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The potentiometric response characteristics of lithium ion-selective membrane electrodes employing three different THF-type 16-crown-4 derivatives as ionophores were investigated. Of the THF- based crown compounds tested, a cis-type isomer of the derivative with only methyl groups (H/CH3) at four bridged positions (cis-THF-I) was found to exhibit the best selectivity for Li+ over other cations. The composition of the cis-THF-I-based PVC-matrix membrane was optimized and its performance was compared with those based on lithium ionophores reported previously. The new cis-THF-I-based membrane exhibits remarkably enhanced selectivities for lithium over other cations: logKpotLi,Na = –2.8, logKpotLi,K = –4.6, logKpotLi,NH4 = –5.4, logKpotLi,Ca = –5.7 and logKpotLi,Mg = –5.4. Furthermore, it was found that serum components do not interfere significantly with the lithium measurements when the cis-THF-I-based electrode is used. The response of the electrode was found to be linear in the clinical range of interest.

A simple and versatile method for the Formation of Arrays of Giant Vesicles with Controlled Size and Composition. The ability of this technique to generate arrays of giant liposomes from a wide range of membrane lipids and protein compositions is demonstrated. The resulting vesicles are utilized for studying protein activity, lipid-protein interactions, and protein-protein interactions.

A major goal in drug design is the improvement of computational methods for docking and scoring. The Community Structure Activity Resource (CSAR) has collected several data sets from industry and added in-house data sets that may be used for this purpose ( www.csardock.org). CSAR has currently obtained data from Abbott, GlaxoSmithKline, and Vertex and is working on obtaining data from several others. Combined with our in-house projects, we are providing a data set consisting of 6 protein targets, 647 compounds with biological affinities, and 82 crystal structures. Multiple congeneric series are available for several targets with a few representative crystal structures of each of the series. These series generally contain a few inactive compounds, usually not available in the literature, to provide an upper bound to the affinity range. The affinity ranges are typically 3-4 orders of magnitude per series. For our in-house projects, we have had compounds synthesized for biological testing. Affinities were measured by Thermofluor, Octet RED, and isothermal titration calorimetry for the most soluble. This allows the direct comparison of the biological affinities for those compounds, providing a measure of the variance in the experimental affinity. It appears that there can be considerable variance in the absolute value of the affinity, making the prediction of the absolute value ill-defined. However, the relative rankings within the methods are much better, and this fits with the observation that predicting relative ranking is a more tractable problem computationally. For those in-house compounds, we also have measured the following physical properties: logD, logP, thermodynamic solubility, and pK(a). This data set also provides a substantial decoy set for each target consisting of diverse conformations covering the entire active site for all of the 58 CSAR-quality crystal structures. The CSAR data sets (CSAR-NRC HiQ and the 2012 release) provide substantial, publically available, curated data sets for use in parametrizing and validating docking and scoring methods.

Radio frequency identification (RFID) systems compliant to the EPCglobal Generation 2 (Gen2) passive UHF RFID protocol are being deployed in a broad range of applications including access control, automated tolling, personal identification, anti-counterfeiting, and supply chain management. With the broad applications and the demand for ever increasing amounts of on-tag functionality, security on the tag has become a critical enabling functionality in many applications. To address this growing marketplace need, EPCglobal is developing a standard security framework within which security functionality may be integrated seamlessly into the Gen2 protocol. We review the proposed Gen2 security framework and introduce example cryptographic suites to illustrate how to utilize this framework to provide a range of security functionality. We analyze the security of the Gen2 protocol and this new functionality in the context of timing-based attacks. We conclude that the tight communication timings specified in the Gen2 protocol mitigate timing-based attacks; however, the loose timing implementations on commercial interrogators and limited timing enforcement on tags lesson the effectiveness of the specified timing constraints. Further, we conclude that the new security framework allows for the efficient integration of secure functionality that, as specified, is resistant to timing-based attacks; however, we caution that using the delayed response of the new Gen2 security functionality creates new vulnerabilities to timing based attacks such as relay attacks and man-in-the-middle attacks.

Individuals with Mowat-Wilson syndrome (MWS; OMIM#235730) have characteristic facial features, a variety of congenital anomalies such as Hirschsprung disease, and intellectual disabilities caused by mutation or deletion of ZEB2 gene. This deletion or cytogenetic abnormality has been reported primarily from Europe, Australia and the United States, but not in Korea. Here we report a patient with characteristic facial features of MWS, developmental delay and spasticity. High resolution microarray analysis revealed 0.9 Mb deletion of 2q22.3 involving two genes: ZEB2 and GTDC1. This case shows the important role of high resolution microarray in patients with unexplained psychomotor retardation and/or facial dysmorphism. Knowledge about the most striking clinical signs and implementation of effective molecular tests like microarray could significantly increase the detection rate of new cases of MWS in Korea. This is the first reported case of MWS in Korea.

