Enzyme-linked immunosorbent assay specific for (1-->6) branched, (1-->3)-beta-D-glucan detection in environmental samples.

(1-->3)-beta-D-Glucans have been recognized as a potential causative agent responsible for bioaerosol-induced respiratory symptoms observed in both indoor and occupational environments. A specific enzyme immunoassay was developed to quantify (1-->6) branched, (1-->3)-beta-D-glucans in environmental samples. The assay was based on the use of a high-affinity receptor (galactosyl ceramide) specific for (1-->3)-beta-D-glucans as a capture reagent and a monoclonal antibody specific for fungal cell wall beta-D-glucans as a detector reagent. The assay was highly specific for (1-->6) branched, (1-->3)-beta-D-glucans (such as that from Saccharomyces cerevisiae) and did not show any response at 200 ng/ml to curdlan, laminarin, pustulan, dextran, mannan, carboxymethyl cellulose, and endotoxins. The detection level was 0.8 ng/ml for baker's yeast glucan and Betafectin. A coefficient of variation of 7.8% was obtained for (1-->3)-beta-D-glucans in house dust samples. Metal working fluids spiked with (1-->3)-beta-D-glucans inhibited the glucan assay. Because the assay is specific for (1-->6) branched, (1-->3)-beta-D-glucans and is sensitive and reproducible, it will be useful for the investigation of health effects from exposure to this class of biologically active molecules.     

Immunologically active (1-->3)-(1-->4)-alpha-D-glucan from Cetraria islandica.

A polysaccharide, Ci-3, resembling isolichenan except with a much higher degree of polymerization, has been isolated from the water extract, as well as from the alkali extract, of the lichen Cetraria islandica (L.) using ethanol fractionation, dialysis, ion-exchange chromatography and gel filtration. The mean M(r) of Ci-3 was determined to be 2000 kD, compared to 6-8 kD reported for isolichenan. The structure of Ci-3 was elucidated and found to be composed of (1-->3)- and (1-->4)-alpha-D-glucopyranosyl units in the ratio of 2:1, using methanolysis, methylation analysis, optical rotation and NMR spectroscopy. The immunomodulating activity of Ci-3 was tested in an in vitro phagocytosis assay and anti-complementary, and proved to be active in both tests.     

An efficient and stereoselective synthesis of beta-D-Arap-(1-->2)-beta-D-Galp-(1-->3)-beta-D-Galp-(1-->4)-alpha-D-Manp, a tetrasaccharide fragment of Leishmania major lipophosphoglycan.

A tetrasaccharide fragment of Leishmania major lipophosphoglycan (which seems to be involved in a biological mechanism for the parasite transmission) has been synthesised using the thioglycoside, trichloroacetimidate and halide-exchange glycosylation procedures and step-wise chain elongation strategy.     

Synthesis of the alpha-L-Araf-(1-->2)-beta-D-Galp-(1-->6)-beta-D-Galp-(1-->6)-[alpha-L-Araf-(1-->2)]-beta-D-Galp-(1-->6)-D-Gal hexasaccharide as a possible repeating unit of the cell-cultured exudates of Echinacea purpurea arabinogalactan.

For the characterization of the supposed epitope of an arabinogalactan, isolated from the extract of the cell-cultured Echinacea purpurea, the title hexasaccharide was synthesized. The whole synthetic route was based on the 6-O-(methoxydimethyl)methyl ether (MIP) protecting group strategy. 2-O-Benzyl-3,4-O-isopropylidene-6-O-(methoxydimethyl)methyl-beta-D-galactopyranosyl-(1-->6)-1,2:3,4-di-O-isopropylidene-alpha-D-galactopyranose was used to prepare the desired glycosyl donor and glycosyl acceptor both carrying a persistent O-benzyl group at position 2'. Reaction of the digalactose donor and the digalactose acceptor resulted in a beta-(1-->6)-linked galactose-containing tetrasaccharide in which OH-2' and OH-2"' were substituted with benzyl groups. Hydrogenolytic removal of the benzyl groups of the tetragalactose compound gave the diol aglycon which was diarabinosylated in one step to furnish the protected target compound, whose deprotection led to the title hexasaccharide. All of the synthesized compounds were characterized by 1H and 13C NMR spectra, as well as by MALDI-TOF mass-spectrometry measurements.     

