Evaluation of interferon gamma (IFN-γ) of <Emphasis Type="Italic">Labeo rohita</Emphasis> as an immunomodulator: in vitro expression model

Interferon gamma (IFN-γ) or type II interferon is a cytokine that is critical for innate and adaptive immunity against viral and some bacterial and protozoal infections. The importance of IFN-γ in the immune system lies in its ability to inhibit viral replication directly and most importantly from its immunomodulatory effects. Previously, we successfully co-administered IFN-γ along with GAPDH gene of Edwardsiella tarda as bicistronic DNA vaccine in Labeo rohita. In order to ascertain the individual role of IFN-γ, the present study involves cloning and expression of 552-bp IFN-γ open-reading frame (ORF) of L. rohita in striped snakehead (SSN-1) cell line using eukaryotic expression vector system (pQE-TriSystem) followed by transfection in peripheral blood lymphocytes (PBMCs) to evaluate its immunomodulatory ability in comparison to polyinosinic-polycytidylic acid (Poly I:C)-treated PBMCs. The 18.7-kDa protein, expressed in the pQE-IFNγ-transfected SSN-1 cells, reacted with anti-His antibody in Western blot confirming it to be recombinant IFN-γ, whereas the relative expression of IFN-γ, iNOS, Mx, and IL-1β genes in PBMCs was quantified at 24 h and 48 h post treatment by qPCR. The comparative kinetics of all four genes showed significantly (p?<?0.05) high upregulation pattern in both pQE-IFNγ-transfected cell group and Poly I:C-treated cell group demonstrating recombinant IFN-γ as an equally efficient inducer like Poly I:C. Thus, our in vitro experiment results highlight the immunomodulatory potential of recombinant IFN-γ as an analogue to synthetic Poly I:C which warranted future studies to further explore the potential of recombinant IFN-γ as an effective vaccine adjuvant against different microbial invasion.     

Verbascoside isolated from Tectona grandis mediates gastric protection in rats via inhibiting proton pump activity

Evidences have suggested that Tectona grandis (TG) attenuates gastric mucosal injury; however its mechanism has not yet been established. The aim of present study was to evaluate the gastroprotective mechanism of ethanolic extract of TG (E-EtOH), butanolic fraction (Fr-Bu) and to identify its active constituents. Anti-ulcer activities were evaluated against cold restraint (CRU) and pyloric ligation (PL) induced gastric ulcer models and further confirmed through H⁺ K⁺-ATPase inhibitory activity. Cytoprotective activity was evaluated in alcohol (AL) induced gastric ulcer model and further through PGE₂ level. E-EtOH and Fr-Bu attenuated ulcer formation in CRU. Moreover E-EtOH and Fr-Bu displayed potent anti-secretory activity as evident through reduced free acidity and pepsin activity in PL, confirmed further by in vitro inhibition of H⁺ K⁺-ATPase activity. In addition cytoprotective potential of E-EtOH and Fr-Bu were apparent with protection in AL model, increased PGE₂ content and enhanced mucin level in PL. Phytochemical investigations of Fr-Bu yielded terpenoides and a phenolic glycoside, verbascoside. The anti-secretory mechanism of verbascoside mediated apparently through inhibition of H⁺ K⁺-ATPase with corresponding decrease in plasma gastrin level, is novel to our finding. Gastroprotection elicited by TG might be through proton pump inhibition and consequent augmentation of the defensive mechanism.     

Standardisation of true cassava seed (TCS) programme with special emphasis on more homogeneous, CMD resistant progenies

