Immunodetectable galactosyltransferase is associated only with human spermatozoa of high buoyant density

Human ejaculated spermatozoa are heterogeneous and can be separated into two distinct populations according to their respective buoyant densities. In order to investigate the functional differences between these two types of spermatozoa, we have searched for the presence of galactosyltransferase. A Western blot of sperm proteins following their electrophoresis was probed with an anti-galactosyltransferase serum revealing that this enzyme is present in human spermatozoa. Furthermore, galactosyltransferase is detectable only in those proteins isolated from the head of high density spermatozoa. These results suggest that ejaculated spermatozoa consist of two populations that are functionally different.     

Isolation and primary structure of rat secretin

A major form of rat secretin was purified to homogeneity from small intestine, being detected with a porcine secretin radioimmunoassay throughout 7 chromatographic steps. The sequence of the heptacosapeptide amide H-S-D-G-T-F-T-S-E-L-S-R-L-Q-D-S-A-R-L-Q-R-L-L-Q-G-L-V-NH2 shows that rat secretin has a glutamine residue in position 14 instead of arginine as in pig secretin.     

Evidence for a novel growth factor in Xenopus oocytes

Xenopus oocytes contain a novel growth factor, as determined by its effect on the anchorage-dependent and -independent growth of various rat cells and on a Xenopus cell line; it is destroyed by trypsin, acidification (pH 2.0), heating (100 degrees C, 3 min), 8 M urea but not by dithiothreitol. Gel filtration of this activity in nondissociating conditions suggests the existence of aggregates and the presence of a very high (approximately 2000 Kd) and a much lower (approximately 30 Kd) form.     

Postulated role of calsequestrin in the regulation of calcium release from sarcoplasmic reticulum

Ca2+ release from heavy sarcoplasmic reticulum (SR) vesicles was induced by 2 mM caffeine, and the amount (A) and the rate constant (k) of Ca2+ release were investigated as a function of the extent of Ca2+ loading. Under both passive and active loading conditions, the A value increased monotonically in parallel to Ca2+ loading. On the other hand, k sharply increased at partial Ca2+ loading, and upon further loading, it decreased to a lower level. Since most of the intravesicular calcium appears to be bound to calsequestrin both under passive and under active loading conditions, these results suggest that the kinetic properties of induced Ca2+ release show significant variation depending upon how much calcium has been bound to calsequestrin at the time of the induction of Ca2+ release. An SR membrane segment consisting of the junctional face membrane (jfm) and attached calsequestrin (jfm-calsequestrin complex) was prepared. The covalently reacting thiol-specific conformational probe N-[7-(dimethylamino)-4-methyl-3-coumarinyl]maleimide (DACM) was incorporated into several proteins of the jfm, but not into calsequestrin. The fluorescence intensity of DACM increased with Ca2+. Upon dissociation of calsequestrin from the jfm by salt treatment, the DACM fluorescence change was abolished, while upon reassociation of calsequestrin by dilution of the salt it was partially restored. These results suggest that the events occurring in the jfm proteins are mediated via the attached calsequestrin rather than by a direct effect of Ca2+ on the jfm proteins. We propose that the [Ca2+]-dependent conformational changes of calsequestrin affect the jfm proteins and in turn regulate the Ca2+ channel functions.     

Modulation of mitochondrial F0F1 catalysis by boundary and bulk phase phospholipids

The importance of boundary and bulk phase phospholipids was studied on a mitochondrial ATPase complex isolated by AH-Sepharose chromatography as described by Dreyfus et al (1984, Anal. Biochem. 142,215-220), this preparation was devoid of the adenine nucleotide carrier. The presence of isoelectric or acidic phospholipids during the purification in the column allows the exchange of tightly bound phospholipids up to 95%. ATP hydrolysis and oligomycin sensitivity were slightly affected by the nature of boundary and bulk phase phospholipids, while Pi-ATP exchange was highly inhibited.     

