Cell adhesion to anosmin via α5β1, α4β1, and α9β1 integrins

Anosmin is an extracellular matrix protein, and genetic defects in anosmin result in human Kallmann syndrome. It functions in neural crest formation, cell adhesion, and neuronal migration. Anosmin consists of multiple domains, and it has been reported to bind heparan sulfate, FGF receptor, and UPA. In this study, we establish cell adhesion/spreading assays for anosmin and use them for antibody inhibition analyses to search for an integrin adhesion receptor. We find that α5β1, α4β1, and α9β1 integrins are needed for effective adhesive receptor function in cell adhesion and cell spreading on anosmin; adhesion is inhibited by both RGD and α4β1 CS1-based peptides. This identification of anosmin-integrin adhesion receptors should facilitate studies of anosmin function in cell and developmental biology.     

Integrin α9β1

Integrins are transmembrane heterodimeric receptors responsible for transducing and modulating signals between the extracellular matrix and cytoskeleton, ultimately influencing cell functions such as adhesion and migration. Integrin α9β1 is classified within a two member sub-family of integrins highlighted in part by its specialized role in cell migration. The importance of this role is demonstrated by its regulation of numerous biological functions including lymphatic valve morphogenesis, lymphangiogenesis, angiogenesis and hematopoietic homeostasis. Compared to other integrins the signaling mechanisms that transduce α9β1-induced cell migration are not well described. We have recently shown that Src tyrosine kinase plays a key proximal role to control α9β1 signaling. Specifically it activates inducible nitric oxide synthase (iNOS) and in turn nitric oxide (NO) production as a means to transduce cell migration. Furthermore, we have also described a role for FAK, Erk and Rac1 in α9β1 signal transduction. Here we provide an over view of known integrin α9β1 signaling pathways and highlight its roles in diverse biological conditions.     

TGF-β1受体1和2在TGF-β1调节细胞增殖中的作用

转化生长因子β1(transforming growth factor-β1,TGF-β1)是一种多功能细胞因子,在细胞增殖、分化、伤口愈合和肿瘤生成转移等过程中均发挥重要调控作用。TGF-β1对细胞增殖的调节可因细胞类型、刺激剂量不同而不同,但其差异调节的机制还不清楚。现普遍认为,TGF-β1在TGFBR2/TGFBR1二聚体参与下通过经典的Smad信号通路抑制增殖,而通过非Smad信号通路促进细胞周期,但是机体是如何调控这种不同增殖调节作用转化的还不明确。TGFBR1和TGFBR2在细胞中的分布和比例变化可能是TGF-β1差异性调控细胞增殖作用的一个重要机制。     

Action of rat liver Galβ1-4GlcNAc α(2-6)-sialyltransferase on Manβ1-4GlcNAcβ-OMe,GalNAcβ1-4GlcNAcβ-OMe,Glcβ1-4GlcNAcβ-OMe and GlcNAcβ1-4GlcNAcβ-OMe as synthetic substrates

Incubation of synthetic Man\1-4GlcNAc-OMe, GalNAc1-4GlcNAc-OMe, Glc1-4GlcNAc-OMe, and GlcNAc1-4GlcNac-OMe with CMP-Neu5Ac and rat liver Gal1-4GlcNAc (2-6)-sialyltransferase resulted in the formation of Neu5Ac2-6Man1-4GlcNAc-OMe, Neu5Ac2-6GalNAc1-4GlcNAc-OMe, Neu5Ac2-6Glc1-4GlcNAc-OMe and Neu5Ac2-6GlcNAc1-4GlcNAc-OMe, respectively. Under conditions which led to quantitative conversion of Gal1-4GlcNAc-OEt into Neu5Ac2-6Gal1-4GlcNAc-OEt, the aforementioned products were obtained in yields of 4%, 48%, 16% and 8%, respectively. HPLC on Partisil 10 SAX was used to isolate the various sialyltrisaccharides, and identification was carried out using 1- and 2-dimensional 500-MHz¹H-NMR spectroscopy.Abbreviations 2D 2-dimensional - CMP cytidine 5-monophosphate - CMP-Neu5Ac cytidine 5-monophospho--N-acetylneuraminic acid - COSY correlation spectroscopy - DQF double quantum filtered - HOHAHA homonuclear Hartmann-Hahn - MLEV composite pulse devised by M. Levitt - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid     