The crystal structure of a new potassium/hydro­nium tungstophosphate, potassium hydro­nium phosphate tungstate, K2.4(H3O)0.6PO4W12O36, obtained by hydro­thermal synthesis at low pH, has been determined by single-crystal X-ray diffraction. Regular PO4 tetrahedra are located on sites of \overline 43m symmetry. 12 W atoms are in a shell around the phosphate anion and are surrounded by O atoms in a distorted octahedral coordination. The close packing of the heteropoly­anionic units forms orthogonally intersecting channels where the statistically distributed potassium/hydro­nium ions are situated.

Background: Angiogenic progress is complex and several factors are involved as a promoter or inhibitor of angiogenesis. Tumor can use the imbalance of pro- and antiangiogenic factors in progression. The CXC chemokine family are consisted of pro- or antiangiogenic factors. Recent studies revealed CXCL10 as an anti-angiogenic factor in response to angiogenic factors like bFGF or VEGF. However, a role of CXCL10 in cancer and angiogenesis is unclear. Here, we investigated the role of CXCL10 on tumor angiogenesis and its mechanisms in renal cell carcinoma. Methods: Human vascular endothelial cell (HUVEC) was used to evaluate the effects of CXCL10 on endothelial cells. MTS assay was performed to assess cell proliferation. Cell cycle analysis was performed using flow cytometry with propidium iodide. RT-PCR and western blot were performed to assess the baseline expression of CXCL10 in a panel of renal cell carcinoma cells. Overexpression of CXCL10 was developed by lentiviral transfection. Quantitative RT-PCR for angiogenic factors (VEGF, PDGF, FGF, MMP9) was performed by angiogenesis multigene screening array. Subcutaneous tumor xenografts were developed using Caki-1 cells to evaluate in vivo effects of CXCL10. Results: CXCL10 inhibited proliferation and tube formation of HUVEC in a dose-dependent manner, while it did not affect in vitro tumor proliferation of renal cell carcinoma cells. Expression of CXCL10 was downregulated in Caki-1, UMRC3 and UMRC6 cells. Overexpression of CXCL10 suppressed expression of angiogenic factors including VEGF-A, PDGF, FGF2 and MMP9 in Caki-1 cells, suggesting that CXCL10 is involved non only as an anti-angiogenic factor but also as a suppressor of critical angiogenic factors in renal cell carcinoma. In vivo Restoration of CXCL10 expression suppresses tumor growth in renal cell carcinoma by suppressing tumor angiogenesis in xenografts. Conclusions: CXCL10 has a role as an anti-angiogenic factor and also as a suppressor of critical angiogenic factors in renal cell carcinoma. Restoration of CXCL10 expression suppresses tumor growth in renal cell carcinoma by impeding expression of other angiogenic factors including VEGF, PDGF, FGF, MMP9 in renal cell carcinoma as well as inducing anti-angiogenic effect on endothelial cells. Our data suggest that targeting CXCL10 might be a novel therapeutic option for advanced renal cell carcinoma. Citation Format: Hyun A Jin, Jun Hyeok Heo, You Hyun Kang, Ki Hong Kim, Kyung Suk Han, Sung Joon Hong. CXCL10 suppresses tumor angiogenesis and impedes expression of critical angiogenic factors in renal cell carcinoma. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr B18.

