Frizzled-7 receptor ectodomain expression in a colon cancer cell line induces morphological change and attenuates tumor growth

Frizzled (FZD) receptors have a conserved N-terminal extracellular cysteine-rich domain that interacts with Wnts and co-expression of the receptor ectodomain can antagonize FZD-mediated signalling. Using the ectodomain as an antagonist we have modulated endogenous FZD7 signalling in the moderately differentiated colon adenocarcinoma cell line, SK-CO-1. Unlike the parental cell line, which grows as tightly associated adherent cell clusters, the FZD7 ectodomain expressing cells display a spread out morphology and grow as a monolayer in tissue culture. This transition in morphology was associated with decreased levels of plasma membrane-associated E-cadherin and β-catenin, localized increased levels of vimentin and redistribution of α6 integrin to cellular processes in the FZD7 ectodomain expressing cells. The morphological and phenotype changes induced by FZD7 ectodomain expression in SK-CO-1 cells is thus consistent with the cells undergoing an epithelial-to-mesenchymal-like transition. Furthermore, initiation of tumor formation in a xenograft tumor growth assay was attenuated in the FZD7 ectodomain expressing cells. Our results indicate a pivotal role for endogenous FZD7 in morphology transitions that are associated with colon tumor initiation and progression.

嗜盐古菌启动子DNA片段的功能检测

将来源于嗜盐古菌染色体DNA的启动子片段RM07或RM13插入到启动子探针载体pYLZ_2的报告基因lacZ之前,通过β_半乳糖苷酶酶活性的检测,进一步确证RM07和RM13片段在大肠杆菌(Escherichia coli)中的启动功能。同时用微量热技术检测了大肠杆菌DH5α及其重组菌株在LB培养基中37℃生长过程的热输出功率。T2(pYLZ_2)、TE07(pYL726)、TE07_2(pYL702)、TE131(pYL131)和TE132(pYL132)菌株的生长速率分别比大肠杆菌DH5α降低了6.5

