An electrogenic proton pump in plasma membranes from the cellular slime mould Dictyostelium discoideum

Plants and fungi possess an outwardly directed plasma membrane proton pump that may regulate intracellular pH. We provide the first demonstration that amoebae of the slime mould Dictyostelium discoideum also possess a similar proton pump. It can be assayed either as an ATPase activity in highly purified plasma membranes or as a proton pump, after solubilization and reconstruction into liposomes. The pump is inhibited by vanadate, diethylstilbestrol (DES) and miconazole but not by azide or ouabain. The proton pump described here may represent the target for the action of DES and miconazole, both of which have previously been shown to induce stalk cell formation during the in vitro development of Dictyostelium.     

Resolution of multiple fluorescence lifetimes in heterogeneous systems by phase-modulation fluorometry

Procedures are described for the treatment of phase and modulation lifetime data in fluorescent systems having multiexponential decay. All computer procedures (called FIT programs) arise from the lifetime resolution theory for phase-modulation measurements (Weber, G (1981) J. Phys. Chem. 85, 949–953). The programs most successful in resolving heterogeneous lifetimes use a Monte Carlo approach in which phase and modulation lifetime data at three modulation frequencies are simultaneously utilized. These programs are shown to have more utility than the final closed form procedure presented by Weber (1981). The FIT routines are simple and require little computer time while yielding excellent results. To illustrate the applicability of these programs, defined binary (carbazole and pyrene) and ternary systems (carbazole, pyrene and POPOD) were examined. In most cases, the resolved lifetimes were within 5% of the independently measured value and the fractional fluorescence contributions were within 10% of that expected. These results demonstrate that phase-modulation measurements analyzed by appropriate computer programs are capable of solving for lifetimes in both binary and, in selected cases, ternary systems. An example is given from the recent literature (Dalbey, R., Weiel, J. and Yount, R.G. (1983) Biochemistry 22, 4696–4706) in which the above programs allowed the resolution of both binary and ternary lifetimes of a dansyl label on myosin, where Förster energy transfer was occuring. These lifetimes] were used to quantify changes in distances between two activity-related thiols on myosin upon the addition of Mg-ATP or its analogs.     

Albumin extinction followed by de novo methylation of its gene in somatic hybrids of a rat hepatoma

We have previously identified an Msp I site at the 5′ end of the rat albumin gene whose undermethylation is necessary but not sufficient for stable albumin expression in rat hepatoma cells [1]. We have also shown that the extinction of albumin expression in somatic hybrids is not the result of methylation at this site, since for two different crosses, rapid extinction was found to occur in the absence of any de novo methylation of the previously active gene[2]. In the present study, we examine albumin expression and albumin gene methylation for independent hybrid clones isolated from crosses between albumin expressing rat hepatoma cells and cells of two different non-expressing lines. The cells from hybrid clones of both crosses are characterized by stable extinction of albumin expression. Moreover, we find that de novo methylation of the “extinguished” albumin gene can occur in somatic hybrids, but only some weeks after the gene has ceased to be expressed.     

A method facilitating the demonstration of synergism in the tissue-responses to mixed peptide stimuli

A methodology for the pharmacodynamical assay of mixtures of 2 spasmogenic peptides is described, illustrated and discussed, with reference to a method intended for the chemists (P. Job, 1927). This allows a quick appreciation of the existence of synergism, of its own order of magnitude and of that of the doses for which it is significant. Also given is a fast way of curve-fitting the dose response to individual peptides, according to Clark's approximation.     

Prolactin increases the density of striatal dopamine receptors in normal and hypophysectomized male rats

Administration of prolactin to adult male rats, by s.c. injection, significantly increases the density of the striatal dopamine (DA) receptors, without altering the apparent affinity of the receptors for [³H]spiroperidol. Larger doses of prolactin are required to increase the density of the striatal DA receptors in hypophysectomized rates compared to normal rats. These results suggest that prolactin might be the common mediator of the increase in striatal DA receptor density produced by either estrogen or haloperidol administration. Monitoring and/or altering prolactin levels might be informative in neurologic or psychiatric disorders involving striatal DA neurotransmission.     

Five 3β, 6α-dihydroxysterols in organ-pipe cactus

The sterol diol fraction from the lipids of organ-pipe cactus, Stenocereus thurberi, was separated into five compounds: macdougallin, peniocerol, cyclostenol, stenocereol and thurberol. The last three compounds have not been described before. All compounds were characterized by physical and spectroscopic properties.     

The structures of garcinones a,b and c: Three new xanthones from Garcinia mangostana

Three new tetraoxygenated xanthones (garcinones A, B and C), each disubstituted with C₅-units, have been isolated from the chloroform extract of the fruit-hulls of Garcinia mangostana. Their structures were established by a combination of spectral interpretation and chemical correlation.     

Quantitation and interaction of glycosaminoglycans with Alcian blue in dimethyl sulfoxide solutions.

An improved method is described for the quantitation of glycosaminoglycans separatedon cellulose acetate, stained with Alcian blue, and dissolved in a dimethyl sulfoxide solution. Standard curves are shown for all eight glycosaminoglycans. It is shown that absorption at the Alcian blue orthochromatic E_max is depressed under conditions which favor formation of dye-glycosaminoglycan complexes. The interaction between Alcian blue and the eight glycosaminoglycans was studied in dimethyl sulfoxide solutions of varying composition. It was shown that the extent of complex formation depends both on the glycosaminoglycan and the composition of the dimethyl sulfoxide solution. A dimethyl sulfoxide solution which contains 0.094 m H₂SO₄ is described which maximizes dye-glycosaminoglycan dissociation and thus the absorbance. Also, an improved staining method is described which improves dye uptake by the glycosaminoglycans and consequently increases the sensitivity of glycosaminoglycan quantitation.     

Secondary structure of mouse and rabbit α- and β-globin mRNAs: Differential accessibility of α and β initiator AUG codons towards nucleases

The nucleotide sequence from the 5′ terminus inward of one third of mouse α- and β^maj-globin messenger RNAs has been established. In addition, using 5′ ³²P end-labeled mRNAs as substrates and S1 and T1 nucleases as probes for single-stranded regions, the secondary structures of mouse and rabbit α- and β-globin mRNAs have been analyzed. Our results indicate that the AUG initiator codon in both mouse and rabbit β-globin mRNA is quite susceptible to cleavage with S1 and T1 nucleases, suggesting that it resides in a single-stranded exposed region. In contrast, the initiator AUG in the α-globin mRNA of both species is inaccessible to cleavage, indicating that it is either buried by tertiary structure or is base-paired. Since the rate of initiation of protein synthesis with β-globin mRNA in rabbit reticulocyte is 30–40% faster than for α-globin mRNA, these results imply a possible correlation between the differential rates of initiation with these two mRNAs and the accessibility of the respective AUG initiator codons.