Background: Immunotherapy based on BCG has been a standard therapy to prevent recurrence and progression in bladder cancer. Activation of immune checkpoints by binding PD-1 to its ligands, PD-L1, is vital for physiologic regulation of immune system. Tumor cells can co-opt the PD-1 pathway to escape from immunosurveillance mechanism. We investigated expression of immune checkpoints and their effects on T cells in bladder cancer. Methods: A panel of transitional cell carcinoma cell lines (UC-3,T24,KU1919 and 253J) and normal human bladder urothelial cells (SVHUC1) were used to evaluate expression of immune checkpoints. Western blot analysis was used for protein expressions from cell lines and qPCR for RNA expression from human bladder tissues. Human bladder cancer/normal paired tissues were obtained at two time points; surgical resection and then after BCG immunotherapy. Immunohistochemical staining was performed to assess infiltration and distribution of CD4+ and CD8+ T cells in bladder tumor tissues and normal bladder tissues. Immune cells were isolated and T cells were isolated by FACS sorting after immune cell isolation from tumor/normal tissues. Results: PD-L1 proteins were highly expressed in bladder cancer cell lines compared to normal bladder epithelial cell line (SVHUC1). In paired human samples (cancer and normal tissues), PD-L1 were also highly expressed in bladder cancer tissues compared with paired normal tissues. Real time RT-PCR confirmed the high expression of PD-L1 in bladder cancer tissues. Interestingly, normal epithelial tissues regenerated after surgical resection of the tumor and BCG immunotherapy had low PD- after surgical resection and BCG immunotherapy while recurred tumor showed high expression of PD-L1. Isolation of TIL and immunohistochemical staining with CD4/CD8 showed that T cells were more infiltrated in bladder cancer tissues compared to paired normal tissues. Mouse bladder tumor model expressing PD-L1 showed increased CD3+CD4+ T cells and Treg cells. Conclusion: PD-L1 are highly expressed in bladder cancer. CD3+CD4+ T cells and Treg cells are highly infiltrated in human and mouse bladder cancer tissues. Our data suggest that PD-L1 might be involved in tumor recurrence after BCG immunotherapy and might be a novel targets in bladder cancer. Citation Format: Jun Hyeok Heo, Hyun A. Jin, You Hyun Kang, Ki Hong Kim, Sung Joon Hong, Kyung Seok Han. Expression of PD-L1 and BCG immunotherapy in non-muscle invasive bladder cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr A16.

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) induces apoptosis selectively in cancer cells while sparing normal cells. However, many cancer cells are resistant to TRAIL-induced cell death. In this study, we examined whether Aurora B, which is frequently overexpressed in cancer cells, is associated with TRAIL resistance. The protein levels of Aurora B were higher in TRAIL-resistant cancer cell lines than in TRAIL-sensitive cancer cell lines. Exogenously expressed Aurora B attenuated TRAIL-induced apoptosis in the tested TRAIL-sensitive cancer cell lines, whereas the small interfering RNA-mediated suppression of Aurora B expression stimulated TRAIL-mediated apoptosis in the tested TRAIL-resistant cancer cell lines. Furthermore, combined treatment with TRAIL and ZM447439, a specific inhibitor of Aurora B, synergistically induced apoptosis in various TRAIL-resistant cancer cells, suggesting that this combined regimen may represent an attractive strategy for effectively treating TRAIL-resistant malignant cancers. Mechanistically, the inhibition of Aurora B activity in various cancer cells commonly downregulated survivin protein levels and potentiated the activation of caspase-3. In addition, Aurora B inhibition induced mitotic catastrophe, which also contributed to the sensitization of cells to TRAIL-mediated apoptosis. Interestingly, forced overexpression of Aurora B increased the protein levels of survivin, but not those of a non-phosphorylatable survivin mutant in which threonine 117 was replaced by alanine, indicating that phosphorylation of survivin is required for this effect. Furthermore, TRAIL-induced apoptosis in MDA-MB-435S cells was attenuated by wild-type survivin but not by the non-phosphorylatable survivin mutant. Collectively, our results demonstrate that Aurora B confers TRAIL resistance to cancer cells via phosphorylation of survivin.

The crystallization and structural characterization of bovine liver catalase (BLC) has been intensively studied for decades. Forms I and II of BLC have previously been fully characterized using single-crystal X-ray diffraction. Form III has previously been analyzed by electron microscopy, but owing to the thinness of this crystal form an X-ray crystal structure had not been determined. Here, the crystal structure of form III of BLC is presented in space group P2(1)2(1)2(1), with unit-cell parameters a = 68.7, b = 173.7, c = 186.3 Å. The asymmetric unit is composed of the biological tetramer, which is packed in a tetrahedron motif with three other BLC tetramers. This higher resolution structure has allowed an assessment of the previously published electron-microscopy studies.

Tumor necrosis factor-related apoptosis-induced ligand (TRAIL) induces apoptosis selectively in cancer cells while sparing normal cells. However, many cancer cells are resistant to TRAIL-induced cell death. Here, we report that paxilline, an indole alkaloid from Penicillium paxilli, can sensitize various glioma cells to TRAIL-mediated apoptosis. While treatment with TRAIL alone caused partial processing of caspase-3 to its p20 intermediate in TRAIL-resistant glioma cell lines, co-treatment with TRAIL and subtoxic doses of paxilline caused complete processing of caspase-3 into its active subunits. Paxilline treatment markedly upregulated DR5, a receptor of TRAIL, through a CHOP/GADD153-mediated process. In addition, paxilline treatment markedly downregulated the protein levels of the short form of the cellular FLICE-inhibitory protein (c-FLIPS) and the caspase inhibitor, survivin, through proteasome-mediated degradation. Taken together, these results show that paxilline effectively sensitizes glioma cells to TRAIL-mediated apoptosis by modulating multiple components of the death receptor-mediated apoptotic pathway. Interestingly, paxilline/TRAIL co-treatment did not induce apoptosis in normal astrocytes, nor did it affect the protein levels of CHOP, DR5 or survivin in these cells. Thus, combined treatment regimens involving paxilline and TRAIL may offer an attractive strategy for safely treating resistant gliomas.

