Complete genome sequence of cnidium virus 2, a novel cytorhabdovirus isolated from Cnidium officinale in South Korea.

High-throughput sequencing identified a cytorhabdovirus, tentatively named "cnidium virus 2" (CnV2), in Cnidium officinale, and Sanger sequencing confirmed the genome sequence. CnV2 is 13,527 nucleotides in length and contains seven open reading frames in the order 3'-N-P-3-4-M-G-L-5', separated by intergenic regions. The full-length nucleotide sequence of CnV2 shares 19.4-53.8% identity with other known cytorhabdovirus genome sequences. The N, P, P3, M, G, and L proteins share 15.8-66.7%, 11-64.3%, 11.1-80.5%, 10.8-75.3%, 12.3-72.1%, and 20-72.7% amino acid sequence identity, respectively, with the cognate deduced protein sequences from known cytorhabdoviruses. CnV2 is related to other members of the genus Cytorhabdovirus, with sambucus virus 1 being the closest relative. Thus, CnV2 should be classified as a new member in the genus Cytorhabdovirus of the family Rhabdoviridae.

Complete genome sequence of a novel member of the genus Polerovirus from Cnidium officinale in South Korea.

The complete genome sequence of a novel virus found infecting Cnidium officinale, which we have named "cnidium polerovirus 1" (CnPV1), is 6,090 nucleotides in length, similar to those of other poleroviruses. Seven open reading frames (ORF0-5 and ORF3a) were predicted in this genome. CnPV1 shares 32.4%-38.9% full-length nucleotide sequence identity with other known polerovirus genome sequences. The putative P0, P1-2, P3-5, P3, and P4 proteins share 11.3%-19.5%, 37.1%-49.8%, 26.7%-39.5%, 40.8%-49.7%, and 40.8%-49.7% amino acid sequence identity, respectively, with homologous inferred protein sequences from known poleroviruses. Phylogenetic analysis of P1-2 and P3 sequences places CnPV1 with other members of the genus Polerovirus, indicating that it should be classified in a new distinct species.

8-Methoxypsoralen Induces Apoptosis by Upregulating p53 and Inhibits Metastasis by Downregulating MMP-2 and MMP-9 in Human Gastric Cancer Cells.

Furanocoumarin 8-methoxypsoralen (8-MOP) is the parent compound that naturally occurs in traditional medicinal plants used historically. 8-MOP has been employed as a photochemotherapeutic component of Psoralen + Ultraviolet A (PUVA) therapy for the treatment of vitiligo and psoriasis. Although the role of 8-MOP in PUVA therapy has been studied, little is known about the effects of 8-MOP alone on human gastric cancer cells. In this study, we observed anti-proliferative effect of 8-MOP in several human cancer cell lines. Among these, the human gastric cancer cell line SNU1 is the most sensitive to 8-MOP. 8-MOP treated SNU1 cells showed G1-arrest by upregulating p53 and apoptosis by activating caspase-3 in a dose-dependent manner, which was confirmed by loss-of-function analysis through the knockdown of p53-siRNA and inhibition of apoptosis by Z-VAD-FMK. Moreover, 8-MOPinduced apoptosis is not associated with autophagy or necrosis. The signaling pathway responsible for the effect of 8-MOP on SNU1 cells was confirmed to be related to phosphorylated PI3K, ERK2, and STAT3. In contrast, 8-MOP treatment decreased the expression of the typical metastasis-related proteins MMP-2, MMP-9, and Snail in a p53-independent manner. In accordance with the serendipitous findings, treatment with 8-MOP decreased the wound healing, migration, and invasion ability of cells in a dose-dependent manner. In addition, combination treatment with 8-MOP and gemcitabine was effective at the lowest concentrations. Overall, our findings indicate that oral 8-MOP has the potential to treat early human gastric cancer, with fewer side effects.

G-749 Promotes Receptor Tyrosine Kinase TYRO3 Degradation and Induces Apoptosis in Both Colon Cancer Cell Lines and Xenograft Mouse Models.