模拟生物膜的电化学研究

介绍了生物膜的组成、结构和一些基本性质，详细描述了各种生物膜模型（支撑磷脂双层膜、非支撑磷脂双层膜、泡囊等等）的制备方法。概要地总结了模拟生物膜的各个领域的研究情况，着重评述了模拟生物膜在电化学、生物传感器、膜片钳、图案化领域的研究进展。采用电化学、各种谱学以及扫描探针显微镜等方法对支撑双层磷脂膜、磷脂浇铸膜等不同的模拟生物膜体系进行了研究。同时还应用分子生物学的方法进行了基因的克隆及其融合蛋白的表达和纯化；另外对生物分子相互结合的动力学问题做了有意义的探索。主要结果如下：1两性霉素在支撑双层磷脂膜上的离子通道行为两性霉素是一种治疗系统性真菌感染的有效药物。通过在电极表面构筑双层磷脂膜作为两性霉素的人工靶向性，用通道离子作为探针分子，检测在两性霉素存在下通道的关闭与打开，建立了两性霉素与支撑双层磷脂膜的相互作用模型。2基于磷脂膜包被的活性肤一微过氧化物酶11的过氧化氢生物传感器首次将活性肤一微过氧化物酶11包被于磷脂膜内制成过氧化氢生物传感器。这一方法使过氧化氢的检侧限大大降低，同时也提高了检测灵敏度。磷脂膜为生物活性分子提供了一个天然的生物相容性的环境，为生物活性分子的有效固定和行使功能提供了良好的场所。3树枝状大分子诱导支撑双层磷脂膜表面的缺陷形成用电化学的方法研究了一系列的不同大小的树枝状大分子与支撑双层磷脂膜的相互作用。研究发现只有较大的分子才能在磷脂膜表面产生缺陷，而且这种诱导产生缺陷的能力还依赖于枝状大分子的结构和表面的电荷密度。4乳链菌肤与支撑双层磷脂膜的相互作用乳链菌肤是一种带正电荷的抗菌肤，它作用于带相反电荷的革兰氏阳性菌的细胞膜表面。通过在电极表面构筑双层磷脂膜来模拟细胞膜表面，使这一复杂的相互作用变得易于用电化学的手段来研究。实验结果表明，乳链菌肤在膜表面形成孔洞是其在膜表面的富集过程，随着乳链菌肤在膜中的浓度的增加，其协同性导致膜表面的孔洞增大，从而最终导致膜的完全被破坏。5电化学结合光谱的方法研究微过氧化物酶11与磷脂膜的相互作用用电化学和光谱的方法研究微过氧化物酶11与阳离子泡囊的相互作用。阳离子泡囊的引入使微过氧化物酶11由在溶液中的无规卷曲向有序的alpha-螺旋转变。这种构型的转变大部分是依靠于短肤分子与膜表面的静电相互作用。6用分子探针指示脂质体诱导的DNA的构型转变用甲基绿分子作为探针分子来检钡（脂质体诱导的DNA分子构型的转变。通过引入探针分子，检测到脂质体诱导DNA分子构型由B到C构型转变。探针分子的引入使DNA分子与脂质体这一复杂的相互作用变得简化。7果蝇热休克蛋白22基因的克隆及其融和蛋白的表达、纯化用PCR方法以果蝇的cDNA为模板扩增出热休克蛋白22基因（hsp22）片段，将其插入原核载体pUCm一T中在大肠杆菌中扩增，再将基因切下插入到原核表达载体pET（28a夕中在大肠杆菌中表达出hsp22:6*His融合蛋白，融合蛋白大小约23KDa，可以用Ni2＋-NTA亲合层析纯化。该蛋白可用来获得hsp22蛋白特异性抗体，从而为检测其在体内的表达以及研究hsp22的功能奠定基础。通过一系列的分子生物学技术获得了融合蛋白，为下一步进行定点突变认为设计所需要的蛋白质序列奠定了基础，为蛋白质折叠和分子识别提供了上游的研究对象和体系。8整体联系的生物分子结合的动力学研究我们讨论了在全局联系的情况下讨论了生物分子表面结合的动力学。对于配体和受体之间相对快速的与没有活化的过程的结合的动力学，具有很好的近似性。当动力学上的驱动力很大的时候，全局联系的观点具有很恰当的适应性。结合的动力学时间常数与温度之间呈“U”字型曲线。在高温的时候时间常数增加是由于在高温下结合变得非常不稳定；而在低温的时候，时间常数随着温度的下降而增加，这是由于陷入了局域的最小值状态。结合的能量地形面的波动也导致动力学随温度的变化而变化。

Chemical Constituents of a Marine-Derived Endophytic Fungus Penicillium commune G2M

Cultivation of the endophytic fungus Penicillium commune, which was isolated from the semi-mangrove plant Hibiscus tiliaceus, afforded one new compound 1-O-(2,4-dihydroxy-6-methylbenzoyl)-glycerol (1) along with thirteen known products, including 1-O-acetylglycerol (2), N-acetyltryptophan (3), 3-indolylacetic acid methyl ester (4), 1-(2,4-dihydroxy-3,5-dimethylphenyl)ethanone (5), 2-(2,5-dihydroxyphenyl)acetic acid (6), (4R,5S)-5-hydroxyhexan-4-olide (7), thymidine (8), uracil (9), thymine (10), ergosterol (11), beta-sitosterol (12), beta-daucosterol (13), and ergosta-7,22-dien-3 beta,5 alpha,6 beta-triol (14). The structures of these compounds were established by detailed NMR spectroscopic analysis, as well as by comparison with literature data or with authentic samples.

