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.     

Essentiality of Boron for Dinitrogen Fixation in Anabaena sp. PCC 7119

The relationship between the requirement for boron and the form of N supplied in nutrient media to cyanobacterium Anabaena sp. PCC 7119 was investigated. When cells were grown in a medium which contained nitrate or ammonium-N, boron deficiency in the nutrient media did not inhibit growth or change cell composition. However, when cells were dependent on N₂ fixation, the lack of boron inhibited growth (i.e. growth ceased after 96 hours under these conditions). Additionally, boron-deficient cells showed a significant decrease in their content of phycobiliproteins and chlorophyll and accumulated carbohydrates within 24 hours of removing boron from the nutrient media. Inhibition of photosynthetic O₂ evolution accompanied the decrease in photosynthetic pigments. Boron deficiency symptoms were relieved when either boron or combined N was added to boron-deficient cultures. The degree of recovery depended upon the age of the cultures. Assays of nitrogenase activity showed that, after 2 hours of growth, nitrogenase activity of boron-deficient cells was inhibited by 40%. After 24 hours a total inactivation of nitrogenase activity was observed in boron-deficient cells. These results strongly suggest an involvement of boron in N₂ fixation in cyanobacteria.     

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.     

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.     

Ontogeny of endocrine responses to methadone in rats

The effects of methadone (METH) on serum levels of prolactin (PRL), growth hormone (GH), corticosterone (CS) and TSH were determined in developing rats. METH increased PRL, GH and CS and decreased TSH at all ages tested, but the time course and magnitude of these effects changed during ontogeny. METH effects on day 10 were lower in magnitude than those observed in adults. In 20 day old pups, METH effects on GH and CS were comparable to those of adults, but TSH effects were still blunted. METH effects on hormone secretion in both 10 and 20 day old pups lasted longer than those observed in adults. Naloxone blocked all hormonal responses in adults, but did not completely block METH effects on CS secretion in 10 day old pups.     

Different structure of polytene chromosomes ofPhaseolus coccineus suspensors during early embryogenesis

Summary The pattern of DNA and RNA puffs in pair VII of polytene chromosomes has been investigated in the suspensor ofPhaseolus coccineus during early embryo development. The pattern of³H-TdR and³H-U incorporation has been also detected. Collected data indicate that: 1. both heterochromatic regions, p₁₁ and q_(111+112), of chromosome pair VII, organize large DNA puffs; 2. DNA puffs of both regions are specific of different embryo differentiation steps; 3. a seasonal influence on the DNA puffing seems also to be present, as demonstrated by the comparison of the results collected in two different crops; 4. the incorporation experiment by³H-TdR evidences that not all DNA puffs show clustered labeling; 5. the RNA puffing of the two regions seems also to be specific of determined embryo stages.     

The fate of newly made DNA in Escherichia coli mutants thermosensitive in DNA synthesis

Summary E. coli mutants exist in which DNA synthesis is thermosensitive. In one class of these mutants DNA synthesis stops immediately if a critical temperature (42°C) is reached. When DNA replication in such mutants is followed by ³H thymidine incorporation at 33°C, it is found that 1. only the newly made DNA is degraded at 42°C, 2. the discontinuously replicated DNA is lost predominantly at 42°C, 3. 1–3% of the chromosomal DNA is rendered acid soluble at 42°C without concomitant loss of viability of the cells at 33°C.Replication of phage DNA is inhibited in the same mutant at 42°C. However, when DNA synthesis is followed in infected cells at 33°C it is found that 1. no degradation of specific DNA seems to occur at 42°C in the early phase of infection, 2. replicating DNA molecules in the late phase of infection are completed at 42°C before DNA synthesis comes to a halt.