G-749 is an FLT3 kinase inhibitor that was originally developed as a treatment for acute myeloid leukemia. Some FLT3 kinase inhibitors are dual kinase inhibitors that inhibit the TAM (Tyro3, Axl, Mer) receptor tyrosine kinase family and are used to treat solid cancers such as non-small cell lung cancer (NSCLC) and triple-negative breast cancer (TNBC). AXL promotes metastasis, suppression of immune response, and drug resistance in NSCLC and TNBC. G-749, a potential TAM receptor tyrosine kinase inhibitor, and its derivative SKI-G-801, effectively inhibits the phosphorylation of AXL at nanomolar concentration (IC50 = 20 nM). This study aimed to investigate the anticancer effects of G-749 targeting the TAM receptor tyrosine kinase in colon cancer. Here, we demonstrate the potential of G-749 to effectively inhibit tumorigenesis by degrading TYRO3 via regulated intramembrane proteolysis both in vitro and in vivo. In addition, we demonstrated that G-749 inhibits the signaling pathway associated with cell proliferation in colon cancer cell lines HCT15 and SW620, as well as tumor xenograft mouse models. We propose G-749 as a new therapeutic agent for the treatment of colon cancer caused by abnormal TYRO3 expression or activity.

Bioequivalence for a Fixed-Dose Combination Formulation of Bazedoxifene and Cholecalciferol Compared With the Corresponding Single Entities Given Together.

A fixed-dose combination (FDC) formulation of bazedoxifene 20 mg and cholecalciferol 8 mg was developed to increase medication compliance and convenience for osteoporosis patients. This study was conducted to demonstrate bioequivalence by comparing the pharmacokinetic (PK) profiles and tolerability of an FDC tablet and the individual component tablets. A randomized, open-label, single-dosing, 2-treatment, 2-period, 2-sequence crossover study was conducted in 52 healthy subjects. All subjects were randomly assigned to 2 sequences, and they received FDC tablets of bazedoxifene and cholecalciferol and individual component tablets. Serial blood samples for PK evaluation were collected up to 24 hours predose and 120 hours postdose, and the PK parameters were estimated by noncompartmental methods. Throughout the study, tolerability was assessed based on adverse events, vital signs, and clinical laboratory tests. Of the enrolled 52 subjects, 47 subjects completed the study. The results, the geometric mean ratios (GMRs) and 90% confidence intervals (90%CIs), of bazedoxifene Cmax and AUC0-t for FDC to single entities given together were 0.98 (0.91-1.05) and 1.02 (0.97-1.07), respectively. The GMRs (90%CIs) of cholecalciferol Cmax and AUC0-t for FDC to single entities given together were 0.96 (0.91-1.00) and 0.94 (0.90-0.99), respectively. Overall, the GMRs (90%CIs) of the PK parameter of bazedoxifene and cholecalciferol fell within the conventional bioequivalence range of 0.8-1.25. There were no clinically significant differences in the safety profile between the 2 treatments. In conclusion, this study confirmed the development of a new FDC drug by demonstrating that the FDC formulation of bazedoxifene and cholecalciferol is biologically equivalent to the coadministered individual formulations.

CSNK1G2 differently sensitizes tamoxifen-induced decrease in PI3K/AKT/mTOR/S6K and ERK signaling according to the estrogen receptor existence in breast cancer cells.