Metastasis is a major cause of death in cancer patients. Our previous studies showed that pinosylvin, a naturally occurring trans-stilbenoid mainly found in Pinus species, exhibited a potential cancer chemopreventive activity and also inhibited the growth of various human cancer cell lines via the regulation of cell cycle progression. In this study, we further evaluated the potential antimetastatic activity of pinosylvin in in vitro and in vivo models. Pinosylvin suppressed the expression of matrix metalloproteinase (MMP)-2, MMP-9 and membrane type 1-MMP in cultured human fibrosarcoma HT1080 cells. We also found that pinosylvin inhibited the migration of HT1080 cells in colony dispersion and wound healing assay systems. In in vivo spontaneous pulmonary metastasis model employing intravenously injected CT26 mouse colon cancer cells in Balb/c mice, pinosylvin (10 mg/kg body weight, intraperitoneal administration) significantly inhibited the formation of tumor nodules and tumor weight in lung tissues. The analysis of tumor in lung tissues indicated that the antimetastatic effect of pinosylvin coincided with the down-regulation of MMP-9 and cyclooxygenase-2 expression, and phosphorylation of ERK1/2 and Akt. These data suggest that pinosylvin might be an effective inhibitor of tumor cell metastasis via modulation of MMPs.

The first synthesis of 2′-deoxy-2′-fluoro-4′-selenoarabinofuranosyl pyrimidines as potent anticancer agents was accomplished using the DAST fluorination as a key step. It was first revealed that selenium atom participated in the DAST fluorination of 4′-selenonucleosides and that conformational bias induced by bulky selenium acted as a decisive factor in the DAST fluorination. Among compounds tested, 2′-F-4′-seleno-ara-C (4a) exhibited highly potent anticancer activity in all cancer cell lines tested and was more potent than ara-C (1).

Objectives Quantitative fluorescence polymerase chain reaction (QF-PCR) is a rapid and reliable method for screening common aneuploidies, but it is not an accustomed way of testing in Korea. Our objectives were to investigate QF-PCR as a means for prenatal aneuploidy screening and to evaluate the short tandem repeat (STR) markers in a Korean population. Method Three formats of QF-PCR assays that utilize an Elucigene kit (Tepnel Diagnostics), which contains 7 primer pairs located on chromosome 21, 16 primers on chromosomes 21, 18, and 13, or 26 primers on chromosomes 21, 18, 13, X, and Y were performed. Results Eight hundred of prenatal samples were screened and the results were compared to that of the results obtained with conventional cytogenetics. The 31 of 33 (93%) autosomal and sex chromosome aneuploidies were detected excluding one case of maternal cell contamination and one case of mosaicism respectively; there were no false positives. Interestingly, submicroscopic duplication of the D13S634 marker was observed in 1.6% of cases. Conclusion For prenatal aneuploidy screening, QF-PCR was proved to be efficient and reliable. However, considering the high frequency of triallelic patterns of one STR marker that has not been found in other populations, careful evaluation is recommended in each STR marker when it is applied to different populations. Copyright © 2009 John Wiley & Sons, Ltd.

Dibenzocyclooctadiene lignans isolated from Schisandra chinensis showed antiproliferative effects in various human cancer cells. The methoxy groups at C-3, C-4, C-3', and C-4', the hydroxyl group at C-8', and the stereo-configuration of the biphenyl ring and the angeloyl group might have influence on these activities. Additional studies indicate that one of mechanism of action of an active compound schizantherin C in A549 human lung cancer cells was related to the inhibition of cell cycle progression in G0/G1 phase.

Human A3 adenosine receptor (A3AR) agonists showed the anti-tumor activity in various in vitro and in vivo studies. The present study investigates the anti-proliferative effect of a novel adenosine analog 2-chloro-N6-(3-iodobenzyl)-4'-thioadenosine-5'-N-methyluronamide (thio-Cl-IB-MECA) in A549 human lung cancer cells. Thio-Cl-IB-MECA induced arrest of cell cycle progression in G0/G1 phase at lower concentrations (up to 20 microM) and apoptotic cell death at a higher concentration (80 microM), which were manifested by down-regulation of cyclin D1, c-myc, and CDK4, activation of caspase-3 and -9, and cleavage of poly(ADP-ribose) polymerase (PARP). The activation of Akt-mediated signaling was also inhibited by treatment with thio-Cl-IB-MECA. These data might suggest the potential therapeutic value of an adenosine analog in the treatment of human lung cancer.

