Differential Regulation by Calmodulin of Basal, GTP-, and Dopamine-Stimulated Adenylate Cyclase Activities in Bovine Striatum

The concentration requirements of calmodulin in altering basal, GTP-, and dopamine-stimulated adenylate cyclase activities in an EGTA-washed particulate fraction from bovine striatum were examined. In the bovine striatal particulate fraction, calmodulin activated basal adenylate cyclase activity 3.5-fold, with an EC50 of 110 nM. Calmodulin also potentiated the activation of adenylate cyclase by GTP by decreasing the EC50 for GTP from 303 +/- 56 nM to 60 +/- 10 nM. Calmodulin did not alter the maximal response to GTP. The EC50 for calmodulin in potentiating the GTP response was only 11 nM as compared to 110 nM for activation of basal activity. Similarly, calmodulin increased the maximal stimulation of adenylate cyclase by dopamine by 50-60%. The EC50 for calmodulin in eliciting this response was 35 nM. These data demonstrate that calmodulin can both activate basal adenylate cyclase and potentiate adenylate cyclase activities that involve the activating GTP-binding protein, Ns. Mechanisms that involve potentiation of Ns-mediated effects are much more sensitive to calmodulin than is the activation of basal adenylate cyclase activity. Potentiation of GTP-stimulated adenylate cyclase activity by calmodulin was apparent at 3 and 5 mM MgCl2, but not at 1 or 10 mM MgCl2. These data further support a role for calmodulin in hormonal signalling and suggest that calmodulin can regulate cyclic AMP formation by more than one mechanism.     

Non-mendelian inheritance of mitochondrial DNA and ribosomal DNA in the myxomycete, Didymium iridis

Summary The inheritance of both the mitochondrial DNA (mtDNA) and the nuclear-encoded extrachromosomal ribosomal DNA (rDNA) has been studied in the myxomycete, Didymium iridis, by DNA-DNA hybridization of labeled probes to total DNA at various stage of the life cycle. Both the mtDNA and rDNA populations rapidly become homogeneous in individuals, but there is a qualitative difference in the patterns of inheritance of these two molecules. One parental rDNA type was preferentially inherited in all crosses; selective replication of this molecule is tentatively proposed as the mechanism of inheritance. In contrast, either parental mtDNA type could be inherited. Since the inherited population of parental mtDNA molecules are not partitioned into cells in this coenocytic organism, no known mechanism of inheritance can explain the rapid and apparently random loss of one parental mtDNA type in individuals.     

Dynamic instability of individual microtubules analyzed by video light microscopy: rate constants and transition frequencies

We have developed video microscopy methods to visualize the assembly and disassembly of individual microtubules at 33-ms intervals. Porcine brain tubulin, free of microtubule-associated proteins, was assembled onto axoneme fragments at 37 degrees C, and the dynamic behavior of the plus and minus ends of microtubules was analyzed for tubulin concentrations between 7 and 15.5 microM. Elongation and rapid shortening were distinctly different phases. At each end, the elongation phase was characterized by a second order association and a substantial first order dissociation reaction. Association rate constants were 8.9 and 4.3 microM-1 s-1 for the plus and minus ends, respectively; and the corresponding dissociation rate constants were 44 and 23 s-1. For both ends, the rate of tubulin dissociation equaled the rate of tubulin association at 5 microM. The rate of rapid shortening was similar at the two ends (plus = 733 s-1; minus = 915 s-1), and did not vary with tubulin concentration. Transitions between phases were abrupt and stochastic. As the tubulin concentration was increased, catastrophe frequency decreased at both ends, and rescue frequency increased dramatically at the minus end. This resulted in fewer rapid shortening phases at higher tubulin concentrations for both ends and shorter rapid shortening phases at the minus end. At each concentration, the frequency of catastrophe was slightly greater at the plus end, and the frequency of rescue was greater at the minus end. Our data demonstrate that microtubules assembled from pure tubulin undergo dynamic instability over a twofold range of tubulin concentrations, and that the dynamic instability of the plus and minus ends of microtubules can be significantly different. Our analysis indicates that this difference could produce treadmilling, and establishes general limits on the effectiveness of length redistribution as a measure of dynamic instability. Our results are consistent with the existence of a GTP cap during elongation, but are not consistent with existing GTP cap models.     

