Efficacy of short-synthetic antifungal peptides on pathogenic Aspergillus flavus.

The efficacies of three short synthetic antifungal peptides were tested for their inhibitory action on pathogenic fungi, Aspergillus flavus. The sequences of the short synthetic peptides are PPD1- FRLHF, 66-10-FRLKFH, 77-3- FRLKFHF, respectively. These test peptides inhibited fungal growth and showed a membranolytic activity. The fungal biomass and ergosterol levels were significantly low in peptides treated samples. Further, the fungal cell wall component chitin was also found to be lower in peptides treated samples. Scanning electron microscopic images also showed highly wrinkled fungal mycelia. Significant membrane permeabilisation as well as potassium ion leakage was also observed in fungal samples treated with peptides. To assess the membrane damage, the uptake of Sytox green dye was employed. At tested concentration, peptides induced fungal membrane damage as evidenced by the green fluorescence. Further, at tested concentration, these peptides induced an oxidative stress in A.flavus as evidenced by an increase in the ROS production, malondialdehyde levels, increase in the antioxidant enzymes - superoxide dismutase, catalase with concomitant decrease in the reduced glutathione content. Additionally, a growth dependent reduction in aflatoxin levels were also observed in peptides treated samples. Docking studies on the interaction of the peptides with a trans-membrane protein calcium ATPase of A. flavus showed that all the peptides were able to bind to the protein with high z rank score. The activity of the calcium ATPase was significantly decreased in peptides treated fungal samples, thereby validating the docking results. Among all the tested peptides, 77-3 peptide exhibited the maximal membrane damage property.

Anti-inflammatory activity of anti-hyperlipidemic drug, fenofibrate, and its phase-I metabolite fenofibric acid: in silico, in vitro, and in vivo studies.

Fenofibrate, an anti-hyperlipidemic drug and its phase-I biotransformed metabolite fenofibric acid, was studied for COX-1 (PDB ID: 3N8Y) and COX-2 (PDB ID: 1PXX) inhibition potentials in silico and in vitro for their effects on human recombinant COX-2 enzyme isolated from a Baculovirus expression system in sf21 cells (EC 1.14.99.1) using a conventional spectrophotometric assay. Furthermore, the compounds were also screened for their anti-inflammatory potentials in vivo using carrageenan-induced paw oedema method in Wistar rats. The test compounds fenofibric acid, fenofibrate, and the standard drug diclofenac exhibited binding energies of - 9.0, - 7.2, and - 8.0 kcal mol-1, respectively, against COX-2 and - 7.2, - 7.0, and - 6.5 kcal mol-1, respectively, against COX-1. In in vitro studies, both the test compounds inhibited COX-2 enzyme activity. Fenofibric acid showed an IC50 value of 48 nM followed by fenofibrate (82 nM), while diclofenac showed an IC50 value of 58 nM. Furthermore, under in vivo conditions in carrageenan-induced paw oedema rodent model, fenofibric acid exhibited relatively potent anti-inflammatory activity compared with fenofibrate. Hence, we conclude that fenofibric acid and fenofibrate are not only anti-hyperlipidemic but also shows potent anti-inflammatory activity, which may have an additional impact in the treatment of diabetic complications, viz., hyperlipidemia and inflammation leading to atherosclerosis.

Morphology and metal binding characteristics of a natural polymer-kondagogu (Cochlospermum gossypium) gum.

Kondagogu (Cochlospermum gossypium) gum (KG), a natural tree exudate, was investigated for its morphological, adsorption and metal interaction behavior with various toxic heavy metals (Pb, Cd, Ni, Cr and Fe). SEM, AFM and TEM techniques were used to study the morphological changes occurring after metal adsorption onto the biopolymer structure. The degree of biosorption of metals on KG biopolymer surfaces was assessed by small-angle X-ray scattering analysis. EDXA spectrum revealed that the ion-exchange mechanism plays a major role in the binding process between KG and metal ions. The higher electron density observed in the KG-Cd complex suggests that Cd is strongly bound to KG compared to the other metals. This work provides a potential platform for developing a hydrocolloid-based nanogel for bioremediation of environmental contaminants.

MUC1, CK20, and CDX2 immunohistochemical markers can sub-classify periampullary carcinomas into pancreaticobiliary, intestinal, and mixed subtypes.

