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Differential Centrifugation (differential + centrifugation)
Selected AbstractsProstate-specific membrane antigen and its truncated form PSM,THE PROSTATE, Issue 5 2009Petra Ml, ochová Abstract BACKGROUND Prostate specific membrane antigen (PSMA) is a type II transmembrane protein overexpressed in prostate cancer as well as in the neovasculature of several non-prostatic solid tumors. In addition to full-length PSMA, several splice variants exist in prostatic tissue. Notably, the N-terminally truncated PSMA variant, termed PSM,, is prevalent in healthy prostate, and the ratio of PSMA/PSM, mRNA has been shown to correlate with cancer progression. The widely accepted hypothesis is that the PSM, protein is a translation product arising from the alternatively spliced PSM, mRNA. METHODS Differential ultracentrifugation, cell surface biotinylation, Western blotting, and enzyme activity measurement were used to study the origin and localization of the PSMA/PSM, variants in prostatic (LNCaP; lymph-node carcinoma of the prostate) and non-prostatic (HEK293) cell lines. These experiments were further complemented by analysis of the N -glycosylation patterns of the PSMA/PSM, proteins and by site-directed mutagenesis. RESULTS We identified PSM, protein expression in both the LNCaP cell line and a non-cancerous HEK293 human cell line transfected with a plasmid encoding full-length PSMA. Differential centrifugation revealed that PSM, is localized predominantly to the cytosol of both these cell lines and is proteolytically active. Furthermore, the PSM, protein is N -glycosylated by a mixture of high-mannose and complex type oligosaccharides and therefore trafficked beyond the cis -Golgi compartment. CONCLUSIONS Our data suggest that the PSM, protein is likely not generated by alternative splicing of the PSMA gene but by different mechanism, probably via an endoproteolytic cleavage of the full-length PSMA. Prostate 69:471,479, 2009. © 2008 Wiley-Liss, Inc. [source] Comparison of the effectiveness of five extraction methods for Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum from potato tubersEPPO BULLETIN, Issue 2 2001J. Martin In the EU Control Directives, the recommended extraction procedure for testing potatoes for Clavibacter michiganensis subsp. sepedonicus and Ralstonia solanacearum comprises incubation followed by differential centrifugation. This method can be qualified as complex because of the number of different steps required. This study evaluates five different extraction methods for each bacterium from both a technical point of view and for the quality of the results. Results showed that in the case of C. m. sepedonicus the clarification step should be avoided. The incubation/shaking method with three subsamples gives at least as satisfactory results as the official EU procedure. It also has other advantages, facilitating immunofluorescence readings due to the reduced quantity of plant debris, and improving the speed and the reliability of the analysis. [source] Substrate Channelling in a Creatine Kinase System of Rat Skeletal Muscle Under Various pH ConditionsEXPERIMENTAL PHYSIOLOGY, Issue 1 2003M. Gregor The aim of this study was to evaluate myofibrillar creatine kinase (CK) activity and to quantify the substrate channelling of ATP between CK and myosin ATPase under different pH conditions within the integrity of myofibrils. A pure myofibrillar fraction was prepared using differential centrifugation. The homogeneity of the preparation and the purity of the fraction were confirmed microscopically and by enzymatic assays for contaminant enzyme activities. The specific activity of myofibrillar CK reached 584 ± 33 nmol PCr min,1 mg,1 at pH 6.75. Two methods were used to detect CK activity: (1) measurement of direct ATP production, and (2) measurement of PCr consumption. This method of evaluation has been tested in experiments with isolated creatine kinase. No discrepancy in CK activity between the methods was observed in the pH range tested (6.0-7.5). However, the same procedures resulted in a significant discrepancy between the amounts of reacted PCr and produced ATP within the pure myofibrillar fraction. This discrepancy represents the portion of ATP produced by the CK reaction, which is preferentially channelled to the myosin ATPase before diffusing into the bulk solution. The maximum evaluated difference reached 42.3 % at pH 6.95. The substrate