Biochemical and antioxidant alterations in fresh water bivalve, Lamellidens marginalis exposed to malachite green dye

Aim: To determine acute toxicity (96 hrs) of malachite green for Lamellidens marginalis and to assess it’s biochemical consequences and antioxidant response against control. Methodology: Fresh water bivalve, Lamellidens marginalis were collected from Rajaram tank, Kolhapur and acclimatized for 7 days. To ascertain the acute effect (96 hrs) of malachite green dye, static bioassay was conducted at 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2 ppm on fresh water bivalve, Lamellidens marginalis. The rate of mortality was recorded and LC0 and LC50 values for 96 hrs were calculated. Estimation of biomolecules like protein, glycogen and lipid and antioxidant activity in different tissues of the organism was estimated. Results: The LC0 and LC50 values were determined by static bioassay method. The effects of LC0 and LC50 concentrations of MG were evaluated by examining the biochemical profile and antioxidant response against control in different tissues like gill, mantle, hepatopancreas and gonad. The maximum significant decrease in glycogen (-29%) (-40.86) and protein (-31.73%) (-43.63%) content was observed in gills at both the concentrations LC0 and LC50 respectively as compared to other tissues. There was highly significant depletion in protein content of all the tissues. Lipid content in all tissues at LC0 concentration showed moderately significant depletion (p <0.01), while at LC50 concentration highly significant depletion (p< 0.001) was recorded. From SOD, CAT and GPx enzymes, SOD enzyme activity increased significantly (p < 0.001) in all tissues at LC50 concentration. Gill and heptopancreas showed significant increase in antioxidant enzymes response at both, LC0 and LC50 groups. Interpretation: Malachite dye induces toxicity, by lowering biochemical content and inducing antioxidant enzyme activity.

Bacterial expression, preparation and identification of polyclonal antibody against Escherichia coli FtsZ / 生物工程学报

To prepare polyclonal antibody (PcAb) against Escherichia coli filamentous thermosensitive protein Z (Ec-FtsZ), the artificially synthesized gene fragment coding Ec-FtsZ was subcloned into pET-22b(+) plasmid, and Ec-FtsZ protein was expressed in E. coli BL21(DE3) cell under an optimal bacterial expression condition. Then Ec-FtsZ protein was purified by HisTrap affinity chromatography, and the GTPase (Guanosine triphosphatase) activity of purified Ec-FtsZ protein was further analyzed by malachite green assay. Subsequently, the purified Ec-FtsZ protein was used to immunize rat subcutaneously for preparation of anti-Ec-FtsZ PcAb. The results of enzyme-linked immunosorbent assay (ELISA), Western blotting analysis and immunofluorescence assay showed that the titer of PcAb was 1:256 000, and PcAb exhibited a perfect antigenic specificity against purified and endogenous Ec-FtsZ protein. All these data indicated that the anti-Ec-FtsZ PcAb is successfully prepared, which can be used for further cellular function study and biochemical analysis of Ec-FtsZ protein in vivo.

Reator anaeróbio tipo UASB conjugado com processo Fenton para remoção de cor e demanda química de oxigênio de água residuária sintética de indústria têxtil / Anaerobic reactor type UASB conjunction with process Fenton to remove color and chemical oxygen demand textile industry of synthetic wastewater

RESUMO A contaminação por despejos de efluentes industriais têxteis tem sido uma preocupação emergente de pesquisadores e ambientalistas, pois esses apresentam composição extremamente heterogênea e grande quantidade de material tóxico e recalcitrante, o que dificulta seu tratamento. Durante o processamento têxtil, uma ampla gama de corantes é liberada e alguns desses, como os azo corantes, que se caracterizam pela função azo (-N=N-) ligada a grupos aromáticos e podem ser tóxicos, carcinogênicos e/ou mutagênicos. Em vista disso, esta pesquisa teve como principal objetivo avaliar os benefícios da utilização de um reator anaeróbio tipo reator anaeróbico de fluxo ascendente com manta de lodo (UABS), seguido de processo oxidativo avançado (POA) do tipo Fenton na degradação de cor e demanda química de oxigênio (DQO) de efluente sintético de indústria têxtil. Com os resultados, foram verificadas remoções de DQO em torno de 82,0% para o reator UASB e de 95,6% para o conjunto. A cor alcançou 96,1% de remoção no reator UASB e 100,0% ao final do processo.

