Survey of veterinery drug residues, environmental contaminants and mycotoxins of cultured shirmp in Iran

Economical achievement of optimal growth in developing countries may lead to sustainable poverty reduction. Agricultural activities play an important role in economy and human being welfare, which leads to establishment of food security and quality. Aquaculture products in developing countries share 51.4 percent of total agricultural production and 241 percent in developed countries. Therefore undoubtedly food production by means of quality and quantity has to be increased .The history of shrimp production goes back to 500 years ago. Today 50 countries of the world produce shrimp. In Islamic Republic of Iran shrimp production started since 1992 in the coastal region of Persian Gulf. The shrimp culture farms can be classified in to 4 different categories; extensive, semi-extensive, intensive and super intensive. Global ecological maintenance is one of the major concerns of authorities Human manipulation of nature is the most destructive activity. Industrial sewage leakage in to the rivers and water sources is a big issue that causes reduction in the aquatic population. Heavy metals have an inhibitory effect in the production and growth of sea life. Human intake of food treated with anti-microbial cause's allergy, hypersensitivity and develops microbial resistance. Organochlorine compounds contamination may found in hepatopancreatic tissue of aquatic products, Arsenic may transfer to man via plant and animal product contamination.

C-terminal residues of skeletal muscle calsequestrin are essential for calcium binding and for skeletal ryanodine receptor inhibition

Bioactive compounds through anaerobic digestion of heterotrophic microalgae residues

Several important biomolecules are available into anaerobically digested effluents that were obtained from the biodiesel production process using heterotrophically grown microalga Chlorella protothecoides. Defatted microalgae residues and crude glycerol may undergo anaerobic digestion, separately and in admixture, providing methane/hydrogen and a digestate exploitable for agriculture applications. Furthermore, industrial interesting bioactive compounds such as polyphenols provided with antioxidant activity can be obtained. Anaerobic process offers a promising chance and can be advantageously combined with algae lipid-extraction techniques in order to make it more sustainable.

Accumulation, persistence and effects of indospicine residues in camels fed Indigofera plant

Indospicine (L-2-amino-6-amidinohexanoic acid) is a natural hepatotoxin found in all parts of some Indigofera plants such as I. linnaei and I. spicata. Several studies have documented a susceptibility to this hepatotoxin in different species of animals, including cattle, sheep, dogs and rats, which are associated with mild to severe liver disease after prolonged ingestion. However, there is little published data on the effects of this hepatotoxin in camels, even though Indigofera plants are known to be palatable to camels in central Australia. The secondary poisoning of dogs after prolonged dietary exposure to residual indospicine in camel muscle has raised additional food safety concerns. In this study, a feeding experiment was conducted to investigate the in vivo accumulation, excretion, distribution and histopathological effects of dietary indospicine on camels. Six young camels (2 – 4 year old), weighing 270 − 390 kg were fed daily a roughage diet consisting of Rhodes grass hay and lucerne chaff, supplemented with Indigofera and steam flaked barley. Indigofera (I. spicata) was offered at 597 mg DM/kg body weight (bw)/day designed to deliver 337 µg indospicine/kg bw/day, and fed for a period of 32 days. Blood and muscle biopsies were collected over the period of the study. Concentrations of indospicine in the plasma and muscle biopsy samples were quantitated by validated ultra-performance liquid chromatography−tandem mass spectrometry (UPLC−MS/MS). The highest concentrations in plasma (1.01 mg/L) and muscle (2.63 mg/kg fresh weight (fw)) were found at necropsy (day 33). Other tissues were also collected at necropsy and analysis showed ubiquitous distribution of indospicine, with the highest indospicine accumulation detected in the pancreas (4.86 ± 0.56 mg/kg fw) and liver (3.60 ± 1.34 mg/kg fw); followed by the muscle, heart and kidney. Histopathological examination of liver tissue showed multiple small foci of predominantly mononuclear inflammatory cells. After cessation of Indigofera intake, indospicine present in plasma in the remaining 3 camels had a longer terminal elimination half-life (18.6 days) than muscle (15.9 days), and both demonstrated mono-exponential decreases.

