Expression and Function of Siglec-8 in Human Eosinophils, Basophils, and Mast Cells

Siglec-8, the eighth member of the sialic acid-binding, immunoglobulin [Ig]-like lectin family, was initially discovered as a cell surface protein selectively expressed on human eosinophils. It is now know to also be expressed by mast cells and basophils. Siglec-8 engagement with specific antibodies causes apoptosis via caspase and mitochondrial-dependent pathways. For mast cells, inhibition of mediator release, but no apoptosis, is observed. Siglec-F is the closest mouse paralog to Siglec-8, and both selectively bind the sulfated glycan 6’-sulfo-sialyl Lewis X. Antibodies to Siglec-F reduce blood and tissue eosinophil numbers in vivo. This suggests that Siglec-8 may be a useful future therapeutic target for allergic and other eosinophilic disorders.

Glycosylation is an Androgen-Regulated Process Essential for Prostate Cancer Cell Viability

Steroid androgen hormones play a key role in the progression and treatment of prostate cancer, with androgen deprivation therapy being the first-line treatment used to control cancer growth. Here we apply a novel search strategy to identify androgen-regulated cellular pathways that may be clinically important in prostate cancer. Using RNASeq data, we searched for genes that showed reciprocal changes in expression in response to acute androgen stimulation in culture, and androgen deprivation in patients with prostate cancer. Amongst 700 genes displaying reciprocal expression patterns we observed a significant enrichment in the cellular process glycosylation. Of 31 reciprocally-regulated glycosylation enzymes, a set of 8 (GALNT7, ST6GalNAc1, GCNT1, UAP1, PGM3, CSGALNACT1, ST6GAL1 and EDEM3) were significantly up-regulated in clinical prostate carcinoma. Androgen exposure stimulated synthesis of glycan structures downstream of this core set of regulated enzymes including sialyl-Tn (sTn), sialyl Lewis(X) (SLe(X)), O-GlcNAc and chondroitin sulphate, suggesting androgen regulation of the core set of enzymes controls key steps in glycan synthesis. Expression of each of these enzymes also contributed to prostate cancer cell viability. This study identifies glycosylation as a global target for androgen control, and suggests loss of specific glycosylation enzymes might contribute to tumour regression following androgen depletion therapy.

单细胞凝胶电泳技术检测碳离子束和X射线照射对Lewis肺癌细胞的DNA损伤

比较研究了89.63MeV/u的碳离子束和6MeV的X射线照射Lewis肺癌细胞所致的细胞克隆存活和DNA损伤效应,以探讨单细胞凝胶电泳检测细胞辐射敏感性及重离子束治疗肿瘤的优势。结果表明,在10%细胞存活水平上碳离子束的相对生物学效应(Relative biological effectiveness,RBE)值达到1.77。单细胞凝胶电泳检测损伤DNA尾部百分含量(Tail DNA,TD)和Olive尾矩(Olive tail moment,OTM)的剂量效应曲线表明,X射线的剂量效应曲线为线性,而碳离子束诱导出一个包含线性和指数项的双阶段效应曲线。碳离子束辐照剂量大于8Gy后TD和OTM都存在饱和效应。在2Gy的剂量点,高传能线密度(LET)碳离子束比X射线产生更低的存活分数和更高的初始OTM。本研究提示:在Lewis肺癌细胞中,碳离子束照射比X射线产生更为强烈的细胞致死和DNA损伤效应,可使肿瘤治疗具有更高效率。

Papel da fucana sulfatada FucSulf I na interação entre células tumorais e o endotélio in vitro

