Crystallization and preliminary X-ray diffraction studies of a pea lectin-methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside complex

The seed lectin isolated from garden peas (Pisum sativum) has been co-crystallized with methyl 3,6-di-O-(alpha-D-mannopyranosyl)-alpha-D-mannopyranoside in the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions a = 64.3 A, b = 73.4 A and C = 108.5 A. The asymmetric unit contains one pea lectin dimer (alpha 2 beta 2). The crystals are suitable for high-resolution structure analysis.     

The adjuvant activity of lactoferrin in the generation of DTH to ovalbumin can be inhibited by bovine serum albumin bearing alpha-D-mannopyranosyl residues

Lactoferrin (LF) is an iron-binding glycoprotein present in the cytoplasmic granules of neutrophils and in external secretions of mammals. Although the biological role of human and bovine lactoferrin has been extensively studied, there is still uncertainty as to the nature and function of lactoferrin receptors. We recently determined that methyl-alpha-D-mannopyranoside given intraperitoneally (i.p.) could suppress the adjuvant activity of LF in the generation of delayed-type hypersensitivity (DTH) to ovalbumin (OVA). We concluded that the lactoferrin effects in DTH are mediated by carbohydrate-recognizing receptors like the mannose receptor (MR). This study indicates that subcutaneous (s.c.) administration of very small doses of the Man-bovine serum albumin (Man-BSA) complex, together with a sensitizing dose of the antigen, gives the same effects as i.p. administration of methyl-alpha-D-mannopyranoside. The latter is also a blocker of MR, although of a much lower affinity to the receptor than Man-BSA. The blocking of the adjuvant effect of LF by the Man-BSA complex (when given together with the sensitising dose of antigen) suggests that the function of antigen-presenting cells in the skin (presumably immature dendritic cells expressing MR) is inhibited. The results of our study indicate that a receptor with an affinity for mannose is essential for the mediation of adjuvant lactoferrin function in the generation of DTH.     

Preparation and supramolecular properties of unadulterated glycosyl liposomes from a bis(alpha-D-mannopyranosyl)-[60]fullerene conjugate

The bis(alpha-D-mannopyranosyl)-[60]fullerene conjugate 3 was prepared by thermal coupling of C60 and either 2-azidoethyl 2,3,4,6-tetra-O-acetyl- or 2,3;4,6-di-O-isopropylidene-alpha-D-mannopyranoside (Scheme). Compound 3 was found to readily self-assemble. Dynamic-light-scattering (DLS) and atomic-force microscopy (AFM) experiments supported that the amphiphilic compound gives rise to nano-sized supramolecular structures during sugar deprotection (Ac-group removal) performed in MeOH/CH2Cl2 solution. Encapsulation studies with an aqueous suspension of 3 showed that the self-assembling structure envelopes Ba2+ and the fluorescent dye Acridine Red during its formation, which indicates that it resembles a bilayer vesicle or an unadulterated liposome with an inner hollow space. In addition to this notable property, the unique molecular geometry of the spatially arranged mannosyl surface residues of 3 gives rise to strong binding of the carbohydrate-recognizing lectin Con A. Hence, the polar amphiphilic end of 3 mimics the structure of 3,6-branched tri-alpha-D-mannoside (6; Fig. 3), a natural ligand of the Con A protein.     

Synthesis P1-dolichyl P2-alpha-D-mannopyranosyl pyrophosphate. The acid and alkaline hydrolysis of polyisoprenyl alpha-D-mannopyranosyl mono- and pyrophosphate diesters.

P1-Dolichyl P2-ALPHA-D-mannopyranosyl pyrophosphate (9) has been chemically synthesized by a method developed for the corresponding citronellyl derivative, which also contains a saturated alpha isoprene residue. In each case, the P1-polyisoprenyl P2-diphenyl pyrophosphate was treated with 2,3,4,6-tetra-O-acetyl-alpha-D-mannopyranosyl phosphate to give a fully acetylated pyrophosphate diester, which was purified chromatographically and subsequently deacetylated. The citronellyl and dolichyl pyrophosphate diesters were compared with the previously synthesized citronellyl and dolichyl alpha-D-mannopyranosyl phosphate, respectively, by chromatography and by hydrolysis experiments. Good separations of the monophosphate from the corresponding pyrophosphate were achieved by silica gel tlc in a variety of solvent systems. Brief dilute acid hydrolysis of both the mono- and pyrophosphate diesters gave D-mannose and no alpha-D-mannosyl phosphate, the other products being polyprenyl phosphate and pyrophosphate, respectively. When the polyprenyl alpha-D-mannopyranosyl mono- and pyrophosphate diesters were treated with hot dilute alkali, the major products were polyprenyl phosphate and substances arising from the breakdown of D-mannose, indicating that the alpha-D-mannosyl phosphate bond was the most labile linkage in both compounds. However, the formation of a small proportion of free dolichol indicated that alpha-D-mannosyl phosphate was also formed to a minor extent. The interpretation of the results of the alkaline hydrolysis was complicated by the instability of D-mannose under basic conditions, it being almost completely degraded by even a brief treatment.     

Stereo- and biochemical profiles of the 5-6- and 6-6-junction isomers of alpha-D-mannopyranosyl [60]fullerenes

The 5-6- and 6-6-junction isomers of alpha-D-mannopyranosyl [60]fullerene were studied by means of circular dichroism (CD), deuterium labeling, 1H-NMR, molecular-dynamics (MD) calculations, and a lectin-binding assay. The CD spectra of the O-acetylated derivatives allowed clear discrimination of the isomers, while the 1H-NMR spectra, with assistance from deuterium labeling and MD calculations, served to disclose the unique conformation and molecular geometry of each acetylated isomer in chloroform solution. The deprotected 5-6- and 6-6-isomers, which gave colloidal suspensions in aqueous mixtures, displayed marked activity in blocking lectin-induced hemagglutination by concanavalin A.     

