87 research outputs found
Mitochondrial proteomics: analysis of a whole mitochondrial extract with two-dimensional electrophoresis
Mitochondria are complex organelles, and their proteomics analysis requires a
combination of techniques. The emphasis in this chapter is made first on
mitochondria preparation from cultured mammalian cells, then on the separation
of the mitochondrial proteins with two-dimensional electrophoresis (2DE),
showing some adjustment over the classical techniques to improve resolution of
the mitochondrial proteins. This covers both the protein solubilization, the
electrophoretic part per se, and the protein detection on the gels, which makes
the interface with the protein identification part relying on mass
spectrometry
Silver staining of proteins in polyacrylamide gels
Silver staining is used to detect proteins after electrophoretic separation
on polyacrylamide gels. It combines excellent sensitivity (in the low nanogram
range) with the use of very simple and cheap equipment and chemicals. It is
compatible with downstream processing, such as mass spectrometry analysis after
protein digestion. The sequential phases of silver staining are protein
fixation, then sensitization, then silver impregnation and finally image
development. Several variants of silver staining are described here, which can
be completed in a time range from 2 h to 1 d after the end of the
electrophoretic separation. Once completed, the stain is stable for several
weeks
Muscle and tendon adaptations to moderate load eccentric vs. concentric resistance exercise in young and older males.
Resistance exercise training (RET) is well-known to counteract negative age-related changes in both muscle and tendon tissue. Traditional RET consists of both concentric (CON) and eccentric (ECC) contractions; nevertheless, isolated ECC contractions are metabolically less demanding and, thus, may be more suitable for older populations. However, whether submaximal (60% 1RM) CON or ECC contractions differ in their effectiveness is relatively unknown. Further, whether the time course of muscle and tendon adaptations differs to the above is also unknown. Therefore, this study aimed to establish the time course of muscle and tendon adaptations to submaximal CON and ECC RET. Twenty healthy young (24.5βΒ±β5.1Β years) and 17 older males (68.1βΒ±β2.4Β years) were randomly allocated to either isolated CON or ECC RET which took place 3/week for 8Β weeks. Tendon biomechanical properties, muscle architecture and maximal voluntary contraction were assessed every 2Β weeks and quadriceps muscle volume every 4Β weeks. Positive changes in tendon Young's modulus were observed after 4Β weeks in all groups after which adaptations in young males plateaued but continued to increase in older males, suggesting a dampened rate of adaptation with age. However, both CON and ECC resulted in similar overall changes in tendon Young's modulus, in all groups. Muscle hypertrophy and strength increases were similar between CON and ECC in all groups. However, pennation angle increases were greater in CON, and fascicle length changes were greater in ECC. Notably, muscle and tendon adaptations appeared to occur in synergy, presumably to maintain the efficacy of the muscle-tendon unit
A novel malaria vaccine candidate antigen expressed in Tetrahymena thermophila
Development of effective malaria vaccines is hampered by the problem of producing correctly folded Plasmodium proteins for use as vaccine components. We have investigated the use of a novel ciliate expression system, Tetrahymena thermophila, as a P. falciparum vaccine antigen platform. A synthetic vaccine antigen composed of N-terminal and C-terminal regions of merozoite surface protein-1 (MSP-1) was expressed in Tetrahymena thermophila. The recombinant antigen was secreted into the culture medium and purified by monoclonal antibody (mAb) affinity chromatography. The vaccine was immunogenic in MF1 mice, eliciting high antibody titers against both N- and C-terminal components. Sera from immunized animals reacted strongly with P. falciparum parasites from three antigenically different strains by immunofluorescence assays, confirming that the antibodies produced are able to recognize parasite antigens in their native form. Epitope mapping of serum reactivity with a peptide library derived from all three MSP-1 Block 2 serotypes confirmed that the MSP-1 Block 2 hybrid component of the vaccine had effectively targeted all three serotypes of this polymorphic region of MSP-1. This study has successfully demonstrated the use of Tetrahymena thermophila as a recombinant protein expression platform for the production of malaria vaccine antigens
Iranian joint registry (iranian national hip and knee arthroplasty registry)
Periodic evaluation and monitoring the health and economic outcome of joint replacement surgery is a common and popular process under the territory of joint registries in many countries. In this article we introduce the methodology used for the foundation of the National Iranian Joint Registry (IJR) with a joint collaboration of the Social Security Organization (SSO) and academic research departments considering the requirements of the Iran's Ministry of Health and Education. ΓΒ©BY THE ARCHIVES OF BONE AND JOINT SURGERY
Efficient Nuclear Transport of Structurally Disturbed Cargo: Mutations in a Cargo Protein Switch Its Cognate Karyopherin