Studies on (beta,1-->5) and (beta,1-->6) linked octyl Gal(f) disaccharides as substrates for mycobacterial galactosyltransferase activity.

The emergence of multi-drug resistant (MDR) strains of Mycobacterium tuberculosis (MTB) and the continuing pandemic of tuberculosis emphasizes the urgent need for the development of new anti-tubercular agents with novel drug targets. The recent structural elucidation of the mycobacterial cell wall highlights a large variety of structurally unique components that may be a basis for new drug development. This publication describes the synthesis, characterization, and screening of several octyl Galf(β,1→5)Galf and octyl Galf(β,1→6)Galf derivatives. A cell-free assay system has been utilized for galactosyltransferase activity using UDP[¹⁴C]Galf as the glycosyl donor, and in vitro inhibitory activity has been determined in a colorimetric broth microdilution assay system against MTB H37Ra and three clinical isolates of Mycobacterium avium complex (MAC). Certain derivatives showed moderate activities against MTB and MAC. The biological evaluation of these disaccharides suggests that more hydrophobic analogues with a blocked reducing end showed better activity as compared to totally deprotected disaccharides that more closely resemble the natural substrates in cell wall biosynthesis.     

Recombinant hemoglobin(alpha 29leucine --> phenylalanine, alpha 96valine --> tryptophan, beta 108asparagine --> lysine) exhibits low oxygen affinity and high cooperativity combined with resistance to autoxidation.

Using our hemoglobin expression system in Escherichia coli, we have constructed three recombinant hemoglobins (rHbs) with amino acid substitutions located in the alpha(1)beta(1) and alpha(1)beta(2) subunit interfaces and in the distal heme pocket of the alpha-chain: rHb(alphaV96W, betaN108K), rHb(alphaL29F, alphaV96W, betaN108K), and rHb(alphaL29F). rHb(alphaV96W, betaN108K) exhibits low oxygen affinity and high cooperativity and also ease of autoxidation of the heme iron atoms from the Fe2+ state to the Fe3+ state. It has been reported by Olson and co-workers [Carver et al., (1992) J. Biol. Chem. 267, 14443-14450; Brantley et al. (1993) J. Biol. Chem. 268, 6995-7010] that a mutation at position 29 (B10, helix notation), e.g. , Leu --> Phe, can inhibit the autoxidation of the heme iron of myoglobin. We have introduced such a mutation into our rHb having low oxygen affinity and high cooperativity. This triply mutated rHb(alphaL29F, alphaV96W, betaN108K) is stabilized against autoxidation and azide-induced oxidation compared to the double mutant, rHb(alphaV96W, betaN108K), but still exhibits low oxygen affinity and good cooperativity. According to electron paramagnetic resonance results, the oxidized form of the triple mutant shows a high ratio of an anionic form of bishistidine hemichrome. Previous reports have suggested that this form does not have water present at the distal heme pocket. (1)H nuclear magnetic resonance spectra of the triple mutant in the ferric state also exhibit spectral features characteristic of hemichrome-type signals. We have carried out a series of biochemical measurements to characterize these three interesting rHbs and to compare them to human normal adult hemoglobin. These results provide new insights into the structure-function relationship of hemoglobin with amino acid substitutions in the alpha(1)beta(1) and alpha(1)beta(2) interfaces and in the heme pockets.     

Reversible accumulation of (1-->3,1-->4)-beta-glucan endohydrolase in wheat leaves under sugar depletion.

A (1-->3,1-->4)-beta-D-glucan endohydrolase [(1-->3,1-->4)-beta-glucanase, EC 3.2.1.73] was detected in wheat (Triticum aestivum L.) leaves by Western analyses and activity measurements. This enzyme is able to degrade the (1-->3,1-->4)-beta-glucans present in the cell walls of cereals and other grass species. In wheat, enzyme levels clearly increased during leaf development, reaching maximum values at full expansion and then decreasing upon leaf ageing. To test whether the abundance of (1-->3,1-->4)-beta-glucanase might be controlled by the carbohydrate status, environmental and nutritional conditions capable of altering the leaf soluble sugar contents were used. Both the activity and enzyme protein levels rapidly and markedly increased when mature leaves were depleted of sugars (e.g. during extended dark periods), whereas elevated carbohydrate contents (e.g. following continuous illumination, glucose supply in the dark or nitrogen deficiency during a light/dark cycle) caused a rapid decrease in (1-->3,1-->4)-beta-glucanase abundance or prevented its accumulation in the leaves. The physiological significance of (1-->3,1-->4)-beta-glucanase accumulation under sugar depletion remains to be elucidated.     