Para formar metástases, as células tumorais devem se desprender do tumor primário e migrar através do endotélio num processo denominado intravasamento. Uma vez na circulação, elas devem aderir ao endotélio do tecido alvo e extravasar para o novo sítio de colonização, onde irão proliferar. A interação das células tumorais com o endotélio é mediada por selectinas, seguida pela interação com integrinas. As células tumorais apresentam um padrão anormal de glicosilação, expressando ligantes de selectinas, formados por polissacarídeos fucosilados, como sialyl Lewis a/x. Durante o processo metastático, células tumorais secretam diversos fatores de crescimento. Além de modular diferentes tipos celulares que constituem o microambiente tumoral, estes fatores de crescimento também atuam nas células tumorais de forma autócrina, ativando vias de sinalização envolvidas na proliferação e migração celular. Polissacarídeos sulfatados como a heparina, podem atuar como inibidores de P e L-selectinas, além de se ligar a fatores de crescimento, impedindo a ativação de seus receptores. Neste trabalho, avaliamos o papel de fucanas sulfatadas extraídas de diferentes espécies de invertebrados marinhos (L. variegatus, S. franciscanus, S. pallidus, A. lixula e S. droebachiensis) na modulação da interação entre células tumorais com o endotélio in vitro e comparamos seu efeito com o da heparina. Também avaliamos o papel destas moléculas na proliferação de células tumorais. Para isso, utilizamos duas linhagens tumorais de próstata (DU-145 e PC-3) e culturas primárias de células endoteliais de veia umbilical humana (HUVECs). Ao avaliar o efeito das fucanas na adesão das células tumorais às HUVECs, observamos que todas as fucanas testadas inibiram a adesão da linhagem DU-145 à monocamada endotelial, enquanto apenas a fucana extraída da espécie L. variegatus (FucSulf I) e da espécie S. franciscanus inibiram a adesão da linhagem PC-3. A FucSulf I foi uma das fucanas que apresentou maior potencial inibitório nas duas linhagens e foi a única que inibiu a adesão da linhagem DU-145 à matriz subendotelial, não interferindo na adesão da linhagem PC-3. A FucSulf I mostrou-se capaz de diminuir também a migração transendotelial das linhagens tumorais DU-145 e PC-3. A heparina mostrou efeito significativo apenas nos ensaios de transmigração, inibindo este evento de forma similar a FucSuf I. Sabe-se que o VEGF aumenta a permeabilidade endotelial, facilitando a passagem de células tumorais através do vaso. Observamos que as duas linhagens secretam VEGF e que a FucSulf I se liga a este fator. Estes dados sugerem que a interação da FucSuf I com o VEGF pode impedir a ação deste fator nas células endoteliais, diminuindo a migração transendotelial das células tumorais testadas. Também verificamos que a FucSulf I inibiu a proliferação das linhagens celulares na ausência de fatores exógenos ou na presença de soro fetal bovino ou VEGF. Por fim, avaliamos que a FucSulf I interfere na ativação de proteínas específicas de vias de sinalização disparadas por fatores de crescimento. A FucSulf I inibe a ativação da AKT na linhagem PC-3, enquanto nas células DU-145 observamos uma inibição da ativação da ERK. Esses dados indicam que a FucSulf I modula diversas etapas da progressão tumoral e pode ser um potencial candidato para o uso em terapias antitumorais

Population-based study reveals new risk-stratification biomarker panel for Barrett's esophagus

BACKGROUND & AIMS: The risk of progression of Barrett's esophagus (BE) to esophageal adenocarcinoma (EAC) is low and difficult to calculate. Accurate tools to determine risk are needed to optimize surveillance and intervention. We assessed the ability of candidate biomarkers to predict which cases of BE will progress to EAC or high-grade dysplasia and identified those that can be measured in formalin-fixed tissues. METHODS: We analyzed data from a nested case-control study performed using the population-based Northern Ireland BE Register (1993-2005). Cases who progressed to EAC (n = 89) or high-grade dysplasia =6 months after diagnosis with BE were matched to controls (nonprogressors, n = 291), for age, sex, and year of BE diagnosis. Established biomarkers (abnormal DNA content, p53, and cyclin A expression) and new biomarkers (levels of sialyl Lewis(a), Lewis(x), and Aspergillus oryzae lectin [AOL] and binding of wheat germ agglutinin) were assessed in paraffin-embedded tissue samples from patients with a first diagnosis of BE. Conditional logistic regression analysis was applied to assess odds of progression for patients with dysplastic and nondysplastic BE, based on biomarker status. RESULTS: Low-grade dysplasia and all biomarkers tested, other than Lewis(x), were associated with risk of EAC or high-grade dysplasia. In backward selection, a panel comprising low-grade dysplasia, abnormal DNA ploidy, and AOL most accurately identified progressors and nonprogressors. The adjusted odds ratio for progression of patients with BE with low-grade dysplasia was 3.74 (95% confidence interval, 2.43-5.79) for each additional biomarker and the risk increased by 2.99 for each additional factor (95% confidence interval, 1.72-5.20) in patients without dysplasia. CONCLUSIONS: Low-grade dysplasia, abnormal DNA ploidy, and AOL can be used to identify patients with BE most likely to develop EAC or high-grade dysplasia.