Inhibitory effects and mechanisms of high molecular-weight phlorotannins from Sargassum thunbergii on ADP-induced platelet aggregation

We evaluated the effects of high molecular-weight phlorotannins from Sargassum thunbergii (STP) on ADP-induced platelet aggregation and arachidonic acid (AA) metabolism in New Zealand white rabbits and Wistar rats. The inhibition of STP on platelet aggregation was investigated using a turbidimetric method, and the levels of the terminal products of AA metabolism were measured using the corresponding kits for maleic dialdehyde (MDA), thromboxane B-2 (TXB2) and 6-keto-prostaglandin F-1 alpha (6-keto-PGF(1 alpha)) by colorimetry and radioimmunoassay, as appropriate. We found that STP could inhibit ADP-induced platelet aggregation, and the inhibitory ratio was 91.50% at the STP concentration of 4.0 mg/mL. Furthermore, STP markedly affected AA metabolism by decreasing the synthesis of MDA (P < 0.01) and increasing the synthesis of 6-keto-PGF(1 alpha), thus changing the plasma TXB2/6-keto-PGF(1 alpha) balance when the platelets were activated (P < 0.01). Therefore, STP altered AA metabolism and these findings

Ergosterimide, a new natural Diels-Alder adduct of a steroid and maleimide in the fungus Aspergillus niger

Ergosterimide (1), a natural Diels-Alder adduct of ergosteroid and maleimide, was characterized from the culture extract of Aspergillus niger EN-13, an endophytic fungus isolated from the marine brown alga Colpomenia sinuosa. In addition, four known steroids including (22E,24R)-ergosta-5,7,22-trien-3 beta-ol (2), (22E,24R)-ergosta-4,6,8(14),22-tetraen-3one (3), (22E,24R)-5 alpha,8 alpha-epidioxyergosta-6,22-dien-3 beta-ol (4), and (22E,24R)-ergosta-7,22dien-3 beta,5 alpha,6 beta-triol. (5) were also isolated and identified. The structures of these compounds were elucidated by extensive analysis of 1D and 2D NMR and IR spectra and MS data. The plausible biosynthetic pathway of 1 was also discussed. To the best of our knowledge, 1 is the first natural Diels-Alder adduct of steroid and maleimide reported so far. (C) 2007 Elsevier Inc. All rights reserved.

Sesquiterpenes from red alga Laurencia tristicha

Three naturally new sesquiterpenes named 10-hydroxyepiaplysin, 10-hydroxyaplysin and 10-hydroxybromoepiaplysin have been isolated from Laurencia tristicha. On the basis of the spectroscopic techniques their structures were elucidated as (3S, 3 alpha R, 8 beta S)-(-)-2, 3, 3 alpha, 8 beta-tetrahydro-7-bromo-3-hydroxy-3, 3 alpha, 6,8 beta-tetramethyl-1H-cyclopenta[b]benzofuran, (3R, 3 alpha R, 8 beta S)(-)-2,3,3 alpha, 8 beta-tetrahydro-7-bromo-3-hydroxy-3, 3 alpha,6,8 beta-tetratnethyl-1H-cyclopenta[b]benzofuran and (3S, 3 alpha R, 8 beta S)-(-)-2, 3,3 alpha,8 beta-tetrahydro-3-hydroxy-3,3 alpha,6,8 beta-tetrainethyl-1H-cyclopenta[blbenzofuran, respectively.

Crystallographic and NMR studies on sterols from green alga Chaetomorpha basiretorsa Setchell

Chemical investigation of the ethanol extract of the marine green alga Chaetomorpha basiretorsa Setchell led to the isolation of a new sterol stigmast-4,28-dien-3 alpha 6 beta-diol 1 in addition to the five known sterols of beta-lawsaritol 2, saringosterol 3, 24-hydroperoxy-24-vinyl - cholesterol 4, beta-stigmasterol 5, 29-hydroxystigmasta-5, 24(28) -dien-3 beta-ol 6. Compounds were isolated by normal phase silica gel and Sephadex LH - 20 gel colum chromatography, reverse phase HPLC and recrystalization. Their structures were elucidated by spectroscopic methods including MS, IR 1D/2D NMR and X-ray analysis. Cytotoxicity of compounds was screened by using the standard WIT method. All these compounds were isolated from the green alga Chaetomorpha basiretorsa Setchell for the first time and they were inactive (50% inhibitory concentration was greater than 10 mu g /cm(3)) against KB, Bel -7402, PC - 3M, Ketr 3 and MCF - 7 cell lines.

