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      Insights into the proteomic profile and gene expression of Lutzomyia longipalpis-derived Lulo cell line

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          Abstract

          BACKGROUND

          Lutzomyia longipalpis-derived cell line (Lulo) has been suggested as a model for studies of interaction between sandflies and Leishmania.

          OBJECTIVES

          Here, we present data of proteomic and gene expression analyses of Lulo cell related to interactions with Leishmania (Viannia) braziliensis.

          METHODS

          Lulo cell protein extracts were analysed through a combination of two-dimensional gel electrophoresis and mass spectrometry and resulting spots were further investigated in silico to identify proteins using Mascot search and, afterwards, resulting sequences were applied for analysis with VectorBase.

          RESULTS

          Sixty-four spots were identified showing similarities to other proteins registered in the databases and could be classified according to their biological function, such as ion-binding proteins (23%), proteases (14%), cytoskeletal proteins (11%) and interactive membrane proteins (9.5%). Effects of interaction with L. (V.) braziliensis with the expression of three genes (enolase, tubulin and vacuolar transport protein) were observed after an eight-hour timeframe and compared to culture without parasites, and demonstrated the impact of parasite interaction with the expression of the following genes: LLOJ000219 (1.69-fold), LLOJ000326 (1.43-fold) and LLOJ006663 (2.41-fold).

          CONCLUSIONS

          This set of results adds relevant information regarding the usefulness of the Lulo cell line for studies with Leishmania parasites that indicate variations of protein expression.

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          Most cited references54

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          Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4

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            In-gel digestion for mass spectrometric characterization of proteins and proteomes.

            In-gel digestion of proteins isolated by gel electrophoresis is a cornerstone of mass spectrometry (MS)-driven proteomics. The 10-year-old recipe by Shevchenko et al. has been optimized to increase the speed and sensitivity of analysis. The protocol is for the in-gel digestion of both silver and Coomassie-stained protein spots or bands and can be followed by MALDI-MS or LC-MS/MS analysis to identify proteins at sensitivities better than a few femtomoles of protein starting material.
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              Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250.

              An improved procedure for staining of proteins following separation in polyacrylamide gels is described which utilizes the colloidal properties of Coomassie Brilliant Blue G-250 and R-250. The new method is based on addition of 20% v/v methanol and higher concentrations of ammonium sulfate to the staining solution previously described. The method combines the advantage of much shorter staining time with high sensitivity, a clear background not requiring destaining, stepwise staining, and stable fixation after staining. The method has been applied to staining of polyacrylamide gels after sodium dodecyl sulfate-electrophoresis and isoelectric focusing in carrier ampholyte-generated pH gradients.
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                Author and article information

                Journal
                Mem Inst Oswaldo Cruz
                Mem Inst Oswaldo Cruz
                mioc
                Memórias do Instituto Oswaldo Cruz
                Instituto Oswaldo Cruz, Ministério da Saúde
                0074-0276
                1678-8060
                26 October 2020
                2020
                : 115
                : e200113
                Affiliations
                [1 ]Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Laboratório de Biologia Molecular e Doenças Endêmicas, Rio de Janeiro, RJ, Brasil
                [2 ]Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Laboratório de Genômica Funcional e Bioinformática, Rio de Janeiro, RJ, Brasil
                [3 ]Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Plataforma de Microscopia Eletrônica Rudolf Barth, Rio de Janeiro, RJ, Brasil
                [4 ]Fundação Oswaldo Cruz-Fiocruz, Instituto Oswaldo Cruz, Laboratório de Biologia Estrutural, Rio de Janeiro, RJ, Brasil
                [5 ]Facultad de Ciencias Agropecuarias, Programa de Medicina Veterinaria, Universidad de La Salle, Bogotá, Colombia
                Author notes
                + Corresponding author: calves@ 123456ioc.fiocruz.br

                All authors contributed to writing, reviewing and editing the manuscript. LMCC, DPP, PSGF, GDL, FSS, PRC, RMMS, SCR, LML, OCM and MCW conceived, designed the analyses and collected the data. LMCC, BASP, FJB and CRA performed the manuscript conceptualisation and final review.

                Author information
                http://orcid.org/0000-0001-8703-426X
                Article
                00345
                10.1590/0074-02760200113
                7586444
                33111757
                cfeb40aa-7109-460a-8476-50ddf74b2bc0

                This is an open-access article distributed under the terms of the Creative Commons Attribution License

                History
                : 13 March 2020
                : 24 September 2020
                Page count
                Figures: 5, Tables: 1, References: 47
                Categories
                Original Article

                lulo cell,leishmania (viannia) braziliensis,proteomic,rt-qpcr

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