• Title/Summary/Keyword: 2-DE

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Solubilization of Proteins from Human Lymph Node Tissue and Two-Dimensional Gel Storage

  • De Marqui, Alessandra Bernadete Trovo;Vidotto, Alessandra;Polachini, Giovana Mussi;De Mattos Bellato, Claudia;Cabral, Hamilton;Leopoldino, Andreia Machado;De Gois Filho, Jose Francisco;Fukuyama, Erica Erina;Settanni, Flavio Aurelio Parente;Cury, Patricia Maluf;Bonilla-Rodriguez, Gustavo Orlando;Palma, Mario Sergio;Tajara, Eloiza Helena
    • BMB Reports
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    • v.39 no.2
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    • pp.216-222
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    • 2006
  • In the present study, we compared six different solubilization buffers and optimized two-dimensional electrophoresis (2-DE) conditions for human lymph node proteins. In addition, we developed a simple protocol for 2-D gel storage. Efficient solubilization was obtained with lysis buffers containing (a) 8M urea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM Tris base, 65 mM DTT(dithiothreitol) and 0.2% carrier ampholytes; (b) 5M urea, 2M thiourea, 2% CHAPS, 2% SB 3-10 (N-decyl-N, N-dimethyl-3-ammonio-1-propanesulfonate), 40mM Tris base, 65 mM DTT and 0.2% carrier ampholytes or (c) 7M urea, 2M thiourea, 4% CHAPS, 65 mM DTT and 0.2% carrier ampholytes. The optimal protocol for isoelectric focusing (IEF) was accumulated voltage of 16,500 Vh and 0.6% DTT in the rehydration solution. In the experiments conducted for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), best results were obtained with a doubled concentration (50 mM Tris, 384 mM glycine, 0.2% SDS) of the SDS electrophoresis buffer in the cathodic reservoir as compared to the concentration in the anodic reservoir (25 mM Tris, 192 mM glycine, 0.1% SDS). Among the five protocols tested for gel storing, success was attained when the gels were stored in plastic bags with 50% glycerol. This is the first report describing the successful solubilization and 2D-electrophoresis of proteins from human lymph node tissue and a 2-D gel storage protocol for easy gel handling before mass spectrometry (MS) analysis.

Molecular Characterization of Trypanosoma cruzi Tc8.2 Gene Indicates Two Differential Locations for the Encoded Protein in Epimastigote and Trypomastigote Forms

  • Kian, Danielle;Lancheros, Cesar Armando Contreras;Damiani, Igor Alexandre Campos;Fernandes, Tamiris Zanforlin Olmos;Pinge-Filho, Phileno;dos Santos, Marcia Regina Machado;da Silveira, Jose Franco;Nakamura, Celso Vataru;da Silva, Joao Santana;Yamada-Ogatta, Sueli Fumie;Yamauchi, Lucy Megumi
    • Parasites, Hosts and Diseases
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    • v.53 no.4
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    • pp.483-488
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    • 2015
  • This report describes the molecular characterization of the Tc8.2 gene of Trypanosoma cruzi. Both the Tc8.2 gene and its encoded protein were analyzed by bioinformatics, while Northern blot and RT-PCR were used for the transcripts. Besides, immunolocalization of recombinant protein was done by immunofluorescence and electron microscopy. Analysis indicated the presence of a single copy of Tc8.2 in the T. cruzi genome and 2-different sized transcripts in epimastigotes/amastigotes and trypomastigotes. Immunoblotting showed 70 and 80 kDa polypeptides in epimastigotes and trypomastigotes, respectively, and a differential pattern of immunolocalization. Overall, the results suggest that Tc8.2 is differentially expressed during the T. cruzi life cycle.