Steroids from green alga Chaetomorpha basiretorsa Setchell

Six steroids have been isolated from ethanolic extract of green alga Chaetomorpha basiretorsa Setchell by a combination of repeated normal phase silica gel and Sephadex LH-20 gel column chromatography as well as recrystallization. Using spectroscopic methods including MS and NMR, their structures were determined as beta-lawsaritol (1), saringosterol (2), 24-hydroperoxy-24-vinyl-cholesterol (3), beta-stigmasterol (4), stigmast-4-en-3 alpha, 6 beta-diol (5), 29-hydroxystigmasta-5, 24 (28)-dien-3 beta-ol (6). All these compounds were obtained from this genus for the first time and they were inactive (IC50 > 10 mu g /ml) against KB, Bel-7402, PC-3M, Ketr 3 and MCF-7 cell lines.

Modulation de la réponse immunitaire dans le cerveau par la barrière hémato-encéphalique : implication en sclérose en plaques

La sclérose en plaques (SEP) est une maladie inflammatoire du système nerveux central (SNC) caractérisée par une infiltration périvasculaire de cellules mononucléaires, telles que les lymphocytes T CD4+ et CD8+, les lymphocytes B ainsi que les cellules myéloïdes qui comprend les monocytes, les macrophages et les cellules dendritiques (DCs). Ce phénomène d’infiltration est dû à une fragilisation de la barrière hémato-encéphalique (BHE). L’entrée des cellules immunitaires au SNC va mener à la destruction de la gaine de myéline et donc à l’apparition de plaques de démyélinisation. Ainsi, nous avons émis l’hypothèse que la migration des divers sous-types de cellules immunitaires du sang périphérique à travers la BHE est contrôlée par des mécanismes moléculaires distincts et spécifiques à chaque type cellulaire. Afin de répondre à cette hypothèse, quatre différentes études ont été mises sur pieds. En premier lieu, nous démontrons un effet bénéfique des statines sur la BHE en SEP, en diminuant la migration des lymphocytes T et des monocytes, et en diminuant la diffusion de marqueurs moléculaire soluble. Ce phénomène s’opère via la suppression du processus d’isoprenylation, et en empêchant probablement la contraction des cellules endothéliales de la BHE. De plus, nous démontrons que les monocytes qui migrent au SNC en condition inflammé sont en mesures de se différencier en DCs et d’induire une réponse inflammatoire de la part des lymphocytes T CD4+. La migration des monocytes à travers la BHE est contrôlée par une nouvelle molécule d’adhérence nommée Ninjurin-1. Le blocage de Ninjurin-1 conduit à une inhibition spécifique de la migration des monocytes in vitro, ainsi qu’à une amélioration des signes cliniques du modèle animal de la SEP, soit l’encéphalomyélite auto-immune expérimentale (EAE). Finalement, nous démontrons que la migration des lymphocytes T CD8+ au SNC s’effectue via l’intégrine alpha-4. De plus, la majorité des lymphocytes T CD8+ que l’on retrouve dans le liquide céphalo-rachidien de patients SEP, dans le SNC de souris EAE ainsi que dans le SNC de souris infectée au virus de l’hépatite murine portent un phénotype effecteur mémoire. Ces données pourraient expliquer l’émergence de leucoencéphalopathie multifocale progressive observée chez certains patients SEP traités au natalizumab, un anticorps dirigé contre l’intégrine alpha-4. En conclusion, notre étude a permis de démontrer l’importance des monocytes provenant de la périphérie dans le processus inflammatoire prenant part au SNC en SEP. L’inhibition d’entrée de ces cellules pourrait s’avérer bénéfique en SEP tout en permettant l’immuno-surveillance du cerveau, ce que l’anti-alpha-4 intégrine ne permet pas. Les statines pourraient s’avérer une autre option intéressante puisqu’elles agissent sur les processus inflammatoires impliqués dans la SEP.

