type II (general) secretory pathway (IISP).     

Majvinine: A new indole alkaloid of Vinca major

A new alkaloid, majvinine, was isolated from aerial parts of Vinca major. Its structure was determined by chemical and spectroscopic investigations     

Epidemiology and prediction of brown leaf rust of mulberry caused by Peridiopsora mori

Brown leaf rust (BLR) caused by Peridiopsora mori is one of the major foliar diseases of mulberry (Morus sp.) in the subtropical hills of eastern India. The disease appeared in first week of August and continued up to September with maximum severity in second and third week of September. The disease symptoms appeared at atmospheric temperature (27.00–20.07°C), relative humidity (92.14–82.43%), rainfall (11.20 cm) and rainy days (7) of the preceding week. Disease severity (>50 PDI) was observed at temperature (26.29–19.29°C), relative humidity (94.14–80.14%), rainfall (4.12 cm) and number of rainy days (2–3 days). Apparent rate of infection was found high at temperature (27.00–19.83°C), relative humidity (94.67–85.00%), rainfall (4.6 cm) and rainy days (2) of the preceding week. The correlation coefficient between disease severity and average meteorological factors of the preceding 7 days revealed that BLR disease severity showed significant negative correlation with minimum temperature. It was also revealed that contribution of maximum and minimum temperature 42.23% and 35.21%, maximum and minimum relative humidity (RH) 11.23% and 10.69% and rainfall and number of rainy days 0.11% and 0.50%, respectively towards development of BLR disease severity. Multiple regression analysis revealed that average of maximum and minimum temperatures and minimum RH of preceding 7 days were found to maximally influence BLR disease severity.     

Detection of an intermediate during unfolding of bacterial cell division protein FtsZ: loss of functional properties precedes the global unfolding of FtsZ

Using environment-sensitive fluorescence of 1-anilinonaphthalene-8-sulfonic acid, polarization of fluorescein 5'-isothiocyanate-labeled FtsZ, and far-UV circular dichroism spectroscopy, the chemical unfolding of FtsZ was found to proceed through two steps. The first step of the urea-induced unfolding produced an intermediate, which then unfolded at higher concentrations of urea. The intermediate state contains native-like secondary structure and much less tertiary structure compared with the native state. It is distinct from the native state as well as from the unfolded state. Similar to urea-induced unfolding of FtsZ, thermal unfolding of FtsZ also occurs in two steps. The midpoints for the first and second thermal unfolding transitions were found to be 38 +/- 4 and 77 +/- 5 degrees C, respectively. Further, the functional properties of FtsZ are extremely sensitive to urea, guanidium chloride, and sodium dodecyl sulfate. For example, 50% inhibition of the FtsZ assembly and GTP hydrolysis occurred at 0.1 and 0.2 m of urea, respectively. FtsZ lost its functional properties before any significant perturbation in the secondary or tertiary structure was detected by using several fluorescence techniques and far UV-CD indicating preferential local unfolding of the functional region(s). In addition, the unfolded FtsZ regains its ability to polymerize fully upon removal of urea. The data taken together suggest that FtsZ unfolds reversibly through a multistep process, and local responses that inhibit functional properties precede the global transition of FtsZ to the unfolded state.     

Sublethal temperature stress in juvenile Labeo rohita (Ham-Buch.) and Rita rita (Ham.): some physiological changes

Juveniles of fish L. rohita and R. rita subjected to a rapid (5 min) sublethal temperature increase from 28 to 35 degrees C showed significant increase in cortisol and decrease in interrenal ascorbic acid. Hypercholesterolemia, hyperglycemia and hyperlactemia were also evident accompanied by increased blood haemoglobin and haematocrit and stable protein levels. Compensatory responses were initiated within 72 hr in both the fishes. R. rita recovered more quickly indicating it to be more resistant to the heat stress than L. rohita. Hence fishes subjected to sublethal temperature stress should be given a metabolic recovery period of 72 hr prior to further stress being applied.     

