Glucan-like synthetic oligosaccharides: iterative synthesis of linear oligo-beta-(1,3)-glucans and immunostimulatory effects

Small reducing and linear oligo-beta-(1,3)-glucans, which are able to act as phytoallexin elicitors or as immunostimulating agents in anticancer therapy, were synthesized according to an iterative strategy that involved a unique key monosaccharidic donor. To avoid anomeric mixtures, the reducing entity of the target oligomers was first locked with benzyl alcohol and further selective deprotection of the 3-OH with DDQ afforded the desired building block as an acceptor. The latter was then used in a second cycle of glycosylation/deprotection to afford the desired disaccharide, and successive reiterations of this process provided the desired oligomers. Unusual conformational behaviors were observed by standard NMR sequences and supported by NOESY studies. Finally, removal of protecting groups afforded free tri-, tetra-, and pentaglucosides in good overall yields. Two oligosaccharides representing linear laminaritetraose and laminaripentaose were compared to the recently described beta-(1,3)-glucan phycarine. Following an intraperitoneal injection, the influx of monocytes and granulocytes into the blood and macrophages into the peritoneal cavity was comparable to that caused by phycarine. Similarly, both oligosaccharides stimulated phagocytic activity of granulocytes and macrophages. Using ELISA, we also demonstrated a significant stimulation of secretion of IL-1beta. Together these results suggest that the synthetic oligosaccharides have similar stimulatory effects as natural beta-(1,3)-glucans.     

The analysis of reaction norms for age and size at maturity using maturation rate models

Reaction norms for age and size at maturity are being analyzed to answer important questions about the evolution of life histories. A new statistical method is developed in the framework of time-to-event data analysis, which circumvents shortcomings in currently available approaches. The method emphasizes the estimation of age- and size-dependent maturation rates. Individual probabilities of maturation during any given time interval follow by integrating maturation rate along the growth curve. The integration may be performed in different ways, over ages or sizes or both, corresponding to different assumptions on how individuals store the operational history of the maturation process. Data analysis amounts to fitting generalized nonlinear regression models to a maturation status variable. This technique has three main advantages over existing methods: (1) treating maturation as a stochastic process enables one to specify a rate of maturation; (2) age and size at which maturation occurs do not have to be observed exactly, and bias arising from approximations and interpolations is avoided; (3) ages at which sizes are measured and maturation status are observed can differ between individuals. An application to data on the springtail Folsomia candida is presented. Models with age-dependent integration of maturation rates were preferred. The analysis demonstrates a significant size dependence of the maturation rate but no age dependence.     

Controlling {beta}-amyloid oligomerization by the use of naphthalene sulfonates: trapping low molecular weight oligomeric species

Aggregation of proteins and peptides has been shown to be responsible for several diseases known as amyloidoses, which include Alzheimer disease (AD), prion diseases, among several others. AD is a neurodegenerative disorder caused primarily by the aggregation of beta-amyloid peptide (Abeta). Here we describe the stabilization of small oligomers of Abeta by the use of sulfonated hydrophobic molecules such as AMNS (1-amino-5-naphthalene sulfonate); 1,8-ANS (1-anilinonaphthalene-8-sulfonate) and bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonate). The experiments were performed with either Abeta-1-42 or with Abeta-13-23, a shorter version of Abeta that is still able to form amyloid fibrils in vitro and contains amino acid residues 16-20, previously shown to be essential to peptide-peptide interaction and fibril formation. All sulfonated molecules tested were able to prevent Abeta aggregation in a concentration dependent fashion in the following order of efficacy: 1,8-ANS < AMNS < bis-ANS. Size exclusion chromatography revealed that in the presence of bis-ANS, Abeta forms a heterogeneous population of low molecular weight species that proved to be toxic to cell cultures. Since the ANS compounds all have apolar rings and negative charges (sulfonate groups), both hydrophobic and electrostatic interactions may contribute to interpeptide contacts that lead to aggregation. We also performed NMR experiments to investigate the structure of Abeta-13-23 in SDS micelles and found features of an alpha-helix from Lys(16) to Phe(20). 1H TOCSY spectra of Abeta-13-23 in the presence of AMNS displayed a chemical-shift dispersion quite similar to that observed in SDS, which suggests that in the presence of AMNS this peptide might adopt a conformation similar to that reported in the presence of SDS. Taken together, our studies provide evidence for the crucial role of small oligomers and their stabilization by sulfonate hydrophobic compounds.     

Thematic review series: The immune system and atherogenesis. Cytokines affecting endothelial and smooth muscle cells in vascular disease

The cellular and extracellular matrix accumulations that comprise the lesions of atherosclerosis are driven by local release of cytokines at sites of predilection for lesion formation, and by the specific attraction and activation of cells expressing receptors for these cytokines. Although cytokines were originally characterized for their potent effects on immune and inflammatory cells, they also promote endothelial cell dysfunction and alter smooth muscle cell (SMC) phenotype and function, which can contribute to or retard vascular pathologies. This review summarizes in vivo studies that have characterized endothelial- and smooth muscle-specific effects of altering cytokine signaling in vascular disease. Although multiple reports have identified cytokines as pivotal players in endothelial and SMC responses in vascular disease, they also have highlighted the need to delineate the critical genes and specific cellular functions regulated by individual cytokine signaling pathways.     

