Circular RNA circ-RELL1 regulates inflammatory response by miR-6873-3p/MyD88/NF-κB axis in endothelial cells.

Endothelial inflammation is an important contributor to the pathology of atherosclerotic cardiovascular disease (ASCVD). Circular RNAs (circRNAs) function and role in endothelium inflammation still unknown. In our present study, we firstly identified that circ-RELL1 plays a proinflammatory role in ox-LDL-induced HUVECs through high-throughput circRNA microarray assays. Knockdown circ-RELL1 can reduce the expression of ICAM1 and VCAM1 in ox-LDL induced endothelium inflammation. Mechanistically, circ-RELL1 directly bound to miR-6873-3p in cytoplasm. Subsequently miR-6873-3p reduced MyD88 (myeloid differentiation primary response 88) protein expression and alleviated MyD88 medicated NF-κB activation. Furthermore, circ-RELL1 can abolish the inhibition of inflammation response by miR-6873-3p. Our findings illustrate a novel regulatory pathway that circ-RELL1 modulate inflammatory response by miR-6873-3p/MyD88/NF-κB axis in ox-LDL induced endothelial cells, which provides a potential therapeutic candidate for endothelium inflammation in atherosclerotic cardiovascular disease.

Efficient Synthesis of UDP-Furanoses via 4,5-Dicyanoimidazole(DCI)-Promoted Coupling of Furanosyl-1-Phosphates with Uridine Phosphoropiperidate.

A P(V)-N activation method based on nucleoside phosphoropiperidate/DCI system has been developed for improved synthesis of diverse UDP-furanoses. The reaction conditions including temperature, amount of activator, and reaction time were optimized to alleviate the degradation of UDP-furanoses to cyclic phosphates. In addition, an efficient and facile phosphoramidite route was employed for the preparation of furanosyl-1-phosphates.

[Catalytic Degradation of Diclofenac Sodium over the Catalyst of 3D Flower-like alpha-FeOOH Synergized with H2O2 Under Visible Light Irradiation].

Three dimensional (3D) flower-like alpha-FeOOH nanomaterials were prepared by oil bath reflux method using FeSO4, urea, ethanol and water, and the products which were characterized by XRD, FT-IR and SEM techniques. The SEM images showed that the 3D flower-like samples consisted of nanorods with a length of 400-500 nm and a diameter of 40-60 nm. The catalytic performance of the samples was evaluated by catalytic degradation of diclofenac sodium using H2O2 as the oxidant under simulated visible light. The results showed that the as-prepared samples presented high efficient catalytic performances, and more than 99% of the initial diclofenac sodium (30 mg x L(-1)) was degraded in 90 min. A radical mechanism can be proposed for the catalytic degradation of diclofenac sodium solution.

Protein phosphatase-1 is targeted to DNA polymerase delta via an interaction with the p68 subunit.

Protein phosphatase-1 (PP1) is a Ser/Thr protein phosphatase that participates in the phosphorylation/dephosphorylation regulation of a diverse range of cellular processes. The PP1 catalytic subunit (PP1) achieves this by its ability to interact with many targeting subunits such that PP1 activity is thereby specified against phosphoprotein substrates in the microvicinity of its targeting subunit. DNA polymerase delta (Pol delta) is a key enzyme in mammalian chromosomal replication. It consists of four subunits, p125, p50, p68, and p12. We identify p68 as a novel PP1 targeting subunit. PP1 was shown to associate with human DNA polymerase delta by affinity chromatography and coimmunoprecipitation assays from mammalian cell lysates and in vitro by pull-down assays. The binding domain for PP1 was identified as the sequence KRVAL, a variant of the canonical RVxF PP1 binding motif. These studies provide the first evidence for the targeting of PP1 to DNA polymerase delta. We also show that CK2 phosphorylates the Pol delta p125, p68, and p12 subunits and that these phosphorylated subunits are substrates for PP1. These findings identify a new role for p68 as a PP1 targeting subunit that implicates PP1 in the dephosphorylation of Pol delta. Our findings also show that CK2 is a strong candidate for the protein kinase involved in the in vivo phosphorylation of p68.

Protein phosphatase-1 inhibitor-3 is an in vivo target of caspase-3 and participates in the apoptotic response.

Inh3 (inhibitor-3) is a potent inhibitor of protein phosphatase-1 that selectively associates with PP1gamma1 and PP1alpha but not the PP1beta isoform. We demonstrate that Inh3 is a novel substrate for caspase-3 and is degraded in vivo during apoptosis induced by actinomycin D. Inh3 was not degraded in apoptotic MCF-7 cells, which lack caspase-3. These experiments establish that Inh3 is a novel physiological substrate of caspase-3. Electroporation of the caspase-3-resistant Inh3-D49A mutant into HL-60 cells resulted in a significant attenuation of apoptosis induced by actinomycin D. These results show that Inh3 degradation contributes to the apoptotic process. Immunofluorescence based examination of the subcellular localizations of Inh3 and PP1gamma1 revealed a major relocalization of the cellular pool of PP1gamma1 from the nucleolus to the nucleus and then to the cytoplasm during actinomycin D-induced apoptosis. A similar redistribution of PP1alpha from the nucleus to the cytoplasm occurred. These results are consistent with an unexpected discovery that significant fractions of the cellular pools of PP1gamma1 and PP1alpha are associated with Inh3 in HL-60 cells. Thus, Inh3 is a major factor in the cellular economy of PP1gamma1 and PP1alpha subunits. The unscheduled relocalization of this large a pool of PP1 subunits and their release from a potent inhibitor could deregulate a diverse range of essential cellular processes and signaling pathways. We discuss the significance of these findings in relation to working hypotheses whereby Inh3 destruction could contribute to the apoptotic process.