Stimulation of the chemiluminescence of human polymorphonuclear leukocytes in various stages of the intraerythrocyte development of Plasmodium falciparum

The synchronized cultures of Plasmodium falciparum were used to stimulate in vitro the chemiluminescence of human polymorphonuclear leukocytes in the presence of immune serum. The schizonts were concentrated by Percoll gradient centrifugation method (density 1.085 and osmolarity 285 mOsmol), and placed in culture, treated 6 hours later by sorbitol. Under incubation at constant temperature and pressure, the rate of synchronization reached 85% for schizonts during 5 replicative cycles. Every asexual stages of Plasmodium falciparum were used separately to stimulate polymorphonuclear leukocytes: merozoites were the most effective, followed by schizonts, trophozoites, and lastly supernatants of cultures containing degradation products of parasites.     

High hydrostatic pressure effects in vivo: changes in cell morphology, microtubule assembly, and actin organization

We present the first study of the changes in the assembly and organization of actin filaments and microtubules that occur in epithelial cells subjected to the hydrostatic pressures of the deep sea. Interphase BSC-1 epithelial cells were pressurized at physiological temperature and fixed while under pressure. Changes in cell morphology and cytoskeletal organization were followed over a range of pressures from 1 to 610 atm. At atmospheric pressure, cells were flat and well attached. Exposure of cells to pressures of 290 atm or greater caused cell rounding and retraction from the substrate. This response became more pronounced with increased pressure, but the degree of response varied within the cell population in the pressure range of 290-400 atm. Microtubule assembly was not noticeably affected by pressures up to 290 atm, but by 320 atm, few microtubules remained. Most actin stress fibers completely disappeared by 290 atm. High pressure did not simply induce the overall depolymerization of actin filaments for, concurrent with cell rounding, the number of visible microvilli present on the cell surface increased dramatically. These effects of high pressure were reversible. Cells re-established their typical morphology, microtubule arrays appeared normal, and stress fibers reformed after approximately 1 hour at atmospheric pressure. High pressure may disrupt the normal assembly of microtubules and actin filaments by affecting the cellular regulatory mechanisms that control cytological changes during the transition from interphase into mitosis.     

Proton and sucrose transport in isolated tonoplast vesicles from sugarcane stalk tissue

Tonoplast vesicles prepared from immature sugarcane ( Saccharum spp., hybrid cv. H65–7052) tissue and purified on a discontinuous dextran gradient take up sucrose. Uptake was stimulated by MgATP. Evidence that the mechanism is linked to proton transport is derived from "pH jump'data and from inhibition of ATP-stimulated sucrose transport by the protonophore carbonyl cyanide m -chlorophenylhydrazone (CCCP) and by the proton-channel blocker of proton-linked ATPases. N. N '-dicyclo-hexylcarbodiimide (DCCD). A saturable phase of sucrose uptake was found at low substrate concentrations, and a linear phase characterized uptake at higher concentrations. Uptake was specific for sucrose, as demonstrated by competition experiments with various sugars. Sucrose uptake by the vesicle fraction was inhibited by KNO₃, protonophores and protein modifying reagents, whereas sodium orthovanadate had no effect. Overall, the evidence suggests an ATP-hydrolysis-dependent tonoplasl antiport for sucrose transport, although a more direct influence of ATP on conformational changes in relevant tonoplast proteins cannot be ruled out.     

