Irradiation influence on the phenoloxidase pathway and an anti-oxidant defense mechanism in Spodoptera litura (Lepidoptera: Noctuidae) and its implication in radio-genetic 'F 1 sterility' and biorational pest suppression tactics.

The present study was conducted to appraise the ontogenic radio-sensitivity of a serious tropical pest, Spodoptera litura (Fabr.). The molecular responses pertaining to the phenoloxidase (PO) pathway and an anti-oxidant defense mechanism were evaluated in order to understand its implication in pest control at pre-harvest and post-harvest intervals. Irradiation exhibited an inverse relationship with age with respect to impact on developmental and transcriptional responses. Transcript abundance of PO cascade enzymes, prophenoloxidase (slppo-2), its activating enzyme (slppae-1) and free-radical scavenging enzymes, superoxide dismutase (slsod) and catalase (slcat) was evaluated upon gamma irradiation alone and the dual-stress of radiation plus microbial challenge. The slppo-2, slppae-1, slsod and slcat transcripts were significantly up-regulated in F 1 L6 larvae (6th-instar) resulting from 100 Gy sub-sterilized male adults and unirradiated female moths. The extent of upregulation was relatively higher in comparison with L6 survivors (6th-instar larvae) developed from irradiated neonates (L1) treated with 100 Gy. Upon Photorhabdus challenge, the transcripts were down-regulated in irradiated L1 suggesting increased larval susceptibility to bacterial infections. Radioresistance increased with the age of the insect, and molecular responses (transcript abundance) of insect defense mechanism were less influenced when older age (F 1 progeny) were irradiated. These findings will help to optimize the gamma dose to be employed in inherited sterility technique for (pre-harvest) pest suppression and (post-harvest) phytosanitation and quarantine, and suggest compatible integration of biorational tactics including nuclear technology.

Cytomodulin-1, a synthetic peptide abrogates oncogenic signaling pathways to impede invasion and angiogenesis in the hamster cheek pouch carcinogenesis model.

Constitutive activation of the various oncogenic signaling pathways plays a pivotal role in promoting malignant transformation. The aim of this study was to investigate the therapeutic potential of a synthetic bioactive heptapeptide cytomodulin-1 (CM-1) against hamster cheek pouch carcinomas based on its influence on the predominant carcinogenic signaling pathways - NF-κB, TGFß, and Wnt/ß-catenin and their downstream target events invasion and angiogenesis. Topical application of CM-1 to DMBA-painted hamsters significantly inhibited activation of the canonical NF-κB pathway by blocking kinase activity of IKKß and increasing the cytosolic accumulation of the inhibitor IκB-α. In addition, CM-1 inactivated IKKß by disrupting IKKß/Nemo interactions. CM-1 also hampered the activation of TGFß and Wnt/ß-catenin signaling by averting the phosphorylation of the key upstream ser/thr kinases TGFß RI and GSK-3ß respectively. Attenuation of these oncogenic signaling pathways by CM-1 also mitigated invasion and angiogenesis by suppressing the expression of pro-invasive matrix metalloproteinases, pro-angiogenic VEGF and HIF-1α and upregulating the anti-angiogenic TIMP-2. Synthetic peptides such as CM-1 that target multiple key molecules in oncogenic signaling pathways and their downstream events are ideal candidate agents for cancer chemotherapy.

Molecular characterization of nucleopolyhedrovirus of three lepidopteran pests using late expression factor-8 gene.

A PCR based method for detection of viral DNA in nucleopolyhedrovirus of three lepidopterans, Spodoptera litura, Amsacta albistriga and Helicoverpa armigera, was developed by employing the late expression factor-8 (lef-8) gene of three NPV using specific primers. The amplicons of 689, 699 and 665 bp were amplified, respectively, and the nucleotide sequences were submitted to GenBank and the accession numbers were obtained. The sequences of lef-8 gene of S. litura NPV and H. armigera NPV matched with those of their respective references in the GenBank database, thereby confirming their identity, however, the sequence of A. albistriga NPV was the first sequence submitted to the GenBank database. The sequence similarity analysis between the three lef-8 gene of NPV sequenced in the present study revealed that there was no significant similarity between them, however A. albistriga NPV and S. litura NPV were found to be closely related. CLUSTAL alignment of the sequences generated revealed general relatedness among NPVs lef-8 gene. The study confirmed that lef-8 gene can be used for quick and correct discriminatory identification of insect viruses.