Tamoxifen (TAM) is a selective estrogen receptor modulator used for breast cancer patients. Prolonged use of tamoxifen is not recommended for some patients. In this study, we aimed to identify molecular targets sensitive to TAM using a genome-wide gene deletion library screening of fission yeast heterozygous mutants. From the screening, casein kinase 1 gamma 2 (CSNK1G2), a serine-/threonine protein kinase, was the most sensitive target to TAM with a significant cytotoxicity in estrogen receptor-positive (ER+) breast cancer cells but with only a slight toxicity in the case of ER- cells. In addition, tumor sphere formation and expression of breast stem cell marker genes such as CD44/CD2 were greatly inhibited by CSNK1G2 knockdown in ER+ breast cancer cells. Consistently, CSNK1G2 altered ERα activity via phosphorylation, specifically at serine (Ser)167, as well as the regulation of estrogen-responsive element (ERE) of estrogen-responsive genes such as CTSD and GREB1. However, ERα silencing almost completely blocked CSNK1G2-induced TAM sensitivity. In ER+ breast cancer cells, combined treatment with TAM and CSNK1G2 knockdown further enhanced the TAM-mediated decrease in phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR)/ribosomal protein S6 kinase (S6K) signaling but not extracellular signal-regulated kinase (ERK) signaling. Inversely, in ER- cells treated with TAM, only ERK and PI3K signaling was altered by CSNK1G2 knockdown. The CK1 inhibitor, D4476, partly mimicked the CSNK1G2 knockdown effect in ER+ breast cancer cells, but with a broader repression ranging from PI3K/AKT/mTOR/S6K to ERK signaling. Collectively, these results suggest that CSNK1G2 plays a key role in sensitizing TAM toxicity in ER+ and ER- breast cancer cells via differently regulating PI3K/AKT/mTOR/S6K and ERK signaling.

Knockdown of vps54 aggravates tamoxifen-induced cytotoxicity in fission yeast.

Tamoxifen (TAM) is an anticancer drug used to treat estrogen receptor (ER)‒positive breast cancer. However, its ER-independent cytotoxic and antifungal activities have prompted debates on its mechanism of action. To achieve a better understanding of the ER-independent antifungal action mechanisms of TAM, we systematically identified TAM-sensitive genes through microarray screening of the heterozygous gene deletion library in fission yeast (Schizosaccharomyces pombe). Secondary confirmation was followed by a spotting assay, finally yielding 13 TAM-sensitive genes under the drug-induced haploinsufficient condition. For these 13 TAM-sensitive genes, we conducted a comparative analysis of their Gene Ontology (GO) 'biological process' terms identified from other genome-wide screenings of the budding yeast deletion library and the MCF7 breast cancer cell line. Several TAM-sensitive genes overlapped between the yeast strains and MCF7 in GO terms including 'cell cycle' (cdc2, rik1, pas1, and leo1), 'signaling' (sck2, oga1, and cki3), and 'vesicle-mediated transport' (SPCC126.08c, vps54, sec72, and tvp15), suggesting their roles in the ER-independent cytotoxic effects of TAM. We recently reported that the cki3 gene with the 'signaling' GO term was related to the ER-independent antifungal action mechanisms of TAM in yeast. In this study, we report that haploinsufficiency of the essential vps54 gene, which encodes the GARP complex subunit, significantly aggravated TAM sensitivity and led to an enlarged vesicle structure in comparison with the SP286 control strain. These results strongly suggest that the vesicle-mediated transport process might be another action mechanism of the ER-independent antifungal or cytotoxic effects of TAM.

Systematic Target Screening Revealed That Tif302 Could Be an Off-Target of the Antifungal Terbinafine in Fission Yeast.

We used a heterozygous gene deletion library of fission yeasts comprising all essential and non-essential genes for a microarray screening of target genes of the antifungal terbinafine, which inhibits ergosterol synthesis via the Erg1 enzyme. We identified 14 heterozygous strains corresponding to 10 non-essential [7 ribosomal-protein (RP) coding genes, spt7, spt20, and elp2] and 4 essential genes (tif302, rpl2501, rpl31, and erg1). Expectedly, their erg1 mRNA and protein levels had decreased compared to the control strain SP286. When we studied the action mechanism of the non-essential target genes using cognate haploid deletion strains, knockout of SAGA-subunit genes caused a down-regulation in erg1 transcription compared to the control strain ED668. However, knockout of RP genes conferred no susceptibility to ergosterol-targeting antifungals. Surprisingly, the RP genes participated in the erg1 transcription as components of repressor complexes as observed in a comparison analysis of the experimental ratio of erg1 mRNA. To understand the action mechanism of the interaction between the drug and the novel essential target genes, we performed isobologram assays with terbinafine and econazole (or cycloheximide). Terbinafine susceptibility of the tif302 heterozygous strain was attributed to both decreased erg1 mRNA levels and inhibition of translation. Moreover, Tif302 was required for efficacy of both terbinafine and cycloheximide. Based on a molecular modeling analysis, terbinafine could directly bind to Tif302 in yeasts, suggesting Tif302 as a potential off-target of terbinafine. In conclusion, this genome-wide screening system can be harnessed for the identification and characterization of target genes under any condition of interest.

