Ca2+ calmodulin-dependent protein kinase activity in the ascomycetes Neurospora crassa

DEAE-cellulose column chromatography of Neurospora crassa soluble mycelial extracts leads to the resolution of three major protein kinase activity peaks designated PKI, PKII, and PKIII.PKII activity is stimulated by Ca²⁺ and Neurospora or brain calmodulin. Maximal stimulation was observed at 2 µM-free Ca²⁺ and 1 µg/ml of the modulator. The stimulatory effect of the Ca²⁺-calmodulin complex was blocked by EGTA and by some calmodulin antagonists such as phenothiazine drugs or compound 48/80.PKII phosphorylates different proteins, among which histone II-A at a low concentration and CDPKS, the synthetic peptide specific for Ca²⁺-calmodulin dependent protein kinases, are the best substrates. Some phosphorylation can be detected in the absence of any exogenous acceptor. PKII activity assayed in the presence of histone II-A or in the absence of exogenous phosphate acceptor (autophosphorylation) co-elute in a DEAE-cellulose column at 0.28 M NaCl. As result of the autophosphorylation reaction of the purified enzyme a main phosphorylated component of 70 kDa was resolved by SDS-polyacrylamide gel electrophoresis. It is possible that this component is an active part of this enzyme.     

Influence of Calcium Ion on Ethanol Tolerance of Saccharomyces bayanus and Alcoholic Fermentation by Yeasts

The addition of Ca²⁺ (as CaCl₂) in optimal concentrations (0.75 to 2.0 mM) to a fermentation medium with a trace contaminating concentration of Ca²⁺ (0.025 mM) led to the rapid production of higher concentrations of ethanol by Saccharomyces cerevisiae, Saccharomyces bayanus, and Kluyveromyces marxianus. The positive effect of calcium supplementation (0.75 mM) on alcoholic fermentation by S. bayanus was explained by the increase in its ethanol tolerance. The ethanol inhibition of growth and fermentation followed the equation μ_xi = μ_oi [1 - (X/X_mi)]ⁿⁱ, where μ_oi and μ_xi are, respectively, the specific growth (i = g) and fermentation (i = f) rates in the absence or presence of a concentration (X) of added ethanol, and X_mi is the maximal concentration of ethanol which allows growth or fermentation. The toxic power is given by n_i. In Ca²⁺ - supplemented medium (0.75 mM), n_g = 0.42 for growth and n_f = 0.43 for fermentation compared with 0.52 and 0.55, respectively, in unsupplemented medium; for both media, X_mg = 10% (vol/vol) and X_mf = 13% (vol/vol). For lethal concentrations of ethanol, the specific death rates were minimal for cells that were grown and incubated with ethanol in medium with an optimal concentration of Ca²⁺, maximal for cells grown and incubated with ethanol in unsupplemented medium, and intermediate for cells grown in unsupplemented medium and incubated with ethanol in calcium-supplemented medium. The effect of Ca²⁺ on the acidification curve of energized cells in the presence of ethanol was found to be closely associated with its protective effect on growth, fermentation, and viability.     

Organic microscopic remains in Miocene lacustrine sediments near Libros (Teruel,Spain)

Siliceous remains from Miocene lacustrine sediments near Libros (Teruel, Spain) are studied. Most of them are sponge spicules and may be assigned to Ephydatia fluviatilis. Some chrysophycean cysts and several diatom genera (Melosira, Cyclotella, Fragilaria, Navicula, Pinnularia, and Cymbella) have also been found.     

Polyamine-sensitive protein kinase from chick intestinal mucosa

Summary A cyclic nucleotide-independent protein kinase which phoshorylates preferentially acidic proteins such as casein or phosvitin was isolated from cytosol of chick duodenal mucosa. The enzyme was purified more than 633 fold to apparent homogeneity by ammonium sulfate fractionation, column chromatography on DEAE-cellulose, phosphocellulose, hydroxylapatite and by sucrose density gradient centrifugation. The native enzyme has a molecular weight of 131000 as measured by gel filtration. The enzyme is a complex protein containing three polypeptides of molecular weight of 39 000, 36 000 and 27 000. It behaves as a complex throughout its purification and gel filtration but its components are readily separated by electrophoresis in denaturing buffer. The 27 000 molecular weight band was selectively autophosphorylated when the enzyme was incubated in the presence of [-³²P]ATP.When casein was used as substrate, physiological concentrations of naturally occurring polyamines such as spermine and spermidine markedly stimulated enzyme activity. However with phosvitin as substrate polyamines were strong inhibitors of the enzyme activity. This contrasting effect on intestinal kinase activity was also apparent using cytoplasmic proteins as endogenous phosphate acceptors. A characterization of this differential effect is presented and some possible physiological implications are discussed.     