Listeriolysin O-liposome-mediated cytosolic delivery of macromolecule antigen in vivo: enhancement of antigen-specific cytotoxic T lymphocyte frequency,activity, and tumor protection

Cytotoxic T lymphocytes (CTLs) are primed by peptide antigens that are endogenously processed in the cytosol and presented in the context of major histocompatibility complex I (MHC I) molecules of antigen-presenting cells (APCs). Exogenous soluble protein antigens do not gain efficient entry into the cytosol of APCs, and therefore requires a special cytosolic delivery method. We have developed such a delivery strategy adopting the well-elucidated cytosol-invading listerial endosomal escape mechanism, and report here an efficient delivery of exogenous whole protein antigen into the cytosol in a mouse model. Co-encapsulation of listeriolysin O (LLO) inside liposome (LLO-liposome) was required for delivery of ovalbumin (OVA) into the cytosol of APCs in primary cultures. LLO-liposome-mediated OVA immunization in mice engendered significantly higher OVA-specific CTL activity and increased antigenic peptide-specific CTL precursor (CTLp) frequency as compared to non-LLO-liposome or soluble OVA immunizations. Interferon-gamma (IFN-gamma) production upon specific stimulation by MHC I-restricted peptide was also significantly stronger by the inclusion of LLO in the liposomes. Rerouting of antigen into the cytosol by LLO-liposomes, however, did not reduce the extent of anti-OVA antibody responses. Moreover, LLO-liposome-antigen vaccination was robust in conferring protection to mice from lethal challenges with antigen-expressing tumor cells. Our study demonstrates a novel delivery system for efficient introduction of exogenous protein into the cytosol in vivo, priming cellular immune responses, which are protective in nature.     

Characterization of IgM of Indian major carps and their cross-reactivity with anti-fish IgM antibodies

Indian major carps (IMC), rohu (Labeo rohita), catla (Catla catla) and mrigal (Cirrhinus mrigala) were immunized with bovine serum albumin and the serum immunoglobulin M (IgM) was purified by affinity chromatography. The heavy and light chain of IgM of all the three species of IMC were about 88 and 26 kDa, respectively. Anti-fish IgM antibody against all the three species were raised in mice and the reaction of anti-fish IgM antibodies with IgM of all the three species of IMC were studied by Western blot. The anti-fish IgM antibodies reacted strongly with the heavy chain of the same species against which it was raised while the reactions with the heavy chain of other species were milder indicating some degree of epitope sharing among the heavy chains of IgM of IMCs. However, there was no cross-reaction with the light chain of any of the IgM.     

Discovery and initial optimization of 5,5′-disubstituted aminohydantoins as potent β-secretase (BACE1) inhibitors

8,8-Diphenyl-2,3,4,8-tetrahydroimidazo[1,5-a]pyrimidin-6-amine (1) was identified through HTS, as a weak (micromolar) inhibitor of BACE1. X-Ray crystallographic studies indicate the 2-aminoimidazole ring forms key H-bonding interactions with Asp32 and Asp228 in the catalytic site of BACE1. Lead optimization using structure-based focused libraries led to the identification of low nanomolar BACE1 inhibitors such as 20b with substituents which extend from the S₁ to the S₃ pocket.     

Novel pyrrolyl 2-aminopyridines as potent and selective human β-secretase (BACE1) inhibitors

The proteolytic enzyme β-secretase (BACE1) plays a central role in the synthesis of the pathogenic β-amyloid in Alzheimer’s disease. Recently, we reported small molecule acylguanidines as potent BACE1 inhibitors. However, many of these acylguanidines have a high polar surface area (e.g. as measured by the topological polar surface area or TPSA), which is unfavorable for crossing the blood–brain barrier. Herein, we describe the identification of the 2-aminopyridine moiety as a bioisosteric replacement of the acylguanidine moiety, which resulted in inhibitors with lower TPSA values and superior brain penetration. X-ray crystallographic studies indicated that the 2-aminopyridine moiety interacts directly with the catalytic aspartic acids Asp32 and Asp228 via a hydrogen-bonding network.     

ERbeta ligands. Part 6: 6H-Chromeno[4,3-b]quinolines as a new series of estrogen receptor beta-selective ligands

A new class of estrogen receptor beta (ERbeta) ligands based on the 6H-chromeno[4,3-b]quinoline scaffold has been prepared. Several C7-substituted analogues displayed high affinity and modest selectivity for ERbeta.