A survey on prevalence rate of aflatoxigenic species of Aspergillus and residues of aflatoxins by ELISA method in cold-water cultural fish (rainbow trout) feeds in Tehran and West Azarbayjan provinces-Iran

Aflatoxins are one kind of fungal toxins produced by species of toxigenic Aspergillus (A. flavus and A. parasiticus) and in other words they are secondary metabolites which are considered as one of the threatening factors of food consumer's health. In this research 96 samples of cold-water cultural fish feed, rainbow trout, during the seasons of spring and summer of 2007 (every fifteenth of the month) were randomized (by simple and stratified random) to determine: 1. The prevalence rate of aflatoxigenic species of Aspergillus in stored feed of cold-water cultural fish in West Azarbayjan cultural fish farms in both seasons (spring and summer); 2. The residues of total aflatoxin in stored feed of fish in cultural fish farms of West Azarbayjan in both seasons by ELISA method; and 3. The residues of that toxin in feed produced in aquatic feed factories in Tehran and West Azarbyjan provinces with the same method. In order to study prevalence rate of toxigenic species of Aspergillus, pour-plate culture method by general medium such as Malt Extract Agar (M.E.A.) and Sabouraud-Dextrose Agar (S.D.A.) and by standard No.997 of Iranian Standard Institute were used. The produced colonies were examined microscopically. To determine the aflatoxins residues, ELISA method using Agra-Quant kit of Romer Lab company, were applied. The results of this survey indicated that only 8.3% of the samples were infected by A. flavus. A. parasiticus was not observed. There were no significant differences between the prevalence rate of AFT and seasons/months, either (P<0.05). Evaluating mean of aflatoxin rate showed that the rates of this variable are lower than the tolerance levels designated by the joint FAO/WHO expert committee (The mean of AFT in all data was lower than 11 ppb). Furthermore, mean of total AFT residues rates of stored feed of various cultural center of West Azarbayjan and Tehran factories were comparable in spring and summer, and no significant differences were observed (P<0.05). But there were significant differences between the total aflatoxin rates in the feed of West Azarbayjan factory and spring and summer (P<0.05), and AFT residues in spring (8.6 ppb) were higher than summer (6.1 ppb). Prevalence rates of AFT in Tehran feed factories (9.2 ppb) are higher than W. Azarbayjan (7.4 ppb). In other words, location was considered as a decisive factor in total AFT rates of samples. Moreover, the results indicated that there was significant difference between total aflatoxin rates of feed and cultural centers (P<0.05). The mean of AFT rates in embankment dam cultural fish farms (6.75 ppb) and multi-functions cultural fish farms (6.25 ppb) was higher than individual cultural pond (4.67 ppb). In conclusion, the finally results of this survey indicated that the lower rates of Aspergillus is not effective on the presence of total aflatoxin rates in trout feed. Due to low levels of aflatoxin rates (lower than 20 ppb), the produced feed of cold-cultural fishes, Rainbow Trout, in Tehran and West Azarbayjan provinces, in spring and summer of 2007, were safe and healthy both for fish and their consumers.

Construção de modelos de interação in silico e in vitro do inibidor do tipo Kunitz de Adenanthera pavonina L. para as enzimas cisteínicas e serínicas