ABSTRACT Contamination by textile industrial wastewater discharges has been an emerging concern of researchers and environmentalists, as they have extremely heterogeneous composition and loads of toxic and recalcitrant material, which complicates treatment. In the textile processing, a wide range of dye is released and some of these, such as dyes, azo, characterized by the feature azo (-N=N-) attached to aromatic groups and may be toxic, carcinogenic and/or mutagenic. In view of this, this research aimed to evaluate the benefits of using an anaerobic reactor type anaerobic reactor upflow sludge blanket (UABS), followed by advanced oxidation process (AOP) type Fenton in color degradation and chemical oxygen demand (COD) of synthetic textile industry effluent. From the results of COD removal was observed at around 82.0% for the UASB reactor and 95.6% for the group. The color reached 96.1% removal in UASB reactor and 100.0% at the end of the process.

Partial removal and detoxification of Malachite Green and Crystal Violet from laboratory artificially contaminated water by Pleurotus ostreatus / Eliminación parcial y desintoxicación de Verde Malaquita y Cristal Violeta del laboratorio con agua contaminada artificialmente por Pleurotus ostreatus / Extração e desintoxicação de Verde Malaquita e Cristal Violeta de água contaminada artificialmente em laboratório por Pleurotus ostreatus

Abstract The triphenylmethane Malachite Green (MG) and Crystal Violet (CV) dyes are cationic dyes and mix with domestic wastewater when dumped; increasing, among others, the chemical and biological oxygen demand and can cause acute toxicity at different trophic levels. Promoting the removal (decolorization) of MG and CV, and laccase activity (54.8 ± 8.9 and 30.6 ± 2.9 UL-1 respectively) by using P. ostreatus viable biomass needed parameters such as pH (4.5 and 6.0), temperature (25 to 30 °C), stirring speed (120 rpm), percentage of inoculum (2% v/v), and dye concentration (20 and 10 mg L-1). In adsorption studies, it was showed that an acidic pH favors the adsorption of both dyes and the model of pseudo-second order describes best the phenomenon of adsorption. Finally, the germination index (GI), using Lactuca sativa seeds for the initial dyes solutions, was < 50%; demonstrating its high phytotoxic effect. When dye solutions were treated with viable biomass, the GI increased, leaving open the possibility to perform future research to determine if the aqueous solutions, post-treated with P. ostreatus, could be used in treatments that generate less toxic water which could be used in processes that do not require potable water.

Resumen Los colorantes trifenilmetánicos Verde Malaquita (MG) y Crystal Violeta (CV) son catiónicos y al ser vertidos se mezclan con aguas residuales domésticas, incrementando, entre otros, la demanda química y biológica de oxígeno; pudiendo causar toxicidad aguda en diferentes niveles tróficos. En este estudio se encontró que los parámetros pH (4,5 y 6,0), temperatura (25 y 30 °C), velocidad de agitación (120 r.p.m.), porcentaje de inóculo (2 % v/v) y concentración de colorante (20 y 10 mgL-1), presentaron un efecto significativo (p < 0.05) para favorecer la remoción (decoloración) de MG y CV, así como la actividad lacasa (54,76 ± 8,91 y 30,59 ± 2,89 UL-1 respectivamente) al utilizar biomasa viable de P. ostreatus. En los estudios de adsorción se evidenció que pH ácidos favorecen la adsorción de ambos colorantes y que el modelo de Pseudo-segundo orden describe mejor el fenómeno de quimisorción. Finalmente los índices de germinación (IG) empleando semillas de Lactuca sativa, para los colorantes iniciales fueron < 50 %; demostrando su efecto fitotóxico elevado. Cuando las soluciones de colorantes fueron tratadas con biomasa viable, el IG aumentó, dejando abierta la puerta para la realización de investigaciones futuras con la intensión de determinar si las soluciones acuosas, postratadas con P ostreatus, pueden ser utilizadas en tratamientos que generen aguas menos tóxicas y que estas puedan ser empleadas en otros procesos que no requieran agua potable.

