P-glycoprotein ATPase from the resistant pest, Helicoverpa armigera: Purification, characterization and effect of various insecticides on its transport function

AbstractHelicoverpa armigera is a major pest of agricultural crops and has developed resistance to various insecticides. A P-glycoprotein (Pgp) with ATPase activity likely to be involved in insecticide resistance was purified and characterized from insecticide-resistant H. armigera. The purification was 18-fold with 3% yield. The optimum pH and temperature were found to be 7.4 and 30–40°C, respectively. Kinetic studies indicated that this enzyme had a Km value of 1.2mM for ATP. Pgp from H. armigera was partially sequenced and found to be homologous to conserved sequences of mammalian Pgps. Pesticides stimulated H. armigera Pgp ATPase activity with a maximum stimulation of up to 40%. Quenching of the intrinsic tryptophan fluorescence of purified Pgp was used to quantitate insecticide binding. Using the high-affinity fluorescent substrate, tetramethylrosamine, transport was monitored in real time in proteoliposomes containing H. armigera Pgp. The presence of Pgp could be one of the reasons for insecticide resistance in this pest

Fishery, biology and stock assessment of spotted seer, Scomberomorus guttatus (Bloch & Schneider) off Andhra Pradesh

The fishery, biology and stock of the spotted seerfish, Scomberomorus guttatus (Bloch & Schneider) along Andhra Pradesh was studied during 2012-2014. The annual average landing of spotted seer during the period along Andhra coast was 2,614 t. Major contributors were trawlnets-1,104 t (42.2%) and gillnets-950 t (36.3%). Catches were higher during August-December. Length-weight relationship varied significantly between males, females and indeterminates. Growth was isometric in males and allometric in females and indeterminates. Overall sex ratio was 1:1.87. Length at first maturity was 38.9 cm. Peak spawning was observed during November-April. Higher Gonadosomatic index coincided with periods of peak spawning. Mature ovaries contained both maturing (0.41 mm-0.70 mm) and matured (0.71 mm-0.90 mm) ova. von Bertalanffy growth equation was Lt = 71.98 [1-e -0.23 (t + 0.0722)]. Natural mortality, fishing mortality and total mortality were 0.54, 0.49 and 1.03. Exploitation ratio was 0.48. Annual total stocks, biomass and Maximum Sustainable Yield were 8,546 t, 5,335 t and 2,747 t. A minor increase in yield and yield/recruit is possible by increasing the present level of fishing by 20%

Reproductive biology of Nemipterus japonicus (Bloch, 1791) in the trawl grounds along the north-east coast of India

The reproductive biology of Nemipterus japonicus (Bloch, 1791) along the north-east coast of India was studied during 2012-2015 based on samples from the trawl landings at Digha, Paradeep, Visakhapatnam and Kakinada. Adults dominated the landings. Significant contribution by females was observed in most months of the year. Size at first maturity for females was 15.1 cm. Peak spawning season was during August to February and September to March in the northern and southern regions. Maturity and gonado-somatic index (GSI) were found to correlate well with sea surface temperature (SST). Fecundity increased with length and weight and varied from 13,176 to 1,30,798. Ova diameter distribution indicated the species to be a multiple/batch spawner

Trophodynamics of Nemipterus japonicus (Bloch, 1791) in the trawl landings along the north-east coast of India

The trophodynamics of Nemipterus japonicus (Bloch, 1791) along the north-east coast of India was studied during 2012-2015 based on specimens collected from trawl landings at Digha, Paradeep (northern region), Visakhapatnam and Kakinada (southern region). Crustaceans were the preferred prey with index of relative importance (IRI) of 50% in the northern region and 42.4% in the southern region. Among crustaceans, Oratosquilla dominated in both the regions followed by crabs and several species of penaeids and nonpenaeids. Finfishes ranked next in importance (IRI% of 32.1 in northern region and 39.9 in southern region) and included Stolephorus spp., silverbellies, Coilia sp., other clupeids, Apogon spp., mackerel, sardines, eels, priacanthids, Bregmaceros sp. and sciaenids. Along northern region, in juveniles and preadults, the diet comprised chiefly of crustaceans (IRI% of 72.27 and 52.76), followed by finfishes (IRI% of 15.37 and 27.65). In the southern region, adult fishes exhibited higher preference for finfishes (IRI% of 47.5 to 62.8). Feeding intensity was low throughout the year with close to half (48.23% in northern region and 43.67% in southern region) of the fishes exhibiting empty or trace stomach conditions which increased with an increase in age and size of fish. High similarity of 75.54% in the northern and 84.19% in the southern region was recorded in the diet contents of smaller and moderate sized adults. Annual and seasonal mean similarity in the diet components between the regions was 69.9 and 53%. Prey contents in juveniles between both the regions exhibited a similarity of 59.5%, while preadults and adults recorded similarities of 62.7 and 68.2% respectively

