Thermodynamic binding studies of bivalent oligosaccharides to galectin-1, galectin-3, and the carbohydrate recognition domain of galectin-3

Galectins are a growing family of animal lectins with common consensus sequences that bind beta-Gal and LacNAc residues. There are at present 14 members of the galectin family; however, certain galectins possess different structures as well as biological properties. Galectin-1 is a dimer of two homologous carbohydrate recognition domains (CRDs) and possesses apoptotic and proinvasive activities. Galectin-3 consists of a C-terminal CRD and an N-terminal nonlectin domain implicated in the oligomerization of the protein and is often associated with antiapoptotic activity. Because many cellular oligosaccharide receptors are multivalent, it is important to characterize the interactions of multivalent carbohydrates with galectins-1 and -3. In the present study, binding of bovine heart galectin-1 and recombinant murine galectin-3 to a series of synthetic analogs containing two LacNAc residues separated by a varying number of methylene groups, as well as biantennary analogs possessing two LacNAc residues, were examined using isothermal titration microcalorimetry (ITC) and hemagglutination inhibition measurements. The thermodynamics of binding of the multivalent carbohydrates to the C-terminal CRD domain of galectin-3 was also investigated. ITC results showed that each bivalent analog bound by both LacNAc residues to the two galectins. However, galectin-1 shows a lack of enhanced affinity for the bivalent straight chain and branched chain analogs, whereas galectin-3 shows enhanced affinity for only lacto-N-hexaose, a naturally occurring branched chain carbohydrate. The CRD domain of galectin-3 was shown to possess similar thermodynamic binding properties as the intact molecule. The results of this study have important implications for the design of carbohydrate inhibitors of the two galectins.     

Fate and effects on yield components of extra applications of nitrogen on spring wheat (Triticum aestivum L.) grown in solution culture

Spring wheat (Triticum aestivum L.) was grown with daily additions of nitrate-N. The relative addition rate of nitrate-N was decreased stepwise, and after 125 days of growth, 58 mg N plant^-1 had been introduced. The fate and effect of an extra addition of nitrate (20 mg N plant^-1) at six different times during the ontogeny (37, 54, 66, 79, 94 and 108 days from sowing) on grain yield and grain protein concentration was investigated. The plants absorbed all or most of the extra nitrate at all stages of development evaluated. Dry matter production of both aerial vegetative parts and grains, but not roots, generally increased as a result of the extra nitrate addition. The increase in grain dry matter was mainly an effect of an increased number of grains per plant. Extra nitrate applications had large effects on grain nitrogen content at all stages, but the effect on main shoot and tiller ears varied depending on the time of application. Early applications, i.e. before anthesis, mainly led to increased yield with unchanged protein concentration whereas late applications also led to increased grain protein concentration. The largest effect on grain nitrogen concentration (25–30% increase) was obtained when the extra nitrate was applied late after sowing, i.e. less than four weeks before final harvest. As the extra dose of nitrate was labelled with ¹⁵N, it was possible to follow the movement of the extra nitrogen addition within the plant. Samples were taken at one and five days after ¹⁵N-addition and at final harvest. There were differences in the movement of ¹⁵N depending on when it was introduced. Generally, net movement of the ¹⁵N-labelled N into the grain increased with age at application until 94 days after sowing when a maximum of 90% of the added ¹⁵N ended up in the grain.Abbreviations RN Relative increase in nitrogen content - RA Relative nitrogen addition rate - RG Relative growth rate - N nitrogen     

The Nature of Selected Prairie Lake and Stream Sediments

The mineralogy and related chemistry of selected lake and stream sediments from the upper Qu'Appelle River basin in southern Saskatchewan, Canada, was examined. The principal crystalline clay minerals present in the colloidal fractions of the sediments are smectite, vermiculite, kaolinite, mica, quartz, and feldspar. The cation-exchange capacity of the sediments varies from 16.3 to 28.8 meq/100 g, and the pH ranges from slightly acid to mildly alkaline. The hydrous oxides of Al, Fe, Mn, and Si are present in significant quantities in both the colloidal and noncolloidal fractions of the sediments; this is significant because these oxides likely exert the dominant chemical control on the movement of many nutrients and pollutants in aquatic environments. Calcium and Mg carbonates comprise a significant fraction of the sediments. Most of the C in the sediments is in the organic form; the organic C content varies from 1.2 to 6.0%. Because crystalline clay minerals, poorly ordered sesquioxides, and organic matter play a vital role in the retention and cycling of nutrients and pollutants in aquatic environments, a knowledge of the nature and properties of sediments is fundamental to efforts to preserve and improve the quality of natural water systems.     

Structural and functional characterization of the 5'-flanking region of the rat and human cytochrome P450 2E1 genes: identification of a polymorphic repeat in the human gene.

Cytochrome P450 2E1 (CYP2E1) is a toxicologically very important enzyme with a high extent of interindividual variability in expression. We sequenced and characterized the 5'-flanking region of the human and rat CYP2E1 genes. The identity between the human and rat sequences (-3.8 kb to +1 kb) was generally between 35 and 60%, and the most similar regions were found in the proximal part of the sequence. Two more distant regions at -1.6 to -2.0 kb and -2.5 to -2. 8 kb in the human sequence were also found to have high identity to the rat sequence. A polymorphic repeat sequence in the human gene was found between -2178 to -1945 bp. The common allele (CYP2E1*1C) contained 6 repeats (each 42-60 bp long) and the rare allele (CYP2E1*1D) had 8 repeats with an allele frequency of 1% among Caucasians and 23% among Chinese. The CYP2E1 5'-flanking regions of the human (-3712 bp to +10 bp) and rat (-3685 bp to +28 bp) genes were ligated in front of a luciferase reporter gene and transfected into rat hepatoma Fao and human hepatoma B16A2 cells. Important species specificity was noted in the control of gene expression and regions of negative and positive cis-acting elements were localized. No difference was seen in the constitutive expression between the two polymorphic forms. The importance of this repeat polymorphism for high and low inducible CYP2E1 phenotypes is discussed.     