The Karyopherin (Kap) family of nuclear transport receptors enables trafficking of proteins to and from the nucleus in a precise, regulated manner. Individual members function in overlapping pathways, while simultaneously being very specific for their main cargoes. The details of this apparent contradiction and rules governing pathway preference remain to be further elucidated. S. cerevisiae Lhp1 is an abundant protein that functions as an RNA chaperone in a variety of biologically important processes. It localizes almost exclusively to the nucleus and is imported by Kap108. We show that mutation of 3 of the 275 residues in Lhp1 alters its import pathway to a Kap121-dependent process. This mutant does not retain wild-type function and is bound by several chaperones. We propose that Kap121 also acts as a chaperone, one that can act as a genetic buffer by transporting mutated proteins to the nucleus
Differences in Immunoglobulin Light Chain Species Found in Urinary Exosomes in Light Chain Amyloidosis (AL)
Renal involvement is a frequent consequence of plasma cell dyscrasias. The most common entities are light chain amyloidosis, monoclonal immunoglobulin deposition disease and myeloma cast nephropathy. Despite a common origin, each condition has its own unique histologic and pathophysiologic characteristic which requires a renal biopsy to distinguish. Recent studies have shown urinary exosomes containing kidney-derived membrane and cytosolic proteins that can be used to probe the proteomics of the entire urinary system from the glomerulus to the bladder. In this study, we analyzed urine exosomes to determine the differences between exosomes from patients with light chain amyloidosis, multiple myeloma, monoclonal gammopathy of undetermined significance, and non-paraproteinemia related kidney disease controls. In patients with light chain amyloidosis, multiple myeloma and monoclonal gammopathy of undetermined significance, immunoreactive proteins corresponding to monomeric light chains were found in exosomes by western blot. In all of the amyloidosis samples with active disease, high molecular weight immunoreactive species corresponding to a decamer were found which were not found in exosomes from the other diseases or in amyloidosis exosomes from patients in remission. Few or no light chains monomeric bands were found in non-paraproteinemia related kidney disease controls. Our results showed that urinary exosomes may have tremendous potential in furthering our understanding of the pathophysiology and diagnosis of plasma cell dyscrasia related kidney diseases
Proteome Serological Determination of Tumor-Associated Antigens in Melanoma
Proteome serology may complement expression library-based approaches as strategy utilizing the patients' immune responses for the identification pathogenesis factors and potential targets for therapy and markers for diagnosis. Melanoma is a relatively immunogenic tumor and antigens recognized by melanoma-specific T cells have been extensively studied. The specificities of antibody responses to this malignancy have been analyzed to some extent by molecular genetic but not proteomics approaches. We screened sera of 94 melanoma patients for anti-melanoma reactivity and detected seropositivity in two-thirds of the patients with 2β6 antigens per case detected by 1D and an average of 2.3 per case by 2D Western blot analysis. For identification, antigen spots in Western blots were aligned with proteins in 2-DE and analyzed by mass spectrometry. 18 antigens were identified, 17 of which for the first time for melanoma. One of these antigens, galectin-3, has been related to various oncogenic processes including metastasis formation and invasiveness. Similarly, enolase has been found deregulated in different cancers. With at least 2 of 18 identified proteins implicated in oncogenic processes, the work confirms the potential of proteome-based antigen discovery to identify pathologically relevant proteins
A Novel Anti-CEACAM5 Monoclonal Antibody, CC4, Suppresses Colorectal Tumor Growth and Enhances NK Cells-Mediated Tumor Immunity
Carcinoembryonic antigen (CEA, CEACAM5, and CD66e) has been found to be associated with various types of cancers, particularly colorectal carcinoma, and developed to be a molecular target for cancer diagnosis and therapy. In present study, we generated a novel anti-CEACAM5 monoclonal antibody, namely mAb CC4, by immunizing mice with living colorectal cancer LS174T cells. Immunohistochemical studies found that mAb CC4 specifically and strongly binds to tumor tissues, especially colorectal adenocarcinoma. In xenografted mice, mAb CC4 is specifically accumulated in tumor site and remarkably represses colorectal tumor growth. In vitro functional analysis showed that mAb CC4 significantly suppresses cell proliferation, migration and aggregation of colorectal cancer cells and also raises strong ADCC reaction. More interestingly, mAb CC4 is able to enhance NK cytotoxicity against MHC-I-deficient colorectal cancer cells by blocking intercellular interaction between epithelial CEACAM5 and NK inhibitory receptor CEACAM1. These data suggest that mAb CC4 has the potential to be developed as a novel tumor-targeting carrier and cancer therapeutic
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