Serines proteinases inhibitors (PIs) are widely distributed in nature and are able to inhibit both in vitro and in vivo enzymatic activites. Seed PIs in than leguminous are classified in seven families, Bowman-Birk and Kunitz type families that most studied representing an important role in the first line of defense toward insects pests. Some Kunitz type inhibitors possess activities serine and cysteine for proteinases named bifunctional inhibitor, as ApTKI the inhibitor isolate from seed of Adenanthera pavonina. The A. pavonina inhibitor presenting the uncommon property and was used for interaction studies between proteinases serine (trypsin) and cysteine (papain). In order to determinate the in vitro interaction of ApTKI against enzymes inhibitor purification was carried cut by using chromatographic techniques and inhibition assays. The 3D model of the bifunctional inhibitor ApTKI was constructed SWISS-MODEL program by homology modeling using soybean trypsin inhibitor (STI, pdb:1ba7), as template which presented 40% of identity to A. pavonina inhibitor. Model quality was evaluated by PROCHECK program. Moreover in silico analyzes of formed complex between the enzymes and ApTKI was evaluated by HEX 4.5 program. In vitro results confirmed the inhibitory assays, where the inhibitor presented the ability to simultaneously inhibit trypsin and papain. The residues encountered in the inhibitor model of folder structural three-dimensional that make contact to enzymes target coud explain the specificity pattern against serine and cysteine proteinases

Dual layer solid phase extraction based on molecular imprinting technology: Seeking a route to enhance selectivity for trace analysis of pesticide residues in olive oil.

Aiming to introduce a multiresidue analysis for the trace detection of pesticide residues belonging to organophosphorus and triazine classes from olive oil samples, a new sample preparation methodology comprising the use of a dual layer of “tailor-made” molecularly imprinted polymers (MIPs) SPE for the simultaneous extraction of both pesticides in a single procedure has been attempted. This work has focused on the implementation of a dual MIP-layer SPE procedure (DL-MISPE) encompassing the use of two MIP layers as specific sorbents. In order to achieve higher recovery rates, the amount of MIP layers has been optimized as well as the influence of MIP packaging order. The optimized DL-MISPE approach has been used in the preconcentration of spiked organic olive oil samples with concentrations of dimethoate and terbuthylazine similar to the maximum residue limits and further quantification by HPLC. High recovery rates for dimethoate (95%) and terbuthylazine (94%) have been achieved with good accuracy and precision. Overall, this work constitutes the first attempt on the development of a dual pesticide residue methodology for the trace analysis of pesticide residues based on molecular imprinting technology. Thus, DL-MISPE constitutes a reliable, robust, and sensitive sample preparation methodology that enables preconcentration of the target pesticides in complex olive oil samples, even at levels similar to the maximum residue limits enforced by the legislation.

An innovative rapid method for analysis of 10 organophosphorus pesticide residues in wheat by HS-SPME-GC-FPD/MSD

Characterization of a new platelet aggregating factor from crotoxin Crotalus durissus cascavella venom

In this article we investigated the platelet aggregating activity of whole crotoxin and its subunits isolated from Crotalus durissus cascavella venom. During the purification protocols of the venom, using HPLC molecular exclusion, we detected the presence of two different serine protease activities in the gyroxin fraction, and another in the crotoxin fraction, which induced strong and irreversible platelet aggregation, in addition to blood coagulation. From crotoxin, we isolated PLA(2), crotapotin (both fractions corresponding approximately 85% of whole crotoxin) and another minor fraction (F20) that exhibited serine protease activity. After a new fractionation on reverse phase HPLC chromatography, we obtained three other fractions named as F201, F202 and F203. F202 was obtained with high degree of molecular homogeneity with molecular mass of approximately 28 kDa and a high content of acidic amino residues, such as aspartic acid and glutamic acid. Other important amino acids were histidine, cysteine and lysine. This protein exhibited a high specificity for BApNA, a Michaelis-Menten behavior with Vmax estimated in 5.64 mu M/min and a Km value of 0.58 mM for this substrate. In this work, we investigated the ability of F202 to degrade fibrinogen and observed alpha and beta chain cleavage. Enzymatic as well as the platelet aggregation activities were strongly inhibited when incubated with TLCK and PMSF, specific inhibitors of serine protease. Also, F202 induced platelet aggregation in washed and platelet-rich plasma, and in both cases, TLCK inhibited its activity. The N-terminal amino acid sequence of F202 presented a high amino acid sequence homology with other thrombin-like proteins, but it was significantly different from gyroxin. These results showed that crotoxin is a highly heterogeneous protein composed of PLA(2), thrombin-like and other fractions that might explain the diversity of physiological and pharmacological activities of this protein.