The propagation of cassava through true seeds (sexual seeds) rather than by clones is a promising option due to its manifold advantages such as enhancing the multiplication rate, keeping the dreaded cassava mosaic disease (CMD) under check, longer seed viability, ease of storage and transport. The high genetic heterogeneity and consequent variation among seedlings is the major stumbling block in sexual propagation. In the present study, a CMD resistant exotic accession MNga-1 and a promising cultivar Ambakadan with profuse fruit setting, seed output and male sterility were identified to be promising parents for the TCS programme. The rate of sexual propagation could be more than 20-fold over the traditional clonal propagation. Seed treatment with 1% KNO₃ or 300 ppm GA promoted uniform seed germination and seedling vigour and reduced the transplanting period from 45 days after planting (DAS) to 30 DAS. Removal of taproots of seedlings while transplanting enhanced tuber development. Tuber yield of first clones (C₁) was significantly superior to that of the seedlings. The dry matter content and starch output of seedlings and first clones were comparable to that of the commercial varieties. Similarly, the HCN and cooking quality of seedlings and first clones were at acceptable levels. In the open pollinated (OP) progenies of the Ambakadan the CMD infection increased drastically due to secondary spread of the pathogen. The hybrid progenies of Ambakadan and the CMD resistant line MNga-1 revealed higher percentage of CMD free seedlings and first clonal progenies in the evaluation trials conducted at CTCRI Thiruvananthapuram and ARS, Peddapuram during 2001–2002 and 2002–2003. Nearly homogeneous hybrid population resistant to CMD could be obtained by systematic roguing at seedling and first clonal stage.     

Ceramide triggers budding of exosome vesicles into multivesicular endosomes

Intraluminal vesicles of multivesicular endosomes are either sorted for cargo degradation into lysosomes or secreted as exosomes into the extracellular milieu. The mechanisms underlying the sorting of membrane into the different populations of intraluminal vesicles are unknown. Here, we find that cargo is segregated into distinct subdomains on the endosomal membrane and that the transfer of exosome-associated domains into the lumen of the endosome did not depend on the function of the ESCRT (endosomal sorting complex required for transport) machinery, but required the sphingolipid ceramide. Purified exosomes were enriched in ceramide, and the release of exosomes was reduced after the inhibition of neutral sphingomyelinases. These results establish a pathway in intraendosomal membrane transport and exosome formation.     

The toxicity of phosphine,methyl bromide, 1,1,1-trichloroethane and carbon dioxide alone and as mixtures to the pupae of red flour beetle,Tribolium castaneum herbst

The toxicity of phosphine, methyl bromide, 1,1,1-trichloroethane (methyl chloroform) and carbon dioxide and mixtures of phosphine + methyl bromide, methyl bromide + methyl chloroform, phosphine + carbon dioxide, and methyl bromide + carbon dioxide to one- to two-day-old pupae of Tribolium castaneum Herbst was studied. Joint action ratios estimated at LD₅₀ and LD₉₀ for a 24-h exposure indicated no enhancement of toxicity for mixtures of phosphine and methyl bromide, or methyl chloroform and methyl bromide on the pupae. Carbon dioxide up to 40% in air enhanced the toxic action of phosphine as well as of methyl bromide. Higher levels of carbon dioxide, however, failed to improve the toxicity of phosphine or methyl bromide proportionately. Carbon dioxide used alone produced a maximum of 11% mortality of the pupae exposed to 10–70% levels for 24 h. The order of toxicity of the fumigants both at LD₅₀ and LD₉₀ was phosphine > methyl bromide > methyl chloroform.     

Influence of phosphine on hatching of Cryptolestes ferrugineus (Coleoptera: Cucujidae), Lasioderma serricorne (Coleoptera: Anobiidae) and Oryzaephilus surinamensis (Coleoptera: Silvanidae)

The hatching and mortality response of 0- to 48-h-old eggs of field strains of the stored-product insects Cryptolestes ferrugineus (Stephens), Lasioderma serricorne (F) and Oryzaephilus surinamensis (L) following phosphine fumigation for 24, 48 or 120 h at 27 (+/- 2) degrees C was investigated. Hatching was delayed and reduced in the first few days in a phosphine-resistant strain of C ferrugineus that was treated with 2.0-7.0 mg litre(-1) doses for 48 h (5-80% mortality) and with 1.0-2.0 mg litre(-1) for 120 h (44-84% mortality). In both the exposures there were significant increases in hatching on later days when compared with the corresponding controls. Developmental delay was, however, not evident in susceptible strains of C ferrugineus, L serricorne and O surinamensis that were exposed to phosphine for 24 h.     