Resumen Os corantes de tipo trifenilmetano Verde Malaquita (VM) e Cristal Violeta (CV) são corantes catiônicos e se misturam com águas residuais domésticas quando descartadas; aumentando, entre outros, as demandas químicas e biológicas de oxigênio, podendo causar toxicidade aguda em diferentes níveis tróficos. Promoveu-se a remoção (descoloração) de VM e CV, e atividade da lacase (54.8 ± 8.9 e 30.6 ± 2.9 UL-1 respectivamente) utilizando como parâmetros necessários para a biomassa viável de P. ostreatus como pH (4,5 e 6,0), temperatura (25 a 30 °C), velocidade de agitação (120 RPM), porcentagem de inócuo (2 % v/v), e concentração de corante (20 e 10 mg L-1). Em estudos de absorção, se demonstrou que um pH mais ácido favorece a absorção de ambos corantes e o modelo de pseudo-segunda ordem descreve melhor o fenômeno da absorção. Finalmente, o índice de germinação (IG), utilizando sementes de Lactuca sativa para as soluções iniciais dos corantes, foi < 50 %; demonstrando assim seu alto efeito fitotóxico. Quando as soluções de corante foram tratadas com a biomassa viável, o IG aumentou, deixando em aberto a possibilidade de realizar futuras investigações para determinar se as soluções aquosas, tratadas com P. ostreatus, poderiam ser utilizadas em tratamentos que gerem águas menos tóxicas, que poderia ser utilizada em processos que não requerem água potável.

Au@PVP Core-Shell Nanoparticles Used as Surface-Enhanced Raman Spectroscopic Substrate to Detect Malachite Green / 分析化学

The core-shell nanopaticles of Au@polyvinyl-pyrrolidone ( PVP) with uniform size and controllabe shell-thickness were prepared by hydrothermal method. The core-shell nanoparticles could be assembled to be the monolayer array on Si substrate relying on the dispersion of core-shell nanoparticles arising from PVP shell. The malachite green ( MG ) absorbed by H-bond could be detected on the array under the electromagnetic enhancement of inner-core Au nanoparticles. Under the conditions of the optimum shell-thickness of Au@PVP and the appropriate absorbed time of MG, the detection of MG could be realized in the linear range from 1 × 10-10 mol/L to 1 × 10-5 mol/L with the correlation coefficient ( R2 ) of 0. 98. The detection limit was 10-12 mol/L. This method was applied to the determination of MG in tilapia fish fillets of Xiagang market. No MG was found in this real sample. The spiked recoveries of the sample ranged from 70. 8% to 126. 0%. This method is simple and accurate, and can be used for detection of MG in the fish.

Synthesis of Novel Hapten and Development of Monoclonal Antibody-based Enzyme-Linked Immunosorbent Assay for Malachite Green in Fish Samples / 分析化学

To produce specific antibodies against malachite green ( MG) , one special hapten was synthesized and characterized, and conjugated to carrier protein as immunogen. The immunogen showed excellent reactogenicity and immunogenicity. One specific monoclonal antibody (mAb, named MG-DA4-C7) with high sensitivity and specificity for MG in indirect competitive enzyme-linked immunoassay ( icELISA ) was screened. The isotype was IgG1 and the light chain was κ type. After optimization of ELISA conditions, the proposed icELISA showed a 50% inhibition value ( IC50 ) of 0. 96 μg/L, a linear range ( IC20-IC80 ) of 0. 1-8. 1 μg/L and a limit of detection ( LOD, IC10 ) of 0. 05 μg/L for determination of MG. The assay showed cross-reactivity of 18. 1%, 26. 5% with crystal violet and brilliant green, respectively, and negligible cross-reactivity with other metabolites of MG (<0 . 1%) . The average recoveries of MG from spiked fish samples were from 87. 3% to 107. 3%. Good correlation (R2=0. 999) was obtained between the results of icELISA and those of liquid chromatography-tandem mass spectrometry analysis. The proposed icELISA is suitable for the determination of MG in fish samples in a simple and sensitive manner.