INTRODUCTION: Pancreaticobiliary subtype of Periampullary carcinoma (PAC) has a poor prognosis in comparison to the intestinal subtype. We assessed the potential of cytokeratins and mucin markers to classify the sub-types of periampullary tumors and compared them with the survival data to identify markers that may predict prognosis. METHODOLOGY: PAC tumor tissues were obtained from 94 patients undergoing Whipples Pancreaticoduodenectomy. Paraffin-embedded tissues were immunostained with cytokeratins CK7, CK20), mucins (MUC1, MUC2, MUC5Ac), and CDX2 antibodies. The survival status of patients was obtained as follow-up up to 5-years of surgery. The Receiver Operating Character Curve (ROC) analysis was used for detecting sensitivity and specificity. The survival data were analyzed using the Kaplan-Meier survival curve. RESULTS: Tumors were initially categorized on the basis of histological classification as pancreaticobiliary (n = 46), intestinal (n = 35) and indeterminate (n = 13). Further, using immunohistochemical markers (MUC1, CK20, and CDX2), we gave systematic classification of IHC-PB (n = 51), IHC-Int (n = 30) and IHC-Mixed (n = 13). The interobserver analysis showed good agreement between histologic and IHC type with a kappa value of 0.554. Combined expression of CK20, MUC1 and CDX2 accurately classify the mixed type of tumor. Overall survival rate and duration were 74.4% and 44.95 ± 2.29 months. Survival analysis for subtypes reveal, pancreaticobiliary tumors have low survival (27.9 ± 1.63 months) than mixed type (35.5 ± 0.45 months) and intestinal-type (52.92 ± 2.18 months). Among these, intestinal-type have better survival. Only TNM Stage III (tumor staging as per American Joint Committee on Cancer classification) and perineural invasion have been associated with predicting poor survival in PAC patients. CONCLUSION: Our results suggest that the combined expression of MUC1, CK20 and CDX2 could serve as markers to diagnose histological inconclusive specimens as mixed subtype tumors.

Synthesis and characterization of cecropin peptide-based silver nanocomposites: Its antibacterial activity and mode of action.

Novel antimicrobial agents with a low propensity to develop resistance by microorganisms have contemporary relevance. In this perspective, the present study reports the green synthesis and characterization of cecropins peptides (D2A21, D2A10, and D4E1) based silver nanocomposites. The effect of pH and concentration of peptides on the formation of nanocomposite material was studied using UV-Vis spectroscopy. The particle size was determined by transmission electron microscopy, which indicated the size in the range of 3 ±â€¯0.4 to 20 ±â€¯5 nm. Fourier-transform infrared spectroscopy studies suggested the involvement of peptides as a capping and reducing agent. Zeta potential analysis suggested that nanocomposite material was more cationic in nature than its native peptides. Nanocomposite material exhibited significantly enhanced antibacterial activity as compared to native peptides and silver nanoparticles with minimum inhibitory concentration (MIC) ranging from 1 to 3 µg mL-1 against both gram-positive and negative test bacteria; whereas the MICs of native peptides were found to be in the range of 4-6 µg mL-1. The mode of action of P-AgNPs was evaluated using scanning electron microscopy, membrane potential, and membrane integrity studies; wherein the nanocomposite material was found to act at the cell membrane level, causing complete loss of membrane potential and resulting in compromised membrane integrity with irreversible damage to the cell as shown by the rapid loss of viability due to membrane disruption, resulting in lysis. Among the three peptides tested, D2A21-silver nanocomposite had maximal antibacterial activity. Taken together; our experimental findings suggested that the peptide-based-silver nanocomposites can be considered as potential antibacterial agents for various biomedical applications.

Purification and characterization of mycoferritin from Aspergillus flavus MTCC 873.

