With a view to understand the molecular basis of sperm motility, we have tried to establish the human sperm proteome by two-dimensional PAGE MALDI MS/MS analysis. We report identification of 75 different proteins in the human spermatozoa. Comparative proteome analysis was carried out for asthenozoospermic and normozoospermic patients to understand the molecular basis of sperm motility. Analysis revealed eight proteins (including one unidentified) with altered intensity between the groups. Differential proteins distributed into three functional groups: 'energy and metabolism' (triose-phosphate isomerase, glycerol kinase 2, testis specific isoform and succinyl-CoA:3-ketoacid co-enzyme A transferase 1, mitochondrial precursor); 'movement and organization' (tubulin beta 2C and tektin 1) and 'protein turnover, folding and stress response' (proteasome alpha 3 subunit and heat shock-related 70 kDa protein 2). It was interesting to note that although the proteins falling in the functional group of 'energy and metabolism' are higher in the asthenozoospermic patients, the other two functional groups contain proteins, which are higher in the normozoospermic samples. Validation of results carried out for proteasome alpha 3 subunit by immunoblotting and confocal microscopy, confirmed significant changes in intensity of proteasome alpha 3 subunit in asthenozoospermic samples when compared with normozoospermic controls. Significant positive correlation too was found between proteasome alpha 3 subunit levels and rapid, linear progressive motility of the spermatozoa. In our understanding, this data would contribute appreciably to the presently limited information available about the proteins implicated in human sperm motility.
Amphotericin B (AMB) is the most widely used polyene antifungal drug for the treatment of systemic fungal infections, including invasive aspergillosis. It has been our aim to understand the molecular targets of AMB in Aspergillus fumigatus by genomic and proteomic approaches. In transcriptomic analysis, a total of 295 genes were found to be differentially expressed (165 upregulated and 130 downregulated), including many involving the ergosterol pathway, cell stress proteins, cell wall proteins, transport proteins, and hypothetical proteins. Proteomic profiles of A. fumigatus alone or A. fumigatus treated with AMB showed differential expression levels for 85 proteins (76 upregulated and 9 downregulated). Forty-eight of them were identified with high confidence and belonged to the above-mentioned categories. Differential expression levels for Rho-GDP dissociation inhibitor (Rho-GDI), secretory-pathway GDI, clathrin, Sec 31 (a subunit of the exocyst complex), and RAB GTPase Ypt51 in response to an antifungal drug are reported here for the first time and may represent a specific response of A. fumigatus to AMB. The expression of some of these genes was validated by real-time reverse transcription-PCR. The AMB responsive genes/proteins observed to be differentially expressed in A. fumigatus may be further explored for novel drug development.Invasive aspergillosis, caused primarily by Aspergillus fumigatus, has emerged as the leading cause of mortality among immunocompromised patients with underlying hematological diseases or bone marrow transplantation (7, 38). Amphotericin B (AMB), a broad-spectrum fungicidal agent, has been widely used to treat patients with invasive aspergillosis. AMB is reported to be fungicidal (MFC/MIC Յ 4) against all A. fumigatus and Aspergillus flavus isolates but not on Aspergillus terreus isolates (25). Its therapeutic use is limited by its toxicity (nephrotoxicity, cytotoxicity, and hepatotoxicity, etc.) in the host (13) and development of resistance in fungal isolates (42). However, use of lipid formulations of AMB, administration by the inhalation route, and development of less toxic analogues have facilitated better therapeutic outcomes (35). In general, it is known that AMB intercalates with ergosterol of the fungal cell membrane and forms pores resulting in leakage of fungal cell components, which leads to death via osmotic collapse (15, 22).It also promotes oxidative damage to cell membranes through generation of reactive oxygen species (ROS) (43) and damage to DNA resulting in loss of cell viability, a characteristic of apoptosis (31).Earlier efforts reported genome-wide expression analysis to understand the mechanism of action and specific effects of AMB and other antifungal drugs on nonfilamentous fungal species, such as Saccharomyces cerevisiae and Candida albicans (1,24,46). However, in an earlier study of interaction of A. fumigatus with voriconazole (11), decreased mRNA expression of ergosterol biosynthesis genes was observed, which was in contrast with previous reports of S....