An optimized micro-assay of myosin II ATPase activity based on the molybdenum blue method and its application in screening natural product inhibitors / 中国天然药物

Myosin II plays multiple roles in physiological and pathological functions through its ATPase activity. The present study was designed to optimize a micro-assay of myosin II ATPase activity based on molybdenum blue method, using a known myosin II ATPase inhibitor, blebbistatin. Several parameters were observed in the enzymatic reaction procedure, including the concentrations of the substrate (ATP) and calcium chloride, pH, and the reaction and incubation times. The proportion of coloration agent was also investigated. The sensitivity of this assay was compared with the malachite green method and bioluminescence method. Additionally, 20 natural compounds were studied for myosin II ATPase inhibitory activity using the optimized method. Our results showed that ATP at the concentration of 5 mmol·L(-1) and ammonium molybdate : stannous chloride at the ratio of 15 : 1 could greatly improve the sensitivity of this method. The IC50 of blebbistatin obtained by this method was consistent with literature. Compound 8 was screened with inhibitory activity on myosin II ATPase. The optimized method showed similar accuracy, lower detecting limit, and wider linear range, which could be a promising approach to screening myosin II ATPase inhibitors in vitro.

Evaluation of four colourimetric susceptibility tests for the rapid detection of multidrug-resistant Mycobacterium tuberculosisisolates

The purpose of this study is to evaluate four rapid colourimetric methods, including the resazurin microtitre assay (REMA), malachite green decolourisation assay (MGDA), microplate nitrate reductase assay (MNRA) and crystal violet decolourisation assay (CVDA), for the rapid detection of multidrug-resistant (MDR) tuberculosis. Fifty Mycobacterium tuberculosisisolates were used in this study. Eighteen isolates were MDR, two isolates were only resistant to isoniazid (INH) and the remaining isolates were susceptible to both INH and rifampicin (RIF). INH and RIF were tested in 0.25 µg/mL and 0.5 µg/mL, respectively. The agar proportion method was used as a reference method. MNRA and REMA were performed with some modifications. MGDA and CVDA were performed as defined in the literature. The agreements of the MNRA for INH and RIF were 96% and 94%, respectively, while the agreement of the other assays for INH and RIF were 98%. In this study, while the specificities of the REMA, MGDA and CVDA were 100%, the specificity of the MNRA was lower than the others (93.3% for INH and 90.9% for RIF). In addition, while the sensitivity of the MNRA was 100%, the sensitivities of the others were lower than that of the MNRA (from 94.1-95%). The results were reported on the seventh-10th day of the incubation. All methods are reliable, easy to perform, inexpensive and easy to evaluate and do not require special equipment.

Rapid detection of multidrug-resistant Mycobacterium tuberculosis using the malachite green decolourisation assay

Early detection of drug resistance in Mycobacterium tuberculosis isolates allows for earlier and more effective treatment of patients. The aim of this study was to investigate the performance of the malachite green decolourisation assay (MGDA) in detecting isoniazid (INH) and rifampicin (RIF) resistance in M. tuberculosis clinical isolates. Fifty M. tuberculosis isolates, including 19 multidrug-resistant, eight INH-resistant and 23 INH and RIF-susceptible samples, were tested. The sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and agreement of the assay for INH were 92.5%, 91.3%, 92.5%, 91.3% and 92%, respectively. Similarly, the sensitivity, specificity, PPV, NPV and agreement of the assay for RIF were 94.7%, 100%, 100%, 96.8% and 98%, respectively. There was a major discrepancy in the tests of two isolates, as they were sensitive to INH by the MGDA test, but resistant by the reference method. There was a minor discrepancy in the tests of two additional isolates, as they were sensitive to INH by the reference method, but resistant by the MGDA test. The drug susceptibility test results were obtained within eight-nine days. In conclusion, the MGDA test is a reliable and accurate method for the rapid detection of INH and RIF resistance compared with the reference method and the MGDA test additionally requires less time to obtain results.

Diphenylmethyl selenocyanate attenuates malachite green induced oxidative injury through antioxidation & inhibition of DNA damage in mice.