The photosynthetic F1-α3β3 and α1β1 catalytic core complexes

Minimal photosynthetic catalytic F₁() core complexes, containing equimolar ratios of the and subunits, were isolated from membrane-bound spinach chloroplast CF₁ and Rhodospirillum rubrum chromatophore RrF₁. A CF₁-₃₃ hexamer and RrF₁-₁₁ dimer, which were purified from the respective F₁() complexes, exhibit lower rates and different properties from their parent F₁-ATPases. Most interesting is their complete resistance to inhibition by the general F₁ inhibitor azide and the specific CF₁ inhibitor tentoxin. These inhibitors were earlier reported to inhibit multisite, but not unisite, catalysis in all sensitive F₁-ATPases and were therefore suggested to block catalytic site cooperativity. The absence of this typical property of all F₁-ATPases in the ₁₁ dimer is consistant with the view that the dimer contains only a single catalytic site. The ₃₃ hexamer contains however all F₁ catalytic sites. Therefore the observation that CF₁-₃₃ can bind tentoxin and is stimulated by it suggests that the F₁ subunit, which is required for obtaining inhibition by tentoxin as well as azide, plays an important role in the cooperative interactions between the F₁-catalytic sites.Abbreviations CF₀F₁ chloroplast F₀F₁ - CF₁ chloroplast F₁ - CF₁ chloroplast F₁ subunit - CF₁ chloroplast F₁ subunit - CF₁() a complex containing equal amounts of the CF₁ and subunits - MF₁ mitochondrial F₁ - RrF₀F₁ Rhodospirillum rubrum F₀F₁ - RrF₁ R. rubrum F₁ - RrF₁ R. rubrum F₁ subunit - RrF₁ R. rubrum F₁ subunit - RrF₁() a complex containing equal amounts of the RrF₁ and subunits - Rubisco Ribulose-1,5-bisphosphate carboxylase - TF₁ thermophilic bacterium PS3 F₁     

IL-1β对体外培养NIT-1β细胞胰岛素分泌的影响

利用小鼠胰岛β细胞系NIT 1细胞,对不同时间、不同浓度IL 1β作用下细胞的生长和胰岛素的分泌进行观察,以研究IL 1β对胰岛β细胞胰岛素分泌的影响,探讨细胞因子对胰岛β细胞功能的抑制作用.结果显示在IL 1β作用下,各组NIT 1细胞分泌胰岛素呈不同程度降低,表明IL 1β对NIT 1β细胞系胰岛素的分泌具有显著抑制作用.     

Construction of linear GlcNAcβ1-6Galβ1-OR type oligosaccharides by partial cleavage of GlcNAcβ1-3(GlcNAcβ1-6)Galβ1-OR sequences with jack bean β-N-acetylhexosaminidase

Radiolabelled GlcNAc beta 1-3(GlcNAc beta 1-6)Gal (1), GlcNAc beta 1-3)GlcNAc beta 1-6)Gal beta 1-OCH3 (4), GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4Glc (7), and GlcNAc beta 1-3(GlcNAc beta 1-6)Gal beta 1-4GlcNAc (10) were cleaved partially with jack bean beta-N-acetylhexosaminidase (EC 3.2.1.30), and the digests were analysed chromatographically. All four oligosaccharides were hydrolysed faster at the (1-6) branch, than at the (1-3) branch, but a high branch specificity was observed only with the glycan 4. The saccharides 1 and 7 resembled each other in the kinetics of the enzyme-catalysed release of their two non-reducing N-acetylglucosamine units, but the glycan 10 was rather different. The partial digestions made it possible to obtain radiolabelled GlcNAc beta 1-6Gal, GlcNAc beta 1-6Gal beta 1-OCH3, GlcNAc beta 1-6Gal beta 1-4Glc, and, in particular, GlcNAc beta 1-6Gal beta 1-4GlcNAc.     

