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P450 1A2 is responsible for the metabolism of clinically important drugs and the metabolic activation of environmental chemicals. Genetic variations of P450 1A2 can influence its ability to perform these functions, and thus, this study aimed to characterize the functional significance of three P450 1A2 allelic variants containing nonsynonymous single nucleotide polymorphisms (P450 1A2*8, R456H; *15, P42R; *16, R377Q). Variants containing these SNPs were constructed and the recombinant enzymes were expressed and purified in Escherichia coli. Only the P42R variant displayed the typical CO-binding spectrum indicating a P450 holoenzyme with an expression level of ∼ 170 nmol per liter culture, but no P450 spectra were observed for the two other variants. Western blot analysis revealed that the level of expression for the P42R variant was lower than that of the wild type, however the expression of variants R456H and R377Q was not detected. Enzyme kinetic analyses indicated that the P42R mutation in P450 1A2 resulted in significant changes in catalytic activities. The P42R variant displayed an increased catalytic turnover numbers (k cat) in both of methoxyresorufin O-demethylation and phenacetin O-deethylation. In the case of phenacetin O-deethylation analysis, the overall catalytic efficiency (k cat/K m) increased up to 2.5 fold with a slight increase of its K m value. This study indicated that the substitution P42R in the N-terminal proline-rich region of P450 contributed to the improvement of catalytic activity albeit the reduction of P450 structural stability or the decrease of substrate affinity. Characterization of these polymorphisms should be carefully examined in terms of the metabolism of many clinical drugs and environmental chemicals.

Adipocyte accumulation is associated with the development of obesity and obesity-related diseases. Interactions of master transcription factors and signaling cascades are required for adipogenesis. Regulation of excessive adipogenic processes may be an attractive therapeutic for treatment of obesity and obesity-related diseases. In this study, we found that atorvastatin exerts an anti-adipogenic activity in 3T3-L1 pre-adipocytes, and that this activity is elevated in combination with metformin. Expression of the adipogenic master regulators PPARγ and C/EBPα, and their target gene aP2, was suppressed by atorvastatin. Furthermore, atorvastatin treatment resulted in increased activation of the key master regulator of cellular energy homeostasis, AMPK. These biological activities of atorvastatin were elevated in combination with metformin. These anti-adipogenic activities were associated with regulation of the STAT3 and TGF-β signaling cascades, resulting in the regulation of the expression of STAT3 target genes, such as KLF5, p53, and cyclin D1, and TGF-β signaling inhibitory genes, such as SMAD7. Our results suggest that combination therapy with atorvastatin and metformin may have therapeutic potential for the treatment of obesity and obesity-related diseases caused by excessive adipogenesis.

Chlorogenic acid, a major polyphenol in edible plants, possesses strong antioxidant activity, anti-lipid peroxidation and anticancer effects. It used for industrial applications; however, this is limited by its instability to heat or light. In this study, we, for the first time synthesized chlorogenic acid glucoside (CHG) via transglycosylation using dextransucrase from Leuconostoc mesenteroides and sucrose. CHG was purified and its structure determined by nuclear magnetic resonance and matrix-associated laser desorption ionization–time-of-flight mass spectroscopy. The production yield of CHG was 44.0% or 141 mM, as determined by response surface methodology. CHG possessed a 65% increase in water solubility and a 2-fold browning resistance and it displayed stronger inhibition of lipid peroxidation and of colon cancer cell growth by MTT assay, compared to chlorogenic acid. Therefore, this study may expand the industrial applications of chlorogenic acid as water-soluble or browning resistant compound (CHG) through enzymatic glycosylation.

Carfilzomib (CFZ) is a peptide epoxyketone proteasome inhibitor approved for the treatment of multiple myeloma (MM). Despite the remarkable efficacy of CFZ against MM, the clinical trials in patients with solid cancers yielded rather disappointing results with minimal clinical benefits. Rapid degradation of CFZ in vivo and its poor penetration to tumor sites are considered to be major factors limiting its efficacy against solid cancers. We previously reported that polymer micelles (PMs) composed of biodegradable block copolymers poly(ethylene glycol) (PEG) and poly(caprolactone) (PCL) can improve the metabolic stability of CFZ in vitro. Here, we prepared the CFZ-loaded PM, PEG-PCL-deoxycholic acid (CFZ-PM) and assessed its in vivo anticancer efficacy and pharmacokinetic profiles. Despite in vitro metabolic protection of CFZ, CFZ-PM did not display in vivo anticancer efficacy in mice bearing human lung cancer xenograft (H460) superior to that of the clinically used cyclodextrin-based CFZ (CFZ-CD) formulation. The plasma pharmacokinetic profiles of CFZ-PM were also comparable to those of CFZ-CD and the residual tumors that persisted in xenograft mice receiving CFZ-PM displayed an incomplete proteasome inhibition. In summary, our results showed that despite its favorable in vitro performances, the current CFZ-PM formulation did not improve in vivo anticancer efficacy and accessibility of active CFZ to solid cancer tissues over CFZ-CD. Careful consideration of the current results and potential confounding factors may provide valuable insights into the future efforts to validate the potential of CFZ-based therapy for solid cancer and to develop effective CFZ delivery strategies that can be used to treat solid cancers.