Co-treatment of birinapant with TRAIL synergistically induces apoptosis by downregulating cFLIP(L) in MDA-MB-453 cell lines.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) has received much attention owing to its ability to specifically induce cell death in cancer. However, several types of cancer, including some forms of breast cancer, are resistant to TRAIL. Various chemotherapeutic agents, phytochemicals, and TRAIL combination therapies have been proposed to resolve TRAIL resistance. Here, we explored the sensitization effect of birinapant on TRAIL-induced apoptosis in the MDA-MB-453 cell line. Although neither birinapant nor TRAIL showed any cytotoxic effect when used alone, apoptosis was induced when birinapant and TRAIL were used together. Our data suggest that the combination of birinapant and TRAIL induces downregulation of FLICE-like inhibitory protein (cFLIP) (L) protein expression. Interestingly, cFLIP(L) overexpression reversed apoptosis caused by co-treatment with TRAIL. Taken together, our results indicate that a combination of birinapant and TRAIL may be a promising treatment for TRAIL-resistant breast cancer.

A randomized, open-label, crossover study to compare the safety and pharmacokinetics of two tablet formulations of tenofovir (tenofovir disoproxil and tenofovir disoproxil fumarate) in healthy subjects.

PURPOSE: This study was performed to compare the pharmacokinetic properties and assess bioequivalence for the test formulation (HUG116 tablet; tenofovir disoproxil) and reference formulation (Viread tablet; tenofovir disoproxil fumarate). MATERIALS AND METHODS: A randomized, open-label, single-dosing, two-treatment, two-period, two-sequence cross-over study was conducted in 50 healthy subjects. All subjects were randomly assigned to one of the two sequences, and they received a single dose of test or reference formulation in the first period and the alternative formulation during the next period under fasting conditions. Serial blood samples for pharmacokinetic evaluation were collected up to 72 hours post dose, and the pharmacokinetic parameters were estimated by noncompartmental methods. Throughout the study, tolerability was assessed based on adverse events, vital signs, and clinical laboratory tests. RESULTS: The test formulation showed similar pharmacokinetic profiles to those of the reference formulation. The geometric mean ratio and 90% confidence interval (CI) of the test formulation to the reference formulation for maximum plasma concentration (Cmax) was 0.93 (0.87 - 0.99), and the corresponding value for the area under the concentration-time curve from time zero to time of last quantifiable concentration (AUCt) was 0.94 (0.89 - 0.99). Both CIs were within the conventional bioequivalence range of 0.8 - 1.25. The tolerability profile was not significantly different between the test and reference formulations. CONCLUSION: This study found that the PKs of the test formulation (HUG116 tablet; tenofovir disoproxil) and reference formulation (Viread tablet; tenofovir disoproxil fumarate) were similar, and the test formulation met the regulatory criteria for assuming bioequivalence with the reference formulation.

Bacteria-derived metabolite, methylglyoxal, modulates the longevity of C. elegans through TORC2/SGK-1/DAF-16 signaling.

Gut microbes play diverse roles in modulating host fitness, including longevity; however, the molecular mechanisms underlying their mediation of longevity remain poorly understood. We performed genome-wide screens using 3,792 Escherichia coli mutants and identified 44 E. coli mutants that modulated Caenorhabditis elegans longevity. Three of these mutants modulated C. elegans longevity via the bacterial metabolite methylglyoxal (MG). Importantly, we found that low MG-producing E. coli mutants, Δhns E. coli, extended the lifespan of C. elegans through activation of the DAF-16/FOXO family transcription factor and the mitochondrial unfolded protein response (UPRmt). Interestingly, the lifespan modulation by Δhns did not require insulin/insulin-like growth factor 1 signaling (IIS) but did require TORC2/SGK-1 signaling. Transcriptome analysis revealed that Δhns E. coli activated novel class 3 DAF-16 target genes that were distinct from those regulated by IIS. Taken together, our data suggest that bacteria-derived MG modulates host longevity through regulation of the host signaling pathways rather than through nonspecific damage on biomolecules known as advanced glycation end products. Finally, we demonstrate that MG enhances the phosphorylation of hSGK1 and accelerates cellular senescence in human dermal fibroblasts, suggesting the conserved role of MG in controlling longevity across species. Together, our studies demonstrate that bacteria-derived MG is a novel therapeutic target for aging and aging-associated pathophysiology.

