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A Rare Case of Trisomy 15pter-q21.2 Due to a De Novo Marker Chromosome

PACANARO, Ade Nubia Xavier; CHRISTOFOLINI, Denise Maria; KULIKOWSKI, Leslie Domenici; BELANGERO, Sintia Iole Nogueira; BELLUCCO, Fernanda Teixeira da Silva; VARELA, Monica C.; KOIFFMANN, Celia P.; YOSHIMOTO, Maisa; SQUIRE, Jeremy A.; SCHIAVON, Adriana V.;
Fonte: WILEY-LISS Publicador: WILEY-LISS
Tipo: Artigo de Revista Científica
ENG
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Supernumerary marker chromosomes (sSMC) may or may not be associated with an abnormal phenotype, depending on the presence of euchromatin, on their chromosomal origin and whether they are inherited. Over 80% of sSMCs are derived from acrocentric chromosomes and half of them include the short arm of chromosome 15. Generally, they appear as bisatellited isodicentric marker chromosomes, most of them are symmetric. These chromosomes are normally originated de novo and are associated with mild to severe intellectual disability but not with physical abnormalities. We report on a patient with an SMC studied using classical and molecular cytogenetic procedures (G and C banding, NOR staining, painting and centromeric fluorescent in situ hybridization (FISH), BAC-FISH, and SKY). The MLPA technique and DNA polymorphic markers were used in order to identify its parental origin. The marker chromosome, monosatellited and monocentric, was found to be derived from a maternal chromosome 15 and was defined as 15pter-q21.2. This is the report of the largest de novo monosatellited 15q marker chromosome ever published presenting detailed cytogenetic and clinical data. It was associated with a phenotype including cardiac defect, absence of septum pellucidum...

Expressão gênica de proteínas de choque térmico como marcador molecular de termotolerância em vacas Nelore; Gene expression of heat shock proteins as molecular marker of thermotolerance in Nellore cows

Hooper, Henrique Barbosa
Fonte: Biblioteca Digitais de Teses e Dissertações da USP Publicador: Biblioteca Digitais de Teses e Dissertações da USP
Tipo: Dissertação de Mestrado Formato: application/pdf
Publicado em 08/07/2015 PT
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45.86%
Objetivou-se com este estudo compreender as dinâmicas das temperaturas corporais em fêmeas da raça Nelore, e relacionar os aspectos fisiológicos da termorregulação com as respostas celulares pela expressão de proteínas de choque térmico. O projeto foi desenvolvido no Laboratório de Biometeorologia e Etologia, da Faculdade de Zootecnia e Engenharia de Alimentos da Universidade de São Paulo, Pirassununga-SP. Foram utilizadas 20 vacas Nelore pluríparas, cíclicas, mantidas em sistema de pastejo. O período experimental precedeu a estação de monta de 2013 e terminou na inseminação artificial. Para classificar os animais quanto ao gerenciamento de calor foi monitorada a temperatura vaginal durante seis dias por meio de 16 data loggers (n=16). Com o intuito de melhor compreender o gerenciamento de calor, outras respostas fisiológicas, nomeadamente temperatura retal, caudal, ocular, frequência respiratória e taxa de sudação foram colhidas durante os quatro dias finais do monitoramento da temperatura vaginal. Para expressão gênica relativa colheram-se amostras de sangue (n=20). Foram realizados choques térmicos in vitro a 38ºC, 40ºC e 42ºC por duas horas. Após tratamento térmico obteve-se o pellet de leucócitos para posterior extração do RNA pelo método TRIzol. Foram escolhidas 10 vacas com os melhores resultados de concentração de RNA...

Caracterização molecular de isolados bacterianos de nódulos e rizosfera de soja em diferentes manejos de cultivo

