Glutathione-S-transferase production in earthworm (Annelida: Eudrilidae) as a tool for heavy metal pollution assessment in abattoir soil

AbstractThe use of direct response of animals to environmental challenges by production of biomarkers is a better tool to assess environmental pollution than the conventional methods. This study aimed to measure Glutathione-S-transferase (GST) in earthworms as tools for assessing heavy metal pollution in abattoir soil. Five (5) replicates each of earthworm species (Libyodrilus violaceous, Eudrilus eugeniae and Alma millsoni), soil and rumen waste samples were collected from three (3) abattoir sites (Lafenwa, Gbonogun and Madojutimi abattoirs), and a control site located within Federal University of Agriculture Abeokuta, beside an undisturbed stream with no rumen waste. Heavy metal (Cu, Zn, Pb, Cd, Co, Cr, Ni and Mn) concentrations in rumen waste, abattoir soils and earthworm tissues were determined using Atomic Absorption Spectrophotometer. The pH and organic matter (OM) concentrations of the rumen waste and abattoir soils were determined by standard methods. GST activities in the earthworm tissues were determined through the conjugation of 1 mM reduced glutathione (GSH) with 1 mM 1-chloro-2,4-dinitrobenzene (CDNB). The rumen waste recorded significantly higher (p ≤ 0.05) % OM, heavy metal concentrations and pH level than in their respective abattoir soils. The mean heavy metal concentrations of Cu, Zn, Pb, Cd and Mn were highest in the tissue of earthworm species obtained from Lafenwa abattoir. A significantly (p ≤ 0.05) higher GST activities were recorded in the tissue of earthworm species obtained from Lafenwa and Gbonogun abattoirs. Libyodrilous violaceus obtained from Lafenwa abattoir recorded the highest GST activity (8.47±1.39) in their tissue followed by the ones from Gbonogun abattoir (8.21±0.85). A significant (p ≤ 0.05) positive correlations was observed between GST activities in earthworm tissues and heavy metal concentrations. GST activities can therefore be used to assess the level of heavy metal pollution in abattoir soils. Rev. Biol. Trop. 64 (2): 779-789. Epub 2016 June 01.

Produccion de beta-glucosidasas por cultivos de bacterias termofilas indigenas del altiplano boliviano / beta-Glucoside production by thermophilic bacteria indigenous the bolivian altiplano culture

Las beta-glucosidasas son enzimas que poseen actividad hidrolitica y transferasa o transglucosidasa. Tienen diversas aplicaciones; en la biosintesis de oligosacaridos, produccion de etanol utilizando residuos agricolas y en la industria de vinos. La aplicacion industrial, sin embargo, requiere estabilidad a temperaturas elevadas, por lo que los microorganismos termofilos tienen gran interes. El proposito de esta investigacion es el de optimizar el medio de cultivo anaerobio de bacterias termofilas, para aumentar la produccion de beta-glucosidasas. Esta enzima es producida por tres aislados bacterianos: FT3, 2B y P5 los cuales fueron aislados de la region andina de Bolivia. El aislado bacteriano FT3 mostro una actividad beta-glucosidasa de 0,35 [UI/mL]. Se tomaron como variables dentro de la optimizacion del medio de cultivo las fuentes de nitrogeno y de carbono, y el pH. Asi tambien se probaron dos sistemas de cultivo: celulas libres y encapsuladas. Empleando extracto de levadura como fuente de nitrogeno se obtuvo una actividad de 0,52 [UI/mL]. En la optimizacion del pH del medio de cultivo se obtuvo una actividad de 0,81 [UI/mL] a pH 5. Como fuente de carbono se eligieron los hidrolizados de paja de trigo y paja de quinoa lleg¨¢ndose a obtener actividades de 1,27 y 1,34 [UI/mL] respectivamente. Se establecio que la localizacion celular de la enzima beta-glucosidasa es extracelular y presenta estabilidad hasta una temperatura de 80 ºC y un pH de 7.

