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Whole Cell Lysates (whole + cell_lysate)

Distribution by Scientific Domains
Life Sciences60%
Medical Sciences40%

Selected Abstracts

Application of immunoproteomics to leptospirosis: towards clinical diagnostics and vaccine discovery

PROTEOMICS - CLINICAL APPLICATIONS, Issue 4 2007
Uraiwan Kositanont
Abstract Each of the currently available methods for serodiagnosis of leptospirosis, including the microscopic agglutination test (MAT), has its own drawback(s) when used in clinical practice. A new diagnostic test is therefore required for an earlier and more accurate diagnosis of leptospirosis. We applied immunoproteomics to define potential immunogens from five serovars of Leptospira reference strains. A leptospiral whole cell lysate from each serovar was used as the antigen to react with IgG and IgM in the sera from four patients with a positive MAT. Sera from four non-leptospirosis patients with a negative MAT were pooled and used as the negative control. 2-D Western blot analysis showed that the degree of immunoreactivity corresponded with the MAT titers. No immunoreactive spots were detected when the pooled control sera were used. A total of 24 protein spots immunoreacted with IgM and/or IgG from patients with leptospirosis. These immunoreactive proteins were identified by MALDI-TOF MS and were classified into five groups, including flagellar proteins, chaperones/heat shock proteins, transport proteins, metabolic enzymes, and hypothetical proteins. More immunoreactive spots were detected with anti-human IgG in the sera of all patients and with all the serovars of leptospires used. Some of the identified proteins immunoreacted only with IgG, whereas the others were detectable with both IgM and IgG. Among the immunoreactive proteins identified, FlaB proteins (flagellin and flagellar core protein) have been shown to have a potential role in clinical diagnostics and vaccine development. These data underscore the significant impact of immunoproteomics in clinical applications. [source]

Narrow-band fractionation of proteins from whole cell lysates using isoelectric membrane focusing and nonporous reversed-phase separations

ELECTROPHORESIS, Issue 7-8 2004
Yi Zhu
Abstract Preparative isoelectric focusing (PIEF) is used to achieve narrow-band fractionation of proteins from whole cell lysates of Escherichia coli (E. coli). Isoelectric membranes create well-defined pH ranges that fractionate proteins by isoelectric point (pI) upon application of an electric potential. A commercial IsoPrime device (Amersham-Pharmacia BioTech) is modified for the PIEF separation to lessen run volumes significantly. Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) analysis of chamber contents indicates that excellent pH fractionation is achieved with little overlap between chambers. PIEF pH fractions are further separated using nonporous reversed-phase high-performance liquid chromatography (NPS-RP-HPLC) and HPLC eluent is analyzed on-line by electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS) for intact protein molecular weight (MW) analysis. The result is a pI versus MW map of bacterial protein content. IEF fractionation down to 0.1 pH units combined with intact protein MW values result in a highly reproducible map that can be used for comparative analysis of different E. coli strains. [source]

Effects of Cyclosporine on Osteoclast Activity: Inhibition of Calcineurin Activity With Minimal Effects on Bone Resorption and Acid Transport Activity,

JOURNAL OF BONE AND MINERAL RESEARCH, Issue 3 2003
John P Williams
Abstract Cyclosporine results in rapid and profound bone loss in transplant patients, an effect ascribed to osteoclasts. Cyclosporine, complexed with the appropriate immunophilin, inhibits calcineurin (the calcium/calmodulin dependent serine/threonine phosphatase) activity. We tested the hypothesis that cyclosporine inhibits calcineurin activity in osteoclasts, resulting in stimulation of osteoclast activity. We compared the effects of cyclosporine A and the calmodulin antagonist, tamoxifen, on bone resorption by avian osteoclasts. Tamoxifen inhibits bone resorption ,60%, whereas cyclosporine A only inhibited bone resorption 12%. One-hour treatment with 100 nM cyclosporine inhibited osteoclast calcineurin activity 70% in whole cell lysates, whereas 10 ,M tamoxifen only inhibited calcineurin activity 25%. We compared the effects of cyclosporine A and tamoxifen on acid transport activity in isolated membrane vesicles and in isolated membrane vesicles obtained from osteoclasts treated with cyclosporine A or tamoxifen under conditions that inhibit calcineurin activity. Direct addition of cyclosporine A in the acid transport assay, or pretreatment of cells with cyclosporine A followed by membrane isolation, had no effect on acid transport activity in membrane vesicles. In contrast, direct addition of tamoxifen to membranes inhibits acid transport activity, an effect that can be prevented by addition of exogenous calmodulin. Furthermore, acid transport activity was also inhibited in membrane vesicles isolated from cells treated with tamoxifen. In conclusion, cyclosporine A inhibits osteoclast calcineurin activity; however, calcineurin inhibition does not correspond to a significant effect on acid transport activity in isolated membrane vesicles or bone resorption by osteoclasts. [source]

Cardioprotection: spotlight on PKG

BRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2007
M V Cohen
Classical ischaemic preconditioning, delayed or second window preconditioning and postconditioning are forms of cardioprotection that are dependent on cell surface receptors, intracellular signalling molecules and kinases that ultimately block formation of the mitochondrial permeability transition. The latter is presumed to cause myocardial necrosis as well as apoptosis, so prevention of its formation upon resumption of perfusion after a prolonged coronary occlusion should be cardioprotective. In all of these forms of cardioprotection, formation of cGMP and activation of protein kinase G (PKG) are recognized to be key steps in the signal transduction pathway. Burley et al. highlight the roles of cGMP and PKG in their comprehensive review. They describe the basic biology of PKG and emphasize its compartmentalization, which may be responsible for the frustration induced by assays for PKG in whole cell lysates and for the spurious conclusions about the role of PKG in cardioprotection. This review will be useful to both the novice and the seasoned investigator. British Journal of Pharmacology (2007) 152, 833,834; doi:10.1038/sj.bjp.0707453; published online 17 September 2007 [source]