Costa, Maira Rejane
Fonte: Universidade Estadual Paulista (UNESP) Publicador: Universidade Estadual Paulista (UNESP)
Tipo: Dissertação de Mestrado Formato: xii, 72 f. : il.
POR
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45.72%
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq); Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES); Pós-graduação em Agronomia (Genética e Melhoramento de Plantas) - FCAV; O crescimento da produção e o aumento da capacidade competitiva da soja brasileira estão associados aos avanços científicos e à disponibilização de tecnologias ao setor produtivo. A fim de maximizar os benefícios da FBN, o estudo da diversidade genética de Bradyrhizobium japonicum e Bradyrhizobium elkanii relacionada com diferentes sistemas de manejo da cultura da soja é imprescindível para entender as interações entre a população de rizóbios presentes no solo com as estirpes de inoculantes em ambientes diferentes. O objetivo deste trabalho foi avaliar a variabilidade genética de rizóbios em solos cultivados com soja sob diferentes sistemas de manejo caracterizados com rotação e sucessão de culturas recomendadas para o estado de Mato Grosso do Sul. A partir de amostras de DNA extraídos destas bactérias foi utilizado o marcador molecular fAFLP para estimar a diversidade genética dos 119 isolados de nódulos de soja, e em seguida realizado o sequenciamento parcial do gene 16S rDNA para definir a posição das bactérias em nível de gênero e...

Detection of Legionella pneumophila in water and biofilm samples by culture and molecular methods from man-made systems in São Paulo - Brazil

Carvalho,Fábio R.S.; Foronda,Annette S.; Pellizari,Vivian H.
Fonte: Sociedade Brasileira de Microbiologia Publicador: Sociedade Brasileira de Microbiologia
Tipo: Artigo de Revista Científica Formato: text/html
Publicado em 01/12/2007 EN
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35.78%
Legionella pneumophila is a pathogenic bacteria associated to aquatic habitat of natural and artificial environments. Clinical cases of legionellosis have been reported in Brazil but there is a lack of information about the incidence and concentration of this bacterium in environmental sources. Thus, the present study was designed to evaluate the occurrence of legionellae in São Paulo city, Brazil, using different methods of detection and identification. Sixty-seven water and biofilm samples from natural reservoirs and man-made systems were collected and analyzed for the presence of Legionella spp by culturing onto a selective medium, coculture in axenic free-living amoebae and direct fluorescent antibody (DFA) assay. Results showed that freshwater of reservoirs did not contain legionellae, Legionella pneumophila was isolated from man-made systems, with predominance of Legionella pneumophila serogroup 1 strains. Although there was no statistical difference among the proposed detection methods, the plate culture method yielded a higher number of L. pneumophila positive samples, followed by amoebic coculture procedure and direct fluorescent antibody assay. Results of PCR and sequencing reactions revealed that application of macrophage infectivity potentiator gene as a molecular marker was an important tool for the identification of environmental isolates of L. pneumophila. The agreement among the three detection methods-when all methods yielded similar results- and the prevalence of a single Legionella species in the sampled man-made systems could suggest that the occurrence of this bacterium had been influenced by the higher concentration of metallic ions dissociated in water of those systems than in natural reservoirs. Thus...

Green Fluorescent Protein as a Marker in Plasmodium berghei Transformation

Sultan, Ali A.; Thathy, Vandana; Nussenzweig, Victor; Ménard, Robert
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/1999 EN
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35.92%
We present a new marker that confers both resistance to pyrimethamine and green fluorescent protein-based fluorescence on the malarial parasite Plasmodium berghei. A single copy of the cassette integrated into the genome is sufficient to direct fluorescence in parasites throughout the life cycle, in both its mosquito and vertebrate hosts. Erythrocyte stages of the parasite that express the marker can be sorted from control parasites by flow cytometry. Pyrimethamine pressure is not necessary for maintaining the cassette in transformed parasites during their sporogonic cycle in mosquitoes, including when it is borne by a plasmid. This tool should thus prove useful in molecular studies of P. berghei, both for generating parasite variants and monitoring their behavior.

In Vivo Tracking of Campylobacter jejuni by Using a Novel Recombinant Expressing Green Fluorescent Protein

