1,721,119 research outputs found
Natural history specimens collected and/or identified and deposited.
No full textNatural history specimen data collected and/or identified by Beat Ernst Leuenberger, http://www.wikidata.org/entity/Q813055. Claims or attributions were made on Bionomia, https://bionomia.net using specimen data from the Global Biodiversity Information Facility, https://gbif.org.http://www.wikidata.org/entity/Q81305
Synthesis and biological evaluation of carbohydrate-mimetics as ligands for Siglecs
Get PDFSialic acid binding immunoglobulin-like lectins (Siglecs) play an important role in the mediation of cell-cell interactions as well as in the regulation of signaling pathways.
They are mainly expressed in the haematopoietic and immune system, with exception of Siglec-4, also called myelin-associated glycoprotein (MAG). It was identified as one of neurite outgrowth inhibitors, playing a crucial role in paraplegia, which is caused by injuries of the central nervous system (CNS) and especially young people suffer from these severe consequences as, for example, the loss of motor functions. The lack of repair of the injured nerve strands originates from the inhibitory environment for axon regeneration in the CNS. SpeciÞc inhibitory proteins, such as MAG block the regrowth of nerve roots. We identiÞed potent small molecule MAG antagonists modifies in the 2- and 5-position. Furthermore, we investigated new neuraminic acid derivatives modiÞed in the 4-position, and the inßuence of various structural modiÞcations on their kinetic and thermodynamic binding properties. In a next step we presented high affinity ligands, which were identified in second-site screenings and optimized them according to medicinal chemistry aspects. All ligands were elucidated with respect to their binding affinity as well as their kinetic and thermodynamic profile.
Siglec-2, also known as CD22, is involved in the regulation and survival of B-cells and has been successfully targeted in cell depletion therapies with antibody-based approaches. Sialic acid derivatives, already known to bind with high affinity to myelin-associated glycoprotein (MAG, Siglec-4), were screened for their binding affinity for CD22 by surface plasmon resonance. The best compound identified was further modified with various hydrophobic substituents at the 2-, 5-, and 9-positions of the sialic acid scaffold, leading to nanomolar derivatives. Furthermore, initial tests regarding drug-like properties of these antagonists demonstrate the required high plasma protein binding yet a lack of oral availability, although its distribution coefficient (log D) is in the required range.
Finally, we investigated a library of sialic acid mimetics with respect to binding towards another member of the Siglec family, namely Sialadhesin and discuss the influence of various structural moieties with regard to the arising selectivity towards these three proteins
Expression and characterization of E-LecEGF for structural study and assay development
Get PDFHuman E-selectin (hE-selectin) is a cell adhesion molecule expressed on the membrane of endothelial cells. It is a C-type lectin whose key role is to mediate the initial rolling and adhering of leukocytes in the leukocyte recruitment in inflammation and metastasis of some cancer cells. It is fundamentally involved in many physiological and pathological processes, and hence is an attractive target for developing anti-inflammation drugs. The lectin and EGF domains of hE-selectin (hE-LecEGF) were identified as the minimum functional unit. Crystal structures of hE-LecEGF complexed with its natural ligand, tetrasaccharide sialyl Lewisx (sLex), as well as NMR studies of hE-selectin/IgG bound with this ligand, have been reported and utilized as the structural basis for the development of potent antagonists for hE-selectin. More potent antagonists with better binding affinity than sLex, such as CGP69669, were reported, but their binding modes in hE-LecEGF remain unknown. To obtain the improved structural information of hE-LecEGF complexed with an antagonist and develop more potent antagonists of hE-selectin are challenging tasks. To meet the demands of the protein for the structure determination and the binding assay, a sufficient amount of pure and active hE-LecEGF is needed. In this thesis, insect cell expression systems were initially used to produce the hE-LecEGF protein. hE-LecEGF was cloned, transiently expressed and characterized in Sf9 and High fiveTM cells. The expression plasmid pFastBacYJSE was constructed for expression of the hE-LecEGF protein fused with a N-terminal Flag tag. The recombinant baculovirus was generated and used in the expression of protein in the suspension culture. Pure hE-LecEGF was obtained by anti-Flag M2 affinity chromatography under the optimized condition. The purified protein was active and glycosylated, as identified by mAb 7A9 and glycan detection, respectively. Unfortunately, the homogeneous hE-LecEGF protein was not obtained after the deglycosylation with PNGase F and N-glycosidase A. hE-LecEGF was then cloned, stably expressed and characterized in CHO K1 cells. Stable subclones CHO-YJES and CHO-YJEGS expressing the hE-LecEGF protein with or without a human IgG1 tag were achieved. The CHO-YJES construct was used for production. The monoclonal anti-E-selectin functional blocking antibody 7A9 (mAb 7A9) was produced, purified and coupled to sepharose for functional purification of the hE-LecEGF protein. Highly pure hE-LecEGF protein was obtained in a one-step purification with an mAb 7A9 coupled column. Page, western-blotting, ELISA, MS and NMR were performed to characterize the hE-LecEGF protein. Pure, monomeric and active hE-LecEGF with the molecular weight of 20.444 kDa was obtained. In contrast to the insect cell expression system, pure, active and
