Inhibition of endogenous RNA polymerase activity from thymus chromatin by transcortin-hydrocortisone complex.

A transortin-hydrocortisone complex has been isolated from human serum by affinity chromatography on oxidized corticosterone coupled to AH-Sepharose 4B. The influence of this complex and of hydrocortisone alone on endogenous RNA polymerase activity from thymus chromatin have been tested. Results show that hydrocortisone alone has no effect on RNA polymerase activity from thymus chromatin. Under the same experimental conditions, The transcortin-hydrocortisone complex induces an important decrease in the incorporation of UMP into RNA. The dose response of thymic RNA polymerase to transcortin-hydrocortisone complex and the effects of alpha-amanitin on this response are also reported.

Specific binding of dexamethasone to plasma membranes from skeletal muscle.

A procedure was developed to isolate plasma membranes from rabbit skeletal muscle. K+-dependent phosphatase activity was used as marker enzyme for plasma membranes and was determined in the presence of CHAPS (3-( (3-cholamidopropyl)dimethylammonio)-1-propanesulfonate), a zwitterionic detergent. Ca2+-ATPase and succinate dehydrogenase activities were used as marker enzymes for sarcoplasmic reticulum and mitochondria, respectively. Electron-microscopy revealed that plasma membranes were in the form of vesicles. Significant proteolysis of membrane proteins was observed during extraction, which was inhibited by EGTA and 20 mM molybdate. SDS-polyacrylamide gel electrophoresis revealed the disappearance of an intense 96 kDa protein band when membranes were purified in the absence of EGTA and molybdate. Specific binding sites for [3H]dexamethasone were identified in plasma membranes after freezing and incubation with CHAPS. Dithiothreitol was essential for steroid binding and ATP increased it. Under standardized assay conditions, binding was complete with 50 min a 37 degrees C. No binding occurred at 0 degrees C, nor if EGTA and molybdate were absent from the extraction medium.

Degradation of protein kinase Malpha by mu-calpain in a mu-calpain-protein kinase Calpha complex.

In previous studies, we isolated and identified a mu-calpain-PKCalpha complex from rabbit skeletal muscle. At the same time we pointed out that an association between mu-calpain and PKCalpha could occur at the level of the plasma membrane of muscle cells, and that PKCalpha could thus be considered as a potential mu-calpain substrate. In the present study, using the mu-calpain-PKCalpha complex as a model, we report that mu-calpain is activated in the combined presence of physiological calcium concentrations (less than 1 microM) and phosphatidylserine. Furthermore our data also show that: (1) there exists a correlation between the appearance of autolyzed mu-calpain forms and PKCalpha hydrolysis which leads to the formation of PKMalpha; (2) in certain experimental conditions, autolyzed mu-calpain forms are able to hydrolyze PKMalpha independently of the presence of diacylglycerol.

Association of calpains 1 and 2 with protein kinase C activities.

Calpains 1 and 2 co-eluted with protein kinase C activities after hydrophobic (phenyl-Sepharose) and anion-exchange (Mono Q) chromatographies of a 100,000 X g supernatant which was defined as cytosol. After centrifugation of the cytosol at 200,000 X g for 16 h, the major part of calpain 1 and of its associated protein kinase C activity was recovered in the pellet, when the major part of calpain 2, also associated to a protein kinase C activity, was present in the resulting supernatant. Polyacrylamide gel electrophoresis of the fractions eluted from the Mono Q column, which contained calpains 1 or 2 and their associated protein kinase C activities, revealed two main bands with a molecular mass of 80 and 28 kDa.

Isolation and identification of a mu-calpain-protein kinase C alpha complex in skeletal muscle.

A mu-calpain-PKC complex was isolated from rabbit skeletal muscle by ultracentrifugation and by anion-exchange chromatography. The PKC associated to mu-calpain was stimulated by calcium, phosphatidylserine and diacylglycerol, and corresponds to a conventional PKC (cPKC). This complex presents an apparent molecular mass close to 190 kDa and is composed of one mu-calpain molecule and of one cPKC molecule. Using monoclonal antibodies specific for the different cPKC isoforms, the isoenzyme associated to mu-calpain was identified as cPKC alpha. Immunofluorescence staining reveals a co-localization of mu-calpain and cPKC alpha on the muscle fibre plasma membranes.

Calpain 1-protein kinase C complex: effect of calpain inhibitors after dissociation.

A calpain 1-protein kinase C (PKC) complex was isolated from rabbit skeletal muscle by hydrophobic interaction chromatography on phenyl-sepharose and by strong anion exchange chromatography on Q-Sepharose. Calpain 1 and kinase activities were then dissociated on a phenyl-Sepharose matrix using gradients of decreasing ionic strength. The purified PKC obtained corresponded to conventional PKC and was recognized by a monoclonal antibody specific for alpha and beta isotypes. Leupeptin, calpain inhibitor II, and the more selective calpain inhibitors calpeptin and MDL 28170 did not block the activation of the purified PKC by Ca2+ and phosphatidylserine.