Sensor for cysteine based on oxovanadium(IV) complex of Salen modified carbon paste electrode

The electrochemical behavior of a carbon paste electrode modified (CPEM) with N,N&PRIME;-ethylenebis(salicylideneiminato)oxovanadium(IV) complex ([(VO)-O-IV(Salen)]) was investigated as a new sensor for cysteine. Cyclic voltammetry at the modified electrode in 0.1 mol L-1 KCl Solution (pH 5.0) showed a single-electron reduction/oxidation of the Couple VO3+/VO2+. The CPEM with [VO(Salen)] presented good electrochemical stability in a wide pH range (4.0-10.0) and an ability to electrooxidate cysteine at 0.65 V versus SCE. These results demonstrate the viability of the use of this modified electrode as an amperometric sensor for cysteine determination. © 2004 Elsevier B.V. All rights reserved.

Structural Aspects of the Distinct Biochemical Properties of Glutaredoxin 1 and Glutaredoxin 2 from Saccharomyces cerevisiae

Glutaredoxins (Grxs) are small (9-12 kDa) heat-stable proteins that are ubiquitously distributed. In Saccharomyces cerevisiae, seven Grx enzymes have been identified. Two of them (yGrx1 and yGrx2) are dithiolic, possessing a conserved Cys-Pro-Tyr-Cys motif. Here, we show that yGrx2 has a specific activity 15 times higher than that of yGrx1, although these two oxidoreductases share 64% identity and 85% similarity with respect to their amino acid sequences. Further characterization of the enzymatic activities through two-substrate kinetics analysis revealed that yGrx2 possesses a lower Km for glutathione and a higher turnover than yGrx1. To better comprehend these biochemical differences, the pK(a) of the N-terminal active-site cysteines (Cys27) of these two proteins and of the yGrx2-C30S mutant were determined. Since the pK(a) values of the yGrx1 and yGix2 Cys27 residues are very similar, these parameters cannot account for the difference observed between their specific activities. Therefore, crystal structures of yGrx2 in the oxidized form and with a glutathionyl mixed disulfide were determined at resolutions of 2.05 and 1.91 angstrom, respectively. Comparisons of yGrx2 structures with the recently determined structures of yGrx1 provided insights into their remarkable functional divergence. We hypothesize that the substitutions of Ser23 and Gln52 in yGrx1 by Ala23 and Glu52 in yGrx2 modify the capability of the active-site C-terminal cysteine to attack the mixed disulfide between the N-terminal active-site cysteine and the glutathione molecule. Mutagenesis studies supported this hypothesis. The observed structural and functional differences between yGrx1 and yGrx2 may reflect variations in substrate specificity. (C) 2008 Elsevier Ltd. All rights reserved.

Evaluation of solid residues removed from a mangrove swamp in the Sao Vicente Estuary, SP, Brazil

Mangrove swamps are found in estuaries along the coastal plains of tropical regions and have be subjected to heavy occupation and use pressure due to their privileged locations and abundance of biological resources. The present work evaluated the ecological characteristics and solid wastes accumulated in eight areas along the Santos - Sao Vicente Estuary Complex. The superficially deposited residues at each sampling site were collected and subsequently washed, drained, counted, weighed and separated into classes according to their composition and predominant use. The predominant litter type in terms of density was plastic (62.81%) and, by weight, wood (55.53%). The greatest deposition of residues was associated with areas that were less inclined and that had low plant density levels, indicating that the presence of obstacles was not critical for retaining floating residues in mangrove areas. The presence of the most frequently encountered types of solid waste residues could be explained by local activities. (C) 2010 Elsevier Ltd. All rights reserved.

Development of a novel platform for high-throughput gene design and artificial gene synthesis to produce large libraries of recombinant venom peptides for drug discovery

Tese de Doutoramento em Ciências Veterinárias na Especialidade de Ciências Biológicas e Biomédicas