Cell specific loss of polarity-inducing ability by later stage mouse preimplantation embryos

The individual blastomeres of the preimplantation mouse embryo become polarized during the 8-cell stage. Microvilli become restricted to the free surface of the embryo and this region of the membrane shows increased labeling with FITC-Con A and trinitrobenzenesulfonate (TNBS). Previous studies have shown that this polarity develops in response to asymmetric cell-cell contact with stage specific induction competent blastomeres. In the present study, the ability of later stage embryos to induce 8-cell polarization has been investigated. Newly-formed, nonpolar 8-cell stage blastomeres (1/8 cells) were isolated, then aggregated with morulae, inner cell clusters (from morulae), blastocysts, or inner cell masses (ICM) and cultured for 8 hr. Aggregates were then assayed for polarity. The results show a hierarchy of inducing ability, with the ICM and IC cluster possessing greater activity than the morula and polar trophectoderm of the early blastocyst, while the mural trophectoderm shows very little inducing activity. Furthermore, the inducing ability of the polar trophectoderm decreases with complete expansion and hatching of the blastocyst. These results indicate that the ability to induce 8-cell blastomere polarization is retained by the embryo beyond the 8-cell stage and that this ability is lost with further differentiation.     

Solubilization of the bovine cardiac sarcolemmal binding sites for calcium channel blockers

Nonionic and ionic detergents were used to solubilize the bovine cardiac sarcolemmal binding sites for nimodipine and (-)desmethoxyverapamil in the absence of added ligand. Only Chaps, digitonin and sucrose monolauryl ester were able to solubilize the binding sites in a form that bound radioligands. About 45% of each of the membrane-bound high-affinity site was solubilized by 0.4% Chaps (w/v) in the presence of 48% (w/v) glycerol. The solubilized binding sites were destroyed by trypsin or by a 10-min incubation at 50 degrees C. Calcium stimulated nimodipine binding slightly at 0.3 mM and inhibited (-)desmethoxyverapamil binding completely with an IC50 of 1.2 mM. Nimodipine binding was reduced by 20% in the presence of EGTA. The solubilized receptors sedimented in sucrose density gradients with an apparent s20,w of 21 S. An identical sedimentation value was obtained for the cardiac sarcolemmal and skeletal transverse tubulus receptor which were prelabeled with nitrendipine and solubilized by digitonin. Solubilization reduced the affinity of nimodipine for its high-affinity site slightly from 0.35 nM to 1.2 nM and that for its low-affinity site from 33 nM to 130 nM. Solubilization did not affect significantly the specific density of these sites. Binding of nimodipine to the low-affinity site was completely abolished by 0.1 microM nitrobenzylthioinosine. After solubilization only the high-affinity site for (-)desmethoxyverapamil could be measured with tenfold reduced affinity (Kd = 15.3 nM) but unchanged specific density. Binding to the solubilized high-affinity site for nimodipine and (-)desmethoxyverapamil was stereospecific and showed a similar rank order as the particulate binding sites. Binding of nimodipine was inhibited allosterically by phenylalkylamines. Similarly, (+)PN200-110 inhibited allosterically (-)desmethoxyverapamil binding. d-cis-Diltiazem stimulated nimodipine binding at 20 degrees C 1.2-fold, reduced the dissociation rate from 0.018 min-1 to 0.0083 min-1 and had no effect on the association rate (0.173 min-1. nM-1). The Kd calculated from the rate constants was 0.1 nM and in close agreement with the value of 0.49 nM measured under equilibrium conditions in the presence of nitrobenzylthioinosine. In contrast, desmethoxyverapamil increased the dissociation rate of nimodipine to 0.03 min-1. The association and dissociation rate constants for (-)desmethoxyverapamil were 0.024 min-1. nM-1 and 0.025 min-1, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cardiac sarcoplasmic reticulum contains a low-affinity site for phenylalkylamines

