The antifungal drug voriconazole is an efficient inhibitor of brain cholesterol 24S-hydroxylase in vitro and in vivo

Cholesterol 24S-hydroxylase (CYP46A1) is of key importance for cholesterol homeostasis in the brain. This enzyme seems to be resistant toward most regulatory factors and at present no drug effects on its activity have been described. The crystal structures of the substrate-free and substrate-bound CYP46A1 were recently determined (Mast et al., Crystal structures of substrate-bound and substrate-free cytochrome P450 46A1, the principal cholesterol hydroxylase in the brain. Proc. Natl. Acad. Sci. USA. 2008. 105: 9546–9551). These structural studies suggested that ligands other than sterols can bind to CYP46A1. We show here that the antifungal drug voriconazole binds to the enzyme in vitro and inhibits CYP46A1-mediated cholesterol 24-hydroxylation with a Ki of 11 nM. Mice treated with daily intraperitoneal injections of voriconazole for 5 days had high levels of voriconazole in the brain and significantly reduced brain levels of 24S-hydroxycholesterol. The levels of squalene, lathosterol, and HMG-CoA reductase mRNA were reduced in the brain of the voriconazole-treated animals as well, indicating a reduced cholesterol synthesis. Most of this effect may be due to a reduced utilization of cholesterol by CYP46A1. One of the side-effects of voriconazole is visual disturbances. Because CYP46A1 is also expressed in the neural retina, we discuss the possibility that the inhibition of CYP46A1 by voriconazole contributes to these visual disturbances.     

High glucose increase cell cycle regulatory proteins level of mouse embryonic stem cells via PI3-K/Akt and MAPKs signal pathways

This study examined the effects of high glucose on cell proliferation and its related signal pathways using mouse embryonic stem (ES) cells. Here, we showed that high glucose level significantly increased [3H]thymidine incorporation, BrdU incorporation, the number of cells, [3H]leucine, and [3H]proline incorporation in a time-( >3 hr) and dose-(> 25 mM) dependent manner. Moreover, high glucose level increased the cellular reactive oxygen species (ROS), Akt, and mitogen-activated protein kinases (MAPKs) phosphorylation. Subsequently, these signaling molecules involved in high glucose-induced increase of [3H]thymidine incorporation. High glucose level also increased cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK 4 protein levels, which is cell cycle regulatory proteins acting in G1-S phase of cell cycle. Inhibition of phosphatidylinositol 3-kinase (PI3-K) (LY 294002: PI3-kinase inhibitor, 10(-6) M), Akt (Akt inhibitor, 10(-5) M), and p44/42 MAPKs (PD 98059: MEK inhibitor, 10(-5) M) decreased these proteins. High glucose level phosphorylated the RB protein, which was decreased by inhibition of PI3-K and Akt. In conclusion, high glucose level stimulates mouse ES cell proliferation via the PI3-K/Akt and MAPKs pathways.     

Role of RepB in the replication of plasmid pJB01 isolated from Enterococcus faecium JC1

The plasmid pJB01 (GenBank Accession No. AY425961) isolated from the pathogenic bacterium, Enterococcus faecium JC1, is 2235 base pairs in length and consists of a putative double-strand origin (dso), a single-strand origin, a counter-transcribed RNA, and three open reading frames. A comparison of a few replication factors and motifs, bind and nic regions, for replication initiation on the nucleotide sequence level revealed that it belongs to the pMV158 family among RC-replicating plasmids. A runoff DNA synthesis assay demonstrated that nicking occurred between G525 and A526, which is located on the internal loop of a putative secondary structure in the dso. Unlike all the other plasmids of the pMV158 family having two or three direct repeats, pJB01 has three non-tandem direct repeats of 5'-CAACAAA-3' separated by four nucleotides, as the RepB-binding site in the dso. Moreover, the nick site on the internal loop is located at 77 nucleotides upstream from the RepB-binding region. Irrespective of the structural difference of direct repeats from other members of the pMV158 family, we think, it is still a new member of this plasmid family. The introduction of mutations in conserved regions of RepB confirmed that RepB N-moiety is important for nicking/nick-closing activity. Within N-moiety, especially all of the motif R-III, the Y100 in R-IV and Y116 in R-V residues, played particularly critical roles in this activity, however, for its binding, both of the N- and C-moieties of RepB were needed.     