Mixter, Philip F.; Klena, John D.; Flom, Gary A.; Siegesmund, Amy M.; Konkel, Michael E.
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /05/2003 EN
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35.78%
Campylobacter jejuni is a leading cause of food-borne disease in developed countries. The goal of this study was to develop a plasmid-based reporter system with green fluorescent protein (GFP) to facilitate the study of C. jejuni in a variety of niches. C. jejuni transformants harboring the pMEK91 GFP gene (gfp)-containing vector were readily detectable by both fluorescence microscopy and flow cytometry. Given the ease of detecting these organisms, additional experiments were performed in which BALB/c mice were injected intraperitoneally with C. jejuni harboring the gfp-containing vector. Four hours after injection of the mice, flow cytometry analyses determined that C. jejuni synthesizing GFP were predominantly associated with granulocytes. More specifically, the proportion of CD11b+ Gr-1+ lavage neutrophils with green fluorescence ranged from 99.7 to 100%, while the proportion of CD11b+ Gr-1− lavage macrophages ranged from 77.0 to 80.0%. In contrast, few CD11b− CD45R+ B lymphocytes from the lavage of the C. jejuni-injected mice were associated with green-fluorescent C. jejuni (proportions ranged from 0.75 to 0.77%). Cell-free C. jejuni was recovered from tissue homogenates after intraperitoneal injection. Macrorestriction profiling with pulsed-field gel electrophoresis identified a genotypic variant of the C. jejuni F38011 wild-type isolate. In vivo this variant displayed a phenotype identical to that of the wild-type isolate. In summary...

A microarray-based high throughput molecular marker genotyping method: the tagged microarray marker (TAM) approach

Flavell, Andrew J.; Bolshakov, Viacheslav N.; Booth, Allan; Jing, Runchun; Russell, Joanne; Ellis, T. H. Noel; Isaac, Peter
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Publicado em 01/10/2003 EN
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A microarray-based method has been developed for scoring thousands of DNAs for a co-dominant molecular marker on a glass slide. The approach was developed to detect insertional polymorphism of transposons and works well with single nucleotide polymorphism (SNP) markers. Biotin- terminated allele-specific PCR products are spotted unpurified onto streptavidin-coated glass slides and visualised by hybridisation of fluorescent detector oligonucleotides to tags attached to the allele- specific PCR primers. Two tagged primer oligonucleotides are used per locus and each tag is detected by hybridisation to a concatameric DNA probe labelled with multiple fluorochromes.

A Phosphoribosylanthranilate Transferase Gene Is Defective in Blue Fluorescent Arabidopsis thaliana Tryptophan Mutants 1

Rose, Alan B.; Casselman, Amy L.; Last, Robert L.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /10/1992 EN
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35.78%
An Arabidopsis thaliana gene encoding phosphoribosylanthranilate transferase is shown to be the gene that is defective in blue fluorescent trp1 mutant plants. This gene, named PAT1, was isolated using an A. thaliana cDNA clone that suppressed an Escherichia coli trpD− mutation. The PAT1 coding region is homologous to those for the phosphoribosylanthranilate transferases from many microorganisms. Unlike other genes involved in aromatic amino acid biosynthesis in A. thaliana, PAT1 appears to be a single-copy gene. PAT1 was demonstrated to be the gene that is defective in blue fluorescent trp1 mutants by two methods: genetic complementation in transgenic plants and genetic mapping studies. This is the first report of cloning a plant phosphoribosylanthranilate transferase gene. The PAT1 gene should prove useful as a selectable marker for transformation or a visible reporter of gene expression when used in conjunction with trp1 plants.

Quenching of Superoxide Radicals by Green Fluorescent Protein

Bou-Abdallah, Fadi; Chasteen, N. Dennis; Lesser, Michael P.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
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35.9%
Green fluorescent protein (GFP) is a widely used in vivo molecular marker. These proteins are particularly resistant, and maintain function, under a variety of cellular conditions such as pH extremes and elevated temperatures. Green fluorescent proteins are also abundant in several groups of marine invertebrates including reef-forming corals. While molecular oxygen is required for the post-translational maturation of the protein, mature GFPs are found in corals where hyperoxia and reactive oxygen species (ROS) occur due to the photosynthetic activity of algal symbionts. In vitro spin trapping electron paramagnetic resonance and spectrophotometric assays of superoxide dismutase (SOD)-like enzyme activity show that wild type GFP from the hydromedusa, Aequorea victoria, quenches superoxide radicals (O2•−) and exhibits SOD-like activity by competing with cytochrome c for reaction with O2•−. When exposed to high amounts of O2•− the SOD-like activity and protein structure of GFP are altered without significant changes to the fluorescent properties of the protein. Because of the distribution of fluorescent proteins in both the epithelial and gastrodermal cells of reef-forming corals we propose that GFP, and possibly other fluorescent proteins...