Spectroscopic and interfacial properties of myoglobin/surfactant complexes

The complexes of horse myoglobin (Mb) with the anionic surfactant sodium dodecyl sulfate (SDS), and with the cationic surfactants cetyltrimethylammonium chloride (CTAC) and decyltrimethylammonium bromide (DeTAB), have been studied by a combination of surface tension measurements and optical spectroscopy, including heme absorption and aromatic amino acid fluorescence. SDS interacts in a monomeric form with Mb, which suggests the existence of a specific binding site for SDS, and induces the formation of a hexacoordinated Mb heme, possibly involving the distal histidine. Fluorescence spectra display an increase of tryptophan emission. Both effects point to an increased protein flexibility. SDS micelles induce both the appearance of two more heme species, one of which has the features of free heme, and protein unfolding. Mb/CTAC complexes display a very different behavior. CTAC monomers have no effect on the absorption spectra, and only a slight effect on the fluorescence spectra, whereas the formation of CTAC aggregates on the protein strongly affects both absorption and fluorescence. Mb/DeTAB complexes behave in a very similar way as Mb/CTAC complexes. The surface activity of the different Mb/surfactant complexes, as well as the interactions between the surfactants and Mb, are discussed on the basis of their structural properties.     

Sexually dimorphic expression of secreted frizzled-related (SFRP) genes in the developing mouse Müllerian duct

In developing male embryos, the female reproductive tract primordia (Müllerian ducts) regress due to the production of testicular anti-Müllerian hormone (AMH). Because of the association between secreted frizzled-related proteins (SFRPs) and apoptosis, their reported developmental expression patterns and the role of WNT signaling in female reproductive tract development, we examined expression of Sfrp2 and Sfrp5 during development of the Müllerian duct in male (XY) and female (XX) mouse embryos. We show that expression of both Sfrp2 and Sfrp5 is dynamic and sexually dimorphic. In addition, the male-specific expression observed for both genes prior to the onset of regression is absent in mutant male embryos that fail to undergo Müllerian duct regression. We identified ENU-induced point mutations in Sfrp5 and Sfrp2 that are predicted to severely disrupt the function of these genes. Male embryos and adults homozygous for these mutations, both individually and in combination, are viable and apparently fertile with no overt abnormalities of reproductive tract development.     

Biochemical and Spectroscopic Characterization of the Human Mitochondrial Amidoxime Reducing Components hmARC-1 and hmARC-2 Suggests the Existence of a New Molybdenum Enzyme Family in Eukaryotes

The mitochondrial amidoxime reducing component mARC is a newly discovered molybdenum enzyme that is presumed to form the catalytical part of a three-component enzyme system, consisting of mARC, heme/cytochrome b₅, and NADH/FAD-dependent cytochrome b₅ reductase. mARC proteins share a significant degree of homology to the molybdenum cofactor-binding domain of eukaryotic molybdenum cofactor sulfurase proteins, the latter catalyzing the post-translational activation of aldehyde oxidase and xanthine oxidoreductase. The human genome harbors two mARC genes, referred to as hmARC-1/MOSC-1 and hmARC-2/MOSC-2, which are organized in a tandem arrangement on chromosome 1. Recombinant expression of hmARC-1 and hmARC-2 proteins in Escherichia coli reveals that both proteins are monomeric in their active forms, which is in contrast to all other eukaryotic molybdenum enzymes that act as homo- or heterodimers. Both hmARC-1 and hmARC-2 catalyze the N-reduction of a variety of N-hydroxylated substrates such as N-hydroxy-cytosine, albeit with different specificities. Reconstitution of active molybdenum cofactor onto recombinant hmARC-1 and hmARC-2 proteins in the absence of sulfur indicates that mARC proteins do not belong to the xanthine oxidase family of molybdenum enzymes. Moreover, they also appear to be different from the sulfite oxidase family, because no cysteine residue could be identified as a putative ligand of the molybdenum atom. This suggests that the hmARC proteins and sulfurase represent members of a new family of molybdenum enzymes.     

Adenylyl cyclase type-VIII activity is regulated by G(betagamma) subunits

The Ca2+-activated adenylyl cyclase type VIII (AC-VIII) has been implicated in several forms of neural plasticity, including drug addiction and learning and memory. It has not been clear whether Gi/o proteins and G-protein coupled receptors regulate the activity of AC-VIII. Here we show in intact mammalian cell system that AC-VIII is inhibited by mu-opioid receptor activation and that this inhibition is pertussis toxin sensitive. Moreover, we show that G(betagamma) subunits inhibit AC-VIII activity, while constitutively active alphai/o subunits do not. Different Gbeta isoforms varied in their efficacies, with Gbeta1gamma2 or Gbeta2gamma2 being more efficient than Gbeta3gamma2 and Gbeta4gamma2, while Gbeta5 (transfected with gamma2) had no effect. As for the Ggamma subunits, Gbeta1 inhibited AC-VIII activity in the presence of all gamma subunits tested except for gamma5 that had only a marginal activity. Moreover, cotransfection with proteins known to serve as scavengers of Gbetagamma dimers, or to reduce Gbetagamma plasma membrane anchorage, markedly attenuated the mu-opioid receptor-induced inhibition of AC-VIII. These results demonstrate that Gbetagamma (originating from agonist activation of these receptors) and probably not Galphai/o subunits are involved in the agonist inhibition of AC-VIII.