Mode of action studies on a Manduca sexta diuretic hormone

The mode of action of a diuretic hormone from pharate adult Manduca Sexta heads, which triggers fluid loss in M. sexta larvae and Pieris rapae adults, was studied. In vivo, Mas-DH (M. sexta diuretic hormone) decreased fluid absorption from larval recta, and increased levels of the second messenger cAMP in recta and Malpighian tubules (Mt) from larvae, and in fat body of larvae and adult M. sexta. In vitro, Mas-DH triggered minor changes in fluid loss from adult Mt, but did not affect levels of cAMP in Mt from larvae, pharate adults, or adults, though it elevated cAMP levels in fat body of these stages. © 1992 Wiley-Liss, Inc.     

''Cross-talk'' between phospholipase C and adenylyl cyclase involves regulation of G-protein levels in GH3 rat pituitary cells.

We have investigated the possibility that adenylyl cyclase (AC) activity and membrane protein levels of the -subunits of the stimulatory and inhibitory G-proteins of AC (G_s and G_i−2) in cultured prolactin-producing rat pituitary adenoma cells (GH₃ cells) are modulated by phospholipase C (PLC)-generated second messengers. Pretreatment of cells (6–48 h) with ionomycin (1 μM) or 1-oleoyl-2-acetylglycerol (OAG; 1μM) showed that ionomycin regulated G_s levels in a time-dependent, biphasic manner; a two-fold increase followed a 40% initial reduction, while OAG lowered G_s levels by more than 50% at all time-points. G_i−2 levels remained unchanged by both pretreatments. OAG, but not ionomycin, increased basal AC activity without increasing enzyme protein levels. Alterations in AC responsiveness to peptide hormones (e.g. thyroliberin and vasoactive intestinal peptide) correlated to membrane G_s protein -subunit content. These results demonstrate the involvement of G-protein translation regulation as one mechanism of ‘cross-talk’ between the PLC- and AC-dependent signalling pathways.     

Molecular organisation of the malate synthase genes of Aspergillus nidulans and Neurospora crassa

Summary A soybean nodulin cDNA clone (E41) hybrid-selected mRNA for three in vitro translation products with apparent molecular weights of 26 kDa, 25 kDa and 24 kDa. Based on Southern analysis of soybean genomic DNA, combined with mapping and sequencing of genomic clones, we identified four genes that are related to E41, one of which was identified to be the previously characterized N-20 gene. Our data indicate the linkage of three of the genes, of which one is a truncated version and suggest that they originated by gene duplication combined with deletion and conversion. The genes are highly expressed and we postulate that the sequence conservation in the 5 and 3 flanking regions of all four genes, has a functional role in their expression. Hybrid-selected translation products of E41 are not immunoprecipitable with antibody to the soluble fraction of nodules suggesting that they are membrane associated. The N-20 gene, which is a member of this gene subfamily, showed sequence similarity to four previously characterized nodulin genes and a phylogenetic tree is proposed based on the extent of sequence similarity.     

Capitalist Philanthropy and Russian Revolutionaries: The Jesup North Pacific Expedition (1897-1902)

This review of the Jesup North Pacific Expedition, still the most important expedition in American anthropology, gives an idea of the goals and hazards of fieldwork around 1900, the pitfalls of international research, the tensions between anthropologists and host populations, the careers of early anthropologists, the role of private philanthropy, and the character of anthropology at the turn of the century. Franz Boas was the Expedition's linchpin. His organization of the Expedition, the way he handled problems, and his personal concerns reveal aspects of his view of anthropology and some of his basic attitudes.     

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.     

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)