CP-61,405, a novel polycyclic pyrrolether antibiotic produced byStreptomyces routienii Huang sp. nov.

Summary CP-61,405 (C₂₆H₂₉N₂O₇Na) is a novel polycyclic pyrrolether antibiotic produced by a new species,Streptomyces routienii Huang sp. nov. (ATCC 39446). Recovery, fractionation and purification were achieved using standard procedures. The crystalline form includes the CP-61,405 sodium salt, m.p. 334–335°C, [] _D ^25°C +315°C (c=1, chloroform). The structure is shown below. CP-61,405 was co-produced with the polycyclic ether antibiotics salinomycin and epi-17-deoxy-(0–8)-salinomycin. It exhibited activity in vitro against gram-positive and anaerobic bacteria, efficacy against poultry coccidia and stimulation in vitro of propionic acid production.     

Evaluation of two fluorescent test kits for detection of selectedBacteroides species in clinical specimens

Two pools of isothiocyanate fluorescein-conjugated rabbit antisera toBacteroides fragilis, B. thetaiotaomicron, B. vulgatus, B. distasonis, B. ovatus, and toB. melaninogenicus, subsp.melaninogenicus andintermedius, andB. asaccharolyticus, respectively, were used in immunofluorescent (IF) tests of smears of 122 clinical specimens from a variety of sources, including abscesses and wounds. Smears of broth medium of 14 blood cultures were also tested. Bacteria of the species and subspecies mentioned were detected by the IF tests in 100 and by culture studies in 106 of the 136 speciments tested. Results of the culture studies indicated that 6 tests were false-negative by the IF test, while no false-positive tests were found. The sensitivities of the two test kits were 97% and 85%, respectively, while their specificities were 100%. The predicted value for a positive test with any of the two pools of antisera was 100%, while the predicted value for a negative test was 93% and 97%, respectively. The use of the two test kits (Fluoretec F and M) was found to be a valuable means for a rapid diagnosis of human infections with theBacteroides species mentioned.     

Synthesis of oligosaccharides with oligoethylene glycol spacers and their conversion into glycoconjugates usingN,N,N′,N″-tetramethyl(succinimido)uronium tetrafluoroborate as coupling reagent

Glycosides of glucose and lactose with di- and tetraethylene glycols, transformed into bifunctional (alcohol, ester) spacer molecules, have been synthesized. After deprotection, these spacer glycosides, containing a free carboxyl group, have been transformed efficiently into glycoconjugates usingN,N,N,N-tetramethyl(succinimido)uronium tetrafluoroborate (TSTU) for the formation of an active ester.     

CP-82,996, a novel diglycoside polyether antibiotic related to monensin and produced byActinomadura sp.

Summary A new polyether antibiotic CP-82,996 (C₅₀H₈₆O₁₆) was isolated by solvent extraction from the fermentation broth ofActinomadura sp. (ATCC 63764). Following purification by silica gel column chromatography and crystallization, the structure of CP-82,996 was determined by a single crystal X-ray analysis. The structure is closely related to monensin, but is unique in that it contains two sugar groups, whereas monensin has none. The¹H and¹³C NMR chemical shifts and assignments for CP-82, 996 were elucidated, and they were compared with those determined previously for monensin. CP-82,996 is active against certain Gram-positive bacteria, and is a very potent anticoccidial agent. It effectively controlled chicken coccidiosis caused by severalEimeria species at 5–10 ppm in feed, and is 10–20 times more potent than monensin.     

Intervening with urinary tract infections using anti-adhesives based on the crystal structure of the FimH-oligomannose-3 complex

Background

Escherichia coli strains adhere to the normally sterile human uroepithelium using type 1 pili, that are long, hairy surface organelles exposing a mannose-binding FimH adhesin at the tip. A small percentage of adhered bacteria can successfully invade bladder cells, presumably via pathways mediated by the high-mannosylated uroplakin-Ia and α3β1 integrins found throughout the uroepithelium. Invaded bacteria replicate and mature into dense, biofilm-like inclusions in preparation of fluxing and of infection of neighbouring cells, being the major cause of the troublesome recurrent urinary tract infections.

Methodology/Principal Findings

We demonstrate that α-d-mannose based inhibitors of FimH not only block bacterial adhesion on uroepithelial cells but also antagonize invasion and biofilm formation. Heptyl α-d-mannose prevents binding of type 1-piliated E. coli to the human bladder cell line 5637 and reduces both adhesion and invasion of the UTI89 cystitis isolate instilled in mouse bladder via catheterization. Heptyl α-d-mannose also specifically inhibited biofilm formation at micromolar concentrations. The structural basis of the great inhibitory potential of alkyl and aryl α-d-mannosides was elucidated in the crystal structure of the FimH receptor-binding domain in complex with oligomannose-3. FimH interacts with Manα1,3Manβ1,4GlcNAcβ1,4GlcNAc in an extended binding site. The interactions along the α1,3 glycosidic bond and the first β1,4 linkage to the chitobiose unit are conserved with those of FimH with butyl α-d-mannose. The strong stacking of the central mannose with the aromatic ring of Tyr48 is congruent with the high affinity found for synthetic inhibitors in which this mannose is substituted for by an aromatic group.

Conclusions/Significance

The potential of ligand-based design of antagonists of urinary tract infections is ruled by the structural mimicry of natural epitopes and extends into blocking of bacterial invasion, intracellular growth and capacity to fluxing and of recurrence of the infection.