Econazole Induces p53-Dependent Apoptosis and Decreases Metastasis Ability in Gastric Cancer Cells.

Econazole, a potent broad-spectrum antifungal agent and a Ca2+ channel antagonist, induces cytotoxicity in leukemia cells and is used for the treatment of skin infections. However, little is known about its cytotoxic effects on solid tumor cells. Here, we investigated the molecular mechanism underlying econazole-induced toxicity in vitro and evaluated its regulatory effect on the metastasis of gastric cancer cells. Using the gastric cancer cell lines AGS and SNU1 expressing wild-type p53 we demonstrated that econazole could significantly reduce cell viability and colony-forming (tumorigenesis) ability. Econazole induced G0/G1 phase arrest, promoted apoptosis, and effectively blocked proliferation- and survival-related signal transduction pathways in gastric cancer cells. In addition, econazole inhibited the secretion of matrix metalloproteinase- 2 (MMP-2) and MMP-9, which degrade the extracellular matrix and basement membrane. Econazole also effectively inhibited the metastasis of gastric cancer cells, as confirmed from cell invasion and wound healing assays. The protein level of p53 was significantly elevated after econazole treatment of AGS and SNU1 cells. However, apoptosis was blocked in econazole-treated cells exposed to a p53-specific small-interfering RNA to eliminate p53 expression. These results provide evidence that econazole could be repurposed to induce gastric cancer cell death and inhibit cancer invasion.

Optimization of a microarray for fission yeast.

Bar-code (tag) microarrays of yeast gene-deletion collections facilitate the systematic identification of genes required for growth in any condition of interest. Anti-sense strands of amplified bar-codes hybridize with ~10,000 (5,000 each for up- and down-tags) different kinds of sense-strand probes on an array. In this study, we optimized the hybridization processes of an array for fission yeast. Compared to the first version of the array (11 µm, 100K) consisting of three sectors with probe pairs (perfect match and mismatch), the second version (11 µm, 48K) could represent ~10,000 up-/down-tags in quadruplicate along with 1,508 negative controls in quadruplicate and a single set of 1,000 unique negative controls at random dispersed positions without mismatch pairs. For PCR, the optimal annealing temperature (maximizing yield and minimizing extra bands) was 58°C for both tags. Intriguingly, up-tags required 3 higher amounts of blocking oligonucleotides than down-tags. A 1:1 mix ratio between up- and down-tags was satisfactory. A lower temperature (25°C) was optimal for cultivation instead of a normal temperature (30°C) because of extra temperature-sensitive mutants in a subset of the deletion library. Activation of frozen pooled cells for >1 day showed better resolution of intensity than no activation. A tag intensity analysis showed that tag(s) of 4,316 of the 4,526 strains tested were represented at least once; 3,706 strains were represented by both tags, 4,072 strains by up-tags only, and 3,950 strains by down-tags only. The results indicate that this microarray will be a powerful analytical platform for elucidating currently unknown gene functions.

Arginase II activity regulates cytosolic Ca2+ level in a p32-dependent manner that contributes to Ca2+-dependent vasoconstriction in native low-density lipoprotein-stimulated vascular smooth muscle cells.