Moderate exercise improves leucocyte function and decreases inflammation in diabetes

P>The genesis and progression of diabetes occur due in part to an uncontrolled inflammation profile with insulin resistance, increased serum levels of free fatty acids (FFA), proinflammatory cytokines and leucocyte dysfunction. In this study, an investigation was made of the effect of a 3-week moderate exercise regimen on a treadmill (60% of VO(2max), 30 min/day, 6 days a week) on inflammatory markers and leucocyte functions in diabetic rats. The exercise decreased serum levels of tumour necrosis factor (TNF)-alpha (6%), cytokine-induced neutrophil chemotactic factor 2 alpha/beta (CINC-2 alpha/beta) (9%), interleukin (IL)-1 beta (34%), IL-6 (86%), C-reactive protein (CRP) (41%) and FFA (40%) in diabetic rats when compared with sedentary diabetic animals. Exercise also attenuated the increased responsiveness of leucocytes from diabetics when compared to controls, diminishing the reactive oxygen species (ROS) release by neutrophils (21%) and macrophages (28%). Exercise did not change neutrophil migration and the proportion of neutrophils and macrophages in necrosis (loss of plasma membrane integrity) and apoptosis (DNA fragmentation). Serum activities of creatine kinase (CK) and lactate dehydrogenase (LDH) were not modified in the conditions studied. Therefore, physical training did not alter the integrity of muscle cells. We conclude that moderate physical exercise has marked anti-inflammatory effects on diabetic rats. This may be an efficient strategy to protect diabetics against microorganism infection, insulin resistance and vascular complications.

RNS derivation of N-point disk amplitudes from the revisited S-matrix approach

Recently, in [7] we proposed a revisited S-matrix approach to efficiently find the bosonic terms of the open superstring low energy effective lagrangian (OSLEEL). This approach allows to compute the alpha'(N) terms of the OSLEEL using open superstring n-point amplitudes in which n is considerably lower than (N + 2) (which is the order of the required amplitude to obtain those alpha'(N) terms by means of the conventional S-matrix approach). In this work we use our revisited S-matrix approach to examine the structure of the scattering amplitudes, arriving at a closed form for them. This is a RNS derivation of the formula first found by Mafra, Schlotterer and Stieberger [21], using the pure spinor formalism. We have succeeded doing this for the 5, 6 and 7-point amplitudes. In order to achieve these results we have done a careful analysis of the kinematical structure of the amplitudes, finding as a by-product a purely kinematical derivation of the BCJ relations (for N = 4, 5, 6 and 7). Also, following the spirit of the revisited S-matrix approach, we have found the alpha' expansions for these amplitudes up to alpha'(6) order in some cases, by only using the well known open superstring 4-point amplitude, cyclic symmetry and tree level unitarity: we have not needed to compute any numerical series or any integral involving polylogarithms, at any moment. (C) 2014 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/3.0/).

Augmented nitric oxide production and up-regulation of endothelial nitric oxide synthase during cecal ligation and perforation