Synthesis and conformational and NMR studies of alpha-D-mannopyranosyl and alpha-D-mannopyranosyl-(1----2)-alpha-D-mannopyranosyl linked to L-serine and L-threonine.

alpha-D-Mannopyranosyl and alpha-D-mannopyranosyl-(1----2)-alpha-D- mannopyranosyl linked to L-serine and L-threonine have been synthesised as model substances for the linkage region in certain O-linked glycoproteins. Metropolis Monte Carlo simulations were performed with a modified version of the GESA program, to yield theoretical NOEs and interatomic distances as ensemble-average values, and these were compared with results from steady-state NOE experiments. The NOEs were determined as ensemble-average and as global minimum values. NMR chemical shift differences, obtained for signals of the glycopeptides relative to those of the respective monomers, were interpreted in terms of short inter-residue atomic distances as found within the global minima, and on the basis of averaged distances derived from Monte Carlo simulations.     

Synthesis of allyl 2-O-(alpha-L-arabinofuranosyl)-6-O-(alpha-D-mannopyranosyl)-beta-D-mannopyranoside, a unique plant N-glycan motif containing arabinose

The synthesis of the trisaccharide allyl 2-O-(alpha-L-arabinofuranosyl)-6-O-(alpha-D-mannopyranosyl)-beta-D-mannopyra-noside is reported. Stereoselective glycosylation at C-6 of a non-protected allyl beta-mannoside with the acetylated alpha-D-mannosyl bromide gave the alpha-(1 --> 6)-disaccharide as the main product and the crystalline 3,6-branched trisaccharide as minor compound. Further glycosylation of the 2,3 diol (1 --> 6) disaccharide with L-arabinofuranosyl bromide furnished a mixture of 3-O- and 2-O-alpha-L-Ara-trisaccharides from which the title compound was isolated.     

Adaptive phosphate uptake behaviour of phytoplankton to environmental phosphate fluctuations

When phytoplankton growth in lakes is limited by the available phosphate, the external phosphate concentration fluctuates around a threshold value at which available energy is insufficient to drive phosphate incorporation into a polyphosphate pool. As a result, occasional increases in the external concentration are experienced by phytoplankton as a series of phosphate pulses. Based on [(32) P] phosphate uptake experiments with lake phytoplankton, we show that a community is able to process information about the experienced pattern of phosphate pulses via a complex regulation of the kinetic and energetic properties of cellular phosphate uptake systems. As a result, physiological adaptation to alterations of ambient phosphate concentration depends on the pattern of phosphate fluctuations to which the community had been exposed during its previous growth. In this process, the entire community exhibits coherent uptake behaviour with respect to a common threshold value. Thereby, different threshold values result from different antecedent pulse patterns, apparently unrestrained by the amount of previously stored phosphate. The coherent behaviour observed contradicts the basic assumptions of the competitive exclusion principle and provides an alternative perspective for explaining the paradoxical coexistence of many phytoplankton species.     

Organic phosphate probes of the avian renal phosphate secretory mechanism

Parathyroid hormone (PTH) stimulates net renal inorganic phosphate (Pi) secretion in domestic fowl (Gallus domesticus). Recent evidence indicates that secreted Pi is derived from a highly sequestered, presumably organic, phosphate pool. A modified Sperber technique was used to survey the response of domestic fowl to unilateral renal portal infusions of organic phosphate compounds that had been implicated in previous studies of Pi secretion. None of the organic phosphate compounds produced a significant unilateral Pi secretory effect. It is concluded that these compounds neither directly stimulate Pi secretion in normal or parathyroidectomized birds, nor are they rate-limiting for the Pi secretory mechanism in birds infused with PTH.     

Effect of dietary phosphate on intestinal calcium and phosphate absorption

Intestinal Ca and P absorption was investigated on rachitic chicks raised on diets with a 1% Ca and 0.3% or 1% P contents. 45Ca and 32P absorption was determined by the technique of the isolated gut sac in vivo. In addition, 32P transport was also measured by the everted gut sac procedure in vitro. Treatment with vit. D3 during 7 days increased the 45Ca absorption in animals fed diets containing 0.3% or 1% P. 32P absorption showed an increase after 2 days of treatment and a decrease afterwards. The reduction of 32P absorption was larger in animals fed diet with 1% P. Study of 32P transport with the everted gut sac technique showed an increase after vit. D3 and a loss of intracellular P, regardless the duration of treatment.     

Sodium-dependent phosphate co-transporters

The renal proximal tubular reabsorption of inorganic phosphate (Pi) mediated by sodium-dependent phosphate (Na+/Pi) co-transporters plays a critical role in the maintenance of Pi homeostasis. Two nonhomologous Na+/Pi co-transporters (type I and type II) have been identified in the renal cortex of various species. The role of the type I co-transporter in Pi regulation remains to be clarified. Type II co-transporters play a major role in the regulation of renal Pi reabsorption by dietary Pi and parathyroid hormone, which regulate the rapid endocytosis/exocytosis of the transporters. Type III Na+/Pi co-transporters, which are expressed in a wide variety of tissues and are regulated by changes in the Pi concentration, have recently been described. The presence of a novel Pi-regulating hormone called 'phosphatonin' has been postulated in studies of the mechanisms of X-linked hypophosphatemic rickets and oncogenic osteomalacia. The regulation of phosphatonin and Na+/Pi co-transporters may provide novel pharmacological approaches to the treatment of these diseases.