The fungus Aspergillus flavus MTCC 873, a non-toxigenic isolate demonstrated its capability to synthesize mycoferritin (MF) upon induction with iron in yeast extract sucrose (YES) medium. The molecular mass, yield, iron and carbohydrate contents of the MF were 440 kDa, 0.015 mg/g of wet mycelia, 0.8 and 30.4%, respectively. Native gel-electrophoresis revealed a band corresponding to dimeric form of equine spleen ferritin (ESF). Subunit analysis by SDS-PAGE revealed a single protein band with an apparent molecular mass of 24 kDa, suggesting similar sized subunits in the structure of apoferritin shell. Immunological cross-reactivity was observed with the anti-fish liver ferritin. Transmission electron microscopy (TEM) revealed an apparent particle size of 100 A. N-terminal amino acid sequence of MF revealed a sequence of SLPLQDYA, which showed identities with other eukaryotic ferritin sequences. The spectral characteristics (UV/VIS, fluorescence and circular dichroic spectra) were similar to ESF. The fungus, unlike A. parasilicus 255 (non-toxigenic) was incapable of producing allatoxins, when grown in YES media.

Antiaflatoxigenic effects of selected antifungal peptides.

Aflatoxins are potent carcinogenic mycotoxins produced as secondary metabolites mainly by the fungi Aspergillus flavus and Aspergillus parasiticus. Control measures to curtail the contamination of aflatoxin in food products is still a challenge. Although there are several reports on the antifungal peptides, there is no specific study on the action of antifungal peptides on aflatoxin synthesis. This work details the effect of four antimicrobial peptides (AMPs) - PPD1 (FRLHF), 66-10 (FRLKFH), 77-3 (FRLKFHF) and D4E1 (FKLRAKIKVRLRAKIKL) on the aflatoxin production by A. flavus and A. parasiticus. Results of the investigations suggests that AMPs at near minimum inhibitory concentrations (MIC) were effectively inhibiting aflatoxins, without hindering the growth of the fungi. These AMPs, at concentrations near MIC, induced membrane permeabilisation, without inducing cellular leakage. The involvement of oxidative stress for the aflatoxin synthesis was reversed by the antioxidant nature of the peptides as evidenced by the 2,2-diphenyl-1-picrylhydrazyl (DPPH) and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid (ABTS) assay, reactive oxygen species production, malondialdehyde and antioxidant enzymes analysis. Quantitative real time polymerase chain reaction (RT-qPCR) analysis of the aflatoxin gene cluster showed that 'aflR' and its downstream genes expressions were significantly down regulated. Conidiation of the fungi were negatively influenced by the peptides as evidenced by scanning electron microscopy analysis and RT-qPCR. mRNA levels of Manganese-superoxide dismutase (Mn-SOD) showed a decrease in the expression in RT-qPCR. The effect of these peptides on aflatoxin inhibition provides insight into their use as novel antiaflatoxigenic molecules.

Degradation of the insecticidal toxin produced by Bacillus thuringiensis var. kurstaki by extracellular proteases produced by Chrysosporium sp.

AIMS: Some Cry proteins produced by the soil bacterium Bacillus thuringiensis (Bt) or by transgenic Bt plants persist in agricultural soils for an extended period of time, which may pose a hazard for nontarget soil organisms. The aims of our study were to screen for soil fungi capable of degrading the Cry1Ac toxin and to identify the mechanisms that lead to the inactivation of this protein. METHODS AND RESULTS: Of the eight fungal strains screened, only one, Chrysosporium sp., was found to produce extracellular proteases capable of degrading the 66-kDa Cry1Ac at the N-terminal end of amino acid 125 (alanine). The proteolytic products of the Cry1Ac toxin did not exhibit any insecticidal activity against Helicoverpa armigera, in contrast to its high toxicity exhibited in the native form. CONCLUSIONS: Proteases elaborated by the Chrysosporium sp. degrade the Cry1Ac toxin in a way that it looses its insecticidal activity against H. armigera. SIGNIFICANCE AND IMPACT OF THE STUDY: Chrysosporium sp., a specific soil micro-organism capable of producing proteases that degrade the Cry1Ac toxin into inactive products under controlled conditions is being reported for the first time. Application of this observation needs to be further tested in field conditions.

Compositional analysis and rheological properties of gum kondagogu (Cochlospermum gossypium): a tree gum from India.