Although arginase II (ArgII) is abundant in mitochondria, Ca2+-accumulating organelles, the relationship between ArgII activity and Ca2+ translocation into mitochondria and the regulation of cytosolic Ca2+ signaling are completely unknown. We investigated the effects of ArgII activity on mitochondrial Ca2+ uptake through mitochondrial p32 protein (p32m) and on CaMKII-dependent vascular smooth muscle cell (VSMC) contraction. Native low-density lipoprotein stimulation induced an increase in [Ca2+]m as measured by CoCl2-quenched calcein-AM fluorescence, which was prevented by Arg inhibition in hAoSMCs and reduced in mAoSMCs from ArgII-/- mice. Conversely, [Ca2+]c analyzed with Fluo-4 AM was increased by Arg inhibition and ArgII gene knockout. The increased [Ca2+]c resulted in CaMKII and MLC 20 phosphorylation, which was associated with enhanced vasoconstriction activity to phenylephrine (PE) in the vascular tension assay. Cy5-tagged siRNA against mitochondrial p32 mRNA (sip32m) abolished mitochondrial Ca2+ uptake and induced activation of CaMKII. Spermine, a polyamine, induced mitochondrial Ca2+ uptake and dephosphorylation of CaMKII and was completely inhibited by sip32m incubation. In mAoSMCs from ApoE-null mice fed a high-cholesterol diet (ApoE-/- +HCD), Arg activity was increased, and spermine concentration was higher than that of wild-type mice. Furthermore, [Ca2+]m and p32m levels were elevated, and CaMKII phosphorylation was reduced in mAoSMCs from ApoE-/- +HCD. In vascular tension experiments, an attenuated response to vasoconstrictors in de-endothelialized aorta from ApoE-/- +HCD was recovered by incubation of sip32m. ArgII activity-dependent production of spermine augments Ca2+ transition from the cytosol to the mitochondria in a p32m-dependent manner and regulates CaMKII-dependent constriction in VSMCs.

Resveratrol is an arginase inhibitor contributing to vascular smooth muscle cell vasoconstriction via increasing cytosolic calcium.

The contractility of vascular smooth muscle cells (VSMCs) controls the lumen diameter of vessels, thus serving a role in regulating blood pressure and organ blood flow. Although arginases are known to have numerous effects in the biological activities of VSMCs, the effects of arginase II on the constriction of VSMCs has not yet been investigated. When conducting a natural products screen for an inhibitor against arginase, the present study identified that a relatively high concentration of resveratrol (RSV) exhibited arginase inhibitory activity. Therefore, the present study investigated whether RSV could regulate VSMCs contractions and the underlying mechanism. Arginase inhibition by RSV led to an increase in the concentration of the substrate L­Arg and an accompanying increase in the cytosol Ca2+ concentration [(Ca2+)c] in VSMCs. The increased [Ca2+]c induced by RSV and L­Arg treatments resulted in CaMKII­dependent MLC20 phosphorylation. The effects of RSV on VSMCs were maintained even when VSMCs were pre­treated with sirtinol, an inhibitor of Sirt proteins. In a vascular tension assay with de­endothelialized aortic vessels, vasoconstrictor responses, which were measured using phenylephrine (PE), were significantly enhanced in the RSV­ and L­Arg­treated vessels. Therefore, although arginase inhibition has exhibited beneficial effects in various diseases, care is required when considering administration of an arginase inhibitor to patients with vessels endothelial dysfunction as RSV can induce vessel contraction.

Mutation Analysis of Synthetic DNA Barcodes in a Fission Yeast Gene Deletion Library by Sanger Sequencing.

Incorporation of unique barcodes into fission yeast gene deletion collections has enabled the identification of gene functions by growth fitness analysis. For fine tuning, it is important to examine barcode sequences, because mutations arise during strain construction. Out of 8,708 barcodes (4,354 strains) covering 88.5% of all 4,919 open reading frames, 7,734 barcodes (88.8%) were validated as high-fidelity to be inserted at the correct positions by Sanger sequencing. Sequence examination of the 7,734 high-fidelity barcodes revealed that 1,039 barcodes (13.4%) deviated from the original design. In total, 1,284 mutations (mutation rate of 16.6%) exist within the 1,039 mutated barcodes, which is comparable to budding yeast (18%). When the type of mutation was considered, substitutions accounted for 845 mutations (10.9%), deletions accounted for 319 mutations (4.1%), and insertions accounted for 121 mutations (1.6%). Peculiarly, the frequency of substitutions (67.6%) was unexpectedly higher than in budding yeast (∼28%) and well above the predicted error of Sanger sequencing (∼2%), which might have arisen during the solid-phase oligonucleotide synthesis and PCR amplification of the barcodes during strain construction. When the mutation rate was analyzed by position within 20-mer barcodes using the 1,284 mutations from the 7,734 sequenced barcodes, there was no significant difference between up-tags and down-tags at a given position. The mutation frequency at a given position was similar at most positions, ranging from 0.4% (32/7,734) to 1.1% (82/7,734), except at position 1, which was highest (3.1%), as in budding yeast. Together, well-defined barcode sequences, combined with the next-generation sequencing platform, promise to make the fission yeast gene deletion library a powerful tool for understanding gene function.