[Effect of cortisol and cortisol-transcortin complex on transcription in calf thymus chromatin]. / Effets sur la transcription, du cortisol et du complexe transcortine-cortisol, au niveau de la chromatine du thymus de veau

The influence of transcortin and of cortisol on RNA synthesis in thymus chromatin, have been tested. The experimental results show that cortisol alone has no effect on RNA synthesis. But, in the same conditions of ionic strength, a serious decrease in transcription, induced by cortisol-transcortin complex, is demonstrated.

[Calpains, protein kinase c and development of muscle tissue]. / Calpaines, protéine kinase C et développement du tissu musculaire.

Calpains are Ca2+ -dependent thiol proteases which have been identified in various tissues of eucaryotes, but their physiological function in the cell is uncertain. In the muscle fiber, two types of calpains are present which differ by their calcium sensitivity: calpain 1 and calpain 2, which require for their activity micro and millimolar concentrations of calcium respectively. These calpains are associated with protein kinase C activities in the differentiated fiber. The multinucleate myotube is formed by fusion of mononucleated precursor cells, myoblasts. Calpains have been reported to appear in myoblasts at around the time of fusion. Moreover, an apparent synthesis of 1,2 diacylglycerol, an activator of protein kinase C, was observed during fusion of myoblasts. However, more information is required to incriminate totally protein kinase C and calpains in the mechanism of myoblast fusion.

Induction of protein kinase C down-regulation by the phorbol ester TPA in a calpain/protein kinase C complex.

Using a calpain/protein kinase C (PKC) complex, we were able to reproduce, in vitro, the induction of PKC down-regulation by the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA) which had been previously observed in cells. We show that TPA initiates this phenomenon by promoting a calpain-dependent conversion of PKC to the Ca2+ phospholipid-independent protein kinase M (PKM), at physiological calcium concentrations. This effect of TPA was dependent upon the presence of phosphatidylserine and was observed only when PKC was the substrate for the protease, inactivation of calpain by autolysis not being modified by the presence of TPA. Moreover, PKM generated from the calpain-PKC complex was resistant to calpain, even after addition of TPA. These results suggest that TPA induces a conformational change in PKC, increasing the affinity of the kinase for calpain and consequently permitting its proteolysis for the basal level of calcium in cells.

MR monitoring of laser-induced lesions of the liver in vivo in a low-field open magnet: temperature mapping and lesion size prediction.

The aims of this study were, firstly, to monitor temperature with magnetic resonance (MR) during laser ablations performed in pig livers in vivo in a low-field open scanner (0.23T) and, secondly, to study the feasibility of lesion size prediction. Spin-echo (SE) images of 29 sec acquired during laser applications allowed calculation of temperature maps using T1 and M(0) temperature sensitivity. Temperature was also measured with thermocouples. Images of prediction of tissue damage were calculated using temperature maps and Arrhenius model. T2W sequences were acquired after the ablations. Animals were sacrificed immediately. Lesions were photographed macroscopically. Lesion surfaces were measured and compared in T2W images, temperature images, damage prediction images, and macroscopic pictures. A correlation exists between temperature measured with MR and with thermocouples (rho = 0.878; P < 0.001, Spearman test). Mean surface of predicted damaged tissue is consistent with mean early necrosis measured in macroscopic pictures. Early T2W images underestimate mean necrosis size. J. Magn. Reson. Imaging 2001;13:42-49.

MR temperature measurement in liver tissue at 0.23 T with a steady-state free precession sequence.

MRI can be used for monitoring temperature during a thermocoagulation treatment of tumors. The aim of this study was to demonstrate the suitability of a 3D steady-state free precession sequence (3D Fast Imaging with Steady-State Precession, 3D TrueFISP) for MR temperature measurement at 0.23 T, and to compare it to the spin-echo (SE) and spoiled 3D gradient-echo (3D GRE) sequences. The optimal flip angle for the TrueFISP sequence was calculated for the best temperature sensitivity in the image signal from liver tissue, and verified from the images acquired during the thermocoagulation of excised pig liver. Factors influencing the accuracy of the measured temperatures are discussed. The TrueFISP results are compared to the calculated values of optimized SE and 3D GRE sequences. The accuracy of TrueFISP in the liver at 0.23 T, in imaging conditions used during thermocoagulation procedures, is estimated to be +/-3.3 degrees C for a voxel of 2.5 x 2.5 x 6 mm(3) and acquisition time of 18 s. For the SE and GRE sequences, with similar resolution and somewhat longer imaging time, the uncertainty in the temperature is estimated to be larger by a factor of 2 and 1.2, respectively.