Molecular mechanism of sulfated carbohydrate recognition: structural and biochemical studies of the cysteine-rich domain of mannose receptor

Mannose receptor (MR) is widely expressed on macrophages, immature dendritic cells, and a variety of epithelial and endothelial cells. It is a 180 kD type I transmembrane receptor whose extracellular region consists of three parts: the amino-terminal cysteine-rich domain (Cys-MR); a fibronectin type II-like domain; and a series of eight tandem C-type lectin carbohydrate recognition domains (CRDs). Two portions of MR have distinct carbohydrate recognition properties: Cys-MR recognizes sulfated carbohydrates and the tandem CRD region binds terminal mannose, fucose, and N-acetyl-glucosamine (GlcNAc). The dual carbohydrate binding specificity allows MR to interact with sulfated and nonsulfated polysaccharide chains, and thereby facilitating the involvement of MR in immunological and physiological processes. The immunological functions of MR include antigen capturing (through binding non-sulfated carbohydrates) and antigen targeting (through binding sulfated carbohydrates), and the physiological roles include rapid clearance of circulatory luteinizing hormone (LH), which bears polysaccharide chains terminating with sulfated and non-sulfated carbohydrates.

We have crystallized and determined the X-ray structures of unliganded Cys-MR (2.0 Å) and Cys-MR complexed with different ligands, including Hepes (1.7 Å), 4SO_4-N-Acetylgalactosamine (4SO_4-GalNAc; 2.2 Å), 3SO_4-Lewis^x (2.2 Å), 3S04-Lewis^a (1.9 Å), and 6SO_4-GalNAc (2.5 Å). The overall structure of Cys-MR consists of 12 anti-parallel β-strands arranged in three lobes with approximate three fold internal symmetry. The structure contains three disulfide bonds, formed by the six cysteines in the Cys-MR sequence. The ligand-binding site is located in a neutral pocket within the third lobe, in which the sulfate group of ligand is buried. Our results show that optimal binding is achieved by a carbohydrate ligand with a sulfate group that anchors the ligand by forming numerous hydrogen bonds and a sugar ring that makes ring-stacking interactions with Trpll7 of CysMR. Using a fluorescence-based assay, we characterized the binding affinities between CysMR and its ligands, and rationalized the derived affinities based upon the crystal structures. These studies reveal the mechanism of sulfated carbohydrate recognition by Cys-MR and facilitate our understanding of the role of Cys-MR in MR recognition of its ligands.

Chemical characterization of oligosaccharides in the milk of six species of New and Old World monkeys.