Cytochrome P450 2A6 (P450 2A6) is the major enzyme responsible for the oxidation of coumarin, nicotine, and tobacco-specific nitrosamines in human liver. In this study, the catalytic turnover of coumarin oxidation was improved by directed-evolution analysis of P450 2A6 enzyme. A random mutant library was constructed using error-prone polymerase chain reaction (PCR) of the open reading frame of the P450 2A6 gene and individual mutant clones were screened for improved catalytic activity in analysis of fluorescent coumarin 7-hydroxylation. Four consecutive rounds of random mutagenesis and screening were performed and catalytically enhanced mutants were selected in each round of screening. The selected mutants showed the sequentially accumulated mutations of amino acid residues of P450 2A6: B1 (F209S), C1 (F209S, S369G), D1 (F209S, S369G, E277K), and E1 (F209S, S369G, E277K, A10V). E1 mutants displayed approximately 13-fold increased activity based on fluorescent coumarin hydroxylation assays at bacterial whole cell level. Steady-state kinetic parameters for coumarin 7-hydroxylation and nicotine oxidation were measured in purified mutant enzymes and indicated catalytic turnover numbers (kcat) of selected mutants were enhanced up to sevenfold greater than wild-type P450 2A6. However, all mutants displayed elevated Km values and therefore catalytic efficiencies (kcat/Km) were not improved. The increase in Km values was partially attributed to reduction in substrate binding affinities measured in the analysis of substrate binding titration. The structural analysis of P450 2A6 indicates that F209S mutation is sufficient to affect direct interaction of substrate at the active site.

Cytochrome P450 4A11 (CYP4A11) is a fatty acid hydroxylase enzyme expressed in human liver. It catalyzes not only the hydroxylation of saturated and unsaturated fatty acids, but the conversion of arachidonic acid to 20-hydroxyeicosatetraenoic acid (20-HETE), a regulator of blood pressure. In this study, we performed a directed evolution analysis of CYP4A11 using the luminogenic assay system. A random mutant library of CYP4A11, in which mutations were made throughout the entire coding region, was screened with luciferase activity to detect the demethylation of luciferin-4A (2-[6-methoxyquinolin-2-yl]-4,5-dihydrothiazole-4-carboxylic acid) of CYP4A11 mutants in Escherichia coli. Consecutive rounds of random mutagenesis and screening yielded three improved CYP4A11 mutants, CP2600 (A24T/T263A), CP2601 (T263A), and CP2616 (A24T/T263A/V430E) with ~3-fold increase in whole cells and10-fold increase in purified proteins on the luminescence assay. However, the steady state kinetic analysis for lauric acid hydroxylation showed the significant reductions in enzymatic activities in all three mutants. A mutant, CP2600, showed a 51% decrease in catalytic efficiency (k cat/K m) for lauric acid hydroxylation mainly due to an increase in K m. CP2601 and CP2616 showed much greater reductions (75%) in the catalytic efficiency due to both a decrease in k cat and an increase in K m. These decreased catalytic activities of CP2601 and CP2616 can be partially attributed to the changes in substrate affinities. These results suggest that the enzymatic activities of CYP4A11 mutants selected from directed evolution using a luminogenic P450 substrate may not demonstrate a direct correlation with the hydroxylation activities of lauric acid.

Aberrantly activated signal transducer and activator of transcription (STAT) proteins are implicated with human cancers and represent essential roles for cancer cell survival and proliferation. Therefore, the development of small-molecule inhibitors of STAT signaling bearing pharmacological activity has therapeutic potential for the treatment of human cancers. In this study, we identified sophoraflavanone G as a novel small-molecule inhibitor of STAT signaling in human cancer cells. Sophoraflavanone G inhibited tyrosine phosphorylation of STAT proteins in Hodgkin's lymphoma and tyrosine phosphorylation of STAT3 in solid cancer cells by inhibiting phosphorylation of the Janus kinase (JAK) proteins, Src family tyrosine kinases, such as Lyn and Src, Akt, and ERK1/2. In addition, sophoraflavanone G inhibited STAT5 phosphorylation in murine-bone-marrow-derived pro-B cells transfected with translocated Ets Leukemia (TEL)-JAKs and cytokine-induced rat pre-T lymphoma cells, as well as STAT5b reporter activity in TEL-JAKs and STAT5b reporter systems. Sophoraflavanone G also inhibited nuclear factor-κB (NF-κB) signaling in multiple myeloma cells. Furthermore, sophoraflavanone G inhibited cancer cell proliferation and induced apoptosis by regulating the expression of apoptotic and anti-apoptotic proteins. Our data suggest that sophoraflavanone G is a novel small-molecule inhibitor of STAT signaling by targeting upstream signals of STATs that may have therapeutic potential for cancers caused by persistently activated STAT proteins.