Combined assessment of EGFR pathway-related molecular markers and prognosis of NSCLC patients

Ruiz, M I Galleges; Floor, K; Steinberg, S M; Grünberg, K; Thunnissen, F B J M; Belien, J A M; Meijer, G A; Peters, G J; Smit, E F; Rodriguez, J A; Giaccone, G
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
EN
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45.82%
The purpose of this study is to evaluate the prognostic value of the combined assessment of multiple molecular markers related to the epidermal growth factor receptor (EGFR) pathway in resected non-small cell lung cancer (NSCLC) patients. Tumour specimens of 178 NSCLC patients were collected and analysed for EGFR and KRAS mutation status by DNA sequencing, and for EGFR copy number by fluorescent in situ hybridisation. Tissue microarrays were generated and used to determine the expression of multiple EGFR pathway-related proteins by immunohistochemistry. We analysed the association between each marker and patient prognosis. Univariate analyses for each clinical variable and each molecular marker were performed using Kaplan–Meier curves and log-rank tests. From these results, we selected the variables KRAS mutations and expression of cytoplasmic EGFR, granular pERK, nuclear pSTAT3, cytoplasmic E-cadherin and cytoplasmic pCMET to enter into a Cox proportional hazards model, along with stage as the strongest clinical variable related with prognosis. Of the EGFR-related markers evaluated here, the markers EGFR, pERK, pSTAT3, E-cadherin, pCMET and mutations in KRAS were associated with survival when analysed in combination in our patient cohort...

Localization of the Dantrolene-binding Sequence near the FK506-binding Protein-binding Site in the Three-dimensional Structure of the Ryanodine Receptor*

Wang, Ruiwu; Zhong, Xiaowei; Meng, Xing; Koop, Andrea; Tian, Xixi; Jones, Peter P.; Fruen, Bradley R.; Wagenknecht, Terence; Liu, Zheng; Chen, S. R. Wayne
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
EN
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35.78%
Dantrolene is believed to stabilize interdomain interactions between the NH2-terminal and central regions of ryanodine receptors by binding to the NH2-terminal residues 590–609 in skeletal ryanodine receptor (RyR1) and residues 601–620 in cardiac ryanodine receptor (RyR2). To gain further insight into the structural basis of dantrolene action, we have attempted to localize the dantrolene-binding sequence in RyR1/RyR2 by using GFP as a structural marker and three-dimensional cryo-EM. We inserted GFP into RyR2 after residues Arg-626 and Tyr-846 to generate GFP-RyR2 fusion proteins, RyR2Arg-626-GFP and RyR2Tyr-846-GFP. Insertion of GFP after residue Arg-626 abolished the binding of a bulky GST- or cyan fluorescent protein-tagged FKBP12.6 but not the binding of a smaller, nontagged FKBP12.6, suggesting that residue Arg-626 and the dantrolene-binding sequence are located near the FKBP12.6-binding site. Using cryo-EM, we have mapped the three-dimensional location of Tyr-846-GFP to domain 9, which is also adjacent to the FKBP12.6-binding site. To further map the three-dimensional location of the dantrolene-binding sequence, we generated 10 FRET pairs based on four known three-dimensional locations (FKBP12.6, Ser-437-GFP, Tyr-846-GFP, and Ser-2367-GFP). Based on the FRET efficiencies of these FRET pairs and the corresponding distance relationships...

Pyrroloquinoline Quinone Biosynthesis Gene pqqC, a Novel Molecular Marker for Studying the Phylogeny and Diversity of Phosphate-Solubilizing Pseudomonads ▿ †

Meyer, Joana Beatrice; Frapolli, Michele; Keel, Christoph; Maurhofer, Monika
Fonte: American Society for Microbiology Publicador: American Society for Microbiology
Tipo: Artigo de Revista Científica
Publicado em /10/2011 EN
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45.77%
Many root-colonizing pseudomonads are able to promote plant growth by increasing phosphate availability in soil through solubilization of poorly soluble rock phosphates. The major mechanism of phosphate solubilization by pseudomonads is the secretion of gluconic acid, which requires the enzyme glucose dehydrogenase and its cofactor pyrroloquinoline quinone (PQQ). The main aim of this study was to evaluate whether a PQQ biosynthetic gene is suitable to study the phylogeny of phosphate-solubilizing pseudomonads. To this end, two new primers, which specifically amplify the pqqC gene of the Pseudomonas genus, were designed. pqqC fragments were amplified and sequenced from a Pseudomonas strain collection and from a natural wheat rhizosphere population using cultivation-dependent and cultivation-independent approaches. Phylogenetic trees based on pqqC sequences were compared to trees obtained with the two concatenated housekeeping genes rpoD and gyrB. For both pqqC and rpoD-gyrB, similar main phylogenetic clusters were found. However, in the pqqC but not in the rpoD-gyrB tree, the group of fluorescent pseudomonads producing the antifungal compounds 2,4-diacetylphloroglucinol and pyoluteorin was located outside the Pseudomonas fluorescens group. pqqC sequences from isolated pseudomonads were differently distributed among the identified phylogenetic groups than pqqC sequences derived from the cultivation-independent approach. Comparing pqqC phylogeny and phosphate solubilization activity...