Nitric oxide (NO) has been pointed out as being the main mediator involved in the hypotension and tissue injury taking place during sepsis. This study aimed to investigate the cellular mechanisms implicated in the acetylcholine (ACh)-induced relaxation detected in aortic rings isolated from rats submitted to cecal ligation and perforation (CLP group), 6 h post-CLP. The mean arterial pressure was recorded, and the concentration-effect curves for ACh were constructed for endothelium-intact aortic rings in the absence (control) or after incubation with one of the following NO synthase inhibitors: L-NAME (non-selective), L-NNA (more selective for eNOS), 7-nitroindazole (more selective for nNOS), or 1400W (selective for iNOS). The NO concentration was determined by using confocal microscopy. The protein expression of the NOS isoforms was quantified by Western blot analysis. The prostacyclin concentration was indirectly analyzed on the basis of 6-keto-prostaglandin F-1 alpha (6-keto-PGF(1 alpha)) levels measured by enzyme immunoassay. There were no differences between Sham- and CLP-operated rats in terms of the relaxation induced by acetylcholine. However, the NOS inhibitors reduced this relaxation in both groups, but this effect remained more pronounced in the CLP group as compared to the Sham group. The acetylcholine-induced NO production was higher in the rat aortic endothelial cells of the CLP group than in those of the Sham group. eNOS protein expression was larger in the CLP group, but the iNOS protein was not verified in any of the groups. The basal 6-keto-PGF(1 alpha) levels were higher in the CLP group, but the acetylcholine-stimulated levels did not increase in CLP as much as they did in the Sham group. Taken together, our results show that the augmented NO production in sepsis syndrome elicited by cecal ligation and perforation is due to eNOS up-regulation and not to iNOS. (C) 2012 Elsevier Inc. All rights reserved.

Preclinical testing of strategies for therapeutic targeting of human T-cell trafficking in vivo

Naive T cells are migratory cells that continuously recirculate between blood and lymphoid tissues. Antigen-specific stimulation of T cells within the lymph nodes reprograms the trafficking properties of T cells by inducing a specific set of adhesion molecules and chemokine receptors on their surface which allow these activated and effector T cells to effectively and specifically home to extralymphoid organs. The observations of organ-specific homing of T cells initiated the development of therapeutic strategies targeting adhesion receptors for organ-specific inhibition of chronic inflammation. As most adhesion receptors have additional immune functions besides mediating leukocyte trafficking, these drugs may have additional immunomodulatory effects. Therapeutic targeting of T-cell trafficking to the central nervous system is the underlying concept of a novel treatment of relapsing remitting multiple sclerosis with the humanized anti-alpha-4-integrin antibody natalizumab. In this chapter, we describe a possible preclinical in vivo approach to directly visualize the therapeutic efficacy of a given drug in inhibiting T-cell homing to a certain organ at the example of the potential of natalizumab to inhibit the trafficking of human T cells to the inflamed central nervous system in an animal model of multiple sclerosis.

Diversity of structure and function of alpha1alpha6beta3delta GABAA receptors: comparison with alpha1beta3delta and alpha6beta3delta receptors

delta subunit-containing gamma-aminobutyric acid, type A (GABA(A))receptors are expressed extrasynaptically and mediate tonic inhibition. In cerebellar granule cells, they often form receptors together with alpha(1) and/or alpha(6) subunits. We were interested in determining the architecture of receptors containing both subunits. We predefined the subunit arrangement of several different GABA(A) receptor pentamers by concatenation. These receptors composed of alpha(1), alpha(6), beta(3), and delta subunits were expressed in Xenopus oocytes. Currents elicited in response to GABA were determined in the presence and absence of 3alpha,21-dihydroxy-5alpha-pregnan-20-one (THDOC) or ethanol, or currents were elicited by 4,5,6,7-tetrahydroisoxazolo[5,4-c]-pyridin-3-ol (THIP). Several subunit configurations formed active channels. We therefore conclude that delta can assume multiple positions in a receptor pentamer made up of alpha(1), alpha(6), beta(3), and delta subunits. The different receptors differ in their functional properties. Functional expression of one receptor type was only evident in the combined presence of the neurosteroid THDOC with the channel agonist GABA. Most, but not all, receptors active with GABA/THDOC responded to THIP. None of the receptors was modulated by ethanol concentrations up to 30 mm. Several observations point to a preferred position of delta subunits between two alpha subunits in alpha(1)alpha(6)beta(3)delta receptors. This property is shared by alpha(1)beta(3)delta and alpha(6)beta(3)delta receptors, but there are differences in the additionally expressed isoforms.