Untersuchung der N-Acetyl-β-glucosaminidase mit 1-Naphthyl-N-Acetyl-β-glucosaminid

Zusammenfassung Mit 1-Naphthyl-N-acetyl--glucosaminid als Substrat wird die N-Acetyl--glucosaminidase (N-A-Gase) histochemisch (Simultankupplung von 1-Naphthol an Hexazonium-p-rosanilin) und mikrochemisch (fluorometrische Messung von 1-Naphthol) in Ratten-, Mäuseund Meerschweinchenorganen untersucht.Das histochemische Inkubationsmedium enthält 5–12 mg 1-Naphthyl-N-acetyl--glucosaminid (gelöst in NN-Dimethylformamid) und 0,8–1 ml 2% Hexazonium-p-rosanilin in 9 ml 0,1 M Citrat-Puffer, pH 5; das mikrochemische 2 mg/ml dieses Substrats (6 mM) im gleichen Puffer, pH 4,5.Zum Nachweis in situ empfiehlt sich wegen der geringeren Hemmrate (32%) Formol-Fixation, in hochaktiven Geweben auch Glutaraldehyd (54% Inhibition im proximalen Konvolut der Rattenniere). Die histochemische Gesamtaktivität der N-A-Gase ist besser an frischen Schnitten mit semipermeablen Membranen zu erfassen.Nach Fixation in Glutaraldehyd kann das Enzym vor allem bei Ratten in den Lysosomen zahlreicher Organe nachgewiesen werden (u.a. Niere, Nebenhoden, Bronchien, Darm, Uterus und Samenblase), wobei artspezifische Unterschiede bestehen: Über die höchste N-A-Gase-Aktivität verfügen Nebenniere, Nebenhoden und Milz von Meerschweinchen. Weniger Enzym enthalten diese Organe bei Ratten und Mäusen; speziell die Nebenniere ist weitgehend N-A-Gase-frei. Am kräftigsten reagiert die Niere bei Ratten. Geschlechtsspezifische Differenzen sind hier nur mikrochemisch faßbar, in der Niere der Maus aber schon histochemisch. — Reich an N-A-Gase sind Speicheldrüsen, Harnblase und Colon sowie andere Organe mit mucopolysaccharid-haltigen Strukturen.Die mit der Naphthol-AS und 1-Naphthyl-Methode für die N-A-Gase erhaltenen Aktivitätsund Verteilungsmuster entsprechen sich. Die Qualität der intrazellulären Enzymlokalisation mit 1-Naphthyl-N-acetyl--glucosaminid als Substrat kommt den Möglichkeiten des Naphthol-AS-Verfahrens in vielen Organen nahe oder ist ihm gleichwertig.

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Investigation of N-acetyl--glucosaminidase by means of 1-naphthyl-N-acetyl--glucosaminide

Summary Using 1-naphthyl-N-acetyl--gIucosaminide as substrate N-acetyl--glucosaminidase (N-A-Gase) has been investigated histochemically (simultaneous coupling of 1-naphthol and hexazonium p-rosaniline) and microchemically (fluorometric measurement of 1-naphthol) in various organs of rats, mice, and guinea-pigs.The histochemical incubation medium consists of 5–12 mg 1-naphthyl-N-acetyl--glucosaminide (dissolved in NN-dimethyl formamide) and 0.8–1 ml hexazotized p-rosaniline in 9 ml 0.1 M citrate buffer, pH 5; for microchemical purposes 2 mg/ml of this substrate (6 mM) in the same buffer, pH 4.5 were used.For the in situ demonstration of N-A-Gase formol fixation is recommended because of its lower inhibition rate (32%); in highly active tissues glutaraldehyde is also suitable (54% inhibition in the proximal convoluted tubules of the rat kidney). The total activity of N-A-Gase can better be detected in fresh frozen sections in connection with semipermeable membranes.Following fixation in glutaraldehyde the enzyme occurs in the lysosomes of many organs, e.g. kidney, epididymis, bronchi, adrenal gland, intestine, uterus, and vesicular gland, especially in rats. Furthermore spezies-dependent differences exist: the suprarenal gland, epididymis, and spleen of guinea-pigs display the highest amount of N-A-Gase. In rats and mice the enzyme activity of these organs is lower; the adrenal cortex is nearly free of N-A-Gase. — The kidney reacts intensely in rats, the sex differences of which can only be detected by means of microchemistry. In the mouse kidney they are more pronounced. Therefore the histochemical N-A-Gase assay reveals them, too. — Organs containing considerable quantities of mucopolysaccharides, e.g. the submandibular gland, urinary bladder, and colon are also rich in N-A-Gase.The activity and distribution pattern of N-A-Gase obtained with the naphthol AS and 1-naphthyl technique are completely in correspondance with one another. In some cases the quality of the intracellular enzyme localization using naphthol AS-BI N-acetyl--glucosaminide as substrate surpasses the possibilities of the 1-naphthyl derivate, in others the latter one enables identical results.