Hsp27 (HSPB1): a possible surrogate molecular marker for loss of heterozygosity (LOH) of chromosome 1p in oligodendrogliomas but not in astrocytomas

Castro, Gisela N.; Cayado-Gutiérrez, Niubys; Moncalero, Vera L.; Lima, Patricia; De Angelis, Rodolfo Lucero; Chávez, Victor; Cuello-Carrión, F. Darío; Ciocca, Daniel R.
Fonte: Springer Netherlands Publicador: Springer Netherlands
Tipo: Artigo de Revista Científica
EN
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35.77%
In oligodendrogliomas, 1p loss of heterozygosity (LOH) is a predictor of good prognosis and treatment response. In contrast, in uveal melanomas, LOH of chromosome 3 has been linked to poor prognosis and downregulation of Hsp27. In the present study, we have analyzed the expression of heat-shock proteins (Hsps) to characterize subtypes of gliomas and their histopathologic features and to correlate with other molecular markers including LOH of 1p. Biopsies from patients with primary gliomas (n = 65) were analyzed by immunohistochemistry, chromogenic in situ hybridization and fluorescent in situ hybridization and methylation-specific PCR (MSP). Elevated Hsp27 and total Hsp70 expression levels were associated with high-grade astrocytomas (p = 0.0001 and p = 0.01, respectively). In grade III oligodendrogliomas, the Hsp27 levels were significantly higher (p = 0.03). Low O6-methylguanine-DNA methyltransferase (MGMT) expression was associated with grade II astrocytomas. Elevated β-catenin expression was associated with grade III/IV astrocytomas (p = 0.003); p53 (+) tumors were more frequently found in grade III/IV astrocytomas (p = 0,001). LOH on 1p was associated with oligodendroglial tumours. In addition, a higher Hsp27 expression correlated with LOH of 1p (p = 0.017); this was also tested in two glioma cell lines. MSP was successful in only six samples. No significant correlations were found for the other markers. In conclusion...

A slippery molecular assembly allows water as a self-erasable security marker

Thirumalai, Rajasekaran; Mukhopadhyay, Rahul Dev; Praveen, Vakayil K.; Ajayaghosh, Ayyappanpillai
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
Publicado em 05/05/2015 EN
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35.91%
Protection of currency and valuable documents from counterfeit continues to be a challenge. While there are many embedded security features available for document safety, they are not immune to forgery. Fluorescence is a sensitive property, which responds to external stimuli such as solvent polarity, temperature or mechanical stress, however practical use in security applications is hampered due to several reasons. Therefore, a simple and specific stimuli responsive security feature that is difficult to duplicate is of great demand. Herein we report the design of a fluorescent molecular assembly on which water behaves as a self-erasable security marker for checking the authenticity of documents at point of care. The underlying principle involves the disciplined self-assembly of a tailor-made fluorescent molecule, which initially form a weak blue fluorescence (λem = 425 nm, Φf = 0.13) and changes to cyan emission (λem = 488 nm,Φf = 0.18) in contact with water due to a reversible molecular slipping motion. This simple chemical tool, based on the principles of molecular self-assembly and fluorescence modulation, allows creation of security labels and optically masked barcodes for multiple documents authentication.