Human and great ape milks contain a diverse array of milk oligosaccharides, but little is known about the milk oligosaccharides of other primates, and how they differ among taxa. Neutral and acidic oligosaccharides were isolated from the milk of three species of Old World or catarrhine monkeys (Cercopithecidae: rhesus macaque (Macaca mulatta), toque macaque (Macaca sinica) and Hamadryas baboon (Papio hamadryas)) and three of New World or platyrrhine monkeys (Cebidae: tufted capuchin (Cebus apella) and Bolivian squirrel monkey (Saimiri boliviensis); Atelidae: mantled howler (Alouatta palliata)). The milks of these species contained 6-8% total sugar, most of which was lactose: the estimated ratio of oligosaccharides to lactose in Old World monkeys (1:4 to 1:6) was greater than in New World monkeys (1:12 to 1:23). The chemical structures of the oligosaccharides were determined mainly by (1)H-NMR spectroscopy. Oligosaccharides containing the type II unit (Gal(β1-4)GlcNAc) were found in the milk of the rhesus macaque, toque macaque, Hamadryas baboon and tufted capuchin, but oligosaccharides containing the type I unit (Gal(β1-3)GlcNAc), which have been found in human and many great ape milks, were absent from the milk of all species studied. Oligosaccharides containing Lewis x (Gal(β1-4)[Fuc(α1-3)]GlcNAc) and 3-fucosyl lactose (3-FL, Gal(β1-4)[Fuc(α1-3)]Glc) were found in the milk of the three cercopithecid monkey species, while 2-fucosyl lactose (5'-FL, Fuc(α1-2)Gal(β1-4)Glc) was absent from all species studied. All of these milks contained acidic oligosaccharides that had N-acetylneuraminic acid as part of their structures, but did not contain oligosaccharides that had N-glycolylneuraminic acid, in contrast to the milk or colostrum of great apes which contain both types of acidic oligosaccharides. Two GalNAc-containing oligosaccharides, lactose 3'-O-sulfate and lacto-N-novopentaose I (Gal(β1-3)[Gal(β1-4)GlcNAc(β1-6)]Gal(β1-4)Glc) were found only in the milk of rhesus macaque, hamadryas baboon and tufted capuchin, respectively. Further research is needed to determine the extent to which the milk oligosaccharide patterns observed among these taxa represent wider phylogenetic trends among primates and how much variation occurs among individuals or species.

Neoglycolipid probes prepared via oxime ligation for microarray analysis of oligosaccharide-protein interactions

Neoglycolipid technology is the basis of a microarray platform for assigning oligosaccharide ligands for carbohydrate-binding proteins. The strategy for generating the neoglycolipid probes by reductive amination results in ring opening of the core monosaccharides. This often limits applicability to short-chain saccharides, although the majority of recognition motifs are satisfactorily presented with neoglycolipids of longer oligosaccharides. Here, we describe neoglycolipids prepared by oxime ligation. We provide evidence from NMR studies that a significant proportion of the oxime-linked core monosaccharide is in the ring-closed form, and this form selectively interacts with a carbohydrate-binding protein. By microarray analyses we demonstrate the effective presentation with oxime-linked neoglycolipids of (1) Lewis(x) trisaccharide to antibodies to Lewisx, (2) sialyllactose analogs to the sialic acid-binding receptors, siglecs, and (3) N-glycans to a plant lectin that requires an intact N-acetylglucosamine core.

Design, synthesis and the effect of 1,2,3-triazole sialylmimetic neoglycoconjugates on Trypanosoma cruzi and its cell surface trans-sialidase

This work describes the synthesis of a series of sialylmimetic neoglycoconjugates represented by 1,4-disubstituted 1,2,3-triazole-sialic acid derivatives containing galactose modified at either C-1 or C-6 positions, glucose or gulose at C-3 position, and by the amino acid derivative 1,2,3-triazole fused threonine-3-O-galactose as potential TcTS inhibitors and anti-trypanosomal agents. This series was obtained by Cu(I)-catalysed azide-alkyne cycloaddition reaction ('click chemistry') between the azido-functionalized sugars 1-N(3)-Gal (commercial), 6-N(3)-Gal, 3-N(3)-Glc and 3-N(3)-Gul with the corresponding alkyne-based 2-propynyl-sialic acid, as well as by click chemistry reaction between the amino acid N(3)-ThrOBn with 3-O-propynyl-GalOMe. The 1,2,3-triazole linked sialic acid-6-O-galactose and the sialic acid-galactopyranoside showed high Trypanosoma cruzi trans-sialidase (TcTS) inhibitory activity at 1.0 mM (approx. 90%), whilst only the former displayed relevant trypanocidal activity (IC(50) 260 mu M). These results highlight the 1,2,3-triazole linked sialic acid-6-O-galactose as a prototype for further design of new neoglycoconjugates against Chagas' disease. (C) 2011 Elsevier Ltd. All rights reserved.

