Asymmetric synthesis and affinity of potent sialyltransferase inhibitors based on transition-state analogues

Inhibitors that are structurally related to the transition-state model of the proposed SN1-type mechanism of sialyl transfer, exhibit particularly high binding affinities to alpha(2-6)sialyltransferases. Furthermore, replacing the neuraminyl residue with a simple aryl or hetaryl ring and substituting the carboxylate group for a phosphonate moiety, improves both binding affinity and synthetic accessibility. Herein we report on the synthesis and inhibition of a wide range of novel, potent transition-state analogue based alpha(2-6)sialyltransferase inhibitors comprising a planar anomeric carbon, an increased distance between the anomeric carbon and the CMP leaving group, and at least two negative charges. We also present a short, efficient asymmetric synthesis of the most promising benzyl inhibitors, providing rapid access to large quantities of highly potent, stereochemically-pure (>96% de) inhibitors for further biological investigation (e.g.(R)-3b, Ki = 70 nM).     

Asymmetric synthesis and reactivity of potent sialyltransferase inhibitors based on transition-state analogues: Supplementary data

Published in 2003. This revised version was published online in July 2006 with corrections to the Cover Date.     

T-cell-mediated lysis of B cells induced by a CD19xCD3 bispecific single-chain antibody is perforin dependent and death receptor independent

A recently developed bispecific antibody construct, directed against CD19 and CD3 (bscCD19xCD3), induces T-cell-mediated lysis of allogeneic and autologous B cells in a specific and highly efficient manner. Since knowledge of the molecular mechanisms underlying this lysis is limited, a study on bscCD19xCD3-activated T-cell-effector pathways was performed. BscCD19xCD3-induced lysis of target B-cell lines Nalm-6, Daudi, and Raji and of autologous primary B cells is caused by the perforin-dependent granule-exocytosis pathway but not by the death ligands FasL, TRAIL, or TNF-. When activated by bscCD19xCD3 and Raji cells, T cells express FasL mRNA, but incubation of Raji cells with cell-free supernatants from cytotoxicity experiments caused an upregulation of c-Flip_l, possibly accounting for the cells insensitivity toward death-receptor-mediated lysis. In addition to granule exocytosis, Raji cells are lysed by at least one mechanism independent of perforin, which requires transport through the T cells Golgi apparatus.This investigation was supported by the Deutsche Forschungsgemeinschaft, Klinische Forschergruppe, grant no. KFO 105/1.     

Plasticity of human chromosome 3 during primate evolution

Comparative mapping of more than 100 region-specific clones from human chromosome 3 in Bornean and Sumatran orangutans, siamang gibbon, and Old and New World monkeys allowed us to reconstruct ancestral simian and hominoid chromosomes. A single paracentric inversion derives chromosome 1 of the Old World monkey Presbytis cristata from the simian ancestor. In the New World monkey Callithrix geoffroyi and siamang, the ancestor diverged on multiple chromosomes, through utilizing different breakpoints. One shared and two independent inversions derive Bornean orangutan 2 and human 3, implying that neither Bornean orangutans nor humans have conserved the ancestral chromosome form. The inversions, fissions, and translocations in the five species analyzed involve at least 14 different evolutionary breakpoints along the entire length of human 3; however, particular regions appear to be more susceptible to chromosome reshuffling. The ancestral pericentromeric region has promoted both large-scale and micro-rearrangements. Small segments homologous to human 3q11.2 and 3q21.2 were repositioned intrachromosomally independent of the surrounding markers in the orangutan lineage. Breakage and rearrangement of the human 3p12.3 region were associated with extensive intragenomic duplications at multiple orangutan and gibbon subtelomeric sites. We propose that new chromosomes and genomes arise through large-scale rearrangements of evolutionarily conserved genomic building blocks and additional duplication, amplification, and/or repositioning of inherently unstable smaller DNA segments contained within them.     