Protein phosphatase-1 inhibitor-3 is co-localized to the nucleoli and centrosomes with PP1gamma1 and PP1alpha, respectively.

In this study, we show that protein phosphatase-1 (PP1) inhibitor-3 (Inh3) is localized to the nucleoli and centrosomes in interphase HEK 293 cells. Inh3 exhibited a specific co-localization to the nucleoli with PP1gamma1, and to the centrosomes with PP1alpha. These findings indicate that Inh3 may act as a modulator of PP1 functions in the processes of cytokinesis, as well as of nucleolar events. The specificity of the interaction of Inh3 with the PP1 isoforms was also demonstrated in vitro, where Inh3 co-immunoprecipitated with PP1alpha and PP1gamma1, but not with PP1beta. The nuclear localization signal of Inh3 was identified as a N-terminal basic cluster (33RKRK36), while nucleolar localization was shown to be dependent on a C-terminal basic cluster (94HRKGRRR100). The importance of the individual basic residues was quantitatively assessed by site-directed mutagenesis and a novel use of laser scanning cytometry.

Novel aminopeptidase specific for glycine from Actinomucor elegans.

Glycyl aminopeptidase was purified 600-fold from a cell extract of Actinomucor elegans by ammonium sulfate fractionation and sequential chromatography on DEAE-Toyopearl, Toyopearl HW65C, and FPLC-Superdex 200 HR, with recovery of 3.3% of the activity. The enzyme highly specifically hydrolyzed Gly-X (amino acid, peptide, or arylamide) bonds. The enzyme hydrolyzed other amino acid residues but at a rate of less than one fifth that with Gly. The order was Gly >> Ala >> Met > Arg > Ser > Leu. The Km value for glycyl-2-naphthylamide was 0.24 mM. The enzyme was most active at pH 8.0 with glycyl-2-naphthylamide as the substrate and its optimal temperature was 40 degrees C. The enzyme was inhibited by iodoacetic acid, and p-chloromercuribenzoate but not done by diisopropylfluorophosphate, o-phenanthroline, or EDTA. Magnesium and calcium had no effect on enzymic activity, but the activity was suppressed by cadmium, zinc, and copper ions. The molecular mass was estimated to be 320 kDa by gel filtration on FPLC-Superdex 200 HR and 56.5 kDa by SDS-PAGE, so the enzyme probably was a hexamer.

Cloning and sequencing of the Thermus aquaticus glycerol facilitator gene.

The gene glpK, encoding glycerol kinase of Thermus aquaticus has been identified [Biosci. Biotechnol. Biochem., 62 (1998) 2375-2381]. In the present work, the nucleotide sequence of glpFK operon and the gene glpF encoding glycerol facilitator were determined. T. aquaticus GlpF was predicted to contain 272 amino acids with six putative transmembrane segments and two half-membrane-spanning segments that contained the motif Asn-Pro-Ala, respectively. The amino acid residues involved in the discrimination of glycerol were deduced to be Trp44, Tyr182, and Arg188.

Glycerol transport and phosphoenolpyruvate-dependent enzyme I- and HPr-catalysed phosphorylation of glycerol kinase in Thermus flavus.

The genes glpK and glpF, encoding glycerol kinase and the glycerol facilitator of Thermus flavus, a member of the Thermus/Deinococcus group, have recently been identified. The protein encoded by glpK exhibited an unusually high degree of sequence identity (80-6%) when compared to the sequence of glycerol kinase from Bacillus subtilis and a similar high degree of sequence identity (64.8%) was observed when the sequences of the glycerol facilitators of the two organisms were compared. The work presented in this paper demonstrates that T. flavus is capable of taking up glycerol, that glpF and glpK are expressed constitutively and that glucose exerts a repressive effect on the expression of these genes. T. flavus was found to possess the general components of the phosphoenolpyruvate (PEP): sugar phosphotransferase system (PTS) enzyme I and histidine-containing protein (HPr). These proteins catalyse the phosphorylation of T. flavus glycerol kinase, which contains a histidyl residue equivalent to His-232, the site of PEP-dependent, PTS-catalysed phosphorylation in glycerol kinase of Enterococcus casseliflavus. Purified glycerol kinase from T. flavus could also be phosphorylated with enzyme I and HPr from B. subtilis. Similar to enterococcal glycerol kinases, phosphorylated T. flavus glycerol kinase exhibited an electrophoretic mobility on denaturing and non-denaturing polyacrylamide gels that is different from the electrophoretic mobility of non-phosphorylated glycerol kinase. However, in contrast to PEP-dependent phosphorylation of enterococcal glycerol kinases, which stimulated glycerol kinase activity about 10-fold, phosphorylation of T. flavus glycerol kinase caused only a slight increase in enzyme activity.