uniform deglycosylated hE-LecEGF protein was obtained after treatment with PNGase F and purification by a Sepharose Q matrix. A prescreening of the crystallization condition of hE-LecEGF was also performed using a sitting-drop method. Furthermore, a novel cell-free assay “capture-binding assay” was developed with the tag-free hE-LecEGF protein to evaluate the binding activity of the hE-LecEGF protein and the binding affinity of hE-selectin antagonists. The rIC50 of six hE-selectin antagonists was determined. The obtained results were in close agreement with the published results. Compared to the previously unstable polymer assay with hE-selectin/IgG, the capture-binding assay with hE-LecEGF is accurate, sensitive and reproducible. It can correctly evaluate the binding affinities of hE-selectin antagonists. In addition, the antibody BBA1 was used to solve the problem of immobilization of the hE-LecEGF protein on ELISA plates in the assay
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Targeting the liver via the asialoglycoprotein-receptor : synthesis of directed small molecule libraries for the H1-CRD
Get PDFThe ASGP-R, exclusively located on the cell surfaces of hepatocytes, is well known
for removing desialylated glycoproteins with terminal galactose or N -
acetylgalactosamine residues from circulation. However, small high affinity ligands
with more drug-like properties than the natural glycoproteins, that can be used for
specific targeting of the liver, were not yet investigated for the human H1-CRD.
Various small-directed libraries of galactose derivatives and mimics thereof were
synthesized in this work. The design of the ligands was based on the crystal structure
of the H1-CRD[10], and on data from in silico studies[40] as well as from previous SAR
studies for similar receptors. The influence of different types of modifications at the
galacto moiety on the binding affinity, were analyzed by a competitive target-based
assay and Biacore experiments, both performed with immobilized H1-CRD.
Monosaccharide mimics with slightly improved binding affinities for the H1-CRD
relative to GalNAc were found. This work provides experimental data for simple
synthetic ligands of the H1-CRD and therefore gives guidelines for the rational design
of a novel generation of improved ligands.
1.13 Galactose mimics modified at their 6-position.
Galactose mimics based on the 1,5-anhydro-2-deoxy-D-lyxo-hexitol core were
modified at their 6-position by conjugated additions, nucleophilic substitutions and
1,3-dipolar cycloadditions. Compound 3 was used as scaffold. By
substituting its 6-position it was attempted to establish hydrophobic interactions with
the Trp 243. Due to the reduced number of sites with similar reactivity, short synthetic
pathways provided fast access to these ligands.
All the derivatives competed approximately two to three-fold better for H1-CRD than
Gal. Binding affinities comparable to the one of methyl β-Dgalactopyranoside
were obtained. Since for derivatives modified with 1,4-
disubstituted-[1,2,3]-triazoles (45, 50), the behavior was independent of the 4-
substituent at the triazole, the interaction with Trp 243 is possibly restricted to the
triazole ring. The 6-position is directed towards the surrounding water and bulky
substituents could be accommodated without interfering with the binding.
Compound 39, obtained as secondary product from Michael additions and
nucleophilic substitutions, showed a comparable binding affinity as GalNAc (figure
22) in the competitive target-based assay. However, Biacore monitoring of the direct
interaction between 39 and the immobilized H1-CRD did not show a comparable
increase of the binding affinity. Since hexitol 39 has two pairs of free 3,4-hydroxyls in
equatorial and axial arrangement, the difference may come from a local
concentration effect which could convert two weak binders into one highly improved
binder.
In general, galactose mimics modified at the 6-position by hydrophobic groups
showed only a modest improvement of the binding affinity for H1-CRD relative to Gal.
However, bulky groups in the 6-position did not interfere with the binding indicating
that this position can be used to link the targeting device with a possible drug.
1.14 Galactose derivatives and mimics thereof modified only at their 2-
position.
To investigate the role that modifications at the 2-position of the galactose moiety
may play, two families of derivatives were synthesized.