An assessment of performance based incentive system for ASHA Sahyogini in Udaipur, Rajasthan.

OBJECTIVES: To assess the performance based incentive system for ASHA Sahyogini in Udaipur district of Rajasthan. METHODS: This cross-sectional study was conducted in three blocks (one each from rural, urban and tribal area) of Udaipur district during October-December 2008. From each block 60 ASHAs were selected randomly, thus a tola of 180 ASHAs were included. Besides interviewing the ASHAs, focus group discussions were also conducted for primary data collection. The study assessed the performance based incentives system to ASHAs during the last six months. RESULTS: The study revealed that almost 50% ASHA's in the studied blocks were covering population ranging from 1000-1500. All the ASHA has good coordination with local community and they are participating in community meetings regularly. All the ASHAs received incentives for the cases of sterilization; 55.5 percent urban, 85.7 percent rural and 82.7% tribal ASHAs received it on the same day when sterilization was done. Half of the urban, 35% of the rural and 56.7% of tribal ASHAs got incentive less than Rs. 250 in last 6 months (less than 50/- per month). Common cause identified for dissatisfaction was less incentives compared to their work, especially for the ASHA working in tribal areas. CONCLUSION: Timely release of incentives, adequate cooperation from staff such as ANMs, AWW, hospital staff and improved community awareness are needed for better performance of ASHAs.

Variation in metabolites constituent in leaves of downy mildew resistant and susceptible genotypes of pearl millet.

In the present investigation biochemical characterization of pearl millet genotypes was carried at pre (45 DAS) and post-infection (57 DAS i.e. 7 days after infection) stage. Total soluble sugar was greater at pre infection than post infection in downy mildew resistant and susceptible genotypes of pearl millet. Total soluble sugar decreased in all genotypes at 7 days after infection (d.a.i.) except in 7042 S in which it increased 4.6 %. However, total soluble sugar was 2-3 folds more in highly susceptible genotypes (J-2296 and 7042 S) compared to resistant genotypes at 7 d.a.i. but it was decreased as compared to pre infection. The total amino acid content of all genotypes whether resistant or susceptible, finally increased as a result of infection. Moreover, susceptible genotypes registered 2-2.5 % higher amino acid, whereas resistant genotypes possessed 6.2-76 % higher amino acid than their constitutive level. Total chlorophyll and carotenoids content did not show any clear cut difference in resistant and susceptible genotypes at pre-infection stage. However, at post-infection stage a significant decrease in chlorophyll and carotenoids content occurred in susceptible genotypes from pre-infection. Amino acid profiling through HPTLC showed sulphur containing amino acids were higher in resistant genotypes.

Schwannoma of the nasal septum.

Schwannomas of the nasal cavity and paranasal sinuses are very rare. We report the case of a 54-year-old woman with a schwannoma arising from the nasal septum. We discuss the clinical presentation, differential diagnosis, imaging characteristics and treatment of this rarely encountered lesion.

Selective surgicel packing for the treatment of posterior epistaxis.

Rod lens endoscopes provide residents in otolaryngology a means of more accurately identifying the site of bleeding and, when possible, cauterizing the bleeding vessel. Identification of a posterior bleeding point is often difficult and sometimes impossible. Intranasal manipulation for electrocautery is painful, may require general anesthesia, and is associated with complications. We describe a pilot study designed to evaluate selectively packing the bleeding site with Surgicel (oxidized cellulose) to control the hemorrhage without packing the nasal cavity and to reduce patient morbidity and length of stay in the hospital. We describe the technique and present the results of treating 8 patients admitted with acute posterior epistaxis over a 10-month period in 1995-1996.

Synthetic peptides cytomodulin-1 (CM-1) and cytomodulin-2 (CM-2) promote collagen synthesis and wound healing in vitro.

