Project description:In the present study, the whole cell protein extracts of B. abortus and B. melitensis were separated using SDS-PAGE and western blotting was carried out with the antiserum from naturally infected host animals (cow, buffalo, goat and sheep). The proteins bands that matched with western blot signals were excised, trypsin digested and subjected to MALDI identification.
Project description:This study assessed proteomic profile of Candida albicans after serial systemic infection in a murine model. The animals were infected initially by wild-type C. albicans SC5314 (WT) with an inoculum of of 3.5x105 cells via lateral tail vein. Then, five days post-infection, the animals were euthanized and their kidneys were removed, homogenized in lysis buffer, plated on SDA and incubated for 24 h at 35 °C. Colonies recovered from infected kidney were used to prepare inoculum for the subsequent infections as described for WT, totalizing five serial passages (P1-P5) and they were also used to protein extraction. By LC-MS/MS, 479 proteins were identified, with 56 proteins statistically significant in abundance in P1, 29 proteins in P3 and 97 proteins in P4. Regarding biological processes, the majority of proteins were related to carbohydrate metabolism, stress response and amino acid metabolism. The proteins were also categorized according to their potential role in virulence factors such as biofilm production, yeast-to-hyphae transition, phenotypic switching, proteins related to stress response and uncharacterized proteins. Therefore, serial infection associated with proteomic approach enabled to deepen the knowledge about host-pathogen interaction.
Project description:We have previously shown that FGF9 is overexpressed in hypoxia through the IRES located in the 5’UTR. To identify the protein that binds to FGF9 IRES and controls FGF9 protein synthesis in hypoxia, FGF9 IRES RNA was in vitro synthesized and used to pulled-down interacting proteins. The RNA-protein complexes were first visualized by sliver staining, followed by cutting specific bands for protein identification using matrix-assisted laser desorption/ionization time-of-flight mass spectrometer (MALDI-TOF MS).
Project description:Glial cells (microglia and astrocytes) have recently became appreciated as an important target for antidepressant drugs. Here we report on the results of comprehensive proteomic analysis of the alteration in protein profile of rat primary mixed glial culture exposed to imipramine. Two-dimensional differential in gel electrophoresis method allowed to identify 62 proteins regulated by imipramine hydrochloride. Functional analysis revealed the impact of imipramine on the level of proteins involved in oxidative stress. Imipramine upregulated proteins related to glycolysis but downregulated many mitochondrial proteins also enzymes of oxidative phosphorylation. Moreover, imipramine influenced the proteins engaged in phagocytosis and cell migration. Alteration in the level of large number of structural and plasma membrane associated proteins evidenced a widespread cytoskeleton and membrane rearrangement. Imipramine triggered decrease of mitochondrial membrane potential, impairment of protein synthesis and downregulation of chaperon proteins, what could be related to increased apoptosis. Many imipramine regulated proteins, among them chaperons, cathepsins and annexins are evidenced to be engaged in immunity response. Overall these experimental findings suggest that in response to imipramine, glial cells (mainly microglia) undergo a transition toward more quiescent, metabolically less demanding phenotype.
Project description:The current study aimed to detect and identify significant differentially expressed proteins between a virulent and an attenuated Histomonas meleagridis strain which was in vitro co-cultivated with Escherichia coli DH5α. Two-dimensional gel electrophoresis (2-DE) was used for proteome visualization , gel image software for computational detection of significantly up-regulated protein spots and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) for protein identification. The statistical analysis fulfilling two criteria (> or = 3-fold up-regulation and P<0.05) detected 119 differentially expressed protein spots out of which 62 spots were located in gels of the virulent strain and 57 spots in gels of the attenuated strain. The mass spectrometric analysis of 32 spots, up-regulated in gels of the virulent strain, showed that they are of H. meleagridis origin. As opposed to this, the mass spectrometric analysis of 49 protein spots , up-regulated in the gels of the attenuated strain , identified 32 spots as specific to the protozoan. Additionally, the analysis identified a number of E. coli DH5α proteins which were detected as differentially expressed by the computational gel image and statistical analysis.
Project description:Andrographis paniculata (AP) is a medicinal herb used to treat infectious diseases. The medicinal properties are due to presence of andrographolide and other secondary metabolites synthesized via mevalonic acid (MVA) and methyl erythritol phosphate (MEP) pathways. To increase the andrographolide content plants were treated with 50µM Jasmonic acid (JA) for one week. HPLC analysis revealed that one fold increase in the andrographolide content compared to control. To study the signal mechanism underlying in the stress response, samples were subjected to Q-TOF-LC-MS/MS analysis. JA influences the expression of MVA and MEP enzymes in treated (22 enzymes) than in untreated (3 enzymes). We found total 3308 proteins in control and 3994 in elicited sample in which 1393 were commonly found in both. The differential expression revealed 621 proteins were down-regulated and 201 proteins were up regulated. The functional annotation involved 40 metabolic processes where post translational modifications was highly up regulated in treated (5.7%). The total proteome of A.paniculata was reported for the first time. The comparison of elicited and non elicited (control) samples revealed that the elicitation is an integrated process which release variety of bioactive compounds to defend itself from variety of pathogens. The Knowledge obtained from this scrutiny, JA dependent stress adaptive response is likely to have industrial and agricultural impact. It creates a hope towards future for the development of eco-friendly ways of managing pests and tapping into a largely unexplored treasure of plant-derived bioactive metabolites for human use. Particularly in this plant, it is useful for the production of medicines to cure different kinds of diseases and disorders.
Project description:Herein, we constructed a human lectin microarray with 60 human lectin and lectin-like proteins. All of the lectins and lectin-like proteins were purified from yeast, and most showed binding to human glycans. To demonstrate the applicability of the human lectin microarray, human sperm were probed on the microarray and strong bindings were observed for several lectins, including galectin-1, 7, 8, GalNAc-T6, and ERGIC-53 (LMAN1). These bindings were validated by flow cytometry and fluorescence immunostaining. Further, mass spectrometry analysis showed that galectin-1 binds several membrane proteins including HSP90. Finally, functional assays showed that binding of galectin-8 could significantly enhance the acrosome reaction within human sperms. To our knowledge, this is the first construction of a human lectin microarray, and we anticipate it will find wide use for a range of human or mammalian studies, alone or in combination with plant lectin microarrays.
Project description:Expressional alterations and post translational modifications (PTM) of type II collagen can be major cause behind osteo and rheumatoid arthritis. PTM of type II collagen α1 chain (COL2A1) such as hydroxylation of proline (P), lysine and glycosylation of hydroxylysine can act as epitopes resulting COL2A1 as autoantigen in cartilage tissues. Previous study stated proline hydroxylation (Hyp) as an important PTM in type II collagen leading to dysfunctional collagen extracellular matrix assembly in vivo. Here we report for the first time peptide mass fingerprinting (PMF) identification and tandem mass spectrometry based mapping of Hyp PTM in COL2A1 from Capra hircus (C. hircus) using Mascot database. As mascot database does not contain C. hircus COL2A1 sequence information, our identification is based on the homologous COL2A1 from Bos taurus and Homo sapience (above 98 % identity). Findings include identification of new triplet Gly-F-Hyp in C. hircus COL2A1 and well known Gly-X-Y triplet with Hyp present in the X position, instead of Y position. PMF data contains lager number of Hyp in COL2A1 from C. hircus consistent with other collagen sequences. This study suggests positional alteration of Hyp/P in Gly-X-Y triplet may be used for molecular identification and characterization of type II collagen from other sources.