Ca(2+)-dependent structural transitions of the platelet glycoprotein IIb-IIIa complex. Preparation of stable glycoprotein IIb and IIIa monomers

The platelet membrane glycoprotein (GP) IIb-IIIa complex is the receptor for adhesive proteins on activated platelets that mediates platelet aggregation. In the present study, factors affecting the structural stability of the purified GP IIb-IIIa complex and the dissociated subunits were investigated. Purified GP IIb-IIIa was incubated in various Ca2+ concentrations, and the percentage of dissociated subunits was quantitated by sucrose gradient sedimentation. Two Ca(2+)-dependent transitions were observed, one at about 60 microM Ca2+, where half of the complexes became dissociated, and the other at 0.1 microM Ca2+, where half of the dissociated subunits became incapable of reforming heterodimer complexes when higher Ca2+ concentrations were readded. This loss in ability to reform heterodimer complexes was caused primarily by a Ca(2+)-dependent transition in GP IIIa, leading to an apparent unfolding of this subunit, followed by the formation of high molecular weight aggregates. The formation of these aggregates was time- and temperature-dependent and could not be reversed by added Ca2+. Although Mg2+ prevented dissociation of GP IIb-IIIa, it failed to promote reassociation of the dissociated subunits. Based on these findings, conditions were developed for the preparation of dissociated GP IIb and GP IIIa such that 70% of the subunits remained functional in that they retained the ability to reform heterodimer complexes.     

Experimental method for the in vitro testing of the initial stability of cementless hip prostheses

Micromotions at the interface between bone and prosthesis are believed to induce bone resorption and ultimately lead to loosening of the implant. Thus the initial stability achieved by a hip prosthesis is an important factor for the long-term function of the implant. Knowing the biological consequences of the mechanical conditions, it appears to be mandatory to measure the extent of these three-dimensional movements. An in vitro dynamic method for measurement of the micromotion of the femoral component of hip prostheses has been developed. Tests in cemented prostheses have confirmed that the use of cement reduces sinkage and rotation manyfold and have yielded reference values for stability. Comparison with two types of cementless prostheses has shown that certain cementless implants may achieve stability comparable to cemented ones in some load directions.     

Immunocytochemical localization of protein kinases and calmodulin in dog thyroid cells

By immunocytochemical methods, the protein effectors of known intracellular signal molecules were demonstrated in the thyroid follicular cell and their localization investigated. Cyclic AMP protein kinase subunit RII was clearly located in the nucleus. Protein kinase subunits RI and C were in the cytosol and on the apical membrane. Cyclic GMP kinase and calmodulin were mostly found in the cytoplasm and at the apical membrane; they were poorly represented in the nucleus. The only membrane underlined by several markers was the apical membrane, i.e. the site of iodination, oxido reduction and H2O2 generation.     

Assay and purification of a solubilized membrane receptor that binds the lysosomal enzyme α-l-iduronidase

A receptor that binds the lysosomal enzyme α-l-iduronidase via phosphorylated mannose residues on the enzyme has been solubilized from Swarm rat chondrosarcoma membranes using a pH 9.5 buffer containing 0.1% Triton X-100. Detergent-solubilized receptor in crude and purified preparations was measured by assay of bound α-l-iduronidase after adsorbing the receptor-enzyme complex onto insoluble phospholipid vesicles (liposomes). Binding of α-l-iduronidase to the liposomes required receptor and was completely inhibited by mannose 6-phosphate but not glucose 6-phosphate, indicating that the receptor maintained specificity following solubilization. Receptors from rat chondrosarcoma and human diploid fibroblasts were purified to apparent homogeneity using a phosphomannan-Sepharose affinity column. Both had identical molecular weights in polyacrylamide gels containing sodium dodecyl sulfate (M_r = 215,000). Amino acid analysis and two-dimensional gel electrophoresis was carried out on the purified rat chondrosarcoma receptor. Two forms of the receptor with different pI's were observed (pI 5.5 and 6.2). One form (pI 5.5) was made more basic (pI 5.8) by treatment with neuraminidase.     

Isolation and characterization of the retinal-binding component of halorhodopsin

Halorhodopsin (HR) was reconstituted in cell vesicles prepared from Halobacterium halobium strain L-07 by addition of tritium-labelled retinal and subsequently reduced with cyanoborohydride. Lysis of the labelled vesicles in water and dissolution of the cell membranes with 4% SDS allowed the purification of the retinyl protein (RP) by a 3-step procedure. Gel filtration on AcA-44 ultrogel was followed by chromatography on hydroxylapatite and preparative SDS-polyacrylamide gel electrophoresis. This procedure yielded material which migrated as a single band of an apparent mol. wt. of 25 000 on analytical SDS-polyacrylamide gels. The purification was ˜400-fold with an overall yield of ˜15%. Not only the mol. wts. but also the amino acid compositions of the RPs from bacteriorhodopsin (BR) and HR are very similar. Polyclonal antibodies against BR and HR did not, however, crossreact. When the two RPs were partially digested with staphylococcal V8 protease the proteolytic pattern of the retinyl peptides was similar, but not identical: two extra peptides are present in BR. The same kind of differences were found in the h.p.l.c. elution profiles of retinyl peptides produced by subtilisin digestion. Therefore, the two proteins must be different gene products and not modification products of one and the same protein.     