Transforming growth factor-beta1 (TGF-beta1) is believed to be a key player in wound healing, promoting cell proliferation, migration, and matrix synthesis in a variety of cell types. We have designed two peptides, i.e., cytomodulin-1 (CM-1) and cytomodulin-2 (CM-2), to simulate the binding domain of TGF-beta1. In this study we examined the bioactivity of the two synthetic peptides CM-1 and CM-2 on human foreskin fibroblasts (HFF). Synthetic peptides CM-1 and CM-2 in culture media increased wound healing of fibroblasts in an injury model in vitro. In addition, CM-1 and CM-2 enhanced the gene expression of collagen I and increased the production of pro-collagen I peptide in HFF. These results suggest that CM-1 and CM-2 have potentially useful clinical applications in wound healing.

A simple and rapid Agrobacterium-mediated transformation protocol for cotton (Gossypium hirsutum L.): embryogenic calli as a source to generate large numbers of transgenic plants.

A protocol is presented for efficient transformation and regeneration of cotton. Embryogenic calli co-cultivated with Agrobacterium carrying cry1Ia5 gene were cultured under dehydration stress and antibiotic selection for 3-6 weeks to generate several transgenic embryos. An average of 75 globular embryo clusters were observed on selection plates and these embryos were cultured on multiplication medium followed by development of cotyledonary embryos on embryo maturation medium to obtain an average of 12 plants per Petri plate of co-cultivated callus. About 83% of these plants have been confirmed to be transgenic by Southern blot analysis. An efficiency of ten kanamycin-resistant plants per Petri plate of co-cultivated embryogenic callus was obtained. The simplicity of the procedure and the efficiency of the initial material allow transformation of any variety where a single regenerating embryogenic callus line can be obtained. In addition, multiple transformations can be performed either simultaneously or sequentially. The method is extremely simple, reliable, efficient, and much less laborious than any other existing method for cotton transformation.

Relocating expression of vegetative insecticidal protein into mother cell of Bacillus thuringiensis.

Vegetative insecticidal protein (VIP) is a class of insecticidal proteins produced by some strains of Bacillus thuringiensis during the vegetative stage of their growth. Unlike delta-endotoxins which are produced as parasporal inclusion bodies within the cell during sporulation, VIP is secreted into the culture medium. Here we report the relocation of the expression of VIP into the mother cell compartment in a manner similar to well-characterized Cry proteins. Relocation of VIP is directed to mother cell by placing its synthesis under sporulation-dependent promoters, BtI and BtII. The insertion of cry preferred transcription termination sequence at the 3(') region and a STAB-SD sequence at the 5(') region of the gene provided stability to the vip transcript and enhanced its yield. The demonstrated expression of VIP within the cells in the form of inclusion bodies would facilitate development of a suitable formulation for the application of this class of insecticidal proteins in the field.

High-frequency somatic embryo production and maturation into normal plants in cotton (Gossypium hirsutum) through metabolic stress.

A highly efficient somatic embryo production and maturation procedure has been developed to regenerate plantlets from cotton ( Gossypium hirsutum). This procedure involves the acceleration of differentiation through manipulations of nutrient and microenvironment conditions. Embryogenic calli, initiated from hypocotyls or cotyledonary leaf sections on MS medium containing 0.1 mg/l 2,4 dichlorophenoxyacetic acid, 0.5 mg/l kinetin, and 3% maltose produced globular-stage somatic embryos when transferred to hormone-free MS medium supplemented with high concentrations of nitrate. Subculture of globular embryos on hormone-free MS medium led to the development of torpedo- and cotyledonary-stage at a low frequency (two to four per plate) with the majority of embryos lacking further growth or entering into the dedifferentiation stage. Significant improvement in embryogenesis (two- to threefold) was achieved when calli were cultured on 1/5-strength MS medium irrespective of stress treatment. However, the frequency of globular embryos developing into normal plantlets improved considerably (20-24 per plate) when cultured on filter paper placed on MS medium. In this procedure, about 33% of globular embryos not only developed into the cotyledonary stage but rooted simultaneously, eliminating a separate rooting step. More than 70% of cotyledonary embryos developed into normal plantlets when cultured on full- strength MS medium containing 0.05 mg/l gibberellic acid.

Toxicity analysis of N- and C-terminus-deleted vegetative insecticidal protein from Bacillus thuringiensis.

A vegetative insecticidal protein (VIP)-encoding gene from a local isolate of Bacillus thuringiensis has been cloned, sequenced, and expressed in Escherichia coli. The expressed protein shows insecticidal activity against several lepidopteran pests but is ineffective against Agrotis ipsilon. Comparison of the amino acid sequence with those of reported VIPs revealed a few differences. Analysis of insecticidal activity with N- and C-terminus deletion mutants suggests a differential mode of action of VIP against different pests.

