2D-page analysis: a practical guide to principle critical parameters.

Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is a technique that is becoming more and more popular as the secrets of the critical parameters involved in its execution are being continually unravelled. The majority of these parameters are linked to the successful realization of the first dimension. Since the description of the original technique by O'Farrell in 1975, numerous studies have contributed to the improvement of the performance of 2D-PAGE. In this article, solutions to the problems most frequently encountered by 2D-PAGE users are given, such as cathodic drift, focusing at alkaline pH, the solubilization and the loading of the sample and different electrical parameters.

Image analysis of 2D gels: considerations and insights.

2D gel experiments require computer based analysis when the number of gels exceed four and quantitative information is required. Computer based systems should first perform the required tasks and should do so with enough ease and logic that users in a biological laboratory can easily use it on an intermittent basis. Considerations for analysis systems including input device specifications needed, computer hardware configurations and software tools are discussed and compared to the application requirements. The components of this system are then examined as a whole to envision how the total solution should function optimally in an biological laboratory.

Affinity labeling of myoblast surface proteins with 5'-P-fluorosulfonylbenzoyl adenosine: concomitant inhibition of ectoprotein kinase activity and myoblast fusion.

During in vitro myogenesis, mononucleated myoblasts fuse among themselves to form multinucleated myotubes. We have recently reported for the first time in the literature that a Ca2(+)-dependent ectoprotein kinase is responsible for this process, but we had no direct evidence for the role of extracellular ATP. To investigate whether the cells can fuse or not in the absence of this nucleotide, we used a nucleotide affinity label, fluorosulfonylbenzoyl adenosine (FSBA). We report here its use in detecting the nucleotide-binding sites at the cell surface of intact myoblasts in culture. We demonstrate that FSBA blocks fusion by inhibiting the ectoprotein kinase activity of the cells at sublethal concentrations. Radioactive [14C]SBA is incorporated into seven cell surface proteins and into the 48-kDa protein, among others. This species is specific for fusion-competent myoblasts and is implicated in this process. This is the first time that nucleotide-binding molecular species have been identified at the surface of myoblasts.

A cell surface phosphoprotein of 48 kDa specific for myoblast fusion.

The data we present here permit us to affirm that a 48 kDa phosphoprotein is the target of extracellular Ca2+ during fusion. It is detected only in fusion-competent L6 myoblasts and not in the fusion-defective spontaneous stable variants we isolated. The phosphorylation of this protein species can be totally inhibited by culturing myoblasts in a medium containing low Ca2+ concentrations (0.250 mM). However, under such conditions myoblasts do not fuse, but withdraw from the cell cycle and accumulate the muscle isoform of creatine kinase (M-CK). The results we have obtained support the following conclusions: (1) in fusion-competent cells, overall Ca2+-dependent phosphorylation of cell surface proteins appears to be necessary, but is not sufficient by itself for myoblast fusion; (2) the phosphorylation of a 48 kDa protein species is required for cell fusion; and (3) the phosphorylation of this 48 kDa protein is independent of other main events of cellular differentiation.

Spontaneous myoblast fusion is mediated by cell surface Ca2+-dependent protein kinase(s).

Mononucleated myoblasts divide in vitro until they attain confluency and fuse, forming multinucleated myotubes. Fusion is an extracellular Ca2+-dependent process. We used for our studies an established line of skeletal myoblasts (L6) as well as a non-fusing Myo- alpha-amanitin-resistant mutant of this line (Ama102). Our results show that extracellular calcium at concentrations which elicit myoblast fusion activates the phosphorylation of a protein species of 48 kD, present at the surface of mononucleated myoblasts of the fusing wild type (L6). At fusion, as the cells become independent of the extracellular calcium concentration for their further differentiation, this activation can no longer be observed. In fusion inhibition experiments, where we used lowered calcium levels, the phosphorylation of the 48 kD protein band is clearly decreased. When the myoblasts are fed with standard medium, they fuse rapidly and the phosphorylation of the 48 kD species is markedly increased. The above-described phenomenon takes place at the cell surface and is completed in a short time. The use of Myo- mutant showed that it is developmentally regulated. In view of our results, it is reasonable to postulate that Ca2+-activated phosphorylation of the cell surface could be on the basis of spontaneous myoblast fusion.

Influence of whole-body gamma irradiation upon arachidonic acid metabolism in rat platelets.

The effects of whole-body gamma irradiation (8.4 Gy) were studied on arachidonic acid (AA) metabolism in rats' blood platelets, from day D + 1 to day D + 10 after irradiation. AA conversion into thromboxane B2 (TxB2) increased at D + 1 and then gradually decreased to very low values from D + 7 to D + 10. This decrease in the conversion of exogenous AA into TxB2 was due to a lower AA incorporation into platelets and not to a decrease of cyclooxygenase and thromboxane-synthetase activities. AA incorporation into membrane phospholipids of blood platelets was much more decreased than AA incorporation into whole platelets; moreover, the lipid composition of the platelet membranes was markedly modified after irradiation, which must have resulted in structural and functional changes in these membranes; from these effects of whole-body gamma irradiation on platelets, the latter's membranes appeared as a major site of in vivo radiation damage in these cells.