The distribution of the bovine cardiac binding sites for the organic calcium-channel blockers was studied. Crude microsomal membranes were separated into three fractions, which contained mainly membranes derived from sarcolemma, 'junctional' sarcoplasmic reticulum containing transversal tubuli, and free sarcoplasmic reticulum. The high-affinity binding site for the dihydropyridines, determined in the presence of nitrobenzylthioinosine, was enriched 12-fold and 17-fold in sarcolemma and junctional sarcoplasmic reticulum. The binding sites for the phenylalkylamines, determined with [3H]verapamil or [3H](-)desmethoxyverapamil, were enriched 1.5-3.4-fold in sarcolemma and junctional sarcoplasmic reticulum but 6-10-fold in free sarcoplasmic reticulum. The phenylalkylamine-binding site, present in free sarcoplasmic reticulum, was partially destroyed by chymotrypsin or phospholipase A2 and C treatment. Specific binding was proportional to the concentration of the added membrane protein. The binding of (-)desmethoxyverapamil was half-maximally inhibited by 6.5 mM calcium chloride and was optimal in the presence of 5 mM EGTA. In three out of five preparations (-)desmethoxyverapamil bound to a single site with an apparent Kd value of 191 +/- 42.8 nM and a density of 34.5 +/- 7.7 pmol/mg protein. In two out of five preparations an additional high-affinity site (Kd approximately 0.67 nM) was detected. The low-affinity site bound other phenylalkylamines, but stereospecific binding of phenylalkylamines was not observed. Binding of phenylalkylamines to the low-affinity site was inhibited by some but not all calmodulin 'antagonists'. Furthermore dihydropyridines did not affect the binding of (--)desmethoxyverapamil suggesting that the low-affinity site differs considerably from the high-affinity sarcolemmal site. These results suggest that free sarcoplasmic reticulum contains a binding site for phenylalkylamines at a relative high density, which is not related to the high-affinity site present in the voltage-dependent calcium channel.     

The Genetics of the DORSAL-BICAUDAL-D Region of DROSOPHILA MELANOGASTER

The chromosomal region 36C on 2L contains two maternal-effect loci, dorsal (dl) and Bicaudal-D (Bic-D), which are involved in establishing polarity of the Drosophila embryo along the dorsal-ventral and anterior-posterior axes, respectively. To analyze the region genetically, we isolated X-ray-induced dorsal alleles, which we recognized by virtue of the haplo-insufficient temperature-sensitive dorsal-dominant phenotype in progeny of single females heterozygous for a mutagenized chromosome. From the 20,000 chromosomes tested, we isolated three deficiencies, two inversions with breakpoint in dl and one apparent dl point mutant. One of the deficiencies, Df(2L)H20 (36A6,7; 36F1,2) was used to screen for EMS-induced lethal- and maternal-effect mutants mapping in the vicinity of dl and Bic-D. We isolated 44 lethal mutations defining 11 complementation groups. We also recovered as maternal-effect mutations four dl alleles, as well as six alleles of quail and one allele of kelch, two previously identified maternal-effect genes. Through complementation tests with various viable mutants and deficiencies in the region, a total of 18 loci were identified in an interval of about 30 cytologically visible bands. The region was subdivided into seven subregions by deficiency breakpoints. One lethal complementation group as well as the two maternal loci, Bic-D and quail, are located in the same deficiency interval as is dl.     

Double immunocytochemical staining for the in situ study of allotype distribution during an anti-trinitrophenyl immune response in chimeric rabbits

After incubation of tissue sections with anti-allotype-enzyme conjugates, the localization of immunoglobulin-allotype-bearing cells in the lymphoid tissues of conventional and chimeric rabbits could be established. The use of anti-allotype sera bearing distinct enzyme labels allowed simultaneous recognition of B cells producing immunoglobulin of one or the other parental types in heterozygous rabbits, or of B cells from the donor and recipient in chimeras. After immunization of chimeric rabbits with trinitrophenyl-keyhole limpet hemocyanin, anti-trinitrophenyl antibody-forming cells could be demonstrated through the use of a trinitrophenyl-alkaline phosphatase conjugate. Simultaneous incubation of sections with this reagent and with horseradish peroxidase coupled to (donor or recipient) anti-allotype sera made possible the determination of the origin (donor or recipient) of the antibody-forming cells. In agreement with the results of plaque assays and analyses of serum antibodies, all the anti-TNP producing cells were of donor origin when the chimeras had been created through injection of spleen or lymph node cells from trinitrophenyl primed donors. With this study we introduce a simple, direct method for the simultaneous identification of cells that produce antibody of a given allotype and a given specificity, applicable to appropriate studies in heterozygous or chimeric rabbits. The procedure has various advantages over previously reported methods.     

Fusion of bacterial spheroplasts by electric fields

Spheroplasts of Escherichia coli or Salmonella typhimurium were found to fuse in an electric field. We employed the fusion method developed by Zimmermann and Scheurich (1981): Close membrane contact between cells is established by dielectrophoresis (formation of chains of cells by an a.c. field), then membrane fusion is induced by the application of short pulses of direct current. Under optimum conditions the fusion yield was routinely 90%. Fusable spheroplasts were obtained by first growing filamentous bacteria in the presence of cephalexin, then converting these to spheroplasts by the use of lysozyme. The fusion products were viable and regenerated to the regular bacterial form. Fusion of genetically different spheroplasts resulted in strains of bacteria possessing a combination of genetic markers. Fusion could not be achieved with spheroplasts obtained by growing the cells in the presence of penicillin or by using lysozyme on bacteria of usual size.     