     

白细胞介素-1β信号与β细胞功能

由胰岛β细胞功能失调,导致胰岛素分泌的相对或绝对的缺失,进而出现高血糖症状,是糖尿病的重要发病机制.目前认为糖尿病的发病与机体的炎症过程密切相关.作为炎症过程的重要调节因子白细胞介素-1β(IL-1β),通过激活MAPK、NFkB、PKC等信号通路,最终导致b细胞功能失调是糖尿病发生发展的重要原因.IL-1β在介导糖尿病的b细胞功能失调中发挥核心作用.     

Microbial 1β-Hydroxylation of Digitoxigenin

l-Deoxy-l-l-proIino-d-fructose was isolated from flue-cured tobacco leaves in crystalline form. The structure was confirmed by comparison with synthetic compound.     

转化生长因子β1与老年性痴呆

老年性痴呆 (简称ＡＤ)是一种渐进性大脑退行性变性病 ,其发病率随年龄增长而显著升高 ,6 5～ 80岁人群约为 5 % ,80岁以上者可达 2 0 %。我国 6 0岁以上的人口已近1 .3亿 ,ＡＤ即将成为医学和社会面临的严峻问题 ,已引起政府和医学工作者的关注。对ＡＤ的研究在美国、欧州和日本倍受重视 ,并取得了一定成果[1] 。β 淀粉样蛋白 (Ａβ)在中枢神经系统中的沉积是ＡＤ的一个标志 ,并可能是神经退收稿日期 :2 0 0 0 0 5 11作者简介 :吴晓英 ( 196 1— ) ,女 ,硕士 ,讲师。化的一个原因[2 ] 。在脑对损伤的反应中 ,转化生长因子 β1 (ＴＧＦ …     

GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate) Is the Preferred Substrate for Chondroitin N-Acetylgalactosaminyltransferase-1

A deficiency in chondroitin N-acetylgalactosaminyltransferase-1 (ChGn-1) was previously shown to reduce the number of chondroitin sulfate (CS) chains, leading to skeletal dysplasias in mice, suggesting that ChGn-1 regulates the number of CS chains for normal cartilage development. Recently, we demonstrated that 2-phosphoxylose phosphatase (XYLP) regulates the number of CS chains by dephosphorylating the Xyl residue in the glycosaminoglycan-protein linkage region of proteoglycans. However, the relationship between ChGn-1 and XYLP in controlling the number of CS chains is not clear. In this study, we for the first time detected a phosphorylated tetrasaccharide linkage structure, GlcUAβ1–3Galβ1–3Galβ1–4Xyl(2-O-phosphate), in ChGn-1^−/− growth plate cartilage but not in ChGn-2^−/− or wild-type growth plate cartilage. In contrast, the truncated linkage tetrasaccharide GlcUAβ1–3Galβ1–3Galβ1–4Xyl was detected in wild-type, ChGn-1^−/−, and ChGn-2^−/− growth plate cartilage. Consistent with the findings, ChGn-1 preferentially transferred N-acetylgalactosamine to the phosphorylated tetrasaccharide linkage in vitro. Moreover, ChGn-1 and XYLP interacted with each other, and ChGn-1-mediated addition of N-acetylgalactosamine was accompanied by rapid XYLP-dependent dephosphorylation during formation of the CS linkage region. Taken together, we conclude that the phosphorylated tetrasaccharide linkage is the preferred substrate for ChGn-1 and that ChGn-1 and XYLP cooperatively regulate the number of CS chains in growth plate cartilage.     