EGF-induced inhibition of glucose transport is mediated by PKC and MAPK signal pathways in primary cultured chicken hepatocytes

EGF is a regulator of a wide variety of processes in various cell systems. Hepatocytes are important sites in the body's metabolism and function. Glucose transporter 2 (GLUT2) is a major transporter that is expressed strongly in hepatocytes. Therefore, this study examined the effect of EGF on GLUT2 and its related signal cascades in primary cultured chicken hepatocytes. EGF decreased [(3)H]deoxyglucose uptake in a dose- and time-dependent manner (>10 ng/ml, 2 h). AG-1478 (an EGF receptor antagonist) and genistein and herbimycin A (tyrosine kinase inhibitors) blocked the EGF-induced decrease in [(3)H]deoxyglucose uptake, which correlated with the GLUT2 expression level. In addition, the EGF-induced decrease in GLUT2 protein expression was inhibited by staurosporine, H-7, or bisindolylmaleimide I (PKC inhibitors), PD-98059 (a MEK inhibitor), SB-203580 (a p38 MAPK inhibitor), and SP-600125 (a JNK inhibitor), suggesting a role of both PKC and MAPKs (p44/42 MAPK, p38 MAPK, and JNK). In particular, EGF increased the translocation of PKC isoforms (PKC-alpha, -beta(1), -gamma, -delta, and -zeta) from the cytosol to the membrane fraction and increased the activation of p44/42 MAPK, p38 MAPK, and JNK. Moreover, PKC inhibitors blocked the EGF-induced phosphorylation of three MAPKs. In conclusion, EGF decreases the GLUT2 expression level via the PKC-MAPK signal cascade in chicken hepatocytes.     

Redox regulation of RhoA

We have previously shown that redox agents including superoxide anion radical and nitrogen dioxide can react with GXXXXGK(S/T)C motif-containing GTPases (i.e., Rac1, Cdc42, and RhoA) to stimulate guanine nucleotide release. We now show that the reaction of RhoA with redox agents leads to different functional consequences from that of Rac1 and Cdc42 due to the presence of an additional cysteine (GXXXCGK(S/T)C) in the RhoA redox-active motif. While reaction of redox agents with RhoA stimulates guanine nucleotide dissociation, RhoA is subsequently inactivated through formation of an intramolecular disulfide that prevents guanine nucleotide binding thereby causing RhoA inactivation. Thus, redox agents may function to downregulate RhoA activity under conditions that stimulate Rac1 and Cdc42 activity. The opposing functions of these GTPases may be due in part to their differential redox regulation. In addition, the results presented herein suggest that the platinated-chemotherapeutic agent, cisplatin, which is known for targeting nucleic acids, reacts with RhoA to produce a RhoA thiol-cisplatin-thiol adduct, leading to inactivation of RhoA. Similarly, certain arsenic complexes (i.e., arsenate and arsenic trioxide) may inactivate RhoA by bridging the cysteine residues in the GXXXCGK(S/T)C motif. Thus, in addition to redox agents, platinated-chemotherapeutic agents and arsenic complexes may modulate the activity of GTPases containing the GXXXCGK(S/T)C motif (i.e., RhoA and RhoB).     

E2-EPF UCP targets pVHL for degradation and associates with tumor growth and metastasis

The von Hippel-Lindau tumor suppressor, pVHL, forms part of an E3 ubiquitin ligase complex that targets specific substrates for degradation, including hypoxia-inducible factor-1alpha (HIF-1alpha), which is involved in tumor progression and angiogenesis. It remains unclear, however, how pVHL is destabilized. Here we show that E2-EPF ubiquitin carrier protein (UCP) associates with and targets pVHL for ubiquitin-mediated proteolysis in cells, thereby stabilizing HIF-1alpha. UCP is detected coincidently with HIF-1alpha in human primary liver, colon and breast tumors, and metastatic cholangiocarcinoma and colon cancer cells. UCP level correlates inversely with pVHL level in most tumor cell lines. In vitro and in vivo, forced expression of UCP boosts tumor-cell proliferation, invasion and metastasis through effects on the pVHL-HIF pathway. Our results suggest that UCP helps stabilize HIF-1alpha and may be a new molecular target for therapeutic intervention in human cancers.     

Isolation and Characterization of a Lytic Myoviridae Bacteriophage PAS-1 with Broad Infectivity in Aeromonas salmonicida

To search for candidate control agents against Aeromonas salmonicida subsp. salmonicida infections in aquaculture, one bacteriophage (phage), designated as PAS-1, was isolated from the sediment samples of the rainbow trout (Oncorhynchus mykiss) culture farm in Korea. The PAS-1 was morphologically classified as Myoviridae and possessed approximately 48 kb of double-strand genomic DNA. The phage showed broad host ranges to other subspecies of A. salmonicida as well as A. salmonicida subsp. salmonicida including antibiotic-resistant strains. Its latent period and burst size were estimated to be approximately 40 min and 116.7 PFU/cell, respectively. Furthermore, genomic and structural proteomic analysis of PAS-1 revealed that the phage was closely related to other Myoviridae phages infecting enterobacteria or Aeromonas species. The bacteriolytic activity of phage PAS-1 was evaluated using three subspecies of A. salmonicida strain at different doses of multiplicity of infection, and the results proved to be efficient for the reduction of bacterial growth. Based on these results, PAS-1 could be considered as a novel Aeromonas phage and might have potentiality to reduce the impacts of A. salmonicida infections in aquaculture.