Phosphorylation of CKBBP2/CRIF1 by protein kinase CKII promotes cell proliferation

CKII plays a significant role in cell proliferation and cell cycle control. In this report, yeast two-hybrid assay and pull-down assay demonstrate that CKBBP2/CRIF1 associates with the beta subunit of CKII in vitro and in vivo. Recombinant CKBBP2/CRIF1 is phosphorylated in vitro by purified CKII and by CKII inhibitor apigenin-sensitive protein kinase in HEK293 cell extract. Phosphoamino acid analysis and mutational analysis indicate that CKII phosphorylates serine at residue 221 within CKBBP2/CRIF1. Furthermore, serine to alanine mutation at residue 221 abrogates the phosphorylation of CKBBP2/CRIF1 observed in HEK293 cell extract, indicating that CKII is a major kinase that is responsible for phosphorylation of CKBBP2/CRIF1. As compared with the wild-type CKBBP2/CRIF1 or nonphosphorylatable mutant CKBBP2(S221A) (in which the serine-221 is replaced by alanine), overexpression of CKBBP2(S221E) in COS7 cells promotes cell proliferation. Taken together, the present results suggest that CKII may be involved in cell proliferation, at least in part, through the phosphorylation of serine-221 within CKBBP2/CRIF1.     

NF-kappaB inhibition enhances caspase-3 degradation of Akt1 and apoptosis in response to camptothecin

DNA damaging agents, such as camptothecin, and ionizing radiation (IR), can induce both NF-kappaB activation and apoptosis, however, the mechanism of their inter-regulation is not yet clear. In the present study, we discovered that Akt1 is degraded when cells deficient in Ataxia Telangiectasia mutated (ATM) were treated to CPT for apoptosis induction. While CPT-induced NF-kappaB activation could not be detected in ATM-deficient AT5BIVA cells, caspase-3 activation occurred and was even further enhanced by pretreatment with proteasome inhibitor-1 (Pro1), a NF-kappaB inhibitor. In contrast, activation of NF-kappaB but not of caspase-3 by CPT could be found in normal MRC5CV1 cells. NF-kappaB inhibition by Pro1, dominant negative mutant IkappaBalpha (S32/36) or p65 (N250), however, induced the caspase-3 activation in the normal cells, indicating the role of ATM-mediated NF-kappaB activation against cell apoptosis. On the other hand, interestingly, CPT significantly reduced the level of Akt1, this effect further enhanced by Pro1 pretreatment in AT5BIVA cells. In MRC5CV1 cells, however, Akt1 level could be reduced only when CPT and NF-kappaB inhibitors were co-treated to the cells, and this reversed by DEVD-cho treatment, demonstrating the caspase-3-mediated Akt1 degradation. Moreover, although MRC5CV1 cells were much more resistant to CPT compared with AT5BIVA, wortmannin and LY294002 significantly increased the chemosensitivity of MRC5CV1 cells to CPT. Given the accumulating evidences demonstrating Akt as a promising anticancer therapeutic target, all these results suggest that DNA damage induced apoptosis could be regulated by ATM-mediated NF-kappaB activation, and that Akt1 degradation be necessarily required for this apoptotic process.     

The F-actin-microtubule crosslinker Shot is a platform for Krasavietz-mediated translational regulation of midline axon repulsion

Axon extension and guidance require a coordinated assembly of F-actin and microtubules as well as regulated translation. The molecular basis of how the translation of mRNAs encoding guidance proteins could be closely tied to the pace of cytoskeletal assembly is poorly understood. Previous studies have shown that the F-actin-microtubule crosslinker Short stop (Shot) is required for motor and sensory axon extension in the Drosophila embryo. Here, we provide biochemical and genetic evidence that Shot functions with a novel translation inhibitor, Krasavietz (Kra, Exba), to steer longitudinally directed CNS axons away from the midline. Kra binds directly to the C-terminus of Shot, and this interaction is required for the activity of Shot to support midline axon repulsion. shot and kra mutations lead to weak robo-like phenotypes, and synergistically affect midline avoidance of CNS axons. We also show that shot and kra dominantly enhance the frequency of midline crossovers in embryos heterozygous for slit or robo, and that in kra mutant embryos, some Robo-positive axons ectopically cross the midline that normally expresses the repellent Slit. Finally, we demonstrate that Kra also interacts with the translation initiation factor eIF2beta and inhibits translation in vitro. Together, these data suggest that Kra-mediated translational regulation plays important roles in midline axon repulsion and that Shot functions as a direct physical link between translational regulation and cytoskeleton reorganization.     