Synthesis of Galβ1-4GlcNAcβ- and Galβ1-3GalNAcα-O-L-serine Derivatives employing glycosidases

A new approach for the highly specific preparation of L-serine conjugates of lactosamine and Gal1-3GalNAc is described. Thus, the L-serine derivative of lactosamine Gal1-4GlcNAc-O-(N-Z)-Ser-OEt, was obtained from lactose, employing GlcNAc-O-(N-Z)-Ser-OEt as acceptor and a yeast -galactosidase as catalyst Galp 1-3GalNAc-O-(N-Alloc)-Ser-OMe was obtained from lactose, employing GalNAc-O-(N-Alloc)-Ser-OMe as acceptor and -galactosidase from bovine testes as catalyst.     

β分泌酶1降低β肌养蛋白聚糖的蛋白质水平

目的 β分泌酶1（BACE1）是阿尔茨海默病患者脑中淀粉样蛋白（Aβ）产生的关键酶。肌养蛋白聚糖 （dystroglycan,DG）帮助星形胶质细胞的终足锚定在脑血管上,形成一道支持血脑屏障的胶质界限。一项无靶标蛋白质组学研究指出BACE1可能会下调DG的表达水平。本文旨在研究BACE1能否调控DG的蛋白质水平及其可能的调控机制。方法 利用瞬时转染法在HEK-293T细胞系和原代培养的小鼠星形胶质细胞中表达目的蛋白。通过蛋白质免疫印迹分析目标蛋白质的相对水平。利用基因荧光定量和免疫共沉淀技术探索BACE1调控DG的潜在机制。结果 在HEK-293T和原代小鼠星形胶质细胞中引入BACE1会使DG β亚基（β-DG）的蛋白质水平显著降低。在HEK-293T细胞中,β-DG蛋白水平的下降依赖于BACE1的酶活性。结论 在HEK-293T细胞和小鼠星形胶质细胞中,BACE1使β-DG的蛋白质水平下降。     

Tau pathogenesis is promoted by Aβ1-42 but not Aβ1-40

Background

The relationship between the pathogenic amyloid β-peptide species Aβ1–42 and tau pathology has been well studied and suggests that Aβ1–42 can accelerate tau pathology in vitro and in vivo. The manners if any in which Aβ1–40 interacts with tau remains poorly understood. In order to answer this question, we used cell-based system, transgenic fly and transgenic mice as models to study the interaction between Aβ1–42 and Aβ1–40.

Results

In our established cellular model, live cell imaging (using confocal microscopy) combined with biochemical data showed that exposure to Aβ1–42 induced cleavage, phosphorylation and aggregation of wild-type/full length tau while exposure to Aβ1–40 didn’t. Functional studies with Aβ1–40 were carried out in tau-GFP transgenic flies and showed that Aβ1–42, as previously reported, disrupted cytoskeletal structure while Aβ1–40 had no effect at same dose. To further explore how Aβ1–40 affects tau pathology in vivo, P301S mice (tau transgenic mice) were injected intracerebrally with either Aβ1–42 or Aβ1–40. We found that treatment with Aβ1–42 induced tau phosphorylation, cleavage and aggregation of tau in P301S mice. By contrast, Aβ1–40 injection didn’t alter total tau, phospho-tau (recognized by PHF-1) or cleavage of tau, but interestingly, phosphorylation at Ser²⁶² was shown to be significantly decreased after direct inject of Aβ1–40 into the entorhinal cortex of P301S mice.

Conclusions

These results demonstrate that Aβ1–40 plays different role in tau pathogenesis compared to Aβ1–42. Aβ1–40 may have a protective role in tau pathogenesis by reducing phosphorylation at Ser²⁶², which has been shown to be neurotoxic.

     

IL-1β对不同妊娠时期大鼠子宫与胎盘 TGF-β1表达的影响

为探讨妊娠过程中白介素-1β(IL-1β)对转化生长因子-β1(TGF-β1)表达的影响,以妊娠Spreqne-Dawley大鼠为模型,采用宫角注射、逆转录-聚合酶链反应(RT-PCR)和蛋白质印迹方法。检测了妊娠不同时期大鼠子宫和胎盘中IL-1β对TGF-β1 mRNA水平及蛋白水平表达变化的影响。结果显示,植入期和妊娠晚期IL-1β能降低子宫TGF-β1的表达水平,植入后期和妊娠中期能提高TGF-β1的表达水平,对植入前期TGF-β1的表达无显著性影响;IL-1β能抑制妊娠早期胎盘TGF-β1的表达,妊娠晚期能促进其表达。结果提示,在妊娠过程中,IL-1β能够调节TGF-β1的表达,并且这种作用具有阶段特异性和组织特异性。     