Construction of a cosmid library of Spirulina platensis as an approach to DNA physical mapping

Abstract A Spirulina platensis gene library has been constructed using cosmid vector pMMB34. The cosmid bank was controlled for its random gene distribution by colony hybridization. Genes were identified using either homologous or heterologous probes of genes involved in photosynthesis (large and small subunit of d -ribulose 1,5-bisphosphate carboxylase, 32 kDa thylakoid protein, α, β subunits of C-phycocyanin) and protein synthesis (elongation factors EF-Tu, EF-G).     

A new pollen type,C-banded and fluorochrome counterstained chromosomes,and evolution inGuatteria and related genera (Annonaceae)

Guatteria, Guatteriopsis, Guatteriella andHeteropetalum share the same conspicuous pollen type which is new for theSpermatophyta. It is zonoaperturate with a folded aperture region and an extremely reduced exine. First chromosome counts and karyotype analyses forGuatteriopsis (4 species investigated) andGuatteriella (1 species) are identical with those ofGuatteria (19 species seen): 2n = 28. The genome is characterized by diploidization and partly telocentric chromosomes. Sequentially Giemsa C- and fluorochrome banded chromosomes and interphase nuclei are described. The cuticular folding pattern is distinct forHeteropetalum only. Growth forms and ecology are reported for many species. The evolutionary pattern of theGuatteria group is discussed and compared with other genera and families.     

Use of tritium labeled amino acid conjugates of prostaglandins and thromboxanes as labeled ligands in prostanoid radioimmunoassay

Conjugates of prostaglandins and thromboxanes with tritium labeled amino acids were prepared and employed as labeled ligands in porstaglandin and thromboxane radioimmunoassays. Assays for PGF_2α, 15-keto-13, 14-dihydro-PGF_2α, TXB₂ and 15-keto-13, 14-dihydro-TXB₂ were evaluated in comparative studies using either these heterologous ligands or the corresponding homologus tritiated eicosanoid as tracers. Binding properties for the respective antibodies were found to be similar using either tracer.Three biological studies were also conducted, viz. study of the release of TXB₂ during collagen induced platelet aggregation, of 15-keto-13, 14-dihydro-TXB₂ during guinea pig pulmonary anaphylaxis, and of PGF_2α (measured as 15-keto-13, 14-dihydro-PGF_2α in peripheral plasma) during bovine luteolysis. The analyses gave comparable results using either the heterologous or the homologous assay.Thus, this type of labeled prostanoid conjugates may serve as a convenient alternative to homologous tracers in radioimmunoassay. Heterologous tracers may even in certain cases provide the only simple solution to the problem of preparing a labeled ligand of high specific activity.     

Investigation of cellobiohydrolase from trichoderma reesei by small angle X-ray scattering

Summary Trichoderma reesei was grown on sulfite pulp and the major cellobiohydrolase of the culture filtrate was purified to homogeneity. The distance distribution function p(r) measured by the small angle X-ray scattering technique indicates that the enzyme molecule has a rather unusual tadpole like shape with an isotropic head and a long tail. The maximum length is 18 nm and the largest diameter is 4.4 nm.     

Removal of239Pu from mice with 3,4,3 LICAM(C) or N,N′, N″, N‴-tetra-(2,3-dihydroxybenzoyl)-spermine

Summary Male mice SAS/4 were injected i.v. with²³⁹Pu citr(IV) 0.27 µCikg^–1–9.99 kBqkg^–1. After 1 h 30 µmol kg^–1 of 3,4,3 LICAM(C), N, N, N, N-tetra-(2,3-dihydroxybenzoyl)-spermine or Na₃CaDTPA as a reference compound was given intraperitoneally. After 4 days the animals were sacrified and the Pu content in livers, kidneys, femurs and carcasses was determined by the liquid scintillation method. It was found that, as compared with the control, 3,4,3 LICAM(C) removed 83% of the Pu activity deposited in the liver, 71% of that in the femur and 79% of the Pu in the whole body. The Pu content in the kidneys exceeded the control value by about 50%. Na₃CaDTPA removed 96, 86, 40 and 72% of plutonium from the liver, kidneys, femurs and carcasses respectively.Tetra-DHB-spermine caused the excretion of 50, 57 and 39% of Pu from liver, bone and whole body respectively. The retention of Pu in the kidneys was increased to 400% of the control value.     

Autophosphorylation of type 2 casein kinase TS at both its α- and β-subunits: Influence of different effectors

Rat liver casein kinase TS (Ck-TS) having quarternary structure α₂β₂, autophosphorylates at its 25 kDa, β-subunits, incorporating up to 1.2 mol P/mol enzyme. According to their effects on the autophosphorylation pattern the effectors of Ck-TS activity can be grouped into 3 classes: (i) inhibitors, like heparin, which also prevent the autophosphorylation of the β-subunit; (ii) stimulators possessing several amino groups (like spermine) which increase the autophosphorylation at the β-subunit; (iii) stimulators possessing several guanido groups, like protamines and related peptides, which prevent the phosphorylation of the β-subunit, while promoting the autophosphorylation of the 38 kDa α-subunit. In the presence of such polyarginyl effectors the 130 kDa Ck-TS is converted into forms with higher sedimentation coefficient.