Gum kondagogu ( Cochlospermum gossypium) is a tree exudate gum that belongs to the family Bixaceae. Compositional analysis of the gum by HPLC and LC-MS revealed uronic acids to be the major component of the polymer ( approximately 26 mol %). Furthermore, analysis of the gum by GC-MS indicated the presence of sugars such as arabinose (2.52 mol %), mannose (8.30 mol %), alpha- d-glucose (2.48 mol %), beta- d-glucose (2.52 mol %), rhamnose (12.85 mol %), galactose (18.95 mol %), d-glucuronic acid (19.26 mol %), beta- d-galactouronic acid (13.22 mol %), and alpha- d-galacturonic acid (11.22 mol %). Gum kondagogu, being rich in rhamnose, galactose, and uronic acids, can be categorized on the basis of its sugar composition as a rhamnogalacturonan type of gum. The rheological measurements performed on the gum suggest that above 0.6% (w/v) it shows a Newtonian behavior and shear rate thinning behavior as a function of gum concentration. The viscoelastic behavior of gum kondagogu solutions (1 and 2%) in aqueous as well as in 100 mM NaCl solution exhibits a typical gel-like system. The G' (viscous modulus)/ G'' (elastic modulus) ratios of native gum kondagogu (1 and 2%) in aqueous solution were found to be 1.89 and 1.85 and those in 100 mM NaCl to be 1.54 and 2.2, respectively, suggesting a weak gel-like property of the polymer. Crossover values of G' and G'' were observed to be at frequencies of 0.432 Hz for 1% and 1.2 Hz for 2% for native gum in aqueous condition, indicating a predominantly liquid- to solid-like behavior, whereas crossover values of 2.1 Hz for 1% and 1.68 Hz for 2% gum in 100 mM NaCl solution suggest a larger elastic contribution.

Binding Studies of Andrographolide with Human Serum Albumin: Molecular Docking, Chromatographic and Spectroscopic Studies.

BACKGROUND: Human serum albumin acts as a carrier protein to a variety of drugs and aids their transport. Andrographis paniculata, a herbal plant has been used as a source of traditional medicine in the Asian countries. Among the various constituents of this plant, andrographolide is the most active and is being used from centuries in the treatment of many chronic and infectious diseases. OBJECTIVE: The present study was designed to evaluate the interaction and binding affinity of andrographolide with HSA, by molecular docking, chromatographic and spectral studies. METHODS: Andrographolide was docked with crystal structure of human serum albumin (1AO6) using Auto Dock Vina software and the interactions were analyzed by a visualizing software py- MOL. For further characterization and confirmation, andrographolide (3x10-5 M) and HSA (0.001, 0.005, 0.01, 0.02, 0.04 M) sample mixtures were incubated at 37°C for 3h in a metabolic shaker, followed by centrifugation. The supernatant and the filtrate were analyzed by UV spectroscopy, HPLC, CD and FTIR spectral analysis. RESULTS: The docking studies revealed that andrographolide interacted with HSA and formed hydrogen bonds with Trp 214, Arg 218 and Lys 444 amino acid residues. The UV spectral analysis revealed a decrease in the absorption peak of HSA due to its interaction with andrographolide. A new peak was observed at retention time 7.45 min by HPLC analysis and the Bmax was found to be 7.5 ± 0.4 mg protein with a Kd value of 1.89 mM, indicating interaction of andrographolide with HSA. The CD spectra results suggested, a marginal decrease in the negative ellipticity without any significant shift in peak, indicating the stabilization of the HSA-andrographolide complex. The FTIR analysis of the andrographolide-HSA mixture showed a peak at wave number 1637 cm-1 (a shift of amide I groups from 1646 cm-1) and 1016 cm-1 which corresponded to the ligand, confirming the complex formation. CONCLUSION: The molecular docking studies demonstrated the interactions of andrographolide to the crystal structure of HSA. The chromatographic and spectroscopic analysis confirmed the binding of andrographolide with HSA and their complex formation. Overall the present studies conclude the binding of andrographolide to HSA protein, favoring its pharmacokinetics.

Influence of extremely low frequency magnetic fields on Ca2+ signaling and NMDA receptor functions in rat hippocampus.

Extremely low frequency (ELF<300Hz) electromagnetic fields affect several neuronal activities including memory. Because ELF magnetic fields cause altered Ca(2+) homeostasis in neural tissues, we examined their influence on Ca(2+) signaling enzymes in hippocampus and related them with NMDA receptor functions. Hippocampal regions were obtained from brains of 21-day-old rats that were exposed for 90 days to 50Hz magnetic fields at 50 and 100 microT intensities. In comparison to controls, ELF exposure caused increased intracellular Ca(2+) levels concomitant with increased activities of Ca(2+)-dependent protein kinase C (PKC), cAMP-dependent protein kinase and calcineurin as well as decreased activity of Ca(2+)-calmodulin-dependent protein kinase in hippocampal regions. Simultaneous ligand-binding studies revealed decreased binding to N-methyl-D-aspartic acid (NMDA) receptors. The combined results suggest that perturbed neuronal functions caused by ELF exposure may involve altered Ca(2+) signaling events contributing to aberrant NMDA receptor activities.