Signal transduction by membrane receptors in viable electropermeabilized cells: isoproterenol-stimulated cyclic AMP synthesis in C6 glioma cells

The activity of beta-adrenergic receptors at the plasma membrane level was investigated in viable, electropermeabilized C6 glioma cells. Electric field pulses were applied directly to the plated cells without any previous proteinase treatment. The affinity for isoproterenol and the density of the beta-adrenergic receptors, as judged from the number of [3H]CGP-12177 binding sites, were not affected by the electropermeabilization whereas the isoproterenol-stimulated cAMP accumulation was transiently impaired. This decrease in activity is due to an electropermeabilization-induced GTP leak. Normal activity could be obtained either by treating the cells by the electric field in a GTP-containing buffer, or by spontaneous recovery of the cells after the resealing of the plasma membrane, with a delay depending on the temperature. The activity of the receptors was not affected by the structural organization of the membrane associated to its electropermeabilization.     

Entrapment of lipid vesicles and membrane protein-lipid vesicles in gel bead pores

Phospholipid vesicles were entrapped in gel beads of Sepharose 6B and Sephacryl S-1000 during vesicle preparation by dialysis. Egg-yolk phospholipids solubilized with cholate or octyl glucoside were dialysed together with gel beads for 2.5 days in a flat dialysis bag. Some vesicles were formed in gel bead pores and vesicles of sufficient size became trapped. Red cell membrane protein-phospholipid vesicles could be immobilized in the same way. Non-trapped vesicles were carefully removed by chromatographic procedures and by centrifugation. The amount of entrapped vesicles increased with the initial lipid concentration and was dependent on the relative sizes of vesicles and gel pores. The largest amount of trapped vesicles, corresponding to 9.5 mumol of phospholipids per ml gel, was achieved when Sepharose 6B gel beads were dialysed with cholate-solubilized lipids at a concentration of 50 mM. In this case the vesicles had an average diameter of 60 nm and an internal volume of 15 microliters/ml gel. The amount of vesicles trapped in Sephacryl S-1000 gel beads upon dialysis under the same conditions was smaller: 2.2 mumol of phospholipids per ml gel. Probably most of the gel pores were too large to trap such vesicles. Larger vesicles, with an average diameter of 230 nm, were entrapped in the Sephacryl S-1000 matrix in an amount corresponding to 3.0 mumol phospholipids per ml gel upon dialysis of the gel beads and octyl glucoside-solubilized lipids at a concentration of 20 mM. The internal volume of these vesicles was 22 microliters/ml gel. The yield of immobilized phospholipids was up to 19%. The entrapped vesicles were somewhat unstable: 9% of the phospholipids were released during 9 days of storage at 4 degrees C. By the dialysis entrapment method vesicles can be immobilized in the gel beads without using hydrophobic ligands or covalent coupling.     

Expression of vimentin and glial fibrillary acidic protein in human developing spinal cord

Summary Expression of intermediate filament proteins was studied in human developing spinal cord using immunoperoxidase and double-label immunofluorescence methods with monoclonal antibodies to vimentin and glial fibrillary acidic protein (GFAP). Vimentin was found in the processes of radial glial cells in 6-week embryos, while GFAP appeared in vimentin-positive astroglial cells at 8–10 weeks. GFAP and vimentin were present in approximately equal amounts in differentiating astrocytes in 23-week spinal cord. In 30-week fetuses, astrocytes reacted strongly for GFAP, while both the reaction intensity and the number of vimentin-positive cells fluctuated predominantly in the grey matter. No clear-cut transition from vimentin to GFAP was noticed during the development of astrocytes. The majority of ependymal cells in 23-week fetuses contained vimentin but only a few of them reacted for GFAP. The expression of vimentin continued during the whole development of the ependymal layer, in contrast to the reactivity for GFAP which disappeared between the 30th week and term.     

Human enzyme polymorphism in the Canary Islands. III. Tenerife Island population

We analyzed the genetic polymorphism of eight red cell enzymes in samples from different geographical areas of Tenerife and the Iberian peninsula. The gene frequency heterogeneity found within the Tenerife samples was at the same level as that of Tenerife-mainland comparisons. The presence of the Negroid G6PD A+ allele in the Tenerife samples is evidence of an African admixture with a mean estimation of 4.5%.