Histomorphological spectrum of gastrointestinal stromal tumors: an institutional experience

Background: Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the abdominal area. They can involve any portion of the gastrointestinal (GI) tract, omentum, mesentery, retroperitoneum, and other sites. They form 1-2% of the histologic types of gastrointestinal tract tumors. Aims and objectives were to analyze and correlate morphological, clinical and histomorphology features of gastrointestinal tumors presenting at different sites.Methods: This was a retrospective observational study for six years. Medical records of the histopathologically diagnosed GIST cases were reviewed for patient demographics and clinical presentation, and tumor findings were noted.Results: Of the 28 patients, ages ranged from 28 to 80 years. Symptoms ranged from abdominal pain, epigastric discomfort, mass, upper/lower gastrointestinal bleeding, rectal bleeding, anemia, weight loss, and small bowel obstruction. Sites involved were the small bowel, stomach, mesentery, rectum, duodenum, greater omentum, and retroperitoneum. Of 28 cases of GIST, 25 cases showed both c-KIT and DOG-1 positivity, 1 case showed only c-KIT positivity, 1 case showed only DOG-1 positivity, and 1 case was both c-KIT and DOG-1 negative.Conclusions: GISTS are unpredictable mesenchymal tumors. Common sites are the stomach and small gut. Mesenteric and omental GIST are rare. Spindle cell morphology was more commonly present

Preparation of Fluconazole β-Cyclodextrin Complex Ocuserts: In Vitro and In Vivo Evaluation

The main purpose of the present study was to develop ocuserts of Fluconazole β-CD (beta-cyclodextrin) complex and to evaluate both in vitro and in vivo. Fluconazole was made complex with β-CD, and the release rate was controlled by HPMC K4M and ethyl cellulose polymers using dibutyl Phthalate as permeability enhancer. Drug-polymer interactions were studied by Fourier transform infrared spectroscopic studies. The formulated ocuserts were tested for physicochemical parameters of in vitro release and in vivo permeation in rabbits. The optimized formulations (F-5 and F-8) were subjected to stability studies. The formulated ocuserts were found to have good physical characters, thickness, diameter, uniformity in weight, folding endurance, less moisture absorption, and controlled release of drug both in vitro and in vivo. The optimized formulations retained their characteristics even after stability studies. The study clearly showed that this technique was an effective way of formulating ocuserts for retaining the drug concentration at the intended site of action for a sufficient period of time and to elicit the desired pharmacological response

A thiazolo[5,4-: D] thiazole-bridged porphyrin organic framework as a promising nonlinear optical material

Porphyrin-based porous organic frameworks are an important group of materials gaining interest due to their structural diversity and distinct opto-electronic properties. However, these materials are seldom explored for nonlinear optical (NLO) applications. In this work, we investigate a thiazolo[5,4-d]thiazole-bridged porous, porphyrin framework (Por-TzTz-POF) with promising NLO properties. The planar TzTz moiety coupled with integrated porphyrin units enables efficient π-conjugation and charge distribution in the Por-TzTz-POF resulting in a high nonlinear absorption coefficient (β = 1100 cm GW-1) with figure of merit (FoM) σ1/σ0 = 5571, in contrast to analogous molecules and material counterparts e.g. metal-organic frameworks (MOFs; β = ∼0.3-0.5 cm GW-1), molecular porphyrins (β = ∼100-400 cm GW-1), graphene (β = 900 cm GW-1), and covalent organic frameworks (Por-COF-HH; β = 1040 cm GW-1 and FoM = 3534). This journal is © The Royal Society of Chemistry