Epidemiological profile of snake-bite cases from Andhra Pradesh using immunoanalytical approach.

BACKGROUND & OBJECTIVE: Snake-bites are the common cause of morbidity and mortality in tropical countries. In India, there are 216 species of snakes, of which only four are venomous snakes (cobra, krait, Russell's viper and saw scaled viper). This study was undertaken to find out the epidemiological profile of snake-bite incidences in the State of Andhra Pradesh, based on the data collected from State Forensic Science Laboratory, Hyderabad. METHODS: Data from 1379 snake-bite cases were collected from case reports for a 5 yr period (1999- 2003) that included age and sex of the victim, district, month of incidence, time of incident, death of a victim and the time point of analysis. On the basis of the forensic data, specimens were collected from forensic medicine department, during rainy season and were analysed for the venom antigens (cobra and krait) by ELISA method. RESULTS: The peak number of snake-bite cases were seen during June-September. Majority of the cases were observed in the age group 21-50 yr (71%). Higher incidence of snake-bite was recorded in males (76%). Of the 22 cases analysed by the ELISA, 6 tested positive for cobra venom, while 8 cases tested positive for krait venom, the remaining specimens tested negative for both cobra and krait venom. INTERPRETATION & CONCLUSION: Evaluation of forensic specimens (autopsy & biopsy) of human snakebite victims based on specific molecular epidemiological tool like ELISA gives a true estimate of the incidence supplementing clinical and circumstantial evidence.

Use of egg yolk antibody (IgY) as an immunoanalytical tool in the detection of Indian cobra (Naja naja naja) venom in biological samples of forensic origin.

An immunoglobulin Y (IgY) based indirect double antibody sandwich enzyme linked immunosorbent assay (ELISA) was developed for the detection of Indian cobra (Naja naja naja) venom in the biological samples of forensic origin. Polyclonal antibodies were raised and purified from chick egg yolk and rabbit serum. The cobra venom was sandwiched between immobilized affinity purified IgY and the rabbit IgG. The detection concentration of cobra venom was in the range of 0.1 to 300ng. The calibration plot was based on linear regression analysis (y=0.2581x+0.4375, r(2)=0.9886). The limit of detection of the assay was found to be 0.1ng. The coefficient of variation (CV) of different concentrations of working range in inter (n=6) and intra-assay (n=6) was observed to be less than 10%. The recovery of venom was found to be in the range of 80-99%, when different concentrations (0.002, 0.1, 0.2, 1, and 2microg) of cobra venom were spiked to pooled normal human serum (ml(-1)). No cross reactivity was observed with krait and viper venom in the immunoassay system in the concentration range of 0.1-1000ng. The method was initially, validated by analyzing specimens (autopsy) of experimental rats injected with cobra venom (1.2mgkg(-1) body mass). Further, human specimens (autopsy and biopsy) of snake bite victims of forensic origin were also analyzed. The methodology developed may find diagnostic application in forensic laboratories.

Metabolic inhibition of meloxicam by specific CYP2C9 inhibitors in Cunninghamella blakesleeana NCIM 687: in silico and in vitro studies.

Specific inhibitors of Cytochrome P4502C9 enzyme (CYP2C9) viz. clopidogrel, fenofibrate fluvoxamine and sertraline at concentration of 50, 100, 150 and 200 µM were employed to investigate the nature of enzyme involved in bioconversion of meloxicam to its main metabolite 5-OH methyl meloxicam by Cunninghamella blakesleeana. Virtual screening for interaction of specific CYP2C9 inhibitors with human CYP2C9 enzyme was performed by molecular docking using Auto dock vina 4.2 version. The in silico studies were further substantiated by in vitro studies, which indicated fenofibrate to be a potent inhibitor of CYP2C9 enzyme followed by sertraline, clopidogrel and fluvoxamine, respectively. Two-stage fermentation protocol was followed to study metabolism of meloxicam and its inhibition by different CYP2C9 inhibitors. Meloxicam metabolites were identified using HPLC, LC-MS analysis and based on previous reports, as 5-OH methyl meloxicam (M1), 5-carboxy meloxicam (M2) and an unidentified metabolite (M3). All the inhibitors tested in the study showed a clear concentration dependent inhibition of meloxicam metabolism. The results suggest that the enzymes involved in metabolism of meloxicam in C. blakesleeana are akin to mammalian metabolism. Hence, C. blakesleeana can be used as a model organism in studying drug interactions and also in predicting mammalian drug metabolism.