Synthesis and Cations Binding Properties of a New C,N-bipyrazolic Ligand

  • Attayibat, Ahmed;Radi, Smaail;Ramdani, Abdelkrim;Lekchiri, Yahya;Hacht, Brahim;Bacquet, Maryse;Willai, Stephanie;Morcellet, Michel
    • Bulletin of the Korean Chemical Society
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    • v.27 no.10
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    • pp.1648-1650
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    • 2006
  • The synthesis of a new C,N-bipyrazolic ligand with a functionalized donor-group is reported. The binding properties of the ligand and two other ligand of similar structures towards heavy metal ions ($Hg^{2+}$, $Cd^{2+}$, $Pb^{2+}$, $Zn^{2+}$, $Cu^{2+}$) and alkaline metal ions ($K^+$, $Na^{+}$, $Li^+$) were studied by a liquid-liquid extraction process and the extracted cation percentage was determined by atomic absorption measurements. The selectivity of the ligand to Hg(II) has been mentioned in the abstract.

Adsorption of Three Chlorinated Herbicides on Two Activated Carbons: An Example of the Effect of Surface Charge, Pore Diameter and Molecular Size on the Adsorption Process

  • Pila Matias N.;Colasurdo Diego D.;Simonetti Sandra I.;Dodero Gabriela A.;Allegretti Patricia E.;Ruiz Danila L.;Laurella Sergio L.
    • Korean Chemical Engineering Research
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    • v.61 no.1
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    • pp.97-108
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    • 2023
  • Two carbonaceous adsorbents CAT and CARBOPAL were tested for reducing the concentration of the three herbicides in water: 2,4-D (2,4-dichlorophenoxyacetic acid), TCP (2,4,6-trichlorophenol) and metolachlor. Textural and chemical characterization of the adsorbents include nitrogen isotherms, FTIR, titration and thermogravimetric analyses. Adsorption was studied in discontinuous adsorption experiments at different pH values. The experimental adsorption isotherms data were fitted to four theoretical models. Adsorbent characterization reveals that CAT has higher micropore area, lower pore diameter and lower acidity than CARBOPAL. The adsorption is a second-order process and the isotherms best fitted to Sips model. The efficiency of the process depends mainly on the charge of the adsorbate for TCP and 2,4-D, but it depends on the charge of the surface for metolachlor. Adsorption capacity is higher on CAT for 2,4-D and TCP (small molecules), and it is higher on CARBOPAL for metolachlor (large molecules). Theoretical calculations clearly support this assumption.

Optimization of Trichomonas vaginalis Diagnosis during Pregnancy at a University Hospital, Argentina

  • Testardini, Pamela;Vaulet, Maria Lucia Gallo;Entrocassi, Andrea Carolina;Menghi, Claudia;Eliseht, Martha Cora;Gatta, Claudia;Losada, Mirta;Touzon, Maria Sol;Corominas, Ana;Vay, Carlos;Tatti, Silvio;Famiglietti, Angela;Fermepin, Marcelo Rodriguez;Perazzi, Beatriz
    • Parasites, Hosts and Diseases
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    • v.54 no.2
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    • pp.191-195
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    • 2016
  • The aim of this study was to evaluate different methods for Trichomonas vaginalis diagnosis during pregnancy in order to prevent maternal and perinatal complications. A total of 386 vaginal exudates from pregnant women were analyzed. T. vaginalis was investigated by 3 types of microscopic examinations direct wet mount with physiologic saline solution, prolonged May-Grunwald Giemsa (MGG) staining, and wet mount with sodium-acetate-formalin (SAF)/methylene blue method. PCR for 18S rRNA gene as well as culture in liquid medium were performed. The sensitivity and specificity of the microscopic examinations were evaluated considering the culture media positivity or the PCR techniques as gold standard. The frequency of T. vaginalis infection was 6.2% by culture and/or PCR, 5.2% by PCR, 4.7% by culture, 3.1% by SAF/methylene blue method and 2.8% by direct wet smear and prolonged MGG staining. The sensitivities were 83.3%, 75.0%, 50.0%, and 45.8% for PCR, culture, SAF/methylene blue method, and direct wet smear-prolonged MGG staining, respectively. The specificity was 100% for all the assessed methods. Microscopic examinations showed low sensitivity, mainly in asymptomatic pregnant patients. It is necessary to improve the detection of T. vaginalis using combined methods providing higher sensitivity, such as culture and PCR, mainly in asymptomatic pregnant patients, in order to prevent maternal and perinatal complications.