Organic anion transporting polypeptide 1B3 (OATP1B3) was initially considered to be a liver-specific transporter, mediating the uptake of a variety of endogenous and xenobiotic substances. Over the past decade, several investigations reported that OATP1B3 is also expressed across multiple types of cancers. Only recently, our laboratory and others demonstrated the identity of cancer-specific OATP1B3 variants (csOATP1B3) arising from the use of an alternative transcription initiation site, different from the wildtype (WT) OATP1B3 expressed in the normal liver. However, the mechanisms regulating the expression of csOATP1B3 remained unknown. In our current study, we investigated the role of hypoxia and the involvement of hypoxia inducible factor-1α (HIF-1α) in regulating the transcription of csOATP1B3. Our RT-PCR and immunoblotting results indicated that csOATP1B3, but not WT OATP1B3, can be induced in response to ambient or chemical hypoxia (upon exposure to 1% O₂ or cobalt chloride). Reporter assays with deletion and mutated constructs of the csOATP1B3 promoter revealed a functional hypoxia response element (HRE) located in the proximal upstream region. Constructs harboring the HRE displayed the upregulated reporter gene expression in response to hypoxia, but not when mutated. Electrophoretic mobility shift assays using a biotin-labeled csOATP1B3 promoter HRE probe indicated the binding of HIF-1α, which was blocked by an excess of unlabeled csOATP1B3 probe. Furthermore, siRNA-based knockdown of HIF-1α caused a substantial decrease in the expression level of csOATP1B3. Taken together, these findings demonstrate that the transcription of csOATP1B3 is actively engaged during hypoxia, through a commonly utilized pathway involving HIF-1α.

Bone marrow stromal cell antigen 2 (BST-2) is a type II transmembrane protein that is known to be a therapeutic target in several types of cancer. However, despite its clinical importance, the roles of BST-2 expression have remained elusive. Here, we found that BST-2 expression is up-regulated in tamoxifen-resistant MCF-7 human breast cancer (TRM-7) cells, resulting in enhanced invasiveness and migration. Matrigel and wound healing assays also showed that overexpression of BST-2 increased invasion and migration in MCF-7 cells, whereas invasion and migration were decreased by the silencing of BST-2 in TRM-7 cells. In addition, B16F10 cells expressing BST-2 showed increased metastatic melanoma nodule growth in a lung metastasis mouse model. Furthermore, BST-2 expression and promoter activity were regulated by activated signal transducer and activator of transcription 3 (STAT3). Taken together, our results indicate that BST-2 is an important factor in the invasiveness and motility of tamoxifen-resistant breast cancer cells, and that its expression and activity are regulated by activated STAT3. Therefore, regulation of BST-2 is a potential therapeutic target for tamoxifen-resistant breast cancer.

The metabolic activation of many carcinogenic heterocyclic and aryl amines are attributed to hepatic cytochrome P450 enzymes including P450 1A2. Bioactivation mechanism analysis of P450 1A2 in the limited crystallographic data requires the generation of a large number of mutants. In this study, the SRS2 region in P450 1A2 was randomly mutated with random primers designed for the non-wild type using whole-plasmid random mutagenesis, followed the screening of the mutant library. Eight mutants in Asn222 and Glu225 were selected after Escherichia coli genotoxicity assay involving reversion to lac prototrophy as a response to activation of the heterocyclic amine 2-amino-3,5-dimethylimidazo[4,5-f]quinoline (MeIQ). Each mutant E. coli membrane was used to determine k cat and K m values for phenacetin and 7-ethoxyresorufin O-deethylation. Most mutants showed considerably increased activity for phenacetin. The Glu225Asn and Asn222Lys mutants increased catalytic efficiencies (k cat/K m) by 5- and 4-fold, respectively. However, the catalytic efficiencies of 7-ethoxyresorufin O-deethylation were not increased mainly because of the increase of K m values. The Glu225Asn and Asn222Lys mutants showed increased k cat values but exhibited greater increases in K m values, which resulted in lower catalytic efficiencies (k cat/K m) for 7-ethoxyresorufin O-deethylation. These results suggested that structural changes of metabolic enzymes in the process of mutagenesis result in different effects of activity by its substrates.

Streptomyces avermitilis produces clinically useful drugs such as avermectins and oligomycins. Its genome contains approximately 33 cytochrome P450 genes and they seem to play important roles in the biosynthesis of many secondary metabolites. The SAV_7130 gene from S. avermitilis encodes CYP158A3. The amino acid sequence of this enzyme has high similarity with that of CYP158A2, a biflaviolin synthase from S. coelicolor A3(2). Recombinant S. avermitilis CYP158A3 was heterologously expressed and purified. It exhibited the typical P450 Soret peak at 447 nm in the reduced CO-bound form. Type I binding spectral changes were observed when CYP158A3 was titrated with myristic acid; however, no oxidative product was formed. An analog of flaviolin, 2-hydroxynaphthoquinone (2-OH NQ) displayed similar type I binding upon titration with purified CYP158A3. It underwent an enzymatic reaction forming dimerized product. A homology model of CYP158A3 was superimposed with the structure of CYP158A2, and the majority of structural elements aligned. These results suggest that CYP158A3 might be an orthologue of biflaviolin synthase, catalyzing C-C coupling reactions during pigment biosynthesis in S. avermitilis.