Modulation of proteoglycan and collagen profiles in human dermal fibroblasts by high density micromass culture and treatment with lactic acid suggests change to a chondrogenic phenotype.

Cartilage formation during embryonic development and in fracture healing in adult animals involves chondrogenic differentiation of mesenchymal precursors. Here we describe an in vitro model whereby human dermal fibroblasts, considered to be restricted to a fibroblast lineage, are apparently redirected toward a chondrogenic phenotype by high density micromass culture in the presence of lactic acid. Micromass cultures treated with 40 mM lactate exhibited increased levels of Alcian blue staining and sulfate incorporation, indicative of elevated sulfated glycosaminoglycan synthesis. Northern analysis revealed an up-regulation of chondroitin sulfate proteoglycan 1 (aggrecan) and transforming growth factor-beta 1 mRNA and a decrease in type I collagen expression. Type II collagen was detected by reverse transcription-PCR only in experimental cultures. Although the observed changes in biosynthesis and gene expression were consistent with differentiating chondrocytes, the cells displayed an elongated, fibroblast-like morphology. These findings suggest that dermal fibroblasts may be committed to differentiate along a chondrogenic pathway by in vitro culture under specific forcing conditions.

Evidence suggesting that high intake of fluoride provokes nephrolithiasis in tribal populations.

The present study was designed to evaluate the role of fluoride in urolithiasis in humans. Two areas were selected for this purpose, a fluoride endemic area (EA) and a fluoride non-endemic area (NEA). The prevalence of uroliathiasis was 4.6 times higher in EA than in NEA. Furthermore, the prevalence was almost double in subjects with fluorosis than without fluorosis in the endemic area. No relationship was observed between urolithiasis and the duration of fluorosis. The fluoride levels in drinking water ranged from 3.5 to 4.9 ppm in EA and subjects from this area excreted more fluoride. A comparison of normal subjects (NS) from EA and NEA revealed that endemic subjects tend to have slightly higher mean serum thiobarbituric acid reactive substance (TBAR) levels and excrete more oxalate and fluoride than their non-endemic counterparts. The urinary stone formers (SF) from the two areas showed a similar tendency, though again the difference was not significant. Citrate excretion in SF was almost normal in the EA, but NEA SF had significantly lower excretion levels. Urinary stones from endemic patients had higher fluoride, oxalate and calcium levels than those from non-endemic patients. In vitro studies suggested that fluoride did not influence the heterogonous mineralization of calcium oxalate. In conclusion, the data suggest that fluoride in vivo may behave as a mild promoter of urinary stone formation by (a) excretion of insoluble calcium fluoride, (b) increasing oxalate excretion and (c) mildly increasing the oxidative burden.

Anthrax toxin.

Anthrax is primarily a disease of herbivores caused by gram-positive, aerobic, spore-forming Bacillus anthracis. Humans are accidental hosts through the food of animal origin and animal products. Anthrax is prevelant in most parts of the globe, and cases of anthrax have been reported from almost every country. Three forms of the disease have been recognized: cutaneous (through skin), gastrointestinal (through alimentary tract), and pulmonary (by inhalation of spores). The major virulence factors of Bacillus anthracis are a poly-D glutamic acid capsule and a three-component protein exotoxin. The genes coding for the toxin and the enzymes responsible for capsule production are carried on plasmid pXO1 and pXO2, respectively. The three proteins of the exotoxin are protective antigen (PA, 83 kDa), lethal factor (LF, 90 kDa), and edema factor (EF, 89 kDa). The toxins follow the A-B model with PA being the B moeity and LF/EF, the alternative A moeities. LF and EF are individually nontoxic, but in combination with PA form two toxins causing different pathogenic responses in animals and cultured cells. PA + LF forms the lethal toxin and PA + EF forms the edema toxin. During the process of intoxication, PA binds to the cell surface receptor and is cleaved at the sequence RKKR (167) by cell surface proteases such as furin generating a cell-bound, C-terminal 63 kDa protein (PA63). PA63 possesses a binding site to which LF or EF bind with high affinity. The complex is then internalized by receptor-mediated endocytosis. Acidification of the vesicle leads to instertion of PA63 into the endosomal membrane and translocation of LF/EF across the bilayer into the cytosol where they exert their toxic effects. EF has a calcium- and calmodulin-dependent adenylate cyclase activity. Recent reports indicate that LF is a protease that cleaves the amino terminus of mitogen-activated protein kinase kinases 1 and 2 (MAPKK1 and 2), and this cleavage inactivates MAPKK1 and thus inhibits the mitogen-activated protein kinase signal transduction pathway. We describe in detail the studies so far done on unraveling the molecular mechanisms of pathogenesis of Bacillus anthracis.