Cost analysis of molecular techniques for marker assisted selection within a barley breeding program

Fox, R.; Hayden, M.; Mekuria, G.; Eglinton, J.
Fonte: Australian Barley Association Publicador: Australian Barley Association
Tipo: Conference paper
Publicado em //2005 EN
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45.89%
Marker assisted selection (MAS) has become a valuable asset to plant breeders as it provides an alternative strategy to introgress valuable alleles into breeding material. As part of its integration into mainstream breeding, finding reliable low cost, high throughput screening techniques is an important aspect of most implementation programs. With recent advancements in molecular marker techniques and the growing availability of commercial genotyping services, there are now a number of options available to breeders for the implementation of MAS. This paper explores the value of three genotyping methods for marker assisted breeding in barley, namely: the traditional approach using conventional PCR and ethidium bromide stained polyacyrlamide gels, a new technology employing multiplex PCR and fluorescent-based marker detection on an automated DNA fragment analyser, and outsourcing to a commercial service provider. A doubled haploid population screened using these methods are compared in relation to the cost, speed, convenience and reliability of each approach.

Development of a molecular marker linked to late maturity α-amylase (LMA) in wheat; Development of a molecular marker linked to late maturity alpha-amylase (LMA) in wheat

Carter, M.; Mrva, K.; Mares, D.; Wilson, R.; Barclay, I.; Appels, R.; Jones, M.
Fonte: Australasian Plant Breeding Association Publicador: Australasian Plant Breeding Association
Tipo: Conference paper
Publicado em //2002 EN
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45.88%
Late maturity α−amylase (LMA) refers to the synthesis of α−amylase activity, which occurs under particular environmental conditions during the later stages of grain ripening in some genotypes in the absence of rain or sprouting. Grain affected by LMA may have a sound appearance, but because of the high α−amylase activity can be unsuitable for a range of end-product applications. Screening for LMA in wheat cultivars is difficult due to the influence of the environment on the expression of this trait. Therefore, the development of a molecular marker to .tag. this trait for marker assisted selection in wheat breeding programs would be a definite advantage. Bulked segregant analysis (BSA) and fluorescent amplified fragment length polymorphism (AFLP) technologies were used to identify loci linked to LMA in a doubled haploid population derived from wheat (Triticum aestivum L. em. Thell) cultivars Cleo-Inia (LMA source) and Janz (non-LMA). Markers linked to LMA were identified in bulks developed from 10 LMA and 10 non-LMA plants, converted to PCR based markers using radioactive AFLP and validated on other cultivars with known LMA phenotype.

Imaging of Mobile Long-lived Nanoplatforms in the Live Cell Plasma Membrane*

Brameshuber, Mario; Weghuber, Julian; Ruprecht, Verena; Gombos, Imre; Horváth, Ibolya; Vigh, László; Eckerstorfer, Paul; Kiss, Endre; Stockinger, Hannes; Schütz, Gerhard J.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
EN
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35.87%
The plasma membrane has been hypothesized to contain nanoscopic lipid platforms, which are discussed in the context of “lipid rafts” or “membrane rafts.” Based on biochemical and cell biological studies, rafts are believed to play a crucial role in many signaling processes. However, there is currently not much information on their size, shape, stability, surface density, composition, and heterogeneity. We present here a method that allows for the first time the direct imaging of nanoscopic long-lived platforms with raft-like properties diffusing in the live cell plasma membrane. Our method senses these platforms by their property to assemble a characteristic set of fluorescent marker proteins or lipids on a time scale of seconds. A special photobleaching protocol was used to reduce the surface density of labeled mobile platforms down to the level of well isolated diffraction-limited spots without altering the single spot brightness. The statistical distribution of probe molecules per platform was determined by single molecule brightness analysis. For demonstration, we used the consensus raft marker glycosylphosphatidylinositol-anchored monomeric GFP and the fluorescent lipid analog BODIPY-GM1, which preferentially partitions into liquid-ordered phases. For both markers...