Temporal sequence of cell shape changes in cultured rat sertoli cells after experimental elevation of intracellular cAMP

The ability of FSH and pharmacological agents to induce changes in the shape of cultured rat Sertoli cells has been studied by using time-lapse phase-contrast microscopy and scanning electron microscopy (SEM). Morphological studies were combined with an immunocytochemical method for the localization of cAMP in Sertoli cells and the results correlated with determinations of protein-bound cAMP in Sertoli cells. A variable number of Sertoli cells were converted from a flat, epithelial-like morphology into a stellate morphology after exposure to FSH, isobutyl-methylxanthine (MIX), dibutyryl cyclic AMP (db-cAMP) and an FSH-MIX mixture. The morphological changes followed a time- and biological agent-dependent alteration and recovery pattern. While a 120 min exposure to FSH induced shape changes in 38% of the cells, MIX, db-cAMP and FSH-MIX effected shape changes in 75 % of cells. The morphological conversion induced by MIX, db-cAMP and FSH-MIX persisted as long as these biological agents were present in the medium, whereas the effects induced by FSH alone were transient. The flat-to-stellate transition was preceded by an increase in intracellular protein-bound cAMP, a form of cyclic nucleotide which may account for cAMP immunoreactivity observed in morphologically responsive and non-responsive Sertoli cells. From these data and from previous experimental findings of androgen-binding protein (ABP) immunoreactivity in the cytoplasm of responsive and non-responsive Sertoli cells, we conclude that a surge of cAMP triggers a still undefined mechanism by which Sertoli cells modify their shape in coincidence with a progressive depletion of cytoplasmic secretory granules.     

Serotonin binding sites of human blood platelets

The possible use of formaldehyde-fixed platelets to characterize and enumerate the specific receptor sites for 5-hydroxytryptamine was investigated. Equilibrium, pH-dependent capacity and specificity of 5-hydroxytryptamine binding by formaldehyde-fixed platelets were demonstrated. Analysis of binding data revealed two different sites: (i) high affinity with low capacity, and (ii) low affinity with high capacity. The results of binding studies using nonfixed control platelets were comparable with those of formaldehyde-fixed platelets. The versatility of formaldehyde fixation for studies of surface receptors was also shown by demonstrating nearly equal binding affinity for PGE₁ in control and formaldehyde-treated platelets. Our results indicate that formaldehyde fixation is a useful tool for the study of membrane receptor sites especially when active transport of the ligand such as serotonin is a problem.     

Biofeedback efficacy studies

Biofeedback, a field still in its infancy, has developed treatments that have been used with clinical success in the treatment of a number of disorders. Many have expressed their public concern that biofeedback has not lived up to its early promise and that it has not developed treatments that are, in fact, efficacious. A number of factors, which are inherent in biofeedback research, confound the results of clinical efficacy studies of biofeedback treatments. Researchers interested in the efficacy of biofeedback must address several issues: (1) Rejecting the null hypothesis is not equal to proving the null hypothesis (without the use of power analysis); (2) control for nonspecific effects is not equal to a double-blind experimental design; (3) ignorance of a mechanism of action is not equal to a lack of clinical efficacy; (4) the administration of training is not equal to the subject's learning to criterion; (5) untrained therapists are not equal to trained therapists; (6) statistical significance is not equal to clinical significance; and (7) the laboratory setting is not equal to the clinical setting.     

Modification of aspartate before its condensation with dihydroxyacetone phosphate during quinolinic acid formation in Escherichia coli.

A crude enzyme preparation from a nadA mutant of Escherichia coli was used to catalyze the conversion of [14C]aspartic acid into a precursor of quinolinic acid, a key intermediate in the biosynthesis of nicotinamide adenine dinucleotide.     

Phylogeny and radiation of pollination systems in DISA (Orchidaceae)

We studied the patterns of adaptive radiation in Disa, a large orchid genus in southern Africa. A cladogram for 27 species was constructed using 44 morphological characters. Pollination systems were then mapped onto the phylogeny in order to analyze pathways of floral evolution. Shifts from one pollination system to another have been a major feature of the evolutionary diversification of Disa. Unlike many plant genera that are pollinated mainly by a single group of insects, radiation in Disa has encompassed nearly all major groups of pollinating insects; in all, 19 different specialized pollination systems have been found in the 27 species included in this analysis. Another striking pattern is the repeated evolution of broadly similar pollination systems in unrelated clades. For example, butterfly-pollinated flowers have evolved twice; showy deceptive flowers pollinated by carpenter bees, twice; long-spurred flowers pollinated by long-tongued flies, four times; night-scented flowers pollinated by moths, three times; and self-pollination, three times. This suggests that a few dominant pollinator species in a region may be sufficient to generate diversification in plants through repeated floral shifts that never retrace the same pathways.