Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.6.1.44 (AGT)
 770 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Plasmodium falciparum infection kills more than 1 million children annually. Novel drug targets are urgently being sought as multidrug resistance limits the range of treatment options for this protozoan pathogen. PfHT1, the major hexose transporter of P. falciparum is a promising new target. We report detailed structure-function studies on PfHT1 using site-directed mutagenesis approaches on residues located in helix V (Q169N) and helix VII ((302)SGL --> AGT). Studies with hexose analogues in these mutants have established that hexose recognition and permeation are intimately linked to these helices. A "fructose filter" effect results from the Q169N mutation (abolishing fructose uptake but preserving affinity and transport of glucose, as reported in Woodrow, C. J., Burchmore, R. J. S., and Krishna, S. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 9931-9936). Associated changes in competition for glucose uptake by C-2, C-3, and C-6 glucose analogues compared with native PfHT1 indicate subtle alterations in substrate interaction in this mutant. The K(m) values for glucose uptake in helix VII mutants are also similar to native PfHT1. Hydrogen bonding to positions C-5 and C-6 in glucose analogues becomes relatively more important in these mutants compared with native PfHT1. To increase understanding of hexose permeation pathways in PfHT1, we have developed the first three-dimensional model for PfHT1. As predicted for GLUT1, the principal mammalian glucose transporter, PfHT1 contains a main and an auxiliary channel. After modeling, the Q169N mutation leads predominantly to local structural changes, including displacement of neighboring helix IV. The (302)SGL position in helix VII lies in the same plane as Gln-169 in helix V but is also adjacent to the main hexose permeation pathway, consistent with results from experiments mutating this triplet motif. Furthermore, there are obvious structural and functional differences between GLUT1 and PfHT1 that can now be explored in detail using the approaches presented here. The development of specific inhibitors for PfHT1 will also be aided by these insights.
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PMID:Mutational analysis of the hexose transporter of Plasmodium falciparum and development of a three-dimensional model. 1204 21

We report on the use of PDMS multichannels for affinity studies of DNA aptamer-human Immunoglobulin E (IgE) interactions by surface plasmon resonance imaging (SPRi). The sensing surface was prepared with thiol-terminated aptamers through a self-assembling process in the PDMS channels defined on a gold substrate. Cysteamine was codeposited with the thiol aptamers to promote proper spatial arrangement of the aptamers and thus maintain their optimal binding efficiencies. Four aptamers with different nucleic acid sequences were studied to test their interaction affinity toward IgE, and the results confirmed that aptamer I (5'-SH-GGG GCA CGT TTA TCC GTC CCT CCT AGT GGC GTG CCC C-3') has the strongest binding affinity. Control experiments were conducted with a PEG-functionalized surface and IgG was used to replace IgE in order to verify the selective binding of aptamer I to the IgE molecules. A linear concentration-dependent relationship between IgE and aptamer I was obtained, and a 2-nM detection limit was achieved. SPRi data were further analyzed by global fitting, and the dissociation constant of aptamer I-IgE complex was found to be 2.7 x 10(-7) M, which agrees relatively well with the values reported in the literature. Aptamer affinity screening by SPR imaging demonstrates marked advantages over competing methods because it does not require labeling, can be used in real-time, and is potentially high-throughput. The ability to provide both qualitative and quantitative results on a multichannel chip further establishes SPRi as a powerful tool for the study of biological interactions in a multiplexed format.
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PMID:Surface plasmon resonance imaging for affinity analysis of aptamer-protein interactions with PDMS microfluidic chips. 1767 82

The aim of this study was to detect the Mycobacterium species in the sputum samples collected from tuberculosis patients in Elazig province (located in Eastern Anatolia, Turkey), by PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) method. A total of 60 samples from patients (32 male, 28 female) who were diagnosed as tuberculosis by culture positivity at Elazig Tuberculosis Control Dispensary, were included to the study. After DNA extraction and isolation from the samples, gene region encoding for 65 kDa protein of mycobacteria was amplified with specific primers (first step primers: TB1; 5'-GAG ATC GAC TGG AGG ATC C-3' and TB2; 5'-AGC TGC AGC CCA AAG GTG TT- 3', second step primers: TB1 and TB3; 5'-GTG TTG GAC TCC TCG ACG GT-3') by using seminested PCR method. According to hsp65 gene region amplification, 51 (85%) samples yielded positive results, while nine (15%) samples could not be identified. Of 51 samples, 44 (86.3%) were identified as M. tuberculosis complex, four (7.8%) were M.scrofulaceum, two (3.9%) were M. avium and one (1.9%) was M. intracellulare, in the restriction assay by Haelll of the PCR products. In order to identify the species of M. tuberculosis complex, gyrB gene region was amplified in those of 44 samples with specific primers (MTUB-f; 5'-TCG GAC GCG TAT GCG ATA TC-3' and MTUB-r; 5'-ACA TAC AGT TCG GAC TTG CG-3'), and the PCR products were restricted by Rsal and Taql enzymes. In this assay, 34 (77.3%), eight (18.2%), one (2.3%) and one (2.3%) of the 44 M. tuberculosis complex samples were detected as M. tuberculosis, M. bovis, M. microti and M. africanum, respectively. Our data indicated that at least seven different Mycobacterium species were the causative agents of tuberculosis in our region. As a result, researching for species distributions of mycobacteria in all of the parts of Turkey by molecular methods and clarifying their resistance patterns against antituberculous drugs are needed for the effective control of tuberculosis.
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PMID:[Detection of Mycobacterium species distribution in the sputum samples of tuberculosis patients by PCR-RFLP method in Elazig province]. 1768 6