Arginase II Contributes to the Ca2+/CaMKII/eNOS Axis by Regulating Ca2+ Concentration Between the Cytosol and Mitochondria in a p32-Dependent Manner.

Background Arginase II activity contributes to reciprocal regulation of endothelial nitric oxide synthase ( eNOS ). We tested the hypotheses that arginase II activity participates in the regulation of Ca2+/Ca2+/calmodulin-dependent kinase II / eNOS activation, and this process is dependent on mitochondrial p32. Methods and Results Downregulation of arginase II increased the concentration of cytosolic Ca2+ ([Ca2+]c) and decreased mitochondrial Ca2+ ([Ca2+]m) in microscopic and fluorescence-activated cell sorting analyses, resulting in augmented eNOS Ser1177 phosphorylation and decreased eNOS Thr495 phosphorylation through Ca2+/Ca2+/calmodulin-dependent kinase II . These changes were observed in human umbilical vein endothelial cells treated with small interfering RNA against p32 (sip32). Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, fluorescence immunoassay, and ion chromatography, inhibition of arginase II reduced the amount of spermine, a binding molecule, and the release of Ca2+ from p32. In addition, arginase II gene knockdown using small interfering RNA and knockout arginase II -null mice resulted in reduced p32 protein level. In the aortas of wild-type mice, small interfering RNA against p32 induced eNOS Ser1177 phosphorylation and enhanced NO -dependent vasorelaxation. Arginase activity, p32 protein expression, spermine amount, and [Ca2+]m were increased in the aortas from apolipoprotein E (ApoE-/-) mice fed a high-cholesterol diet, and intravenous administration of small interfering RNA against p32 restored Ca2+/Ca2+/calmodulin-dependent kinase II -dependent eNOS Ser1177 phosphorylation and improved endothelial dysfunction. The effects of arginase II downregulation were not associated with elevated NO production when tested in aortic endothelia from eNOS knockout mice. Conclusions These data demonstrate a novel function of arginase II in regulation of Ca2+-dependent eNOS phosphorylation. This novel mechanism drives arginase activation, mitochondrial dysfunction, endothelial dysfunction, and atherogenesis.

Arginase Inhibition Suppresses Native Low-Density Lipoprotein-Stimulated Vascular Smooth Muscle Cell Proliferation by NADPH Oxidase Inactivation.

PURPOSE: Vascular smooth muscle cell (VSMC) proliferation induced by native low-density lipoprotein (nLDL) stimulation is dependent on superoxide production from activated NADPH oxidase. The present study aimed to investigate whether the novel arginase inhibitor limonin could suppress nLDL-induced VSMC proliferation and to examine related mechanisms. MATERIALS AND METHODS: Isolated VSMCs from rat aortas were treated with nLDL, and cell proliferation was measured by WST-1 and BrdU assays. NADPH oxidase activation was evaluated by lucigenin-induced chemiluminescence, and phosphorylation of protein kinase C (PKC) ßII and extracellular signal-regulated kinase (ERK) 1/2 was determined by western blot analysis. Mitochondrial reactive oxygen species (ROS) generation was assessed using MitoSOX-red, and intracellular L-arginine concentrations were determined by high-performance liquid chromatography (HPLC) in the presence or absence of limonin. RESULTS: Limonin inhibited arginase I and II activity in the uncompetitive mode, and prevented nLDL-induced VSMC proliferation in a p21Waf1/Cip1-dependent manner without affecting arginase protein levels. Limonin blocked PKCßII phosphorylation, but not ERK1/2 phosphorylation, and translocation of p47phox to the membrane was decreased, as was superoxide production in nLDL-stimulated VSMCs. Moreover, mitochondrial ROS generation was increased by nLDL stimulation and blocked by preincubation with limonin. Mitochondrial ROS production was responsible for the phosphorylation of PKCßII. HPLC analysis showed that arginase inhibition with limonin increases intracellular L-arginine concentrations, but decreases polyamine concentrations. L-Arginine treatment prevented PKCßII phosphorylation without affecting ERK1/2 phosphorylation. CONCLUSION: Increased L-arginine levels following limonin-dependent arginase inhibition prohibited NADPH oxidase activation in a PKCßII-dependent manner, and blocked nLDL-stimulated VSMC proliferation.