The epimorphic regeneration of zebrafish caudal fin is rapid and complete. We have analyzed the biomechanism of zebrafish caudal fin regeneration at various time points based on differential proteomics approaches. The spectrum of proteome changes caused by regeneration were analyzed among controls (0 h) and 1, 12, 24, 48, and 72 h postamputation involving quantitative differential proteomics analysis based on two-dimensional gel electrophoresis matrix-assisted laser desorption/ionization and differential in-gel electrophoresis Orbitrap analysis. A total of 96 proteins were found differentially regulated between the control nonregenerating and regenerating tissues of different time points for having at least 1.5-fold changes. 90 proteins were identified as differentially regulated for regeneration based on differential in-gel electrophoresis analysis between the control and regenerating tissues. 35 proteins were characterized for its expression in all of the five regenerating time points against the control samples. The proteins identified and associated with regeneration were found to be directly allied with various molecular, biological, and cellular functions. Based on network pathway analysis, the identified proteome data set for regeneration was majorly associated in maintaining cellular structure and architecture. Also the proteins were found associated for the cytoskeleton remodeling pathway and cellular immune defense mechanism. The major proteins that were found differentially regulated during zebrafish caudal fin regeneration includes keratin and its 10 isoforms, cofilin 2, annexin a1, skeletal ␣1 actin, and structural proteins. Annexin A1 was found to be exclusively undergoing phosphorylation during regeneration. The obtained differential proteome and the direct association of the various proteins might lead to a new understanding of the regeneration mechanism. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.014118, 1-19, 2012.Regeneration is an important mechanism found among most of the animals including humans in various tissues and organs towards growth, regrowth, repair, reproduction, and survival. The biomechanism of regeneration has been widely studied but poorly understood because of its different extents in various animals. Understanding the basic molecular mechanism of regeneration in the wound environment is of high significance, because it can lead to an applied possibility of making nonregenerating to a regenerating system.Tissue regeneration in vertebrates is found with extensive capabilities. Regeneration of limbs in urodele and caudal fin in zebrafish are the most projected regeneration studies among vertebrates. Zebrafish regenerates a wide variety of tissue structures including heart, fin, spinal cord, and optic nerve (1-3) based on the characteristic regeneration mechanism involving epithelialization, mesenchymal disorganization, blastema formation, regenerative outgrowth, and termination.A great number of gene families, counting wnt, hox, fgf, fgfr, and msx genes, were shown to...
Gliomas are the most common of the primary intracranial tumors with astrocytomas constituting about 40%. Using clinically and histologically assessed astrocytomas, we have studied their protein profiles using a two-dimensional gel electrophoresis-mass spectrometry approach and identified differentially expressed proteins which may be useful molecular indicators to understand these tumors. Examination of the protein profiles of 27 astrocytoma samples of different grades revealed 72 distinct, differentially expressed proteins belonging to various functional groups such as cytoskeleton and intermediate filament proteins, heat shock proteins (HSPs), enzymes and regulatory proteins. Based on the consistency of their differential expression, 29 distinct proteins could be short-listed and may have a role in the pathology of astrocytomas. Some were found to be differentially expressed in both Grade III and IV astrocytomas while others were associated with a particular grade. A notable observation was underexpression of Prohibitin, a potential tumor suppressor protein, Rho-GDP dissociation inhibitor, Rho-GDI, a regulator of Rho GTPases and HSPs as well as destabilization of glial fibrillary acidic protein, GFAP, major protein of the glial filaments, in Grade III malignant tumors. We attempt to explain glioma malignancy and progression in terms of their combined role.
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