Electrosyntheses of disaccharides from phenyl or ethyl 1-thioglycosides

Constant current electrolyses of the glycosyl donors phenyl and ethyl 2,3,4,6-tetra-O-benzyl-1-thio-β-d-glycopyranoside in dry acetonitrile in the presence of various primary and secondary sugar alcohols, performed in an undivided cell, gave β-linked disaccharide derivatives selectively in good yields. Phenyl 2,3,4,6-tetra-O-benzoyl-1-thio-β-d-glycopyranoside gave the β-glucosides exclusively in good to moderate yields.     

Further studies of the ability of xyloglucan oligosaccharides to inhibit auxin-stimulated growth

The structural features required for xyloglucan oligosaccharides to inhibit 2,4-dichlorophenoxyacetic acid-stimulated elongation of pea stem segments have been investigated. A nonasaccharide (XG9) containing one fucosyl-galactosyl side chain and an undecasaccharide (XG11) containing two fucosyl-galactosyl side chains were purified from endo-β-1,4-glucanase-treated xyloglucan, which had been isolated from soluble extracellular polysaccharides of suspension-cultured sycamore (Acerpseudoplatanus) cells and tested in the pea stem bioassay. A novel octasaccharide (XG8′) was prepared by treatment of XG9 with a xyloglucan oligosaccharide-specific α-xylosidase from pea seedlings. XG8′ was characterized and tested for its ability to inhibit auxin-induced growth. All three oligosaccharides, at a concentration of 0.1 microgram per milliliter, inhibited 2,4-dichlorophenoxyacetic acid-stimulated growth of pea stem segments. XG11 inhibited the growth to a greater extent than did XG9. Chemically synthesized nona- and pentasaccharides (XG9, XG5) inhibited 2,4-dichlorophenoxyacetic acid-stimulated elongation of pea stems to the same extent as the same oligosaccharides isolated from xyloglucan. A chemically synthesized structurally related heptasaccharide that lacked a fucosyl-galactosyl side chain did not, unlike the identical heptasaccharide isolated from xyloglucan, significantly inhibit 2,4-dichlorophenoxyacetic acid-stimulated growth.     

Timing of oral morphogenesis and its relation to commitment to division in Paramecium tetraurelia

The interval between commitment to division and fission in synchronous cell samples is a constant fraction of the cell cycle (0.2) in cell cycles up to 6.5 h in duration. In longer cell cycles this interval has a fixed duration of about 80 min. The point of commitment to division is associated with the six-rowed anlage stage of oral primordium development (stage V). At this stage cells carrying the cc1 mutation are not blocked by transfer to restrictive conditions but rather proceed to division. Stage V is also the stabilization point for oral anlagen. When shifted to restrictive conditions prior to this stage, development is arrested and resorption of anlagen is initiated. The cc1 mutation also blocks contractile vacuole duplication and migration under restrictive conditions. The cc1 gene function is required continuously prior to the transition point. The timing of morphogenetic stages in asynchronous cells is roughly similar to that in synchronous cells. There are, however, significant differences in timing as estimated by the two experimental procedures.     

Synthèse des 2-acé-tamido-2-désoxy-6-O-α-et β-D-xylopyranosyl-α-D-glucopyranose

2-Acetamido-2- deoxy-6-O-, -xylopyranosyl-O-D-glucopyranose has been synthesized in crystalline form by condensation of 2,3,4-tri-O-acetyl-α-D-xylopyranosyl chloride (1) with benzyl 2-acetamido-3,4-di-O-acetyl-2-deoxy-β-D-glucopyranoside (2), followed by O-deacetylation and catalytic hydrogenation. Condensation of 2 with 2,3,4-tri-O-chlorosulfonyl-β-D-xylopyranosyl chloride, followed by dechlorosulfonylation and acetylation, gave benzyl 2-acetamido-3,4-di-O-acetyl-2-deoxy-6-O-(2,3,4-tri-O-acetyl-α-D-xylopyranosyl)β-D-glucopyranoside in crystalline form. O-Deacetylation, followed by catalytic hydrogenation, gave 2-acetamido-2-deoxy-6-O-α-D-xylopyranosyl-α-D-glucopyranose in crystalline form.     

Oligosaccharides corresponding to the regular sequence of heparin: chemical synthesis and interaction with FGF-2.