Arginase II inhibition prevents interleukin-8 production through regulation of p38 MAPK phosphorylation activated by loss of mitochondrial membrane potential in nLDL-stimulated hAoSMCs.

Arginase inhibition exhibits beneficial effects in vascular endothelial and smooth muscle cells. In human aortic smooth muscle cells (hAoSMCs), native low-density lipoprotein (nLDL) induced the production of interleukin-8 (IL-8) that is involved in the pathogenesis of cardiovascular diseases. Therefore, we examined the effect of arginase inhibition on IL-8 production and the underlying mechanism. In hAoSMCs, reverse transcription-PCR, western blotting and immunocytochemistry with MitoTracker confirmed that arginase II was confined predominantly to mitochondria. The mitochondrial membrane potential (MMP) was assessed using tetramethylrhodamine ethyl ester. The MMP decreased upon nLDL stimulation but was restored upon arginase inhibition. MMP loss caused by nLDL was prevented by treatment with the intracellular Ca2+ chelator BAPTA-AM. In mitochondrial Ca2+ measurements using Rhod-2 AM, increased mitochondrial Ca2+ levels by nLDL were inhibited upon preincubation with an arginase inhibitor. Among the polyamines, spermine, an arginase activity-dependent product, caused mitochondrial Ca2+ movement. The nLDL-induced MMP change resulted in p38 mitogen-activated protein kinase (MAPK) phosphorylation and IL-8 production and was prevented by the arginase inhibitors BAPTA and ruthenium 360. In isolated AoSMCs from ApoE-/- mice fed a high-cholesterol diet, arginase activity, p38 MAPK phosphorylation, spermine and mitochondrial Ca2+ levels and keratinocyte-derived chemokine (KC) production were increased compared with wild-type (WT) mice. However, in AoSMCs isolated from arginase II-null mice, increases in MMP and decreases in mitochondrial Ca2+ levels were noted compared with WT and were associated with p38 MAPK activation and IL-8 production. These data suggest that arginase activity regulates the change in MMP through Ca2+ uptake that is essential for p38 MAPK phosphorylation and IL-8 production.

Editor's Highlight: A Genome-wide Screening of Target Genes Against Silver Nanoparticles in Fission Yeast.

To identify target genes against silver nanoparticles (AgNPs), we screened a genome-wide gene deletion library of 4843 fission yeast heterozygous mutants covering 96% of all protein encoding genes. A total of 33 targets were identified by a microarray and subsequent individual confirmation. The target pattern of AgNPs was more similar to those of AgNO3 and H2O2, followed by Cd and As. The toxic effect of AgNPs on fission yeast was attributed to the intracellular uptake of AgNPs, followed by the subsequent release of Ag+, leading to the generation of reactive oxygen species (ROS). Next, we focused on the top 10 sensitive targets for further studies. As described previously, 7 nonessential targets were associated with detoxification of ROS, because their heterozygous mutants showed elevated ROS levels. Three novel essential targets were related to folate metabolism or cellular component organization, resulting in cell cycle arrest and no induction in the transcriptional level of antioxidant enzymes such as Sod1 and Gpx1 when 1 of the 2 copies was deleted. Intriguingly, met9 played a key role in combating AgNP-induced ROS generation via NADPH production and was also conserved in a human cell line.