Fusarium oxysporum is a soil-borne fungus that causes vascular wilts in a wide variety of plant species. Basil is recognized as an ecological niche for Fusarium oxysporum f.sp. basilici (FOB) and this fungus is now present in most countries where basil is cultivated. The rapid identification of the species affecting basil plants is necessary to define a successful method for crop protection. The aim of this study was to develop a PCR method for the rapid detection of Fusarium oxysporum f. sp. basilici in substrates. The specificity of the primers used was tested using the DNA extracted directly from substrate samples. Fusarium oxysporum f.sp. basilici was artificially inoculated with decreasing amounts in a commercial substrate (sphagnum peat moss) and in a mixture with 40% of municipal compost, after steam disinfestation. Basil seeds (cv. Fine verde) were sown in pots that were laid on a bench in the greenhouse. At time 0 and after 7, 14 and 21 days from the inoculation, substrate and root samples were collected and prepared for microbial analysis and for the DNA extraction. DNA extraction was carried out using NucleoSpin Soil Kit (Macherey-Nagel, Germany). PCR amplification for the specific detection was carried out using primer sets Bik 1 (5'-ATT CAA GAG CTA AAG GTC C-3') and Bik 4 (5'-TTT GAC CAA GAT AGA TGC C-3') for the first PCR, while primers Bik 1 + Bik 2 (5'-AAA GGT AGT ATA TCG GAG G-3') for the nested PCR to increase detection sensitivity. Disease incidence was also assessed 21 days after seeding. The results showed the presence of amplified fragments of the expected size when the concentration of F. oxysporum f.sp. basilici was at least 3.5 Log CFU g(-1) by using DNA extract directly from substrate, before roots were infected by the pathogen. The detection of Fusarium oxysporum f. sp. basilici by PCR method developed in this study is certainly simple and fast and can be useful for its reliable detection in substrate samples, but not to guarantee that the substrate is totally free of pathogens.
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PMID:Detection of Fusarium oxysporum f.sp. basilici in substrates and roots by PCR. 2515 41

In the course of a survey to select superior old citrus lines in the area of Siracusa (Sicily, Italy), trees in several blocks of Fortune (Citrus reticulata Blanco), Nova (C. reticulata Blanco), Satsuma (C. unshiu (Macfad.) mandarins Marc.), and Marsh grapefruit (C. paradisi Macfad.) propagated on sour orange (C. aurantium L.) rootstock showed stunting, decline, dieback, and small-sized fruits. Stunting was particularly evident in grapefruit. Declined plants consistently showed pin-holing in the cambial face of sour orange bark below the bud union line, which is often associated with Citrus tristeza virus (CTV) infection. Young shoots from 600 Fortune, 300 Nova, 400 Satsuma, and 20 Marsh grapefruit plants showing decline were analyzed by double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) (Loewe Phytodiagnostica Biochemica, Sauerlach, Germany) and by immunoprinting-ELISA (Agritest Srl Valenzano-Bari-Italy) using CTV specific polyclonal antibodies. All decline tree samples reacted positively with both techniques while healthy greenhouse controls were negative. Total RNA was extracted from 50 of those plants, 25 Fortune and 15 Nova mandarins, 5 Satsuma, and 5 Marsh grapefruit (Qiagen RNeasy Plant minikit, Qiagen S.P.A., Milan, Italy), and tested in reverse transcription-polymerase chain reaction (RT-PCR) using specific primers for genes p20 (forward 5'-CGA GCT TAC TTT AGT GTT A-3' from CTV T36 genomic position 17767-17786 and reverse 5'-TAA TGT CAA ACT GAC CGC from CTV T36 position 18269-18286) and p23 (forward 5'-ACT AAC TTT AAT TCG AAC A-3' from CTV T36 position 18347-18286 and reverse 5'-AAC TTA TTC CGT CCA CTT C-3' from CTV T36 position 19026-19044) (2). In all cases, DNA fragments of the expected size were amplified. Equivalent samples from CTV-free greenhouse control plants did not react in ELISA and yielded no DNA after amplification with the same primers. When the history of the plants in the affected blocks was traced, it was found that all Fortune, Nova, satsuma and Marsh grapefruit trees had been propagated from budwood illegally imported from Spain 10 years before, suggesting the possibility that the imported buds were infected with CTV. The estimated number of infected plants in the area of Siracusa is approximately 10,000, and some evidence suggests that the virus might be spreading in the area (work in progress). Only scattered CTV-infected trees had been detected in Italy previously (1). To our knowledge, this is the first report of an important CTV outbreak in Italy. Additional surveys are being conducted to get a more accurate estimation of the CTV incidence, to determine if the virus is being dispersed by aphid vectors, and to biologically and molecularly characterize the virus strains present in the affected area. Presently, there are approximately 100,000 ha of citrus in Sicily, mostly grown on decline susceptible sour orange rootstock. The presence and potential spread of CTV is a major threat for this citrus industry. References: (1) M. Davino and G. Terranova. Frutticoltura 61:18, 1999. (2) A. Sambade et al. Plant Pathol. 51:257, 2002.
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PMID:The First Citrus tristeza virus Outbreak Found in a Relevant Citrus Producing Area of Sicily, Italy. 3081 70