As a demand for high speed uplink packet services increases, the WCDMA enhanced uplink, also known as high speed uplink packet access (HSUPA), has been specified in release 6 by 3GPR The performance of the HSUPA has been improved using fast scheduling, hybrid ARQ, and shorter TTI. This HSUPA will provide various types of multimedia services, such as realtime video streaming, gaming, VoIP, and FTP. Generally, the performance of HSUPA is dominated by scheduling policy. Therefore, it is required to design a scheduling algorithm considering the traffic characteristics to provide QoS guaranteed services in various traffic environments. In this paper, we propose a scheduling algorithm considering the traffic characteristics to guarantee QoS in a mixed traffic environment. Finally, the performance of the proposed scheduling algorithm is evaluated in terms of average packet delay, packet delay jitter, and system throughput using a system level simulator.

The first tungsten borophosphate compound, K5[W4O8(H2BO4)(HPO4)2(PO4)2]·0.5H2O, has been synthesized under mild hydrothermal conditions, and the crystal structure with a new stoichiometry (B:P rati...

A new prenylated chalcone, 3' ',3' '-dimethylpyrano[3',4']2,4,2'-trihydroxychalcone (1), was isolated from the heartwood of Artocarpus communis. Two flavonoid derivatives, (-)-cycloartocarpin (9) and (-)-cudraflavone A (10), were isolated as new isomers. In addition, eight known flavonoids, isobacachalcone (2), morachalcone A (3), gemichalcones B (4) and C (5), artocarpin (6), cudraflavone C (7), licoflavone C (8), and (2S)-euchrenone a(7) (11), were isolated and identified from this plant for the first time. Compounds 1-4, 6, and 11 exhibited potent inhibitory activity on nitric oxide production in RAW264.7 LPS-activated mouse macrophage cells with IC(50) values of 18.8, 6.4, 16.4, 9.3, 18.7, and 12.3 microM, respectively. The structure of compound 1 was elucidated by spectroscopic data analysis, including 1D and 2D NMR experiments.

Endo-1,3-beta-glucanases hydrolyze internal 1,3-beta-glucosyl linkages. The endo-1,3-beta-glucanase from Arthrobacter sp. was crystallized by the hanging-drop vapour-diffusion method. The crystals belonged to space group P4(1), with unit-cell parameters a = 71.31, c = 60.07 A, and contained one molecule per asymmetric unit. The Matthews coefficient (VM) and the solvent content were 2.35 A3 Da(-1) and 47.63%, respectively. Diffraction data were collected to a resolution of 1.66 A at SPring-8 using a MAR CCD area detector and gave a data set with an overall Rmerge of 5.4% and a completeness of 99.4%.

To survey a diarrhea outbreak in Guangan city and analyze the cause of the disease.The population enrolled in the surveillance came from four different settings and was randomly sampled. Stool specimens collected from diarrhea patients were tested ordinarily for enteric bacteria and further examined for viral pathogens with PAGE, ELISA and RT-PCR.In total, 4,567 persons were surveyed, among them 942 had acute diarrhea (prevalence 20.63%). The incidence was higher in rural area (28.6%) than in urban area (19.6%) (chi-square =22.29, P less than 0.005) with a peak in May 10 through 25 four human caliciviruses were detected from stool specimens by ELISA and RT-PCR in specimens from 4 and 1 patients, respectively.Human calicivirus probably was the cause of this diarrhea outbreak in Guangan city.

Monodehydroascorbate (MDA) radical reductase (EC 1.6.5.4) is an FAD enzyme that catalyzes the univalent reduction of MDA radical to ascorbate using NAD(P)H as an electron donor. The recombinant MDA reductase from cucumber was crystallized using polyethylene glycol 6000 as a precipitant. The crystals belong to space group P2(1), with unit-cell parameters a = 60.8, b = 138.6, c = 61.7 A, beta = 114.5 degrees, and contained two molecules per asymmetric unit. The Matthews coefficient (VM) and the solvent content are 2.46 A3 Da(-1) and 50.0%, respectively. Diffraction data were collected to a resolution of 2.4 A at 100 K using Cu Kalpha radiation with a multi-wire area detector and gave a data set with an overall Rsym of 10.0% and a completeness of 92.5%.