Insight into multi-site mechanisms of glycosyl transfer in (1-->4)beta-D-glycans provided by the cereal mixed-linkage (1-->3),(1-->4)beta-D-glucan synthase.

Synthases of cellulose, chitin, hyaluronan, and all other polymers containing (1-->4)beta-linked glucosyl, mannosyl and xylosyl units have overcome a substrate orientation problem in catalysis because the (1-->4)beta-linkage requires that each of these sugar units be inverted nearly 180 degrees with respect to its neighbors. We and others have proposed that this problem is solved by two modes of glycosyl transfer within a single catalytic subunit to generate disaccharide units, which, when linked processively, maintain the proper orientation without rotation or re-orientation of the synthetic machinery in 3-dimensional space. A variant of the strict (1-->4)beta-D-linkage structure is the mixed-linkage (1-->3),(1-->4)beta-D-glucan, a growth-specific cell wall polysaccharide found in grasses and cereals. beta-Glucan is composed primarily of cellotriosyl and cellotetraosyl units linked by single (1-->3)beta-D-linkages. In reactions in vitro at high substrate concentration, a polymer composed of almost entirely cellotriosyl and cellopentosyl units is made. These results support a model in which three modes of glycosyl transfer occur within the synthase complex instead of just two. The generation of odd numbered units demands that they are connected by (1-->3)beta-linkages and not (1-->4)beta-. In this short review of beta-glucan synthesis in maize, we show how such a model not only provides simple mechanisms of synthesis for all (1-->4)beta-D-glycans but also explains how the synthesis of callose, or strictly (1-->3)beta-D-glucans, occurs upon loss of the multiple modes of glycosyl transfer to a single one.     

Plant members of the alpha1-->3/4-fucosyltransferase gene family encode an alpha1-->4-fucosyltransferase, potentially involved in Lewis(a) biosynthesis, and two core alpha1-->3-fucosyltransferases.

Three putative alpha1-->3/4-fucosyltransferase (alpha1-->3/4-FucT) genes have been detected in the Arabidopsis thaliana genome. The products of two of these genes have been identified in vivo as core alpha1-->3-FucTs involved in N-glycosylation. An orthologue of the third gene was isolated from a Beta vulgaris cDNA library. The encoded enzyme efficiently fucosylates Galbeta1-->3GlcNAcbeta1-->3Galbeta1-->4Glc. Analysis of the product by 400 MHz (1)H-nuclear magnetic resonance spectroscopy showed that the product is alpha1-->4-fucosylated at the N-acetylglucosamine residue. In vitro, the recombinant B. vulgaris alpha1-->4-FucT acts efficiently only on neutral type 1 chain-based glycan structures. In plants the enzyme is expected to be involved in Lewis(a) formation on N-linked glycans.     

Induction of T --> G and T --> A transversions by 5-formyluracil in mammalian cells.

Oxidatively damaged thymine, 5-formyluracil (5-fU), was incorporated into a predetermined site of double-stranded shuttle vectors. The nucleotide sequences in which the modified base was incorporated were 5'-CFTAAG-3' and 5'-CTFAAG-3' (F represents 5-fU), the recognition site for the restriction enzyme AflII (5'-CTTAAG-3'). The 5-fU was incorporated into a template strand of either the leading or lagging strand of DNA replication. The modified DNAs were transfected into simian COS-7 cells, and the DNAs replicated in the cells were recovered and were analyzed after the second transfection into Escherichia coli. The 5-fU did not block DNA replication in mammalian cells. The 5-fU residues were weakly mutagenic, and their mutation frequencies in double-stranded vectors were 0.01-0.04%. The T --> G and T --> A transversions were the mutations found most frequently, suggesting the formation of 5-fU.C and 5-fU.T base pairs, respectively. This is the first report that clearly shows the induction of transversion mutations by an oxidized pyrimidine base in DNA in mammalian cells.