Purification and characterization of mycoferritin from Aspergillus parasiticus (255).

As intracellular iron storage molecules, only hydroxymate type siderophores have been reported in ascomycetes and basidiomycetes. This is the first report documenting the presence of mycoferritin in ascomycetes. The fungus, Aspergillus parasiticus (255), is capable of producing mycoferritin only upon induction with iron in yeast extract sucrose (YES) medium. The same has been purified from Aspergillus sps by application of conventional biochemical techniques. The molecular mass, yield, iron and carbohydrate contents of the HPLC purified protein were 460kDa, 0.012mg/g of wet mycelia, 1.6% and 6.0%, respectively. The iron content was much lower than Mortierella alpina mycoferritin (17%). Native PAGE revealed the presence of trimeric and monomeric forms of ferritin. Subunit analysis by SDS-PAGE showed a single protein subunit of approximately 20kDa suggesting structural simplicity of the apoferritin shell. Variation in amino acid composition was noted upon comparison with ferritins of other species. Interestingly, no phenylalanine could be detected in the mycoferritin of Aspergillus sps. The acidic amino acid content was 1.5-1.6 fold higher than mammalian and fish ferritins. The spectral characteristics (UV/VIS and fluorescence) of mycoferritin were akin to equine spleen ferritin. However, circular dichroic spectra revealed a lower degree of helicity.

Role of mycoferritin from Aspergillus parasiticus (255) in secondary metabolism (aflatoxin production).

Aspergillus parasiticus (255), a non-toxigenic isolate showed the presence of secondary metabolites-aflatoxins (B1, B2, G1, G2) when grown in yeast extract sucrose media but not in basal media, thus demonstrating its toxigenic potential. Native PAGE of the crude protein isolated at different growth periods of A. parasiticus in yeast extract sucrose media containing iron showed prominent expression of mycoferritin from day four onwards. The production of aflatoxins was also maximal on day four, both in the presence and absence of iron. Indicators of oxidative stress metabolites such as reactive oxygen species, thiobarbituric acid reactive species, reduced and oxidized glutathione and antioxidant enzymes like superoxide dismutase and glutathione peroxidase were analyzed both in the presence and absence of iron and the experimental data suggest oxidative stress as a pre-requisite for aflatoxin production. The pro-oxidant role of iron was minimized by induction of mycoferritin and the concomitant alterations in oxidative stress parameters imply an antioxidant role to mycoferritin in secondary metabolism, a finding of significance that has not been reported previously in fungal systems.

Source identification of Indian opium based on chromatographic fingerprinting of amino acids.

Total and free pool of amino acids was determined in Indian opium samples using liquid chromatography (LC) with post-column opthalaldehyde derivatization followed by its fluorimetric detection. The limit of detection (LOD) was found to be in the range of 2-10 pmol with a signal to noise ratio of 3:1 and limit of quantitation (LOQ) was found to be in the range of 7-31 pmol with a signal to noise ratio of 10:1. The recovery of amino acids was found to be in the range of 86-103%. A total of 124 Indian opium samples were collected from the states of Madhya Pradesh (MP), Uttar Pradesh (UP) and Rajasthan (Raj), covering 14 licit opium growing divisions of India were chromatographically fingerprinted for the presence of various amino acids. The amino acids identified in sample hydrosylate included D, T, S, S, G, A, V, I, L, Y, F, H, K and R, while the analysis of free pool of amino acids (80% aqueous ethanol extract) indicated the presence of D, T, S, E, A, V, I, L, Y, H, K respectively. Multiple discriminant analysis was applied to the quantitative total amino acid data to determine an optimal classifier in order to evaluate the source of Indian opium. The foremost amino acid variables that accounted for the true discrimination were identified as D, E, G, A, F and K in evaluating the geographical origin of Indian opium and the predictive value based on the discriminant analysis was found to be 90% in relation to the source of opium samples. Chemometrics performed with amino acid analytical data was used successfully in discriminating the licit opium growing divisions of India into three major groups, viz. groups I, II and III. The methodology developed may find wide application in forensic analysis.