Impact of Genetically Modified Enterobacter cloacae on Indigenous Endophytic Community of Citrus sinensis Seedlings

  • Fernando Dini;Mortatti, Marcelo-Jose;Souza, Andre-Oliveira de;Walter Maccheroni;Joao Lucio;Welington Luiz
    • Journal of Microbiology
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    • v.42 no.3
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    • pp.169-173
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    • 2004
  • Enterobacter cloacae (strain PR2/7), a genetically modified endophyte(GME) in citrus plants, carrying different plasmids (pEC3.0/18, pCelE, pEglA and pGFP), was inoculated into Citrus sinensis seedlings under greenhouse conditions. The impact of this on the indigenous bacterial endophytic community was studied by analyses of 2 different morphologic groups. The germination rates of inoculated seeds were evaluated in greenhouse, and plasmid stability under in vitro conditions. Results demonstrated a great and diverse endophytic community inside plants, and specialization in tissue colonization by some bacterial groups, in different treatments. Shifts in seed germination rate were observed among treatments: in general, the PR2/7 harboring pEglA bacterial- clone significantly reduced seed germination, compared to the PR2/7 harboring pEC3.0/18 clone. This suggests that the presence of the pEglA plasmid changes bacteria-seed interactions. The endophytic community of citrus seedlings changed according to treatment. In seedlings treated with the PR2/7 with pEglA clone, the population of group II decreased significantly, within the context of the total endophytic community. These results indicate that the application of GMEs induces shifts in the endophytic bacterial community of citrus seedlings.

Macroalkoxyamines and macroRAFT agents based on polyethylene for the syntheses of polyolefin based polar block copolymers

  • Lopez R. Godoy;Boisson C.;D'Agosto F.;Spitz R.;Boisson F.;Gigmes D.;Bertin D.
    • Proceedings of the Polymer Society of Korea Conference
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    • 2006.10a
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    • pp.377-377
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    • 2006
  • Alkoxyamine and thiocarbonyl thio end capped polyethylene (PE) chains were synthesized using a direct and simple approach consisting in reacting di(polyethylenyl)magnesium (PE-Mg-PE) chains with a range of nitroxides and disulfides of thiocarbonyl thio compounds. PE-Mg-PE compounds were prepared by a catalyzed chain growth reaction of ethylene on nbutyloctylmagnesium (BOMg) with a neodymocene complex $(C_{5}Me_{5})_{2}NdCl_{2}Li(OEt_{2})_{2}$. Complete characterizations confirm the introduction of the desired end groups. The controlled radical polymerization (NMP and RAFT) of butyl acrylate mediated by these functional polyethylenes was successful.

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Increased Expression of P2RY2, CD248 and EphB1 in Gastric Cancers from Chilean Patients

  • Aquea, Gisela;Bresky, Gustavo;Lancellotti, Domingo;Madariaga, Juan Andres;Zaffiri, Vittorio;Urzua, Ulises;Haberle, Sergio;Bernal, Giuliano
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.5
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    • pp.1931-1936
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    • 2014
  • Background: Gastric cancer (GC) ranks as one of the major causes of mortality due to cancer worldwide. In Chile, it is currently the leading cause of cancer death. Identification of novel molecular markers that may help to improve disease diagnosis at early stages is imperative. Materials and Methods: Using whole-genome DNA microarrays we determined differential mRNA levels in fresh human GC samples compared to adjacent healthy mucosa from the same patients. Genes significantly overexpressed in GC were validated by RT-PCR in a group of 14 GC cases. Results: The genes CD248, NSD1, RAB17, ABCG8, Ephb1 and P2RY2 were detected as the top overexpressed in GC biopsies. P2RY2, Ephb1 and CD248 showed the best sensitivity for GC detection with values of 92.9%, 85.7% and 64.3% (p<0.05), respectively. Specificity was 85.7%, 71.4% and 71.4% (p<0.05), for each respectively.