Background & objectives: Malachite green (MG), an environmentally hazardous material, is used as a non permitted food colouring agent, especially in India. Selenium (Se) is an essential nutritional trace element required for animals and humans to guard against oxidative stress induced by xenobiotic compounds of diverse nature. In the present study, the role of the selenium compound diphenylmethyl selenocyanate (DMSE) was assessed on the oxidative stress (OS) induced by a food colouring agent, malachite green (MG) in vivo in mice. Methods: Swiss albino mice (Mus musculus) were intraperitoneally injected with MG at a standardized dose of 100 μg/ mouse for 30 days. DMSE was given orally at an optimum dose of 3 mg/kg b.w. in pre (15 days) and concomitant treatment schedule throughout the experimental period. The parameters viz. ALT, AST, LPO, GSH, GST, SOD, CAT, GPx, TrxR, CA, MN, MI and DNA damage have been evaluated. Results: The DMSE showed its potential to protect against MG induced hepatotoxicity by controlling the serum alanine aminotransferase and aspartate amino transferase (ALT and AST) levels and also ameliorated oxidative stress by modulating hepatic lipid peroxidation and different detoxifying and antioxidative enzymes such as glutathione-S-transferase (GST), superoxide dismutase (SOD), catalase (CAT), and also the selenoenzymes such as glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) and reduced glutathione level which in turn reduced DNA damage. Interpretation & conclusions: The organo-selenium compound DMSE showed significant protection against MG induced heptotoxicity and DNA damage in murine model. Better protection was observed in pretreatment group than in the concomitant group. Further studies need to be done to understand the mechanism of action.

Isolation and characterization of extracellular thermostable alkaline phosphatase enzyme from bacillus spp.

Alkaline phosphatase (E C 3.1.3.1) belongs to the class of hydrolases and catalyzes the alkaline hydrolysis of a number of phosphoric acid esters, nucleotides etc. Alkaline phosphatase was produced from Bacillus spp, isolated from soil samples. The Bacillus spp. was identified by staining and standard biochemical tests after which screening was done using modified Pikovoskaya’s agar method. Production of alkaline phosphatase using different substrates like calcium phosphate along with casein, starch, glucose and glutamic acid was carried out. High activity was found in calcium phosphate along with the casein. The specific activity of the crude extract was found to be 0.825U and it was subjected to purification by DEAE-Cellulose ion exchange chromatography. Finally,36% recovery was obtained. The molar mass was estimated by using 10% SDS-PAGE and was found to be approximately 84 KD. The optimum activity was at pH 8.8 and temperature of 650C. Alkaline phosphatase activity was enhanced by Mg2++ upto 66% and 80% activity was inhibited by EDTA. Alkaline Phosphatase activity was also confirmed by zymography using malachite green staining method.

Removal of malachite green from water by Firmiana simplex wood fiber / Eliminación de malaquita verde del agua con fibra de madera Firmiana simple

This study shows that wood fiber of Phoenix tree (Firmiana simplex) is an effective adsorbent for malachite green (MG). MG sorption behavior onto the wood adsorbent was investigated in this study. Basic condition was favorable for MG adsorption to the adsorbent. The pseudo second order equation well described MG adsorption onto the wood adsorbent. The Freundlich Isotherm could describe the sorption data. The positive value of AH0 showed that adsorption of malachite green onto the wood adsorbent was endothermic. The negative values of AG at various temperatures indicate the spontaneous nature of the adsorption process.

Recent Research Progress in Fish Ecotoxicology and Aquatic food Safety Technology on Malachite Green / 环境与健康杂志

Malachite green (MG) is a dye which has been widely used as a topical fungicide and antiseptic in aquaculture throughout the world. Now,MG has become a highly controversial compound due to its potential highly residual and toxic characteristics in recent years. In the paper,the advances in toxicity,toxicological mechanism,detection methods,metabolism and removal of malachite green and its metabolite were reviewed,and the problems in aquaculture and the safety of aquaculture technology were discussed,in order to make us be aware of potential hazard of malachite green and provide a dependable evidences of safely breeding in aquatic products.

