Growth and shaping of the fin and limb buds

The development of the fin and limb buds involves a balance of centrifugal (active) and centripetal (passive) mechanical forces, the first of which acts to move the walls of these structures away from each other and the second of which holds them together. When the volume of the mesodermal core increases, the generated force meets with the resistance of the basal membrane, and as a result, the limb bud has a tendency to acquire a cylindrical shape. Collagen fibers, individual mesenchymal cells, and their groups hold together the dorsal and the ventral wall of the limb bud, prevent the movement of these walls away from each other, and in this way direct bud growth along the proximodistal and the anteroposterior axes. The balance of the forces which stretch the ectodermal layer and those which constrain it has also been observed in the development of other body parts.     

Expression of a lipocalin in prokaryote and eukaryote cells: quantification and structural characterization of recombinant bovine beta-lactoglobulin.

In this paper we quantify and characterize the expression of recombinant beta-lactoglobulin (rBLG) in prokaryote and eukaryote cells. In Escherichia coli we used the pET26 vector, which permits the secretion of rBLG in periplasm. We studied the expression of rBLG in COS-7 cells and in vivo in mouse tibialis muscle. The expression of rBLG was measured by two immunoassays specific, respectively, for BLG in its native and denatured conformation. We observed that rBLG was essentially expressed in a denatured form in E. coli even in the periplasm, whereas rBLG in eukaryote cells was found in its native conformation.     

Im-trityl protection of histidine.

A rational attempt to prepare FmocHis(piTrt)OH regiospecifically gave in fact the well-known tau-trityl isomer, and experiments with model systems indicate that the prospects for access to pi-trityl histidine derivatives, which would be of great value for the racemization-free synthesis of histidine-containing peptides, are poor.     

Human acetylcholinesterase. Immunochemical studies with monoclonal antibodies

Monoclonal antibodies were used to investigate the immunochemistry of human erythrocyte acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7). A series of experiments on the sedimentation velocity and Stokes radius of acetylcholinesterase and its immune complexes indicated that each antibody recognized a single high-affinity binding site (epitope) on the monomeric enzyme. Further analysis suggested that the antibody-binding sites were replicated on multimeric enzyme forms but were subject to steric hindrance between nearby IgG molecules or adjacent enzyme subunits. The cellular localization of the epitopes was studied by measuring the binding of monoclonal antibodies to the cholinesterase of intact erythrocytes. The results implied that most of the epitopes are exposed to the external media. However, one antibody failed to bind to intact cells, despite a relatively high affinity for detergent-solubilized antigen, possibly because its epitope is buried in the lipid bilayer.     

Phosphorylation of lens membranes with a cyclic AMP-dependent protein kinase purified from the bovine lens

We report the phosphorylation of lens membranes with a cAMP-dependent protein kinase isolated from bovine lenses. The holoenzyme was eluted from DEAE agarose at less than 100 mM NaCl and from gel filtration columns with a relative molecular weight of 180 000. The regulatory subunit was identified with the affinity label 8-azido-[32P]cAMP. Four focusing variants with relative molecular weights of 49 000 were seen on two-dimensional gels. The catalytic subunit was purified approx. 5000-fold and migrated at 42 000 Mr on SDS gels. Based on these observations, the enzyme is classified as a Type I cAMP-dependent protein kinase. Purified lens plasma membranes were incubated with the holoenzyme or its catalytic subunit in the presence of 32P-labeled ATP. Several membrane proteins, including the major lens membrane polypeptide, MP26, were shown to be substrates for the kinase in this reaction. MP26 appears to be the major component of intercellular junctions in the lens. Studies with protease treatments on labeled membranes appeared to localize the phosphorylation sites to the cytoplasmic side of the membrane.