Tryptophan fluorescence quenching as a binding assay to monitor protein conformation changes in the membrane of intact mitochondria

Intrinsic protein fluorescence is due to aromatic amino acids, mainly tryptophan, which can be selectively measured by exciting at 295 nm. Changes in emission spectra of tryptophan are due to the protein conformational transitions, subunit association, ligand binding or denaturation, which affect the local environment surrounding the indole ring. In this study, tryptophan fluorescence was monitored in intact mitochondria at 333 nm following excitation at 295 nm in presence of insecticides using spectrofluorometer. Methyl-parathion, carbofuran, and endosulfan induced Trp fluorescence quenching and release of cytochrome c when incubated with the mitochondria, except fenvalarate. Mechanism of insecticide-induced mitochondrial toxicity for the tested insecticides has been discussed. Reduction in the intensity of tryptophan emission spectra of mitochondrial membrane proteins in presence of an increasing concentration of a ligand can be used to study the interaction of insecticides/drugs with the intact mitochondria. Furthermore, this assay can be readily adapted for studying protein–ligand interactions in intact mitochondria and in other cell organelles extending its implications for pesticide and pharma industry and in drug discovery

Monitoring biosafety of pharmaceutical drugs, insecticides, and other bioactive molecules to mitochondria

Mitochondria are large cell organelles in plant and animal cells. They convert chemical energy from food in the cell to usable energy using oxygen. This process is called oxidative phosphorylation, which takes place inside the mitochondria. Reactions of Krebs cycle produce a chemical, NADH (Nicotinamide Adenine Dinucleotide, reduced form), which is then used by enzymes present in the inner mitochondrial membrane to generate energy rich adenosine triphosphate (ATP) molecules. These molecules are utilized by the cells whenever energy is required. Therefore, mitochondria are also referred as power houses of the cell. Mitochondria are abundantly present in muscle and brain cells, and sperms to supply ATPs during demand for energy. Monitoring biosafety of pharmaceutical drugs, insecticides, and other bioactive molecules to mitochondria is necessary because some of them interact with the mitochondrial membrane or membrane proteins, thereby affecting the energy levels of cells or even induces mitochondrial-dependent apoptosis (programmed cell death)

Elimination of Gut Microbes with Antibiotics Confers Resistance to Bacillus thuringiensis Toxin Proteins in Helicoverpa armigera (Hubner)

Helicoverpa armigera is one of the most important pests worldwide. Transgenic crops with toxin genes from Bacillus thuringiensis (Bt) have been deployed on a large scale to control this pest. The insecticidal activity of Bt is probably influenced by the insect midgut microbes, which vary across crop hosts and locations. Therefore, we examined the role of gut microbes in pathogenicity of Bt toxins in the H. armigera. Antibiotic cocktail was used for the complete elimination of the H. armigera gut microbes. Activated Cry1Ac, Bt formulation, and transgenic cotton resulted in larval weight loss and increase in mortality, but pretreatment of larvae with antibiotic cocktail significantly decreased larval mortality and increased the larval weight gain. Activated Cry1Ac and Bt formulation inhibited the activity of proteases in midgut of H. armigera larvae but showed no such effect in the larvae pretreated with antibiotic cocktail. Five protease bands in activated Cry1Ac and two in Bt formulation-treated larvae were inhibited but no such effect in the larvae pretreated with antibiotic cocktail. Cry1Ac protein was detected in Bt/Cry1Ac protoxin-fed larval gut extract in the absence of antibiotic cocktail, but fewer in larvae pretreated with antibiotic cocktail. The activity of antioxidant enzymes and aminopeptidases increased in larvae fed on Bt toxin, but there was no significant increase in antioxidant enzymes in larvae reared on toxin protein in combination with antibiotic cocktail. The results suggest that gut microbes exercise a significant influence on the toxicity of Cry1Ac and Bt formulation in H. armigera larvae. The implications of these results have been discussed in relation to development of insect resistance to Bt transgenic crops deployed for pest management