Syntheses of hydroxy substituted 2-phenyl-naphthalenes as inhibitors of tyrosinase

Oxyresveratrol and resveratrol, with hydroxy substituted trans-stilbene structure, exert potent inhibitory effects on cyclooxygenase, rat liver mitochondrial ATPase activity, and tyrosinase. As the isosteres of oxyresveratrol, a new family of hydroxyl substituted phenyl-naphthalenes were synthesized to show excellent inhibition of mushroom tyrosinase. Compound 10, which is isostere of resveratrol, showed IC50 value of 16.52 microM in mushroom tyrosinase activity. As compared to this, the reference compound, resveratrol, showed IC50 value of 55.61 microM. Compound 4, which is isostere of oxyresveratrol, showed IC50 value of 0.49 microM. Among the other three derivatives, compound 13 showed IC50 value of 0.034 microM.     

Amphiphilic effects of dibucaine HCl on rotational mobility of n-(9-anthroyloxy)stearic acid in neuronal and model membranes

We studied dibucaine's effects on specific locations of n-(9-anthroyloxy)palmitic acid or stearic acid (n-AS) within phospholipids of synaptosomal plasma membrane vesicles isolated from bovine cerebral cortex (SPMV) and model membranes. Giant unilamellar vesicles (GUVs) were prepared with total lipids (SPMVTL) and mixture of several phospholipids (SPMVPL) extracted from SPMV. Dibucaine.HCl increased rotational mobility (increased disordering) of hydrocarbon interior, but it decreased mobility (increased ordering) of membrane interface, in both native and model membranes. The degree of rotational mobility in accordance with the carbon atom numbers of phospholipids comprising neuronal and model membranes was in the order at the 16, 12, 9, 6 and 2 position of aliphatic chain present in phospholipids. The sensitivity of increasing or decreasing effect of rotational mobility of hydrocarbon interior or surface region by dibucaine.HCl differed depending on the neuronal and model membranes in the descending order of SPMV, SPMVPL and SPMVTL.     

Heterogeneous rRNA molecules encoded by Streptomyces coelicolor M145 genome are all expressed and assembled into ribosomes

The Streptomyces coelicolor M145 genome harbors six copies of divergent rRNA operons that differ at ~0.2% and ~0.6% of the nucleotide positions in small subunit (SSU) and large subunit (LSU) rRNA genes, respectively. When these rRNA genes are expressed, a single cell may harbor three different kinds of SSU rRNA and five kinds of LSU rRNA. Primer extension analyses revealed that all of the heterogeneous rRNA molecules are expressed and assembled into ribosomes. This finding and the maintenance of the intragenomic variability of rRNA operons imply the existence of functional divergence of rRNA species in this developmentally complex microorganism.     

Antiasthmic effect of fermented Artemisia princeps in asthmic mice induced by ovalbumin

Artemisia princeps Pampanini (AP) was fermented with Bifidobacterium infantis K-525 and its antiasthmic effect investigated. AP and fermented AP (FAP) reduced the IgE level in the blood of ovalbumin-induced asthmic mice. Moreover, FAP reduced the IgE, proinflammatory cytokine IL-6, and IL-4 levels in the trachea, as well as in the lung of the experimental asthmic mice, whereas AP only reduced the IgE and IL-6 levels in the lungs. Nonetheless, AP and FAP both inhibited the mRNA expression of IL-6 and TNF-alpha in IgE-induced RBL-2H3 cells. The in vivo antiasthmic effect of FAP was more potent than that of AP. Therefore, these findings suggest that the enhanced antiasthmic effect of AP after bifidus fermentation was possibly due to the regulation of the proinflammatory cytokine biosynthesis of IL-6 and TNF-alpha.