The action of phosphine against the eggs of phosphine-resistant and -susceptible strains of Rhyzopertha dominica F

The effect of phosphine at exposure periods of 24, 48 and 120 h on hatching and mortality of 0- to 1-day-old eggs of susceptible (TN6) and resistant (FC10) strains of Rhyzopertha dominica of field origin was investigated. The fumigant affected hatching in both the strains. In a 48-h exposure at 27 (+/- 2) degrees C, the LD99 doses for the eggs of TN6 and FC10 were 0.56 and 3.25 mg litre-1, respectively. Significant reduction in hatching was observed in treated batches with progressive increase of phosphine dose in the first 2-3 days. On subsequent days the numbers hatching were often similar to those in controls, and sometimes exceeded control hatch, especially following a 5-day exposure. A critical change in the order of susceptibility of egg and adult stages of the two strains was noticed. In 48-h exposures, eggs of the susceptible strain were more tolerant than their adults, whilst the reverse was true in the resistant strain.     

Development of a DNA marker for distinguishing CMS lines from fertile lines in rice (Oryza sativa L.)

The main objective of the present study was to identify mitochondrial DNA based marker, which can distinguish male sterile and fertile counterparts of the cytoplasmic male sterile (CMS) lines used in production of rice hybrids. Amplified fragment length polymorphism (AFLP) analysis in CMS lines: IR58025A & IR62829A and their respective maintainers: IR58025B & IR62829B identified a polymorphic DNA fragment of about 510 bp size that was present in both CMS (A) and absent in their maintainer (B) lines. Sequencing followed by database analysis of the polymorphic fragment indicated about 97% similarity with mitochondrial NADH gene subunits of rice, maize and wheat. Based on the variable sequence regions, a site specific primer pair (BF-STS-401) was designed. PCR analysis showed that BF-STS-401 could amplify a strong band of 464 bp size in CMS and a faint band of the same size in maintainer line. To act as a positive control and avoid possible errors in PCR, BF-STS-401 was multiplexed with a new primer pair (BF-STS-402), derived from mitochondrial atp9 subunit of rice, producing monomorphic amplification indiscriminately in both CMS and maintainer lines. Both the primer pairs in combination clearly differentiated CMS lines from their corresponding maintainer lines. This primer combination was validated in a set of diverse genotypes consisting of different sources of CMS lines, restorer lines, hybrids, varieties and mixed samples from private seed companies. Our results suggested that the multiplex primer pairs developed in this study can be effectively utilized to assess the genetic purity in commercial seed lots of CMS lines and hybrids of rice.     

Efficient inhibition of the Alzheimer's disease beta-secretase by membrane targeting

beta-Secretase plays a critical role in beta-amyloid formation and thus provides a therapeutic target for Alzheimer's disease. Inhibitor design has usually focused on active-site binding, neglecting the subcellular localization of active enzyme. We have addressed this issue by synthesizing a membrane-anchored version of a beta-secretase transition-state inhibitor by linking it to a sterol moiety. Thus, we targeted the inhibitor to active beta-secretase found in endosomes and also reduced the dimensionality of the inhibitor, increasing its local membrane concentration. This inhibitor reduced enzyme activity much more efficiently than did the free inhibitor in cultured cells and in vivo. In addition to effectively targeting beta-secretase, this strategy could also be used in designing potent drugs against other membrane protein targets.     

Curcumin, a major constituent of turmeric, corrects cystic fibrosis defects

Cystic fibrosis is caused by mutations in the gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). The most common mutation, DeltaF508, results in the production of a misfolded CFTR protein that is retained in the endoplasmic reticulum and targeted for degradation. Curcumin is a nontoxic Ca-adenosine triphosphatase pump inhibitor that can be administered to humans safely. Oral administration of curcumin to homozygous DeltaF508 CFTR mice in doses comparable, on a weight-per-weight basis, to those well tolerated by humans corrected these animals' characteristic nasal potential difference defect. These effects were not observed in mice homozygous for a complete knockout of the CFTR gene. Curcumin also induced the functional appearance of DeltaF508 CFTR protein in the plasma membranes of transfected baby hamster kidney cells. Thus, curcumin treatment may be able to correct defects associated with the homozygous expression of DeltaF508 CFTR.