channelling between myofibrillar-bound CK and myosin ATPase was evaluated under various pH levels within the physiological range and it reached a maximum value in a slightly acidic environment. These results suggest that ATP/ADP flux control by the CK system is more important at lower pH, corresponding to the physiological state of muscle fatigue. [source] Timing and sequence of differentiation of embryonic rat hepatocytes along the biliary epithelial lineageHEPATOLOGY, Issue 3 2003Robbert G. E. Notenboom To study the differentiation of hepatocytes along the biliary epithelial lineage in vivo, embryonic day 14 (E14) rat hepatocytes were isolated by differential centrifugation and transplanted as single-cell suspensions into the spleen of adult syngeneic rats. Hepatocytes and cholangiocytes were identified and their maturation characterized by the level of expression of ,-fetoprotein (AFP), glutamate dehydrogenase (GDH), and carbamoyl phosphate synthetase I (CPS); annexin IV, annexin V, cytokeratin 19 (CK-19), and cystic fibrosis transmembrane conductance regulator (CFTR); and electron microscopy. By correlating morphologic changes with the timing in the expression of these markers, we show that the organization of the transplanted E14 hepatocytes into lobular structures is accompanied by the formation and maturation of bile ducts around these developing lobules. Morphologic differentiation of the emerging bile ducts was accompanied by a gradual loss of hepatocyte markers and a gradual acquisition of cholangiocyte markers, with markers identifying a large-cholangiocyte phenotype appearing latest. Once fully differentiated, the intrasplenic liver lobules developed cholestatic features. The accompanying proliferation of bile ducts was due to cholangiocyte proliferation, but ductular transformation of hepatocytes was also observed. In conclusion, (1) bile duct formation at the interface between hepatocytes and connective tissue is an inherent component of liver development and (2) the susceptibility of developing hepatocytes to bile duct-inducing signals is highest in the fetal liver but that (3) this capacity is not irreversibly lost in otherwise mature hepatocytes. [source] The Phosphodiesterase III Inhibitor Olprinone Decreases Sensitivity of Rat Kupffer Cells to EndotoxinALCOHOLISM, Issue 2004Nobuyuki Enomoto Background: Sensitivity of Kupffer cells to endotoxin [lipopolysaccharide (LPS)] and overproduction of tumor necrosis factor-, (TNF-,) are critical for progression of alcoholic liver injury. Therefore, suppression of TNF-, should prove useful for treatment of alcoholic liver injury. However, a transient increase of intracellular calcium ([Ca2+]i) is required for LPS-induced TNF-, production by the macrophage cell line. The phosphodiesterase III inhibitor olprinone has been shown to suppress [Ca2+]i level in vascular smooth muscle cells. Accordingly, the purpose of this study was to determine whether olprinone could prevent sensitization of Kupffer cells to endotoxin. Methods: Kupffer cells were isolated by collagenase digestion and differential centrifugation. LPS was added to Kupffer cells 24 hr after incubation with or without olprinone (0.1 ,mol/liter). After addition of LPS (10 ,g/ml) to culture media, [Ca2+]i was measured using a fluorescent indicator, fura-2. Results: LPS increased [Ca2+]i of Kupffer cells in control rats from basal levels (28 ± 4 nmol/liter) to 280 ± 14 nmol/liter. This increase was blunted by olprinone (91 ± 8 nmol/liter). Similarly, olprinone diminished the LPS (1 ,g/ml)-induced TNF-, production by Kupffer cells by 30% (2220 ± 116 vs. 1386 ± 199 pg/ml; p < 0.05). Conclusions: These results indicate that olprinone decreases sensitivity of Kupffer cells to endotoxin. [source] Proteomic and functional alterations in brain mitochondria from Tg2576 mice occur before amyloid plaque depositionPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 4 2007Frank Gillardon Dr. Abstract Synaptic dysfunction is an early event in Alzheimer's disease patients and has also been detected in transgenic mouse models. In the present study, we analyzed proteomic changes in synaptosomal fractions from Tg2576 mice that overexpress mutant human amyloid precursor protein (K670N, M671L) and from their nontransgenic littermates. Cortical and hippocampal tissue was microdissected at the onset of cognitive impairment, but before deposition of amyloid plaques. Crude synaptosomal fractions were prepared