Close homolog of L1 modulates area-specific neuronal positioning and dendrite orientation in the cerebral cortex

We show that the neural cell recognition molecule Close Homolog of L1 (CHL1) is required for neuronal positioning and dendritic growth of pyramidal neurons in the posterior region of the developing mouse neocortex. CHL1 was expressed in pyramidal neurons in a high-caudal to low-rostral gradient within the developing cortex. Deep layer pyramidal neurons of CHL1-minus mice were shifted to lower laminar positions in the visual and somatosensory cortex and developed misoriented, often inverted apical dendrites. Impaired migration of CHL1-minus cortical neurons was suggested by strikingly slower rates of radial migration in cortical slices, failure to potentiate integrin-dependent haptotactic cell migration in vitro, and accumulation of migratory cells in the intermediate and ventricular/subventricular zones in vivo. The restriction of CHL1 expression and effects of its deletion in posterior neocortical areas suggests that CHL1 may regulate area-specific neuronal connectivity and, by extension, function in the visual and somatosensory cortex.     

alpha3beta1 integrin modulates neuronal migration and placement during early stages of cerebral cortical development

We show that alpha3 integrin mutation disrupts distinct aspects of neuronal migration and placement in the cerebral cortex. The preplate develops normally in alpha3 integrin mutant mice. However, time lapse imaging of migrating neurons in embryonic cortical slices indicates retarded radial and tangential migration of neurons, but not ventricular zone-directed migration. Examination of the actin cytoskeleton of alpha3 integrin mutant cortical cells reveals aberrant actin cytoskeletal dynamics at the leading edges. Deficits are also evident in the ability of developing neurons to probe their cellular environment with filopodial and lamellipodial activity. Calbindin or calretinin positive upper layer neurons as well as the deep layer neurons of alpha3 integrin mutant mice expressing EGFP were misplaced. These results suggest that alpha3beta1 integrin deficiency impairs distinct patterns of neuronal migration and placement through dysregulated actin dynamics and defective ability to search and respond to migration modulating cues in the developing cortex.     

The VirB type IV secretion system of Bartonella henselae mediates invasion, proinflammatory activation and antiapoptotic protection of endothelial cells

Bartonella henselae is an arthropod-borne zoonotic pathogen causing intraerythrocytic bacteraemia in the feline reservoir host and a broad range of clinical manifestations in incidentally infected humans. Remarkably, B. henselae can specifically colonize the human vascular endothelium, resulting in inflammation and the formation of vasoproliferative lesions known as bacillary angiomatosis and bacillary peliosis. Cultured human endothelial cells provide an in vitro system to study this intimate interaction of B. henselae with the vascular endothelium. However, little is known about the bacterial virulence factors required for this pathogenic process. Recently, we identified the type IV secretion system (T4SS) VirB as an essential pathogenicity factor in Bartonella, required to establish intraerythrocytic infection in the mammalian reservoir. Here, we demonstrate that the VirB T4SS also mediates most of the virulence attributes associated with the interaction of B. henselae during the interaction with human endothelial cells. These include: (i) massive rearrangements of the actin cytoskeleton, resulting in the formation of bacterial aggregates and their internalization by the invasome structure; (ii) nuclear factor kappaB-dependent proinflammatory activation, leading to cell adhesion molecule expression and chemokine secretion, and (iii) inhibition of apoptotic cell death, resulting in enhanced endothelial cell survival. Moreover, we show that the VirB system mediates cytostatic and cytotoxic effects at high bacterial titres, which interfere with a potent VirB-independent mitogenic activity. We conclude that the VirB T4SS is a major virulence determinant of B. henselae, required for targeting multiple endothelial cell functions exploited by this vasculotropic pathogen.     

Mapping of the Physcomitrella patens proteome

The moss Physcomitrella patens is unique among land plants due to the high rate of homologous recombination in its nuclear DNA. The feasibility of gene targeting makes Physcomitrella an unrivalled model organism in the field of plant functional genomics. To further extend the potentialities of this seed-less plant we aimed at exploring the P. patens proteome. Experimental conditions had to be adopted to meet the special requirements connected to the investigations of this moss. Here we describe the identification of 306 proteins from the protonema of Physcomitrella. Proteins were separated by two dimensional electrophoresis, excised form the gel and analysed by means of mass spectrometry. This reference map will lay the basis for further profound studies in the field of Physcomitrella proteomics.