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#27283705   2016/06/10 Save this To Up

A simple microplate-based method for the determination of α-amylase activity using the glucose assay kit (GOD method).

For the first time, a reliable, simple, rapid and high-throughput analytical method for the detection and quantification of α-amylase inhibitory activity using the glucose assay kit was developed. The new method facilitates rapid screening of a large number of samples, reduces labor, time and reagents and is also suitable for kinetic studies. This method is based on the reaction of maltose with glucose oxidase (GOD) and the development of a red quinone. The test is done in microtitre plates with a total volume of 260μL and an assay time of 40min including the pre-incubation steps. The new method is tested for linearity, sensitivity, precision, reproducibility and applicability. The new method is also compared with the most commonly used 3,5-dinitrosalicylic acid (DNSA) method for determining α-amylase activity.

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#26858542   2016/02/09 Save this To Up

Characterization of the plant growth promoting bacterium, Enterobacter cloacae MSR1, isolated from roots of non-nodulating Medicago sativa.

The aim of the present study was to characterize the endophytic bacterial strain designated MSR1 that was isolated from inside the non-nodulating roots of Medicago sativa after surface-sterilization. MSR1 was identified as Enterobacter cloacae using both 16S rDNA gene sequence analysis and API20E biochemical identification system (Biomerieux, France). Furthermore, this bacterium was characterized using API50CH kit (Biomerieux, France) and tested for antibacterial activities against some food borne pathogens. The results showed that E. cloacae consumed certain carbohydrates such as glycerol, d-xylose, d-maltose and esculin melibiose as a sole carbon source and certain amino acids such as arginine, tryptophan ornithine as nitrogen source. Furthermore, MSR1 possessed multiple plant-growth promoting characteristics; phosphate solubility, production of phytohormones acetoin and bioactive compounds. Inoculation of Pisum sativum with MSR1 significantly improved the growth parameters (the length and dry weight) of this economically important grain legume compared to the non-treated plants. To our knowledge, this is the first report addressing E. cloacae which exist in roots of alfalfa growing in Al-Ahsaa region. The results confirmed that E. cloacae exhibited traits for plant growth promoting and could be developed as an eco-friendly biofertilizer for P. sativum and probably for other important plant species in future.

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#26047106   2015/06/06 Save this To Up

Development of a lateral flow test to detect metabolic resistance in Bemisia tabaci mediated by CYP6CM1, a cytochrome P450 with broad spectrum catalytic efficiency.

Cotton whitefly, Bemisia tabaci (Genn.) (Homoptera: Aleyrodidae) is a major sucking pest in many agricultural and horticultural cropping systems globally. The frequent use of insecticides of different mode of action classes resulted in populations resisting treatments used to keep numbers under economic damage thresholds. Recently it was shown that resistance to neonicotinoids such as imidacloprid is linked to the over-expression of CYP6CM1, a cytochrome P450 monooxygenase detoxifying imidacloprid and other neonicotinoid insecticides when recombinantly expressed in insect cells. However over-expression of CYP6CM1 is also known to confer cross-resistance to pymetrozine, an insecticide not belonging to the chemical class of neonicotinoids. In addition we were able to demonstrate by LC-MS/MS analysis the metabolisation of pyriproxyfen by recombinantly expressed CYP6CM1. Based on our results CYP6CM1 is one of the most versatile detoxification enzymes yet identified in a pest of agricultural importance, as it detoxifies a diverse range of chemical classes used to control whiteflies. Therefore we developed a field-diagnostic antibody-based lateral flow assay which detects CYP6CM1 protein at levels providing resistance to neonicotinoids and other insecticides. The ELISA based test kit can be used as a diagnostic tool to support resistance management strategies based on the alternation of different modes of action of insecticides.

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#25533309   2014/12/23 Save this To Up

Iontophoretic and microneedle mediated transdermal delivery of glycopyrrolate.

The objective of this study was to investigate the use of iontophoresis, soluble microneedles and their combination for the transdermal delivery of glycopyrrolate.

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#22507377   2012/06/12 Save this To Up

Optimization of a malachite green assay for detection of ATP hydrolysis by solubilized membrane proteins.

We studied the activity of the fluorescently labeled membrane transporter MalGFK(2), which transports maltose at the expense of ATP hydrolysis. We used a commercially available malachite green assay (SensoLyte MG phosphate assay kit; Anaspec) to quantify the liberated phosphate upon ATP hydrolysis. However, strong variations in phosphate concentration were measured when using the supplier's handling protocol. We optimized the protocol, taking into account the effects mediated by glycerol, SDS, and fluorescent label on the sample. As a result we obtained highly reproducible phosphate concentration values under conditions optimal for solubilized membrane proteins.