Determination of Malachite Green in Water by High Performance Liquid Chromatography / 环境与健康杂志

Objective To develop a sensitive HPLC method for the determination of malachite green in fresh water. Methods The HPLC column of ZORBAX SB-C18 (250 mm?4.6 mm, 5 ?m) was employed and acetonitrile and acetate buffer (0.1 mol/L, pH=4.5, V∶V=80∶20) were taken as the mobile phase. The samples were tested at wave length of 588 nm after post-column oxidation with PbO2 flowed at 1.5 ml/min. Results The average recovery rate were 98.5% and 97.8% respectively at the water concentration of 2.0 ?g/L and 10.0 ?g/L,the relative standard deviations (RSD) were 11.2% and 8.0%,the least detection limit was 0.02 ?g/L. Conclusion The method is stable,sensitive and suitable for the detection of malachite green in water.

Determination of Trace Cadmium (Ⅱ) in Water by Spectrophotometry in Cadmium (Ⅱ)-Potassium Iodide-Malachite Green-Gelatine / 环境与健康杂志

Objective To establish a high sensitive spectrophotometry for determination of trace cadmium in the water. Methods A complicated ion-association complex of Cd(Ⅱ)-potassium iodide-malachite green was formed in the phosphate acid, and the addition of gelatine could enhance the sensitivity of the reaction.The maximum absorption of the ion-association complex was at 680 nm,the effect of experimental conditions such as the reagents concentration,the temperature and the influence of foreign matters were considered.Results In the optimum condition(6.0 ml of 40% potassium iodide-aseorbic acid solution,0.5 ml of 5.0 mol/L phosphate acid solution,0.5 ml of 0.5% gelatine solution,1.5 ml of 1.0?10~(-3)mol/L malachite green solution in a 25ml volumetric flask,diluted with water and mixed well and determined immediately),the linear regression equation was △A=0.011+ 0.957 c,r=0.998 5.Beer's law was obeyed in the range of 0.02 ?g/ml to 0.80 ?g/ml for Cd(Ⅱ)and the limit detection was 0.02 ?g/ ml.The composing ratio of the complex was MG:Cd:I=2:1:4,and its apparent molar absorptivity coefficient was 1.08?10~5 L/(mol? cm).The recovery rates of Cd(Ⅱ)were 97.0%-101.5%,RSDs were 1.36%-3.58%.Conclusion This method is sensitive,simple, rapid and is applicable to the determination of the trace Cd(Ⅱ)in water.

Isolation, Identification and Growth Characteristics of Pseudomonas putida Strain M6 with Malachite Green Decolorization / 微生物学通报

Six bacterial strains with malachite green decolorization ability were isolated from a sediment of aquaculture pond, and strain M6 was selected by further enrichment culture in nutrition broth with malachite green and decolorization rate comparison. The decolorization rate of strain M6 to malachite green was 97.14% in the conditon of 30?C and 150 r/min, and its morphology was observed by gram stain and electronmicroscopy, its physiological and biochemical characteristic was studied by ATB bacteria identification in-strument for identification of bacteria, and its 16S rDNA sequence was determined following PCR amplifi-cation, the sequence was aligned and the phylogenic tree was instructed with those bacterial strains of high identity with strain M6. In addition, its growth characteristics was also studied. The experimental results showed that strain M6 was gram negative and bacilliform with a flagellum at one end. Its size was 0.45 ?m ?0.84 ?m. Its colony produced on common agar plate appeared as round, light blue, dense, hard to choose; 16S rDNA sequence of strain M6 had high identity of 98%～99% with Pseudomonas sp. located in GenBank and strain M6 had the most close relative relation to Pseudomonas putida OW-16 (Locus number: DQ112328.1). Combined the results of the traditional morphological, physiological, biochemical character-istics and 16S rDNA sequence analysis, strain M6 was identified as Pseudomonas putida (Locus number: EU348741.1). Additionally, its growth curve in the condition of 30?C and 150 r/min was as follows: lag phase was 0～4 h, log phase was 4 h～64 h, stationary phase was 64 h～80 h, decline phase was after 80 h. Its best growth conditions were pH 7 and 30?C,and in the rotational speed of 50 r/min to 250 r/min. Its concen-tration increased with the increase in rotational speed.