by differential centrifugation, proteins were separated by 2-D DIGE and identified by MS/MS. Significant alterations were detected in mitochondrial heat shock protein 70 pointing to a mitochondrial stress response. Subsequently, synaptosomal versus nonsynaptic mitochondria were purified from Tg2576 mice brains by density gradient centrifugation. Mitochondrial proteins were separated by IEF or Blue-native gel electrophoresis in the first dimension and SDS-PAGE in the second dimension. Numerous changes in the protein subunit composition of the respiratory chain complexes I and III were identified. Levels of corresponding mRNAs remain unchanged as shown by Affymetrix oligonucleotide array analysis. Functional examination revealed impaired state 3 respiration and uncoupled respiration in brain mitochondria from young Tg2576 mice. By immunoblotting, amyloid-beta oligomers were detected in synaptosomal fractions from Tg2576 mice and reduced glucose metabolism was observed in Tg2576 mice brains by [14C]-2-deoxyglucose infusion. Taken together, we demonstrate alterations in the mitochondrial proteome and function that occur in Tg2576 mice brains before amyloid plaque deposition suggesting that mitochondria are early targets of amyloid-beta aggregates. [source] Isolation, Partial Purification, and Immunogenicity of Flagella from Tritrichomonas foetusTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 3 2005PHILIP F. JEMILOHUN Abstract. Tritrichomonas foetus, the agent of bovine trichomoniasis, is a flagellate protozoan responsible for substantial economic losses to the dairy and calf industries worldwide. As yet, there is no approved treatment nor is there a sensitive diagnostic method. All these problems suggest that immunization is the best control strategy. In view of this, we isolated and partially purified flagella of the parasite by vortex homogenization followed by low-speed differential centrifugation. The resulting enriched flagellar preparation termed "crude flagellar prep" was purified further by sucrose and percoll gradients. Microscopic analysis showed that the flagellar membrane was intact. Analysis by sodium dodecyl-sulfate polyacrylamide gel electrophoresis revealed three prominent protein bands of 42, 49, and >250 kDa, and several minor bands. Immunoblotting of flagellar and whole-cell extracts revealed many flagellar antigens. [source] Microparticles stimulate the synthesis of prostaglandin E2 via induction of cyclooxygenase 2 and microsomal prostaglandin E synthase 1ARTHRITIS & RHEUMATISM, Issue 11 2007Astrid Jüngel Objective Microparticles are small vesicles that are released from activated or dying cells and that occur abundantly in the synovial fluid of patients with rheumatoid arthritis (RA). The goal of these studies was to elucidate the mechanisms by which microparticles activate synovial fibroblasts to express a proinflammatory phenotype. Methods Microparticles from monocytes and T cells were isolated by differential centrifugation. Synovial fibroblasts were cocultured with increasing numbers of microparticles. Gene expression was analyzed by real-time polymerase chain reaction and confirmed by Western blotting and enzyme immunoassay. Arachidonic acid labeled with tritium was used to study the transport of biologically active lipids by microparticles. The roles of NF-,B and activator protein 1 (AP-1) signaling were analyzed with electrophoretic mobility shift assay and transfection with small interfering RNA and I,B expression vectors. Results Microparticles strongly induced the synthesis of cyclooxygenase 2 (COX-2), microsomal prostaglandin E synthase 1 (mPGES-1), and prostaglandin E2 (PGE2). In contrast, no up-regulation of COX-1, mPGES-2, cytosolic PGES, or phospholipase A2 was observed. The induction of PGE2 was blocked by selective inhibition of COX-2. Microparticles activated NF-,B, AP-1, p38, and JNK signaling in synovial fibroblasts. Inhibition of NF-,B, AP-1, and JNK signaling reduced the stimulatory effects. Arachidonic acid was transported from leukocytes to fibroblasts by microparticles. Arachidonic acid derived from microparticles was converted to PGE2 by synovial fibroblasts. Conclusion These results demonstrate that microparticles up-regulate the production of PGE2 in synovial fibroblasts by inducing COX-2 and mPGES-1. These data provide evidence for a novel mechanism by which microparticles may contribute to inflammation and pain in RA. [source] | |||||||||||||