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#21873792   2011/08/29 Save this To Up

[Recombinant expression of hepatoma associated gene and its protein function].

To recombinant express hepatoma associated gene(HTA) and pre-test the function of HTA to determine the role of HTA in the development of liver cancer.

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#20509693   2010/06/16 Save this To Up

An integrated amperometric biosensor for the determination of lactose in milk and dairy products.

An integrated amperometric biosensor for the determination of lactose is reported. The bioelectrode design is based on the use of a 3-mercaptopropionic acid (MPA) self-assembled monolayer (SAM)-modified gold electrode on which the enzymes beta-galactosidase (beta-Gal), glucose oxidase (GOD), peroxidase (HRP) and the mediator tetrathiafulvalene (TTF) are coimmobilized by a dialysis membrane. beta-Gal catalyzes the hydrolysis of lactose, and the produced glucose is catalytically oxidized to gluconic acid and H(2)O(2), which is reduced in the presence of HRP. This enzyme reaction is mediated by TTF, and the reduction of TTF(+) at 0.00 V (vs Ag/AgCl) gives rise to an amperometric signal proportional to the lactose concentration. The biosensor exhibits a good repeatability of the measurement carried out with the same biosensor, a good reproducibility of the responses obtained with different biosensors and a useful lifetime of 28 days. A linear calibration plot was obtained for lactose over the 1.5 x 10(-6) to 1.2 x 10(-4) M concentration range, with a limit of detection of 4.6 x 10(-7) M. The effect of potential interferents (sucrose, lactulose, fructose, arabinose, maltose, galactose, glucose and uric and ascorbic acids) on the biosensor response was evaluated. Furthermore, the bioelectrode exhibits a suitable performance in flow-injection systems in connection with amperometric detection. The developed biosensor was applied to the determination of lactose in milk and other foodstuffs (chocolate, butter, margarine, yogurt, cheese and mayonnaise), and the results obtained were validated by comparison with those provided by using a commercial enzyme test kit.

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#19563395   2009/06/30 Save this To Up

Development of recombinant OmpA and OmpB proteins as diagnostic antigens for rickettsial disease.

In this study the diagnostic potential of Rickettsia conorii recombinant antigens was analyzed. For this, site-specific PCR primers were used to clone the OmpA and OmpB genes of R. conorii into pMAL-c2X plasmids. Six fragments of OmpA and four of OmpB were expressed as fusion proteins with maltose-binding protein in Escherichia coli. OmpA(1350-1784), OmpB(801-1269,) and OmpB(1227-1634) regions from truncated proteins were selected as diagnostic candidate antigens by ELISA using control sera. ELISA results of three antigens were compared to the results obtained by using a commercial ELISA kit which contained whole OmpA and OmpB antigens from R. conorii. For this analysis, 40 serum samples taken from febrile patients and uninfected controls were tested. Of the 20 R. conorii test results which were positive with the commercial kit, 18 were shown to be positive by ELISA using OmpA(1350-1784) (a sensitivity of 90%). The specificity of the ELISA was 100%; all of the 20 samples shown to be negative using the commercial kit were also negative in our assay. The sensitivities of the ELISA using the OmpB(801-1269) and OmpB(1227-1634) were 90% and 95%, respectively. The specificities of the OmpB(801-1269) and the OmpB(1227-1634) were 100% and 95%, respectively. These results suggest that specific regions of OmpA and OmpB effectively detect antibodies against R. conorii, and the truncated recombinant antigens could be used for development of diagnostic tools for rickettsial disease.

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#19081855   2008/12/16 Save this To Up

Highlights and prizes of an international meeting.