A Green Fluorescent Protein with Photoswitchable Emission from the Deep Sea

Vogt, Alexander; D'Angelo, Cecilia; Oswald, Franz; Denzel, Andrea; Mazel, Charles H.; Matz, Mikhail V.; Ivanchenko, Sergey; Nienhaus, G. Ulrich; Wiedenmann, Jörg
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
Publicado em 19/11/2008 EN
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35.78%
A colorful variety of fluorescent proteins (FPs) from marine invertebrates are utilized as genetically encoded markers for live cell imaging. The increased demand for advanced imaging techniques drives a continuous search for FPs with new and improved properties. Many useful FPs have been isolated from species adapted to sun-flooded habitats such as tropical coral reefs. It has yet remained unknown if species expressing green fluorescent protein (GFP)-like proteins also exist in the darkness of the deep sea. Using a submarine-based and -operated fluorescence detection system in the Gulf of Mexico, we discovered ceriantharians emitting bright green fluorescence in depths between 500 and 600 m and identified a GFP, named cerFP505, with bright fluorescence emission peaking at 505 nm. Spectroscopic studies showed that ∼15% of the protein bulk feature reversible ON/OFF photoswitching that can be induced by alternating irradiation with blue und near-UV light. Despite being derived from an animal adapted to essentially complete darkness and low temperatures, cerFP505 maturation in living mammalian cells at 37°C, its brightness and photostability are comparable to those of EGFP and cmFP512 from shallow water species. Therefore, our findings disclose the deep sea as a potential source of GFP-like molecular marker proteins.

Characterization of Fluorescent Siderophore-Mediated Iron Uptake in Pseudomonas sp. Strain M114: Evidence for the Existence of an Additional Ferric Siderophore Receptor

Morris, John; O'Sullivan, Daniel J.; Koster, Margot; Leong, John; Weisbeek, Peter J.; O'Gara, Fergal
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /02/1992 EN
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35.73%
In Pseudomonas sp. strain M114, the outer membrane receptor for ferric pseudobactin M114 was shown to transport ferric pseudobactins B10 and A225, in addition to its own. The gene encoding this receptor, which was previously cloned on pCUP3, was localized by Tn5 mutagenesis to a region comprising >1.6 kb of M114 DNA. A mutant (strain M114R1) lacking this receptor was then created by a marker exchange technique. Characterization of this mutant by using purified pseudobactin M114 in radiolabeled ferric iron uptake studies confirmed that it was completely unable to utilize this siderophore for acquisition of iron. In addition, it lacked an outer membrane protein band of 89 kDa when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. As a result, growth of the mutant was severely restricted under low-iron conditions. However, this phenotype was reversed in the presence of another fluorescent siderophore (pseudobactin MT3A) from Pseudomonas sp. strain MT3A, suggesting the presence of a second receptor in strain M114. Furthermore, wild-type Pseudomonas sp. strain B24 was not able to utilize ferric pseudobactin MT3A, and this phenotype was not reversed upon expression of the M114 receptor encoded on pCUP3. However, a cosmid clone (pMS1047) that enabled strain B24 to utilize ferric pseudobactin MT3A was isolated from an M114 gene bank. Radiolabel transport assays with purified pseudobactin MT3A confirmed this event. Plasmid pMS1047 was shown to encode an outer membrane protein of 81 kDa in strain B24 under iron-limiting conditions; this protein corresponds to a similar protein in strain M114.

Green Fluorescent Protein (GFP) in Vector Systems Played Sense Role of Epigenetic in Plants

Hany A. El-Shemy; Mutasim M. Khalafalla; Masao Ishimoto
Fonte: Nature Preceedings Publicador: Nature Preceedings
Tipo: Manuscript
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The green fluorescent protein (GFP) of jellyfish (_Aequorea victoria_) has significant advantages over other reporter genes, because expression can be detected in living cells without any substrates. Recently, epigenetic phenomena are important to consider in plant biotechnology experiments for elucidate unknown mechanism. Therefore, soybean immature cotyledons were generated embryogenesis cells and engineered with two different gene constructs (pHV and pHVS) using gene gun method. Both constructs contain a gene conferring resistance to hygromycin (_hpt_) as a selective marker and a modified glycinin (11S globulin) gene (_V3-1_) as a target. However, sGFP(_S65T_) as a reporter gene was used only in pHVS as a reporter gene for study the relation between using sGFP(_S65T_) and gene silencing phenomena. Fluorescence microscopic was used for screening after the selection of hygromycin, identified clearly the expression of sGFP(_S65T_) in the transformed soybean embryos bombarded with the pHVS construct. Protein analysis was used to detect gene expression overall seeds using SDS-PAGE. Percentage of gene down regulation was highly in pHV construct compared with pHVS. Thus, sGFP(_S65T_) as a reporter gene in vector system may be play useful role for transgenic evaluation and avoid gene silencing in plants for the benefit of plant transformation system.