Cloning and sequencing of the cDNA encoding the major albumin of Theobroma cacao

The major albumin, a polypeptide of 21 kilodaltons (kDa), from the seeds of cocoa (Theobroma cacao L.), has been identified and partially purified by preparative gel electrophoresis. Some N-terminal sequence was obtained, permitting the construction of an oligonucleotide probe. This probe was used to isolate the corresponding copy DNA (cDNA) clone from a library made from poly(A)⁺ RNA from immature cocoa beans. The cDNA sequence has a single major open reading frame, that translates to give a 221-amino-acid polypeptide of M_r 24003. The existence of a precursor to the 21-kDa polypeptide of this size was confirmed by immunoprecipitation from total poly(A)⁺ RNA translation products. The polypeptide has a hydrophobic signal sequence of 26 amino acids before the mature start, and the mature polypeptide would have an M_r of 21223. The protein sequence is homologous with sequences of the Kunitz protease and -amylase inhibitor family, and the protein probably functions to defend the seed's protein reserves from the digestive enzymes of invading pests. However because the protein comprises 25–30% of the total seed protein it may itself also function as a storage protein. Electron micrographs of immunogold-labelled embryo sections show that the protein is located in membrane-enclosed organelles.Abbreviations cDNA copy DNA - IgG immunoglobulin G - kb kilobase pairs - kDa kilodaltons - M_r relative molecular mass - SDS-PAGE sodium dodecyl sulphate-polyacylamide gel electrophoresis The authors are very grateful to Dr R. Jennings of the Virology Department, Sheffield University Medical School, for help in raising antibodies, and to Dr G. Cope, of the Biological Sciences Electron Microscopy Unit, Sheffield University, for taking the electron micrographs.To whom correspondence should be addressed.     

Structure of equine corticotropin releasing factor

A 41 amino acid peptide, probably identical in structure to human corticotropin releasing factor, was isolated from 70 equine hypothalami by methanol extraction, immunoaffinity chromatography and single step of reverse phase HPLC. The amino acid sequence was determined by gas phase sequence analysis. Probable carboxyl terminal amidation was demonstrated by similar retention times for equine and human corticotropin releasing factor on reverse phase HPLC at pH 8. The likely structure of equine corticotropin releasing factor is: Ser-Glu-Glu-Pro-Pro- Ile-Ser-Leu-Asp-Leu-Thr-Phe-His-Leu-Leu-Arg-Glu-Val-Leu-Glu-Met-Ala-Arg-Ala-Glu-Gln-Leu-Ala-Gln-Gln-Ala-His-Ser-Asn- Arg-Lys-Leu-Met-Glu-Ile-Ile-NH₂. The purified peptide is equipotent with human corticotropin releasing factor in an in vitro bioassay and in a human plasma binding protein assay.     

Sensitivity of some common grain fungi to irradiation on grain and in phosphate-buffered saline

The sensitivity of nine cereal fungi to irradiation on grain and in phosphate-buffered saline (PBS) was investigated. Species of Fusarium and Alternaria were more resistant to irradiation (higher D ₁₀ value) than Aspergillus spp. and Penicillium spp. Generally D ₁₀ values determined on grain were lower than the corresponding values measured in PBS. The plating medium did not significantly affect the D ₁₀ values.     

Characterization of recombinant human renin: Kinetics,pH-stability, and peptidomimetic inhibitor binding

The kinetic behavior andpH-stability of recombinant human renin was analyzed using a new fluorogenic substrate based on the normal P₆-P₃ renin cleavage sequence in human angiotensinogen. The design of this fluorogenic substrate makes possible, for the first time, direct monitoring of the kinetics of proteolytic conversion of prorenin to renin. ThepH-stability profile for renin, measured with the substrate at 25°C, indicated a broad plateau of stability betweenpH 6.0 and 10.0. Analysis of thepH-activity profile of renin for the substrate indicated a minimumK _m (1.8 µM) atpH 7.4 and a maximumV _m betweenpH 7.4 and 8.0. The thermodynamics of the binding of a novel, soluble, peptidomimetic inhibitor to renin indicated it is possible to retain the tight-binding characteristics and enthalpy contributions to binding of larger peptide-derived inhibitors, while reducing inhibitor size and entropic contributions to binding. A novel derivative of the fluorogenic substrate, containing a 3-methyl histidine substitution at the P₂ site, was used to test the recent hypothesis that renin functions by virtue of substrate-directed catalysis.