The beta-glucosidases possess hydrolytic and transferase activity or transglucosidase. They have various applications; such as biosynthesis of oligosaccharides, production of ethanol using agricultural residues and wine industry. However for industrial application, stability to high temperatures is needed. Therefore a great interesting in the thermophile microorganism study exist. The purpose of this research is to optimize the culture medium of thermophilic anaerobic bacteria to increase the production of beta-glucosidase. This enzyme is produced by three isolate bacterial FT3, 2B and P5 which were isolated from the Andean region of Bolivia. FT3 isolate showed beta-glucosidase activity of 0.35 [IU/mL]. In regards to the optimization of culture medium variables such as nitrogen source, carbon source and pH were taken into account and also the combination with free and encapsulated bacterial cells. Yeast extract was the selected source of nitrogen obtaining an activity of 0.52 [IU/ mL]. The optimal pH was 5 obtaining an activity of 0.81 [IU/mL]. The selected carbon source was the hydrolyzed wheat straw and quinoa straw obtaining activities of 1.27 and 1.34 [IU/mL], respectively. The cellular localization of beta-glucosidase enzyme is extracellular and provides stability to temperature of 80 ºC and stability at pH 7.

Gene expression and characterization of 2-keto-3-deoxy-gluconate kinase, a key enzyme in the modified Entner-Doudoroff pathway of Serratia marcescens KCTC 2172

We cloned 2-keto-3-deoxy-gluconate kinase (KDGK), which catalyzes the phosphorylation of 2-keto-3-deoxygluconate (KDG) to 2-keto-3-deoxy-6-phophogluconate (KDPG) from Serratia marcescens KCTC 2172. The nucleotide sequence revealed a single open reading frame containing 1,208 bp and encoding for 309 amino acids, with a molecular weight of 33,993 Da. The enzyme was purified via GST affinity chromatography. The putative KdgT binding site was detected upstream of the initial codon. The KDG kinase utilized 2-ketogluconate (KG) and KDG as substrates. The optimal temperature and pH for KDGK activity were 50ºC and 8.0, respectively.

Expressão e atividade enzimática de glutationa s-transferase em tecidos de fêmeas de Boophilus microplus / Expression and enzymatic activity of glutathione s-transferase in tissues of Boophilus microplus females

Cellular detoxification and excretion enzymes are important to the maintenance of cellular homeostasis. In this work mRNA transcription, protein expression and enzymatic activity of Glutathione S-transferases (GSTs), enzymes involved in the excretion of endo and xenobiotic compounds were analyzed. These parameters are elements believed to protect cells against chemical toxicity and oxidative stress in different tissues (salivary gland, ovary and synganglion) from partially engorged females and engorged females of Boophilus microplus. The results presented showed elevated GST activity in partially engorged females. The enzymatic activity decreased during the preoviposition period in engorged females. GST mRNA transcription was detected in salivary glands and synganglion from partially engorged and engorged females, but not in ovary. The results of this work help to elucidate the role of GST in tick development and assist in the understanding of the importance of GST in tick females during the preparation for oviposition.

Enzimas de detoxificação e excreção celular são importantes para a manutenção da homeostase celular. Neste trabalho foi caracterizada a transcrição de mRNA, a expressão da proteína e a atividade enzimática de glutationa S-transfersases (GSTs), enzimas que atuam em rotas de excreção de substâncias endo e xenobióticas, protegendo as células contra toxicidade química e estresse, em diferentes tecidos (glândula salivar, ovário e singânglio) de fêmeas adultas semi-ingurgitadas e ingurgitadas do carrapato do bovino Boophilus microplus. Os resultados mostraram que a atividade de GST é mais alta em fêmeas semi-ingurgitadas e diminui em fêmeas ingurgitadas de acordo com o final do período de pré-postura. A expressão de mRNA de GST foi detectada em glândulas salivares e singânglios de fêmeas adultas semi-ingurgitadas e ingurgitadas, mas não em ovários. Estes dados podem ajudar a compreender melhor o papel de enzimas antioxidantes durante a preparação das fêmeas do carrapato para a postura.