Normal cells lose their ability to divide after a finite number of cell divisions. Under the influence of Simian virus 40 (SV 40) large T-antigen, which interacts with the cell cycle regulators p53 and pRb, cells enter a phase of an extended pre-immortalized cells. Immortalization-up-regulated protein 1 (IMUP-1) and immortalization-up-regulated protein 2 (IMUP-2) genes have been recently cloned and are known to be involved in SV40-mediated immortalization. However, the roles of IMUP-1 and IMUP-2 are not known in human ovarian epithelial tumors.This study was performed to determine mRNA expression and intracellular localization of the IMUP-1 and IMUP-2 in ovarian epithelial tumors by RT-PCR and immunohistochemical staining using human IMUP-1 and IMUP-2 monoclonal antibodies.IMUP-1 (4.0-fold) and IMUP-2 (2.4-fold) mRNA expression in ovarian epithelial tumors were significantly higher than in normal ovarian tissues (p0.05). The mRNAs of IMUP-1 and IMUP-2 in the ovarian cancer cell lines were about 4.9- and 2.9-fold compared to the normal ovarian cell line, respectively. The subcellular expression of these two proteins in immunohistochemical stains was detected mainly in the nucleus of tumor cells, whereas adjacent normal ovarian stromal cells were faintly or negatively stained with these proteins. However, the staining intensity of IMUP-1 and IMUP-2 in ovarian epithelial tumors were not different between histological types or grades.These results showed the up-regulation of IMUP-1 and IMUP-2 in human ovarian epithelial tumors and suggested that the altered mRNA level of these molecules is possibly associated with ovarian tumorigenesis.

Comparison of the architecture around the active site of soybean beta-amylase and Bacillus cereus beta-amylase showed that the hydrogen bond networks (Glu380-(Lys295-Met51) and Glu380-Asn340-Glu178) in soybean beta-amylase around the base catalytic residue, Glu380, seem to contribute to the lower pH optimum of soybean beta-amylase. To convert the pH optimum of soybean beta-amylase (pH 5.4) to that of the bacterial type enzyme (pH 6.7), three mutants of soybean beta-amylase, M51T, E178Y, and N340T, were constructed such that the hydrogen bond networks were removed by site-directed mutagenesis. The kinetic analysis showed that the pH optimum of all mutants shifted dramatically to a neutral pH (range, from 5.4 to 6.0-6.6). The Km values of the mutants were almost the same as that of soybean beta-amylase except in the case of M51T, while the Vmax values of all mutants were low compared with that of soybean beta-amylase. The crystal structure analysis of the wild type-maltose and mutant-maltose complexes showed that the direct hydrogen bond between Glu380 and Asn340 was completely disrupted in the mutants M51T, E178Y, and N340T. In the case of M51T, the hydrogen bond between Glu380 and Lys295 was also disrupted. These results indicated that the reduced pKa value of Glu380 is stabilized by the hydrogen bond network and is responsible for the lower pH optimum of soybean beta-amylase compared with that of the bacterial beta-amylase.

In spite of the high similarity of amino acid sequence and three-dimensional structure between soybean beta-amylase (SBA) and sweet potato beta-amylase (SPB), their quaternary structure is quite different, being a monomer in SBA and a tetramer in SPB. Because most of the differences in amino acid sequences are found in the surface region, we tested the tetramerization of SBA by examining mutations of residues located at the surface. We designed the SBA tetramer using the SPB tetramer structure as a model and calculating the change of accessible surface area (DeltaASA) for each residue in order to select sites for the mutation. Two different mutant genes encoding SB3 (D374Y/L481R/P487D) and SB4 (K462S added to SB3), were constructed for expression in Escherichia coli and the recombinant proteins were purified. They existed as a monomer in solution, but gave completely different crystals from the native SBA. The asymmetric unit of the mutants contains four molecules, while that of native SBA contains one. The interactions of the created interfaces revealed that there were more intermolecular interactions in the SB3 than in the SB4 tetramer. The substituted charged residues on the surface are involved in interactions with adjacent molecules in a different way, forming a new crystal packing pattern.