Enzymatic syntheses of GlcNAcβ1-2Man and Galβ1-4GlcNAcβ1-2Man as components of complex type sugar chains

GlcNAc1-2Man and GlcNAc1-6Man were synthesized using the reverse hydrolysis activity of -N-acetylglucosaminidase from both jack beans and Bacillus circulans. In turn, Gal1-4GlcNAc1-2Man and Gal1-4GlcNAc1-6Man were synthesized regioselectively using the transglycosylation activity of -galactosidase from Diplococcus pneumoniae and B. circulans, respectively. These di- and trisaccharides are important components of complex type sugar chains and will be used as intermediates in our synthetic studies. Abbreviations: pNp--GlcNAc, p-nitrophenyl 2-acetamido-2-deoxy--D-glucopyranoside; pNp--Gal, p-nitrophenyl -D-galacto-pyranoside     

Hydroxybenzothiazoles as new nonsteroidal inhibitors of 17β-hydroxysteroid dehydrogenase type 1 (17β-HSD1)

17β-estradiol (E2), the most potent estrogen in humans, known to be involved in the development and progession of estrogen-dependent diseases (EDD) like breast cancer and endometriosis. 17β-HSD1, which catalyses the reduction of the weak estrogen estrone (E1) to E2, is often overexpressed in breast cancer and endometriotic tissues. An inhibition of 17β-HSD1 could selectively reduce the local E2-level thus allowing for a novel, targeted approach in the treatment of EDD. Continuing our search for new nonsteroidal 17β-HSD1 inhibitors, a novel pharmacophore model was derived from crystallographic data and used for the virtual screening of a small library of compounds. Subsequent experimental verification of the virtual hits led to the identification of the moderately active compound 5. Rigidification and further structure modifications resulted in the discovery of a novel class of 17β-HSD1 inhibitors bearing a benzothiazole-scaffold linked to a phenyl ring via keto- or amide-bridge. Their putative binding modes were investigated by correlating their biological data with features of the pharmacophore model. The most active keto-derivative 6 shows IC₅₀-values in the nanomolar range for the transformation of E1 to E2 by 17β-HSD1, reasonable selectivity against 17β-HSD2 but pronounced affinity to the estrogen receptors (ERs). On the other hand, the best amide-derivative 21 shows only medium 17β-HSD1 inhibitory activity at the target enzyme as well as fair selectivity against 17β-HSD2 and ERs. The compounds 6 and 21 can be regarded as first benzothiazole-type 17β-HSD1 inhibitors for the development of potential therapeutics.     

IL-1β对L-谷氨酸致痫大鼠大脑皮质、海马PLCβ1表达的影响

目的探讨白细胞介素-1β(Interleukin-1 beta,IL-1β)对L-谷氨酸致痫大鼠大脑PLCβ1表达的影响,为阐明IL-1β在致痫中的作用机制提供资料.方法随机将健康成年SD大鼠分为5组,每组8只,即对照组、Glu组、IL-1β Glu组、IL-1ra IL-1β Glu组和MPEP IL-1β Glu组,采用行为学及Western blot和免疫组织化学方法进行研究.结果行为观察显示,对照组、IL-1ra IL-1β Glu组和MPEP组无痫性发作;Western blot结果显示PLCβ1含量在Glu组和IL-1β Glu组大鼠鼠脑皮质及海马内均较对照组、IL-1ra IL-1β Glu组和MPEP IL-1β Glu组明显增多(P<0.05),而对照组、IL-1ra IL-1β Glu组和MPEP IL-1β Glu组组间无明显差别(P>0.05);免疫组织化学染色显示,PLCβ1免疫反应增强主要表现在海马CA3区和大脑皮质,各组间的变化规律与Western blot检测结果一致.结论 IL-1β对L-谷氨酸致痫有促进作用,mGluR5介导的PLCβ1激活参与了IL-1的促痫机制.