Gene Expression Analysis of Cultured Amniotic Fluid Cell with Down Syndrome by DNA Microarray

Complete or partial triplication of human chromosome 21 results in Down syndrome (DS). To analyze differential gene expressions in amniotic fluid (AF) cells of DS, we used a DNA microarray system to analyze 102 genes, which included 24 genes on chromosome 21, 28 genes related to the function of brain and muscle, 36 genes related to apoptosis, 4 genes related to extracellular matrix, 8 genes related to other molecular function and 2 house-keeping genes. AF cells were collected from 12 pregnancies at 16-18 weeks of gestation in DS (n=6) and normal (n=6) subjects. Our DNA microarray experiments showed that the expressions of 11 genes were altered by at least 2-folds in DS, as follows. Ten genes, COL6A1, CASP5, AKT2, JUN, PYGM, BNIP1, OSF-2, PRSS7, COL3A1, and MBLL were down-regulated and GSTT1 was only up-regulated. The differential expressions of GSTT1 and COL3A1 were further confirmed by semi-quantitative RT-PCR for each sample. The gene dosage hypothesis on chromosome 21 may explain the neurological and other symptoms of DS. However, our results showed that only two genes (COL6A1 and PRSS7), among 24 genes on chromosome 21, were down-regulated in the AF cells of DS. Our data may provide the basis for a more systematic identification of biological markers of fetal DS, thus leading to an improved understanding of pathogenesis for fetal DS.

Glutathione-S-transferases from chloroquine-resistant and -sensitive strains of Plasmodium falciparum: what are their differences?

Glutathione-S-transferases (GSTs) from chloroquine-resistant (CQR, K1) and -sensitive (CQS, T9/94) strains of Plasmodiumfalciparum were studied. The enzymes from both strains exhibited the optimal pH for enzyme catalysis, at pH 7.5, and were stable at temperatures below 60 degrees C. They also showed the highest specific activities toward CDNB and moderate activities to ethacrynic acid (40% of the activity to CDNB) but little or no activity for other substrates. Km and Vmax values for CDNB and GSH, calculated by Lineweaver-Burk plot from both CQR- and CQS-GSTs, were not statistically different (p<0.05). However, the GSTs activity from CQR appeared to be significantly higher than that from CQS. Therefore, we proposed that GSTs from both malarial strains are identical in their functional domain but different in level of gene expression. Furthermore, protein sequence alignment between P. falciparum GST and GSTs from other organisms suggested that the malarial enzyme is closely similar to other GSTs in Sigma, Alpha, Mu and Pi subclasses, probably most to the Alpha group. Characterization of the purified malarial GST in detail would reveal more precise classification and better understanding of its role in malarial detoxification.

Plant products as protective agents against cancer.

Out of various spices and leafy vegetables screened for their influence on the carcinogen-detoxifying enzyme, glutathione-S-transferase (GST) in Swiss mice, cumin seeds, poppy seeds, asafoetida, turmeric, kandathipili, neem flowers, manathakkali leaves, drumstick leaves, basil leaves and ponnakanni leaves increased GST activity by more than 78% in the stomach, liver and oesophagus, - high enough to be considered as protective agents against carcinogenesis. Glutathione levels were also significantly elevated in the three tissues by these plant products. All of them except neem flowers, significantly suppressed (in vivo) the chromosome aberrations (CA) caused by benzo(a)pyrene in mouse bone marrow cells. Multiple CA and exchanges reflecting the severity of damage within a cell were significantly suppressed by these nine plant products. The results suggest that these nine plant products are likely to suppress carcinogenesis and can act as protective agents against cancer.