Objective: Perilipins are phosphoproteins that are localized to the surface of triacylglycerol droplets within adipocytes where they regulate the rate of lipolysis. We sought to determine the effects of severe obesity and depot [omental (Om) vs. subcutaneous (Sc)] on perilipin expression in the adipose tissue of individuals. Research Methods and Procedures: Samples of Om and Sc adipose tissues obtained at surgery from severely obese subjects and fat aspirations from nonobese subjects were analyzed for perilipin protein and mRNA levels by Northern and Western analysis. Results: Perilipin A (periA) was the major perilipin expressed in adipose tissues. periA mRNA relative abundance was significantly lower in Sc adipose tissue from severely obese compared to that from nonobese subjects. Western blotting of adipose tissue extracts showed that periA protein levels expressed relative to tissue protein or fat cell surface area were significantly lower (∼ −40%) in abdominal Sc adipose tissue from severely obese compared to that from nonobese subjects. However, the calculated mass of perilipin per fat cell did not differ between the two groups. Perilipin mRNA levels were higher in Sc compared to Om adipose tissue from obese individuals (p < 0.025; n = 26; 17 women, 9 men); however, periA protein levels did not differ. In addition, perilipin protein, but not mRNA, levels were higher in Sc adipose tissue from obese men than from women (p < 0.025). Discussion: Variations in perilipin expression may contribute to the higher basal lipolytic rates observed in obese compared to nonobese individuals and in obese women compared to obese men.

Tumor necrosis factor-alpha (TNF-alpha) increases adipocyte lipolysis after 6-12 h of incubation. TNF-alpha has been demonstrated to activate mitogen-activated protein (MAP) kinases including extracellular signal-related kinase (ERK) and N-terminal-c-Jun-kinase (JNK) in different cell types. To determine if the MAP kinases have a role in TNF-alpha-induced lipolysis, 3T3-L1 adipocytes were treated with the cytokine (10 ng/ml), in the presence or absence of PD98059 or U0126 (100 micromoles), specific inhibitors of ERK activity. We demonstrated that U0126 or PD98059 blocked TNF-alpha-induced ERK activity and decreased TNF-alpha-induced lipolysis by 65 or 76% respectively. The peroxisome-proliferator-activated receptor gamma (PPARgamma) agonists, rosiglitazone (ros), and 15-deoxy-Delta-(12,14)- prostaglandin J(2) (PGJ2) have been demonstrated to block TNF-alpha-induced lipolysis. Pretreatment of adipocytes with these agents almost totally blocked TNF-alpha-induced ERK activation and reduced lipolysis by greater than 90%. TNF-alpha also stimulated JNK activity, which was not affected by PD98059 or PPARgamma agonist treatment. The expression of perilipin, previously proposed to contribute to the mechanism of lipolysis, is diminished in response to TNF-alpha treatment. Pretreatment of adipocytes with PD98059 or ros significantly blocked the TNF-alpha-induced reduction of perilipin A protein level as determined by Western analysis. These data suggest that activation of the ERK pathway is an early event in the mechanism of TNF-alpha-induced lipolysis.

Multidrug resistance (MDR), a phenomenon in which cells become resistant to a number of drugs, is a growing obstacle for treatment of cancer and bacterial infections. A group of membrane transporters, known as MDR efflux pumps that bind to a wide range of foreign molecules and pump them out of the cell, are suspected to be the main contributors to MDR. P-glycoprotein (Pgp), which is the most well studied MDR efflux pump, has a critical role in the distribution of drugs in the body. As a result, screening the interactions between this protein and candidate drugs has recently become a required step in the FDA approval process. Biophysical characterization of Pgp and its transport activity may provide key information for the design and development of effective therapeutics. Giant unilamellar vesicles (GUV) are excellent models for natural cell membranes and present appealing platforms for biophysical studies of Pgp under well-defined conditions. Here, we reconstitute Pgp from Chinese hamster ovary B30 cells in giant unilamellar vesicles and investigate its ATPase activity as well as its transport activity.In this study, we applied small proteoliposomes with reconstituted Pgp for electroformation of Pgp-containing giant vesicles via our recently reported technique of hydrogel-assisted electroformation. These electroformed giant vesicles were characterized using fluorescence microscopy, immunohistochemistry, and ATPase assays. Fluorescence immunostaining confirmed the presence of Pgp in the membrane of GUVs. ATPase assay demonstrated that Pgp in GUVs retained its ATPase activity upon electroformtion. The specific ATPase activity of Pgp in GUVs was about 640 nmol/mg⋅min, comparable to that of this protein in small proteoliposomes, ∼660 nmol/mg⋅min. Moreover, we are investigating the transport activity of Pgp in these giant liposomes using fluorescence microscopy. The present study may provide valuable insight to the function of this MDR efflux pump.