It has been proposed that oligosaccharides corresponding to the so-called regular region of heparin/heparan sulfate (HS) bind to fibroblast growth factor-2 (FGF-2). In order to explore the molecular basis of FGF/HS interaction, we describe here the chemical synthesis of a tetra and a hexasaccharide, prepared as methyl glycosides, corresponding to the regular sequence of heparin. The strategy relies on the efficient preparation of three building blocks: a seeding block, an elongating block and a capping block. The hexasaccharide inhibited the binding of FGF-2 on its receptor on human aorta vascular smooth muscle cells with an IC50 value (16+/-1.2 microg/mL) close to that of standard heparin (14.8+/-0.5 microg/mL) whereas the tetrasaccharide was much less potent (IC50 = 127+/-10.5 microg/mL). The hexasaccharide and heparin, inhibited in a dose-dependent manner FGF-2 (30 nM) induced proliferation (IC50 = 23.7+/-1.6 and 30.1+/-1.3 microg/mL, respectively). Under the same experimental conditions, the tetrasaccharide only slightly inhibited the mitogenic effect of FGF-2 (IC50 > 100 microg/mL).     

A synthetic heparan sulfate pentasaccharide, exclusively containing L-iduronic acid, displays higher affinity for FGF-2 than its D-glucuronic acid-containing isomers.

It has been suggested that the FGF-2 binding site on heparan sulfate chains is a trisulfated pentasaccharide containing three hexuronic acid units. The configuration at C-5 of two of them being undetermined, we have synthesized the four possible pentasaccharides, and have evaluated their FGF-2 binding affinity through in vitro biological assays. The pentasaccharide containing L-iduronic acid as the sole hexuronic acid showed higher affinity for FGF-2 than the other pentasaccharides, where one hexuronic acid unit at least is D-glucuronic acid.     

Functional CD8+ T cell responses in lethal Ebola virus infection

Ebola virus (EBOV) causes highly lethal hemorrhagic fever that leads to death in up to 90% of infected humans. Like many other infections, EBOV induces massive lymphocyte apoptosis, which is thought to prevent the development of a functional adaptive immune response. In a lethal mouse model of EBOV infection, we show that there is an increase in expression of the activation/maturation marker CD44 in CD4(+) and CD8(+) T cells late in infection, preceding a dramatic rebound of lymphocyte numbers in the blood. Furthermore, we observed both lymphoblasts and apoptotic lymphocytes in spleen late in infection, suggesting that there is lymphocyte activation despite substantial bystander apoptosis. To test whether these activated lymphocytes were functional, we performed adoptive transfer studies. Whole splenocytes from moribund day 7 EBOV-infected animals protected naive animals from EBOV, but not Marburgvirus, challenge. In addition, we observed EBOV-specific CD8(+) T cell IFN-gamma responses in moribund day 7 EBOV-infected mice, and adoptive transfer of CD8(+) T cells alone from day 7 mice could confer protection to EBOV-challenged naive mice. Furthermore, CD8(+) cells from day 7, but not day 0, mice proliferated after transfer to infected recipients. Therefore, despite significant lymphocyte apoptosis, a functional and specific, albeit insufficient, adaptive immune response is made in lethal EBOV infection and is protective upon transfer to naive infected recipients. These findings should cause a change in the current view of the 'impaired' immune response to EBOV challenge and may help spark new therapeutic strategies to control lethal filovirus disease.     

A potent peptidomimetic inhibitor of botulinum neurotoxin serotype A has a very different conformation than SNAP-25 substrate

Botulinum neurotoxin serotype A is the most lethal of all known toxins. Here, we report the crystal structure, along with SAR data, of the zinc metalloprotease domain of BoNT/A bound to a potent peptidomimetic inhibitor (K(i)=41 nM) that resembles the local sequence of the SNAP-25 substrate. Surprisingly, the inhibitor adopts a helical conformation around the cleavage site, in contrast to the extended conformation of the native substrate. The backbone of the inhibitor's P1 residue displaces the putative catalytic water molecule and concomitantly interacts with the "proton shuttle" E224. This mechanism of inhibition is aided by residue contacts in the conserved S1' pocket of the substrate binding cleft and by the induction of new hydrophobic pockets, which are not present in the apo form, especially for the P2' residue of the inhibitor. Our inhibitor is specific for BoNT/A as it does not inhibit other BoNT serotypes or thermolysin.