Orthorhombic tungsten ditelluride (WTe2), with a distorted 1T structure, exhibits a large magnetoresistance that depends on the orientation, and its electrical characteristics changes rom semimetallic to insulating as the thickness decreases. Through polarized Raman spectroscopy in combination with transmission electron diffraction, we establish a reliable method to determine the thickness and crystallographic orientation of few-layer WTe2. The Raman spectrum shows a pronounced dependence on the polarization of the excitation laser. We found that the separation between two Raman peaks at ~90 cm-1 and at 80-86 cm-1, depending on thickness, is a reliable fingerprint for determination of the thickness. For determination of the crystallographic orientation, the polarization dependence of the A1 modes, measured with the 632.8-nm excitation, turns out to be the most reliable. We also discovered that the polarization behaviors of some of the Raman peaks depend on the excitation wavelength as well as thickness, indicating a close interplay between the band structure and anisotropic Raman scattering cross section., 16 pages, 4 figures

Human CYP4A11 is the major ω-hydroxylase of fatty acids in the liver and kidneys. It produces 20-hydroxyeicosatetraenoic acid as well as hydroxylates fatty acids. In this study, we investigated the biochemical properties of three alleles of CYP4A11: W126R, K276T, and S353G. Site-directed mutagenesis of the wild type CYP4A11 was performed, to construct the W126R, K276T, and S353G variant clones. The CYP4A11 wild type and variant constructs were heterologously expressed in Escherichia coli. CO-binding spectra showed the expression of the wild type, K276T and S353G variants, indicating the functional P450 holoenzyme. The W126R variant was not expressed in E. coli. Binding affinities of lauric acid in K276T and S353G variants were stronger than that of wild type. Steady-state kinetics in the hydroxylation reaction of fatty acids were studied. The catalytic efficiencies (kcat/Km) of K276T and S353G variants in the reactions without cytochrome b5 were approximately 2- and 4-fold higher, respectively, than that of wild type, and in the reactions with cytochrome b5 they were approximately 2- and 3-fold higher, respectively. These results suggest that individuals carrying the alleles, K276T and S353G, might exhibit higher catalysis of CYP4A11, which may affect the endogenous metabolic products associated with regulation of blood pressure.

Ethnopharmacological relevance Panax ginseng is one of the most well-known medicinal herbs in Korea and China, which has been used for treatment and prevention of cancer, obesity, diabetes, and cardiovascular diseases. Ginsenosides are the major components of P. ginseng, having a wide range of pharmacological activities. Among the ginsenosides, protopanaxadiol (PPD)-types reportedly have potent anti-cancer effects. Rh2 is PPD-type ginsenoside, and two stereoisomeric forms of Rh2 as 20(S)- and 20(R)-Rh2 were selectively isolated recently. Aim of the study The biological activities of Rh2 ginsenosides are known to depend on their differences in stereochemistry. Colorectal cancer (CRC) is one of the most lethal neoplasm, and cancer-related death is usually associated with metastasis to other organs. We aimed this study to investigate whether 20(S)- and 20(R)-Rh2 can suppress tumor invasion in human CRC cells. Materials and methods 20(S)- and 20(R)-Rh2 were isolated from the roots of ginseng. Human CRC cells were incubated with 20(S)- or 20(R)-Rh2 in the presence or absence of interleukin-6. An MTT assay was used to measure cell viability. Western blot and quantitative real-time PCR analyses were performed to determine levels of expression and phosphorylation. An invasion assay was performed using a Boyden chamber system with the Matrigel-coated membrane to measure cancer cell invasion. Results 20(S)- and 20(R)-Rh2 showed differential cytotoxic activity. Only 20(S)-Rh2 decreased cancer cell viability. Additionally, 20(S)-Rh2 effectively inhibited IL-6-induced signal transducer and activator of transcription 3 (STAT3) phosphorylation and the expression of matrix metalloproteinases (MMPs), including MMP-1, −2, and −9, resulting in inhibition of cancer cell invasion. Interestingly, these pharmacological activities of 20(S)-Rh2 were more potent than those of 20(R)-Rh2. Furthermore, combination treatment showed that 20(S)-Rh2 enhanced the sensitization of doxorubicin-treated anti-cancer activities in CRC cells. Conclusion Our results demonstrated that ginsenoside 20(S)-Rh2 has therapeutic potential for the treatment with CRC and may be valuable as a combination partner with more classic chemotherapeutic agents, such as doxorubicin, to treat CRC.

This study was done to identify the relationship of sexual subjectivity, exposure to sexual content on smartphone and sexual attitudes and to identify predictors of sexual attitudes of undergraduates. This research design for this study was descriptive survey design using quota sampling. Data collection was done using self-questionnaire with 400 university students in C University. The results of this study are following. The sexual attitudes were 2.65 points. The sexual attitudes of the subjects exhibited a positive correlation with opinion reflection(r=.492, p

Esophageal cancer is the third most common cancer of the gastrointestinal tract. Despite new therapies, the prognosis for patients with these cancers remains poor with 5-year survival rates lower than 15%. Recently, immunotherapy has increasingly gained attention as a novel treatment strategy for advanced esophageal cancer.Recent success of immunotherapy in treating other solid tumors has shed light on the utility of these approaches for esophageal cancers. Here, the authors focus on antibody-based, adoptive-cell-therapy-based, and vaccine-based immunotherapies, and briefly address their rationale, clinical data, and implications.Immunotherapy is now established to be a key treatment modality that can improve the outcomes of many cancer patients and appears to be ushering in a new era in cancer treatment. Checkpoint inhibitor drugs have shown preliminary favorable results in esophageal cancer treatment. Adoptive cell therapy and vaccine studies have also shown some promise in various clinical studies. Future endeavors will need to focus on identifying patients who are likely to benefit from immunotherapy, monitoring and managing immune responses and designing optimal combination strategies where immunotherapy agents are combined with other traditional treatment modalities.