A short description selected from the presentations which had been awarded prizes in the 4th International Meeting of Nuclear Medicine, of the Hellenic Society of Nuclear Medicine, in Thessaloniki, Greece, is as follows: Professor L.G. Strauss from Heidelberg received the first prize for his original paper under the title: "Modulation of FDG kinetics in tumors by gene expression". He studied 25 patients with colorectal tumors with dynamic PET-FDG within 2 days prior to surgery and he finally came to the conclusion that angiogenesis has a significant impact primarily on the kinetic data (k1, k3), but also on the global FDG uptake. A detailed analysis of the FDG kinetics can help to classify the grade of angiogenesis in primary colorectal tumors. The cell cycle associated genes have a comparable impact on the FDG kinetics as compared to the angiogenesis related genes. Furthermore, hypoxia was associated with the FDG parameters. Enhanced expression of HIF-1a was primarily associated with an enhanced influx of FDG. The results demonstrated the impact of dedicated groups of genes on the FDG kinetics. Furthermore, if the FDG kinetics are quantitatively analyzed, the expression of certain genes may be predicted from these data. Dr. P. Bouziotis et al. received the second prize for their original paper under the title: "Labeling of bevacizumab, an anti-VEGF monoclonal antibody, with technetium-99m and rhenium-188". The authors were from Athens and Oxford and studied the reduction of the endogenous disulphide bonds of bevacizumab by treatment with 2-mercaptoethanol and TCEP-HCl. The number of generated-SH groups was estimated before each labelling experiment. The results of the present study show that VEGF expression in tumors can be detected and visualized specifically with the anti-VEGF monoclonal antibody bevacizumab, labelled with gamma-emitting radioisotopes. The third prize went to Dr C. H. Tsopelas et al. from Adelaide who presented an original paper under the title: "Evaluation of visceral sensitivity after transient inflammation-An experimental model". Their aim was to characterise the inflammatory response to the transient chemically-induced colitis after instillation of trinitrobenzenesulfonic acid. Inflammation was tested by (99m)Tc-Sn-colloid-leucocytes. They concluded that the Group of Lewis rats compared to Fisher rats, developed a prolonged visceral hyperalgesia and more severe inflammation following colorectal instillation of TNBS/ethanol doses, possibly involving the systemic immune response. The Lewis rat species appears to be a good model of transient colitis, because of its heightened sensitivity to the chemical stimulus, and due to detectable visceral changes long after administration of the above stimulus. Professor A.M. Peters from England, received the fourth prize with his original paper: "New quantitative techniques for investigating and predicting lymphoedema resulting from breast cancer treatment". In lymphoedema from breast cancer treatment he investigated local uptake via putative peripheral lympho-venous communications (LVCs), using intradermally injected labelled red cells. He concluded that his results suggest that protective mechanisms could include i) interstitial proteolysis, ii) increased peripheral trans-endothelial protein transport or iii) development of peripheral LVCs. Other prizes were awarded to: Professor G.P. Bandopadhyaya et al. from New-Delhi for their original paper: "Molecular targetting of infective bacterial maltose binding protein for infection imaging using Tc-99m hydroxypropyl cyclodextrin in patients with knee joint replacement and other prostheses", to Dr P.J. Marsouvanidis et al. from Demokritos Athens and Patras for their original paper: "Synthesis, radiochemistry and preclinical comparison of [(111)In-DOTA(0)]SS-14 and [(111)In-DOTA(0),(D)Trp(8)]SS-14 in AR4-2J cells and Swiss albino mice", to Professor B. Singh et al. from Chandigarh and New Delhi for their original paper: "Efficacy of indigenously developed single vial kit preparation of (99m)Tc-ciprofloxacin in the detection of bacterial infection-An Indian experience" and to Dr. A. Bantis et al. from Alexandroupolis for their original paper: "The prognostic value of serum chromogranin A in patients with advanced prostate cancer".

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#18693547   2008/08/12 Save this To Up

Preliminary studies on the microbiological characterization of lactic acid bacteria in suero costeño, a Colombian traditional fermented milk product.

Suero costeño is a fermented milk product from the Colombian Atlantic coast, which is produced by the spontaneous acidification of raw milk due to the action of environmental microbes during traditional and semi-industrial processes. Eleven fermentations were carried out in experimental settings replicating traditional conditions and changes in concentration among microbial groups involved during the process (Aerobic Mesophilic bacteria, Yeasts, Enterobacteriaceae and Lactic Acid Bacteria (LAB)). LAB plays an important role in the fermentation process, especially during the final stage (24 hours). In addition, yeasts seem to have an effect on fermentation, showing an increase during the first hours of the process, while Enterobacterial counts decreased during fermentation. Thirty six LAB strains were isolated from commercial samples and thirty two were identified using the API 50 CH kit (BioMCrieux). 41% of the strains identified belonged to the species Lb. plantarum, and 19% were Lb. paracasei subsp. paracasei. Sugars fermented by LAB include milk carbohydrates such as D-Lactose, D-Glucose and D-Galactose. Because of their capacity to use other carbohydrates (manose, celobiose, maltose, fructose, ribose, trehalose, salicin, gentiobiose), it would also be possible to use these strains as starter cultures for other fermentations.

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