Compounds 66 to 75 were obtained by 1,3-dipolar cycloadditions performed on an
azido group at the C-2’ linked to the 2-OH of the sugar scaffold. They do not show
improved binding affinity in the competitive assay and the Biacore experiments.
Derivative 72 was the only member of this group to show a binding affinity (IC50 (μM)
= 110 ± 13) comparable to the one of GalNAc. However, a different binding mode
was found by biosensor studies of the direct interaction between this compound and
the immobilized monomer of H1-CRD.
2-N-acyl derivatives of a methyl 6-azido-β-D-galactopyranoside were synthesized on
solid phase by amino acid coupling at the N-2-position of the sugar moiety.
Compounds 98-100 showed improved binding affinities for the H1-CRD relative to
methyl β-D-galactopyranoside. Whereas derivatives acylated with glycine
(98) or valine (100) experienced only a modest improvement of their binding
affinities, the 2-(2-amino propionamido) derivative 99 achieved a binding affinity
approximately 1.5 to 2-fold better than GalNAc and 12-fold better than methyl β-Dgalactopyranoside.
Even though the galactose mimic 46 containing an azido
functionality at the 6-position, competed 2 to 3 times better for binding to the H1-CRD
relative to 3, the 6-azido group present also in derivatives 98-100 does not contribute
to the binding.
N-acyl coupled chains appear to improve the binding affinity for the receptor
depending on the size of the acyl group. The propionamido derivative displayed the
lowest IC50 value and therefore the optimal chain length studied. It is possible that
such N-acyl derivatives allow establishing an optimal interaction with His 256,
through H-bond formation with the hydrogen of the nitrogen of the N-acyl group. This
interaction is known for being responsible for the preferential binding of GalNAc with
respect to Gal. 2-N-acyl derivatives are an excellent starting point for the
development of novel high affinity ligands for the H1-CRD.
1.15 Monosaccharides mimics modified simultaneously at their 2- and 6-
positions.
Methyl β-D-galactosamine-derivatives modified at the 2- and 6-positions were
synthesized to elucidate the hypothesis of an added effect on the binding affinity. A
collection of ten di-substituted galactose mimics was synthesized on solid phase with
excellent yields. Amino acids were first coupled with the N-2-position of a
galactosamine mimic and modified Huisgen 1,3-dipolar cycloadditions were
performed at the 6-position of the monosaccharide mimic.
Results from the competitive target-based assay indicated that all the derivatives
synthesized were better binders for the H1-CRD than methyl β-D-galactopyranoside.
However, all di-substituted compounds showed very similar IC50 values to their
parent derivatives N-acyl monosubstituted (98-100). Improved binding due to the
additional 6-substitution could not be observed. The better-defined orientation of the
monosaccharide in the binding pocket, after establishing additional interactions
through its 2-N-substituent, could be the reason for the loss of a complementary 6-
interaction. In addition, this hypothesis may also explain the behavior observed in the
preliminary IC50 determination for compound 69 relative to 68 (figure 24). In the
presence of the substitution in 2-position, compound 68 did not showed an improved
binding affinity compared to Gal. Lacking good interactions with the protein in the
region of the 2-position of Gal, the substituent at the 6-position in 69 could improve
binding.
Since none of the substituents employed at the 6-position interfered with the binding, a collection of eight derivatives obtained by amino acid coupling with alanine (99 and 104-110), with similar or slightly improved binding affinities than GalNAc was
obtained for the H1-CRD.
Outlook.
The results obtained in this thesis may be applied to produce a new generation of
ligands for the H1-CRD.
First, the synthesis of three 2-substituted galactose derivatives (figure 26) may be
envisaged.
The influence of the one-carbon extension of the N-acyl chain of 99 on the binding
affinity could be studied with compound 112. Between the propionamido derivative
99 and the valine-derivatized compound 100 exists a space to be analyzed for
determining the real optimal length preferred for the N-acyl chain. Moreover, the
absence or replacement of the α-amino group on the N-acyl chains (113) would allow
further exploration of the binding site and the type of established interactions.
Substituents as H, CH3 and OH are suggested in order to determine whether
hydrophobic interactions or H-bond formation are preferred. Additionally, compound
114 will help to establish if the N-acyl derivatives have a preferred binding relative to
their O-counterparts due to specific interactions with His 256.
Second, compound 99 could be employed to synthesize a triantennary ligand[36] for
the H1-CRD. Determination of the IC50 value of the novel multivalent ligand in a
competitive assay and comparison with a triantennary galactose ligand would be of
high interest for the development of liver targeting strategies. Additionally, a
comparative study of its cellular uptake by the hepatocytes would also be required
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Dispelling the Myths Behind First-author Citation Counts
Get PDFWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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