We study two-color high-order harmonic generation in Neon with 790 nm and 1300 nm driving laser fields and observe an extreme-ultraviolet continuum that extends to photon energies of 160 eV. Using a 6-mm-long, high pressure gas cell, we optimize the HHG yield at high photon energies and investigate the effect of ionization and propagation under phase-matching conditions that allow us to control the temporal structure of the XUV emission. Numerical simulations that include the 3D propagation of the two-color laser pulse show that a bright isolated attosecond pulse with exceptionally high photon energies can be generated in our experimental conditions due to an efficient hybrid optical and phase-matching gating mechanism.

Cytochrome P450 2A6 (CYP2A6) catalyzes important metabolic reactions of many xenobiotic compounds, including coumarin, nicotine, cotinine, and clinical drugs. Genetic polymorphisms of CYP2A6 can influence its metabolic activities. This study analyzed the functional activities of six CYP2A6 allelic variants (CYP2A6*5, *7, *8, *18, *19, and *35) containing nonsynonymous single-nucleotide polymorphisms. Recombinant variant enzymes of CYP2A6*7, *8, *18, *19, and *35 were successfully expressed in Escherichia coli and purified. However, a P450 holoenzyme spectrum was not detected for the CYP2A6*5 allelic variant (G479V). Structural analysis shows that the G479V mutation may alter the interaction between the A helix and the F-G helices. Enzyme kinetic analyses indicated that the effects of mutations in CYP2A6 allelic variants on drug metabolism are dependent on the substrates. In the case of coumarin 7-hydroxylation, CYP2A6*8 and *35 displayed increased K(m) values whereas CYP2A6*18 and *19 showed decreased k(cat) values, which resulted in lower catalytic efficiencies (k(cat)/K(m)). In the case of nicotine 5-oxidation, the CYP2A6*19 variant exhibited an increased K(m) value, whereas CYP2A6*18 and *35 showed much greater decreases in k(cat) values. These results suggest that individuals carrying these allelic variants are likely to have different metabolisms for different CYP2A6 substrates. Functional characterization of these allelic variants of CYP2A6 can help determine the importance of CYP2A6 polymorphisms in the metabolism of many clinical drugs.

Vacuum ultraviolet (VUV) mass-analyzed threshold ionization (MATI) spectrum of supersonically cooled methylcyclohexane has been obtained to give the precise adiabatic ionization energy of 9.6958 ± 0.0025 eV for the chair equatorial conformer. Vibrationally resolved MATI spectrum has been analyzed with the aid of density functional theory and Franck–Condon calculations. The MATI spectrum reflects the structural change upon ionization and its origin is discussed by inspecting the shapes of the valence orbitals involved in the ionization process. The spectroscopic implication of the structural interconversion above the certain energy level is discussed with theoretical calculations of molecular structures and energetics.

Candida albicans is responsible for serious fungal infections in humans. Analysis of its genome identified NCP1 gene coding for a putative NADPH-P450 reductase (NPR) enzyme. This enzyme appears to supply reducing equivalents to cytochrome P450 or heme oxygenase enzymes for fungal survival and virulence. In this study, we report the characterization of the functional features of NADPH-P450 reductase from C. albicans. The recombinant C. albicans NPR protein harboring a 6×(His)-tag was expressed heterologously in Escherichia coli, and was purified. Purified C. albicans NPR has an absorption maximum at 453 nm, indicating the feature of an oxidized flavin cofactor, which was decreased by the addition of NADPH. It also evidenced NADPH-dependent cytochrome c or nitroblue tetrazolium reducing activity. This purified reductase protein was successfully able to substitute for purified mammalian NPR in the reconstitution of the human P450 1A2-catalyzed O-deethylation of 7-ethoxyresorufin. These results indicate that purified C. albicans NPR is an orthologous reductase protein that supports cytochrome P450 or heme oxygenase enzymes in C. albicans.

We study high-order harmonics generated by an intense multicycle 800 nm laser pulse in the presence of a weaker 1300 nm mid-infrared (MIR) pulse. Additionally to the previously observed effects of yield enhancement, cutoff extension, and continuum generation, we report here a spectral shift of harmonic peaks controlled by the delay of the MIR pulse. We explain this effect considering the half-cycle to half-cycle behavior of the two-color field.

The human cytochrome P450 4A11 is the major monooxygenase to oxidize the fatty acids and arachidonic acid. The production of 20-hydroxyeicosatetraenoic acid by P450 4A11 has been implicated in the regulation of vascular tone and blood pressure. Oxidation reaction by P450 4A11 requires its reduction partners, NADPH-P450 reductase (NPR). We report the functional expression in Escherichia coli of bicistronic constructs consisting of P450 4A11 encoded by the first cistron and the electron donor protein, NPR by the second. Typical P450 expression levels of wild type and several N-terminal modified mutants was observed in culture media and prepared membrane fractions. The expression of functional NPR in the constructed P450 4A11: NPR bicistronic system was clearly verified by reduction of nitroblue tetrazolium. Membrane preparation containing P450 4A11 and NPR efficiently oxidized lauric acid mainly to ω-hydroxylauric acid. Bicistronic coexpression of P450 4A11 and NPR in E. coli cells can be extended toward identification of novel drug metabolites or therapeutic agents involved in P450 4A11 dependent signal pathways.