Assessment of DNA damage of Lewis lung carcinoma cells irradiated by carbon ions and X-rays using alkaline comet assay

DNA damage and cell reproductive death determined by alkaline comet and clonogenic survival assays were examined in Lewis lung carcinoma cells after exposure to 89.63 MeV/u carbon ion and 6 MV X-ray irradiations, respectively. Based on the survival data, Lewis lung carcinoma cells were verified to be more radiosensitive to the carbon ion beam than to the X-ray irradiation. The relative biological effectiveness (RBE) value, which was up to 1.77 at 10% survival level, showed that the DNA damage induced by the high-LET carbon ion beam was more remarkable than that induced by the low-LET X-ray irradiation. The dose response curves of '' Tail DNA (%)'' (TD) and "Olive tail moment" (OTM) for the carbon ion irradiation showed saturation beyond about 8 Gy. This behavior was not found in the X-ray curves. Additionally, the carbon ion beam produced a lower survival fraction at 2 Gy (SF2) value and a higher initial Olive tail moment 2 Gy (OTM2) than those for the X-ray irradiation. These results suggest that carbon ion beams having high-LET values produced more severe cell reproductive death and DNA damage in Lewis lung carcinoma cells in comparison with X-rays and comet assay might be an effective predictive test even combining with clonogenic assay to assess cellular radio sensitivity

Expression and distribution of cell-surface proteoglycans in the normal Lewis rat molar periodontium

Cell-surface proteoglycans participate in several biological functions such as cell cell and cell-matrix interactions, cell adhesion, the binding to various growth factors as co-receptors and repair. To understand better the expression and distribution of cell-surface proteoglycans in the periodontal tissues, an immunohistochemical evaluation of the normal Lewis rat molar periodontium using panels of antibodies for syndecan-1, -2, -4, glypican and betaglycan was carried out. Our results demonstrated the expression and distribution of all proteoglycans in the suprabasal gingival epithelium, soft and hard connective tissues. Both cellular and matrix localization was evident within the various periodontal compartments. The presence of these cell-surface proteoglycans indicates the potential for roles in the process of tissue homeostasis, repair or regeneration in periodontium of which each function requires further study.

Bishop Lewis Hartman, Reverend Dr. Isaac Higginbotham, Leo Baeck, Bishop Norman B. Nash, and Reverend Frank Jennings Portraits; Groups; Men

Page 57 of the "American Jewish Cavalcade" scrapbook of Leo Baeck in New York found in ROS 10 Folder 3

Copy of bill of costs for Lewis Gomes' denization

Digital image

Lewis Acid-Lewis Base Mediated Metal-Free Hydrogen Activation and Catalytic Hydrogenation

Organocatalysis, the use of organic molecules as catalysts, is attracting increasing attention as one of the most modern and rapidly growing areas of organic chemistry, with countless research groups in both academia and the pharmaceutical industry around the world working on this subject. The literature review of this thesis mainly focuses on metal-free systems for hydrogen activation and organocatalytic reduction. Since these research topics are relatively new, the literature review also highlights the basic principles of the use of Lewis acid-Lewis base pairs, which do not react irreversibly with each other, as a trap for small molecules. The experimental section progresses from the first observation of the facile heterolytical cleavage of hydrogen gas by amines and B(C6F5)3 to highly active non-metal catalysts for both enantioselective and racemic hydrogenation of unsaturated nitrogen-containing compounds. Moreover, detailed studies of structure-reactivity relationships of these systems by X-ray, neutron diffraction, NMR methods and quantum chemical calculations were performed to gain further insight into the mechanism of hydrogen activation and hydrogenation by boron-nitrogen compounds.

Ferrocene-based Lewis acids and Lewis pairs: Synthesis and structural characterization

Optically active Lewis acids and Lewis pairs were synthesized and characterized by multinuclear NMR, UV/Vis spectroscopy and elemental analysis. Optical rotation measurements were carried out and the absolute configuration of the new chiral molecules confirmed by single crystal X-ray diffraction.