Casein kinase I δ/ɛ phosphorylates topoisomerase IIα at serine-1106 and modulates DNA cleavage activity

We previously reported that phosphorylation of topoisomerase (topo) IIα at serine-1106 (Ser-1106) regulates enzyme activity and sensitivity to topo II-targeted drugs. In this study we demonstrate that phosphorylation of Ser-1106, which is flanked by acidic amino acids, is regulated in vivo by casein kinase (CK) Iδ and/or CKI, but not by CKII. The CKI inhibitors, CKI-7 and IC261, reduced Ser-1106 phosphorylation and decreased formation of etoposide-stabilized topo II–DNA cleavable complex. In contrast, the CKII inhibitor, 5,6-dichlorobenzimidazole riboside, did not affect etoposide-stabilized topo II–DNA cleavable complex formation. Since, IC261 specifically targets the Ca²⁺-regulated isozymes, CKIδ and CKI, we examined the effect of down-regulating these enzymes on Ser-1106 phosphorylation. Down-regulation of these isozymes with targeted si-RNAs led to hypophosphorylation of the Ser-1106 containing peptide. However, si-RNA-mediated down-regulation of CKIIα and α′ did not alter Ser-1106 phosphorylation. Furthermore, reduced phosphorylation of Ser-1106, observed in HRR25 (CKIδ/ homologous gene)-deleted Saccharomyces cerevisiae cells transformed with human topo IIα, was enhanced following expression of human CKI. Down-regulation of CKIδ and CKI also led to reduced formation of etoposide stabilized topo II–DNA cleavable complex. These results provide strong support for an essential role of CKIδ/ in phosphorylating Ser-1106 in human topo IIα and in regulating enzyme function.     

ELECTRON MICROSCOPY OF SPORES OF BACILLUS MEGATERIUM WITH SPECIAL REFERENCE TO THE EFFECTS OF FIXATION AND THIN SECTIONING

Resting spores of Bacillus megaterium appear uniformly opaque and undifferentiated under the electron microscope. Germinated spores and spores which have lost their dipicolinic acid underwent characteristic changes in structure. Spores fixed with KMnO₄ lose their dipicolinic acid. Spores fixed with OsO₄ under certain conditions retain their dipicolinic acid. When conventional sectioning procedures are used with either method of fixation, abnormal spore structure is produced as a result of the solution of cellular constitutents. Dry sections of unfixed spores embedded in methacrylate reveal the spore structure in a more normal state. Indirect evidence has been obtained for the existence of a penetration barrier at or near the outer edge of the cortex.     

Kinetic analysis of genetic complementation in heterokaryons of propionyl CoA carboxylase-deficient human fibroblasts.

We studied genetic complementation of propionyl CoA carboxylase (PCC) deficiency in cultures of polyethylene glycol (PEG)-induced heterokaryons, using mutant fibroblast lines assigned to five mutant classes, designated bio, pcc A, pcc B, pcc C, and pcc BC. By measuring PCC activity directly in extracts of fused cells or indirectly in intact cells by [1-14C]propionate utilization, we confirmed the nonlinear nature of the PCC deficiency complementation map described by Gravel et al. [1]. When we studied the kinetics of complementation, we detected three distinct patterns using the [1-14C]propionate utilization assay. When either pcc A or pcc C lines were fused to bio cells, 14C-fixation increased to half of the maximally restored values within 4 hrs. In pcc A x pcc C crosses or in pcc A x pcc B crosses, however, complementation was much slower. In fusions between pcc B and pcc C cells, a third pattern was elicited; complementation was incomplete, maximum restoration of PCC activity begin less than 20% of that observed in other complementing crosses. From these data and previous biochemical evidence, we suggest (1) that the bio and pcc mutations affect different genes; (2) that complementation between pcc A and either pcc B, pcc C, or pcc BC lines is intergenic and involves subunit exchange and synthesis of new PCC molecules; and (3) that complementation between pcc B and pcc C mutants is interallelic.     

Survey of Chemical Compounds Tested In Vitro against Rumen Protozoa for Possible Control of Bloat

Over 170 chemical agents were screened for antiprotozoal action in bovine ruminal fluid. Compounds were tested at 0.1 and 0.05% concentrations. Tested compounds included inorganic compounds, antibiotics, biocides, neuromuscular agents, arsenicals, plant and animal hormones, antimalarials, surface-active agents, anthelmintics, and many others. The most active compounds were cupric sulfate, nickel sulfate, nitrofurazone, hydrogen peroxide, dodecyl sodium sulfate, pelargonic acid, iodoacetic acid, 1-diethylaminoethylamino-4-methylthiaxanthrone, sodium arsanilate, sodium arsenate, bismuth glycolyl arsanilate, 1-β-hydroxyethyl-2-methyl-5-nitroimidazole, and p-nitroaniline. Copper ion was not particularly effective against entodinia; nickel ion had no effect on holotrichs. Hydrogen peroxide and iodoacetic acid were effective at a concentration of 0.005%. Anionic surface-active agents were very effective, especially long-chain sulfates and phosphates. These antiprotozoal agents warrant further in vivo studies for possible use in treating or curing bloat in ruminants.     

Myostatin inhibits cell proliferation and protein synthesis in C2C12 muscle cells

Myostatin mutations in mice and cattle are associated with increased muscularity, suggesting that myostatin is a negative regulator of skeletal muscle mass. To test the hypothesis that myostatin inhibits muscle cell growth, we examined the effects of recombinant myostatin in mouse skeletal muscle C2C12 cells. After verification of the expression of cDNA constructs in a cell-free system and in transfected Chinese hamster ovary cells, the human recombinant protein was expressed as the full-length (375-amino acid) myostatin in Drosophila cells (Mst375D), or the 110-amino acid carboxy-terminal protein in Escherichia coli (Mst110EC). These proteins were identified by immunoblotting and were purified. Both Mst375D and Mst110EC dose dependently inhibited cell proliferation (cell count and Formazan assay), DNA synthesis ([3H]thymidine incorporation), and protein synthesis ([1-14C]leucine incorporation) in C2C12 cells. The inhibitory effects of both proteins were greater in myotubes than in myoblasts. Neither protein had any significant effects on protein degradation or apoptosis. In conclusion, recombinant myostatin proteins inhibit cell proliferation, DNA synthesis, and protein synthesis in C2C12 muscle cells, suggesting that myostatin may control muscle mass by inhibiting muscle growth or regeneration.