Conformational isomers of alkanethiols are isolated in the molecular beam, and the conformer-specific ionization dynamics have been investigated using vacuum ultraviolet mass-analyzed threshold ionization (MATI) spectroscopy. Only a single conformer of ethanethiol is observed to give the adiabatic ionization potential (IP) of 9.2922 +/- 0.0007 eV for the gauche conformer. For isopropanethiol, IP is found to be 9.1426 +/- 0.0006 for the trans conformer and 9.1559 +/- 0.0006 eV for the gauche conformer. Only two major conformational isomers are identified for 1-propanethiol, giving an IP of 9.1952 +/- 0.0006 for the trans-gauche conformer and 9.2008 +/- 0.0006 eV for the gauche-gauche conformer. The tert-butanethiol, as expected, has a single conformer with an IP of 9.0294 +/- 0.0006 eV. For 1-butanethiol, there are a number of conformers, and the assignment of the MATI bands to each conformer turns out to be nontrivial. The spectral simulation using the Franck-Condon analysis based on the density functional theory (DFT) calculations has been used for the identification of each conformational isomer in the MATI spectrum. Each conformer undergoes its unique structural change upon ionization, as revealed in the vibration resolved MATI spectrum, providing the powerful method for the spectral identification of a specific conformational isomer. The conformer specificity in the ionization-driven structural change reflects the role of the electron of the highest occupied molecular orbital (HOMO) in the conformational preference.

CYP107W1 from Streptomyces avermitilis is a cytochrome P450 enzyme involved in the biosynthesis of macrolide oligomycin A. A previous study reported that CYP107W1 regioselectively hydroxylated C12 of oligomycin C to produce oligomycin A, and the crystal structure of ligand free CYP107W1 was determined. Here, we analyzed the structural properties of the CYP107W1-oligomycin A complex and characterized the functional role of the Trp178 residue in CYP107W1. The crystal structure of the CYP107W1 complex with oligomycin A was determined at a resolution of 2.6 A. Oligomycin A is bound in the substrate access channel on the upper side of the prosthetic heme mainly by hydrophobic interactions. In particular, the Trp178 residue in the active site intercalates into the large macrolide ring, thereby guiding the substrate into the correct binding orientation for a productive P450 reaction. A Trp178 to Gly mutation resulted in the distortion of binding titration spectra with oligomycin A, whereas binding spectra with azoles were not affected. The Gly178 mutant's catalytic turnover number for the 12-hydroxylation reaction of oligomycin C was highly reduced. These results indicate that Trp178, located in the open pocket of the active site, may be a critical residue for the productive binding conformation of large macrolide substrates.

The study was done to identify social support network, activities of daily living, depression and health-related quality of life of male elders and to clarity the relationship among these variables. The data were collected via self- report questionnaire from 194 male elders who visited the senior welfare center in City G from December 20, 2014 to January 31, 2015. The social support network of the subjects was 35.48 points; the activities of daily living, 20.83 points; depression, 2.93 points; and health-related quality of life, 0.83 points. The health-related quality of life of the subjects exhibited a positive correlation with social support network (r=.23, p=.001) and with activities of daily living (r=.40, p

Streptomyces avermitilis contains 33 cytochrome P450 genes in its genome, many of which play important roles in the biosynthesis process of antimicrobial agents. Here, we characterized the biochemical function and structure of CYP107W1 from S. avermitilis, which is responsible for the 12-hydroxylation reaction of oligomycin C. CYP107W1 was expressed and purified from Escherichia coli. Purified proteins exhibited the typical CO-binding spectrum of P450. Interaction of oligomycin C and oligomycin A (12-hydroxylated oligomycin C) with purified CYP107W1 resulted in a type I binding with Kd values of 14.4 ± 0.7 μM and 2.0 ± 0.1 μM, respectively. LC–mass spectrometry analysis showed that CYP107W1 produced oligomycin A by regioselectively hydroxylating C12 of oligomycin C. Steady-state kinetic analysis yielded a kcat value of 0.2 min−1 and a Km value of 18 μM. The crystal structure of CYP107W1 was determined at 2.1 A resolution. The overall P450 folding conformations are well conserved, and the open access binding pocket for the large macrolide oligomycin C was observed above the distal side of heme. This study of CYP107W1 can help a better understanding of clinically important P450 enzymes as well as their optimization and engineering for synthesizing novel antibacterial agents and other pharmaceutically important compounds.

Ar clustering dynamics around the metal-benzene sandwich complex, bis(eta (6)-benzene)chromium: Cr(Bz) 2, is found to occur in two distinct regimes. The shift of the ionization potential (IP) upon the addition of Ar is measured to be 151 cm (-1), and it is constant until the number of Ar solvents ( n) becomes 6. The IP shift per Ar is found to be suddenly decreased to 82 cm (-1) for the clusters of n = 7-12. The cluster distribution indicates that the n = 6 cluster is most populated in the molecular beam. These experimental findings with the aid of ab initio calculation indicate that the first six Ar solvent molecules are attached to top and bottom of Cr(Bz) 2 to give the robust structure for the Cr(Bz) 2-Ar 6 cluster whereas the next six Ar molecules are gathered on the side of the solute core to give the highly symmetric structure of the Cr(Bz) 2-Ar 12 cluster.