In this work, we report on a self-forming barrier process in Cu–Mn alloys. Cu–Mn alloy films were directly deposited onto low-k substrates by co-sputtering and then subjected to an annealing treatment at various temperatures. X-ray diffraction patterns obtained for the Cu–Mn alloys showed Cu(111), Cu(200), and Cu(220) peaks, while transmission electron microscopy images revealed that a uniform Mn-based interlayer self-formed at the Cu–Mn/low-k interface after annealing. In order to evaluate the barrier properties of the Mn-based interlayer, thermal stability measurements were carried out with the low-k dielectrics. The Cu–Mn alloy showed improved thermal stability when compared to a pure Cu reference sample. The chemical composition of the self-formed interlayers on the low-k substrates was ultimately investigated by X-ray photoelectron spectroscopy analysis. Our results show that the composition of the self-formed interlayers depends on the oxide and carbon concentrations in the low-k material.

The first tungsten borophosphate compound, K5[W4O8(H2BO4)(HPO4)2(PO4)2]·0.5H2O, has been synthesized under mild hydrothermal conditions, and the crystal structure with a new stoichiometry (B:P rati...

The authors synthesized a Cu–Al alloy by employing alternating atomic layer deposition (ALD) surface reactions using Cu and Al precursors, respectively. By alternating between these two ALD surface chemistries, the authors fabricated ALD Cu–Al alloy. Cu was deposited using bis(1-dimethylamino-2-methyl-2-butoxy) copper as a precursor and H2 plasma, while Al was deposited using trimethylaluminum as the precursor and H2 plasma. The Al atomic percent in the Cu–Al alloy films varied from 0 to 15.6 at. %. Transmission electron microscopy revealed that a uniform Al-based interlayer self-formed at the interface after annealing. To evaluate the barrier properties of the Al-based interlayer and adhesion between the Cu–Al alloy film and SiO2 dielectric, thermal stability and peel-off adhesion tests were performed, respectively. The Al-based interlayer showed similar thermal stability and adhesion to the reference Mn-based interlayer. Our results indicate that Cu–Al alloys formed by alternating ALD are suitable seed layer materials for Cu interconnects.

The crystal structure of potassium tetratin(II) tris­(ortho­phosphate), KSn4(PO4)3, obtained by hydro­thermal synthesis, has been determined from single-crystal X-ray diffraction data. The structure consists of [Sn4(PO4)3] units sharing common corners, which leads to a three-dimensional network structure and generates cages along the c direction. The cages are occupied by K atoms on threefold axes.

To achieve an optimum cold cathode emitter, well-aligned TiO2/Ti nanotube arrays (TNAs) were synthesized by anodic oxidation and doped with carbon by pyrolysis of C2H2 at 550 °C. By controlling the carbon doping content, the field emission (FE) properties of carbon-doped TiO2/Ti nanotube arrays (C-TNAs) were optimized. Compared with the high turn-on field of 19.19 V/μm from pure TNAs, the turn-on field of C-TNAs was decreased to 11.60, 6.35, 4.10, and 5.77 V/μm when the doping content of carbon was increased to 0.62, 0.82, 1.81, and 3.31 at. %, respectively. Obviously, the FE properties of TNAs were dramatically improved and optimized by adjusting the carbon doping content, which can be attributed to the typical tubular morphology, an enhanced conductivity, and a reduced work function.

Chronic pain is often associated with changes in the immune responses, which highlights the need for the aggressive pain control to obtain a better prognosis. This study examined splenic NK cell cytotoxicity in an attempt to assess the possible changes in the immune function under chronic neuropathic pain after a partial transsection of the sciatic nerve. Methods: After confirming tactile allodynia in response to the von Frey filament, a modified lactate dehydrogenase (LDH) release assay was used to determine the cytotoxic activity of splenic NK cells on the YAC-1 cell line in C3H/HeN (H-2k) mice (n = 6). NK cells as effector cells were mixed with YAC-1 cells as target cells (1 × 104/100μl), resulting in an effector-target ratio of 1：25, 1：50, 1：100 in the culture medium. Results: At 1 and 2 weeks after the nerve injury, all the subjects showed significant mechanical sensitivity compared with those observed before surgery. The percentage of NK cell cytotoxicity of the neuropathic mice increased significantly 1 week after the nerve injury but decreased within 2 weeks compared with the normal mice. Conclusions: In terms of the altered NK cell cytotoxicity, neuropathic pain can cause changes in the normal performance of the immune function.

Lymphoma is the third most common childhood malignancy after leukemia and brain tumor. In contrast to adult non-Hodgkin's lymphoma, that occurring in children is usually found extranodally. The most common site is the abdomen, including the gastrointestinal tract, kidney and pancreas, and the next most common is the extranodal head and neck. Gastrointestinal non-Hodgkin's lymphoma in adults is usually considered to be MALT lymphoma, a distinct B-cell type, but the occurrence of this variety in the small bowel of children is relatively rare. We report a case of high-grade MALT lymphoma occurring in a nine-year-old boy who presented with ileoileal intussusception.