Structural wrinkles and the genomic regulatory sites of eukaryotes

Summary Calculations of DNA angular parameters in 50 eukaryotic sequences reveal regions of large conformational deviations from ideal DNA around regulatory sites. Frequently, discrete peaks of structural variation are present upstream of genes. Known regulatory regions often include variants of consensus sequences. Thus, imprecise sequences and structures are recognized within large genomic stretches. The existence of structurally wrinkled regions in the vicinity of regulatory sequences is likely to facilitate greatly their recognition by proteins and enzymes.     

Characterization of the binding sites for nimodipine and (-)-desmethoxyverapamil in bovine cardiac sarcolemma

The bovine cardiac sarcolemmal binding sites for the dihydropyridine nimodipine and the phenylalkylamine (-)-desmethoxyverapamil were studied. The density of the nimodipine and (-)-desmethoxyverapamil binding sites increased 8.3-fold and 3.4-fold with the sarcolemma. The binding sites for both compounds were destroyed by trypsin. Nimodipine bound in the presence of 1 mM free calcium to a high-affinity and a low-affinity site with apparent Kd values of 0.35 +/- 0.09 nM (n = 9) and 33 +/- 6.0 nM (n = 9) and with apparent densities of 0.3 +/- 0.05 pmol/mg (n = 9) and 8.2 +/- 1.0 pmol/mg (n = 9). The binding to the high-affinity site was abolished by 1 mM EGTA. The binding sites were specific for dihydropyridines. The (-)-isomers of several phenylalkylamines inhibited nimodipine binding by an apparent allosteric mechanism. (-)-Desmethoxyverapamil bound in the presence of 5 mM EGTA to a high-affinity and a low-affinity site with apparent Kd values of 1.4 +/- 0.3 nM (n = 6) and 171 +/- 26 nM (n = 6) and with apparent densities of 0.16 +/- 0.02 pmol/mg (n = 6) and 13.6 +/- 2.7 pmol/mg (n = 6). The binding to both sites was inhibited by calcium with a half-maximal concentration of 4.3 mM. The binding sites were specific for the other phenylalkylamines and had a higher affinity for the (-)-isomers than for the (+)-isomers. Nimodipine inhibited the binding of (-)-desmethoxyverapamil by an apparent allosteric mechanism. d-cis-Diltiazem inhibited non-competitively the binding of (-)-[3H]desmethoxyverapamil with a Ki of 3.7 microM. Diltiazem up to concentrations of 10 microM did not affect the amount of nimodipine bound at equilibrium at 20 degrees C. However, but in agreement with this result, diltiazem decreased threefold at 20 degrees C the dissociation and association rates for the high-affinity nimodipine receptor. These rates were only marginally affected at 4 degrees C and 37 degrees C. d-cis-Diltiazem reversed in a competitive manner the inhibition of nimodipine binding elicited by the addition of (-)-desmethoxyverapamil with a Ka value of 1.6 microM. The amount of nimodipine bound was inhibited by 50% by the adenosine uptake inhibitors nitrobenzylthioinosine and hexobendine with apparent median inhibitory concentrations of 1 nM and 3 nM, respectively. Nitrobenzylthioinosine completely abolished binding of nimodipine to the low-affinity site, but did not affect binding to the high-affinity site.(ABSTRACT TRUNCATED AT 400 WORDS)     

A histochemical study of the changes occurring in the protein-carbohydrate composition of the cumulus-oocyte complex and zona pellucida in immature mice in response to gonadotrophin stimulation

Summary A histochemical account is presented of the changes that occur in the protein—carbohydrate composition of the cumulus—oocyte complex in immature mice after gonadotrophin treatment. The distribution and nature of the glycosaminoglycans (GAG) present was established by enzymic digestion of tissue sections with testicular orStreptomyces hyaluronidase prior to staining with periodic—acid Schiff (PAS) or Alcian Blue. Treatment with exogenous gonadotrophins [pregnant mare's serum and human chorionic gonadotrophin (hCG)] induced gross changes in the appearance of the zona pellucida (and in the histochemical staining of the cumulus—oocyte complex). A reduction was observed in the amount of PAS-positive material present within the zona pellucida of oocytes located in large Graafian follicles examined 40 h after stimulation with pregnant mare's serum. After the injection of hCG, the zona pellucida was further depleted of PAS-positive naterial. Most of the PAS-positive material became confined to the plasma membrane of the oocyte, while the oocyte itself also became increasingly PAS-positive. All the GAGs disappeared from zona pellucida within 4 h of hCG stimulation. The changes observed in the protein—carbohydrate composition of the zona pellucida in preovulatory oocytes immediately prior to ovulation may be a prerequisite for successful sperm-egg interactions.