CYP2D6 is responsible for the oxidative metabolism of 20-25 % of clinical drugs and its genetic polymorphisms can significantly influence the drug metabolism. In this study, we analyzed the functional activities of four nonsynonymous single nucleotide polymorphisms from CYP2D6*52 allele, which were recently found, and one found frequently in CYP2D6 alleles. Recombinant variant enzymes of E418K, S486T, and R296C were successfully expressed in Escherichia coli and purified. However, a CYP holoenzyme spectrum of P34S variant was not detected in E. coli whole cell level. Structural analysis indicated that P34S mutation seemed to perturb a highly conserved proline-rich N-terminus of CYP2D6. Steady state kinetic analyses showed the significant reductions of enzymatic activities in E418K and R296C variants. In the case of bufuralol 1'-hydroxylation, a novel mutant, E418K, showed 32 % decrease in catalytic efficiency (k cat/K m) mainly due to the decrease of k cat value. R296C showed much greater reduction in the catalytic efficiency (9 % of wild-type) due to both of a decrease of k cat value and an increase of K m value. In the case of dextromethorphan O-demethylation, E418K showed both of a decrease of k cat value and an increase K m value to result in ~43 % reduction of catalytic efficiency. A highly decreased catalytic efficiency (~6 % of wild-type) in the mutant of R296C also was observed mainly due to the dramatic change of k cat value of dextromethorphan O-demethylation. These results suggested that individuals carrying these allelic variants are likely to have the altered metabolic abilities of many clinical drugs therefore, these polymorphisms of CYP2D6 should be much concerned for reliable drug treatment.

Induction of liver cytochrome P450 2B1 by beta-ionone was investigated in male and female Sprague Dawley rats. Administration of beta-ionone subcutaneously 72 and 48 hr before sacrificing the animals not only significantly induced the liver microsomal activity of pentoxyresorufin O-dealkylase, but also clearly increased in the level of cytochrome P450 2B1 protein. The induction of cytochrome P450 2B1 by beta-ionone was much greater in male rats than in female rats. A slot blot analysis showed that the mRNA level was increased from 6 hr after treatment with beta-ionone in male rats and from 12 hr after treatment in female rats. Taken together, the present results indicate for the first time that the induction of cytochrome P450 2B1 by beta-ionone might be regulated by the accumulation of mRNAs.

OATP1B3 is a member of the OATP (organic anion transporting polypeptides) superfamily, responsible for mediating the transport of numerous endogenous and xenobiotic substances. Although initially reported to be exclusively expressed in the liver, several studies reported that OATP1B3 is frequently expressed in multiple types of cancers and may be associated with differing clinical outcomes. However, a detailed investigation on the expression and function of OATP1B3 protein in cancer has been lacking. In this study, we confirmed that colon and pancreatic cancer cells express variant forms of OATP1B3, different from OATP1B3 wild-type (WT) expressed in the normal liver. OATP1B3 variant 1 (V1), the most prevalent form among the variants, contains alternative exonic sequences (exon 2a) instead of exons 1 and 2 present in OATP1B3 WT. The translated product of OATP1B3 V1 is almost identical to OATP1B3 WT, with exception to the first 28 amino acids at the N-terminus. Exogenous expression of OATP1B3 V1 revealed that OATP1B3 V1 undergoes post-translational modifications and proteasomal degradation to a differing extent compared to OATP1B3 WT. OATP1B3 V1 showed only modest transport activity toward cholecystokin-8 (CCK-8, a prototype OATP1B3 substrate) in contrast to OATP1B3 WT showing a markedly efficient uptake of CCK-8. Consistent with these results, OATP1B3 V1 was localized mainly in the cytoplasm with a much lower extent of trafficking to the surface membrane compared to OATP1B3 WT. In summary, our results demonstrate that colon and pancreatic cancer cells express variant forms of OATP1B3 with only limited transport activity and different subcellular localization compared to OATP1B3 WT. These observed differences at the molecular and functional levels will be important considerations for further investigations of the biological and clinical significance of OATP1B3 expression in cancer.

The discovery of NF-κB signaling pathways has greatly enhanced our understanding of inflammatory and immune responses. In the canonical NF-κB pathway, the proteasomal degradation of IκBα, an inhibitory protein of NF-κB, is widely accepted to be a key regulatory step. However, contradictory findings have been reported as to whether the immunoproteasome plays an obligatory role in the degradation of IκBα and activation of the canonical NF-κB pathway. Such results were obtained mainly using traditional gene deletion strategies. Here, we have revisited the involvement of the immunoproteasome in the canonical NF-κB pathway using small molecule inhibitors of the immunoproteasome, namely UK-101 and LKS01 targeting β1i and β5i, respectively. H23 and Panc-1 cancer cells were pretreated with UK-101, LKS01 or epoxomicin (a prototypic inhibitor targeting both the constitutive proteasome and immunoproteasome). We then examined whether these pretreatments lead to any defect in activating the canonical NF-κB pathway following TNFα exposure by monitoring the phosphorylation and degradation of IκBα, nuclear translocation of NF-κB proteins and DNA binding and transcriptional activity of NF-κB. Our results consistently indicated that there is no defect in activating the canonical NF-κB pathway following selective inhibition of the immunoproteasome catalytic subunits β1i, β5i or both using UK-101 and LKS01, in contrast to epoxomicin. In summary, our current results using chemical genetic approaches strongly support that the catalytic activity of the immunoproteasome subunits β1i and β5i is not required for canonical NF-κB activation in lung and pancreatic adenocarcinoma cell line models.