Hydrophobic residues Phe552, Phe554, Ile562, Leu566, and Ile574 are required for oligomerization of anthrax protective antigen.

Anthrax protective antigen (PA) plays a central role in facilitating the entry of active toxin components, namely, lethal factor and edema factor, into the cells. PA is also the main immunogen of both human and veterinary vaccine against anthrax. During host cell intoxication, protective antigen binds to the receptors on cell surface, gets proteolytically activated, oligomerizes to form a heptamer and binds to lethal factor or edema factor. The complex, formed by binding of lethal factor or edema factor to oligomerized PA, is internalized by receptor-mediated endocytosis. Acidification of the endosome results in the insertion of the heptamer into the membrane, thereby forming a pore through which lethal factor or edema factor can translocate into the cytosol. In this study we have identified hydrophobic residues, Phe552, Phe554, Ile562, Leu566, and Ile574, which are required for oligomerization of anthrax protective antigen. Mutation of these conserved residues to alanine impaired the oligomerization of protective antigen. Consequently, these mutants became nontoxic in combination with lethal factor and edema factor. Therapeutic importance of these mutants and their potential as vaccine candidates is discussed.

Purification of anthrax edema factor from Escherichia coli and identification of residues required for binding to anthrax protective antigen.

The structural gene for anthrax edema factor (EF) was expressed in Escherichia coli under the control of a powerful T5 promoter to yield the 89-kDa recombinant protein that reacted with anti-EF antibodies. Recombinant EF was purified to homogeneity by a two-step procedure involving metal chelate affinity chromatography and cation-exchange chromatography. From 1 liter of culture, 2.5 mg of biologically active EF was easily purified. This is the first report of purification of anthrax EF from E. coli. EF purified from E. coli was biologically and functionally as active as its Bacillus anthracis counterpart. The recombinant protein could compete with lethal factor for binding to protective antigen. Sequence analysis revealed a stretch of seven amino acids, Val Tyr Tyr Glu Ile Gly Lys, present both in EF (residues 136 to 142) and lethal factor (residues 147 to 153). To investigate the role of these seven residues in binding to protective antigen, the residues were individually mutated to alanine in EF. Mutations in residues Tyr137, Tyr138, Ile140, and Lys142 of EF specifically blocked its interaction with anthrax protective antigen. The adenylate cyclase activity of the mutants remained unaffected. The results suggested that residues Tyr137, Tyr138, Ile140, and Lys142 are required for binding of EF to anthrax protective antigen, which facilitates its entry into susceptible cells.

Rapid purification of recombinant anthrax-protective antigen under nondenaturing conditions.

Anthrax-protective antigen is the central moiety of the anthrax toxin complex that mediates the entry of the other two toxin components, lethal factor and edema factor into the cells. It is also the main immunogen of the cell-free vaccine against anthrax. However, in addition to PA, the vaccine contains trace amounts of other culture-derived proteins that contribute to the side effects of the vaccine like pain, edema, erythrema, etc. Thus there is a need to develop high-resolution purification methods to purify PA to homogeneity. In this study we have presented a purification strategy for rapid purification of recombinant protective antigen under nondenaturing conditions, which ensures that not only biological activity but also the conformational integrity of immunological epitopes is well-preserved. The protein was purified to homogeneity in a two-step purification procedure that takes just 6 h for completion. Three milligrams of recombinant protective antigen obtained from 1-liter culture was comparable to B. anthracis protective antigen in terms of functional and biological activity. Moreover, the immunogenicity elicited by the purified protein in mice was also studied. The studies reported here are part of continuing research that aims to provide a safe and efficacious alternative to the current vaccine against anthrax.