Bioprospecting of gum kondagogu (Cochlospermum gossypium) for bioremediation of uranium (VI) from aqueous solution and synthetic nuclear power reactor effluents.

An ecofriendly green chemistry method using a natural biopolymer, Gum Kondagogu (GK) for the removal of U (VI) from aqueous, simulated nuclear effluents was studied. The adsorption characteristic of GK towards U (VI) from aqueous solution was studied at varied pH, contact time, adsorbent dose, initial U (VI) concentration and temperature using UV-Visible spectroscopy and ICP-MS. Maximum adsorption was seen at pH 4, 0.1% GK with 60 min contact time at room temperature. The GK- U (VI) composite was characterized by FT-IR, zeta potential, TEM and SEM-EDAX. The Langmuir isotherm was found to be 487 mg of U (VI) g(-1) of GK. The adsorption capacity and (%) of U (VI) was found to be 490 ± 5.4 mg g(-1) and 98.5%. Moreover adsorption of U (VI) by GK was not influenced by other cations present in the simulated effluents. The adsorbed U (VI) was efficiently stripped from composite using 1 M HCl.

Quantitation of epsilon-amino group using amino acids as reference standards by trinitrobenzene sulfonic acid. A simple spectrophotometric method for the estimation of hapten to carrier protein ratio.

A simple, sensitive and direct spectrophotometric method is presented for the determination of epsilon-amino groups of L-lysine present in carrier proteins, using the free amino acids L-lysine and L-glutamic acid as reference standards, and 2,4,6-trinitrobenzene 1-sulfonic acid (TNBS) reagent. The amount of epsilon-amino group present in the carrier protein after coupling with hapten is directly quantitated using the standard curve generated by the difference in absorbance observed with L-lysine and L-glutamic acid after their reaction with TNBS reagent. Spectral analysis of L-lysine and L-glutamic acid (27.36 nmol) derivatives of TNBS at 335 nm showed that TNP-L-lysine had twice the absorbance of TNP-L-glutamic acid, since TNBS reagent interacts equally with the alpha-amino and epsilon-amino groups present in L-lysine and the alpha-amino group of L-glutamic acid, respectively. The relationship between absorbance and concentration of epsilon-amino groups (up to 16 micrograms/ml) was found to be linear. The number of epsilon-amino groups of lysine present in carrier proteins such as BSA, HSA, thyroglobulin and the enzyme, horseradish peroxidase, were analyzed by the present method and were found to be similar to the reported values. Various carrier protein-hapten conjugates (protein-mycotoxin/vitamin/steroid hormone conjugates) were made and analyzed by the method developed in order to determine their mole to mole ratio.

Dip-strip method for monitoring environmental contamination of aflatoxin in food and feed: use of a portable aflatoxin detection kit.

Aflatoxin contamination of food and feed have gained global significance due to its deleterious effect on human and animal health and its importance in the international trade. The potential of aflatoxin as a carcinogen, mutagen, teratogen, and immunosuppressive agent is well documented. The problem of aflatoxin contamination of food and feed has led to the enactment of various legislation. However, meaningful strategies for implementation of this legislation is limited by nonavailability of simple, cost-effective method for screening and detection of aflatoxin under field conditions. Keeping in mind the analytical constraints in developing countries, a simple-to-operate, rapid, reliable, and cost-effective portable aflatoxin detection kit has been developed. The important components of the kit include a hand-held UV lamp (365 nm, 4 W output), a solvent blender (12,000 rpm) for toxin extraction, and adsorbent-coated dip-strips (polyester film) for detecting and quantifying aflatoxin. Analysis of variance indicates that there were no significant differences between various batches of dip-strips (p > 0.05). The minimum detection limit for aflatoxin B1 was 10 ppb per spot. The kit may find wide application as a research tool in public health laboratories, environmental monitoring agencies, and in the poultry industry.