Ionization of equatorial and axial conformational isomers of the chair-bromocyclohexane is investigated with use of the vacuum ultraviolet mass-analyzed threshold ionization (MATI) spectroscopic technique. Two distinct ionization energies of 9.8308 ± 0.0025 and 9.8409 ± 0.0025 eV are determined for equatorial or axial conformers, respectively. From the conformer-selective vibrational analysis, it is found that the equatorial conformer undergoes a drastic structural change upon ionization especially along the C-Br distortion mode, whereas the axial conformer shows the modest change along the ring-puckering mode with ionization, corresponding to the reaction coordinate for the conformational interconversion. Density functional theory (DFT) calculations with and without considering the spin-orbit coupling provide the appropriate mode assignments for the vibrational bands active in the ionization spectra. Natural bond orbital (NBO) analysis is carried out to give insights into the contribution of the anomeric effect to the structure-energy relationship in each conformational isomer.

The human genome contains approximately 13 orphan cytochrome P450 (P450, CYP) genes, of which the apparent function or substrate has not been identified. However, they seem to possess their own biological relevance in some tissues or developmental stages. Here, we characterized the heterologously expressed CYP2W1, an orphan P450 enzyme. The recombinant CYP2W1 protein containing a 6 × (His) -tag at Nterminus has been expressed in Escherichia coli and purified. Expression level of CYP2W1 holoenzyme was around 500 nmol P450 holoenzyme per liter culture medium. The reduced CO difference spectrum of CYP2W1 showed a maximum absorption at 449 nm. CYP2W1 indicated the significant induction to bioactivate Trp-P-1, MeIQ, and IQ in E. coli DJ701 tester strain. However, the bioactivation of B[α]P, and NNK by CYP2W1 was relatively low. The model structure of CYP2W1 suggested the characteristic P450 folds with the lengths and orientations of the individual secondary elements. The F-G loop is situated on the distal side of heme to accommodate the flexibility of active site of CYP2W1. These studies can provide useful information for the finding of its biological roles and structure-function relationships of an orphan CYP2W1 enzyme.

The A (2)A(1)--X (2)B(1) transitions of H(2)S(+) above the barrier to linearity have been investigated with the energy resolution high enough to identify individual rotational transition lines for the first time. The rotational cooling of the cation is achieved either by the direct ionization or mass-analyzed threshold ionization (MATI) technique employed in the vacuum-ultraviolet laser excitation of the jet-cooled H(2)S. Subsequent photoexcitation leads to the H(2)S(+)--H(2)+S(+) dissociation and the S(+) product yield taken as a function of the excitation energy gives the photofragment excitation (PHOFEX) spectra. The combined use of MATI and PHOFEX techniques greatly simplifies the spectrum allowing the accurate identification of the rotationally resolved bands which is otherwise a formidable task due to the intrinsic complexity of the A (2)A(1)--X (2)B(1) transition. Highly excited states of A(0,7,0), A(0,8,0), and A(0,9,0) vibronic levels with different K quantum numbers which are located above the barrier to linearity are thoroughly investigated. The bent-to-quasilinear transition of H(2)S(+) above the barrier to linearity shows the characteristics of the Renner-Teller effect, showing the large A rotational constant and strong intensity borrowing of the highly vibrationally excited ground levels such as X(0,23,0) or X(0,24,0) in the dipole-allowed excitation. Spectroscopic parameters of term values, rotational, and spin-orbit coupling constants are precisely determined in this work, providing the most quantitative spectroscopic structure of the H(2)S(+) to date. Quantum-state dependent photodissociation dynamics are also discussed from spectral features of PHOFEX.

Archaebacteria Sulfolobus acidocaldarius contains the highly thermophilic cytochrome P450 enzyme (CYP119). CYP119 possesses stable enzymatic activity at up to 85 degrees C. However, this enzyme is still considered as an orphan P450 without known physiological function with endogenous or xenobiotic substrates. We characterized the regioselectivity of lauric acid by CYP119 using the auxiliary redox partner proteins putidaredoxin (Pd) and putidaredoxin reductase (PdR). Purified CYP119 protein showed a tight binding affinity to lauric acid (K(d)=1.1+/-0.1 microM) and dominantly hydroxylated (omega-1) position of lauric acid. We determined the steady-state kinetic parameters; k(cat) was 10.8 min(-1) and K(m) was 12 microM. The increased ratio to omega-hydroxylated production of lauric acid catalyzed by CYP119 was observed with increase in the reaction temperature. These studies suggested that the regioselectivity of CYP119 provide the critical clue for the physiological enzyme function in this thermophilic archaebacteria. In addition, regioselectivity control of CYP119 without altering its thermostability can lead to the development of novel CYP119-based catalysts through protein engineering.