“de novo” trisomy 1q32→1qter and monosomy 3p25→3pter

Summary The syndrome of familial lymphedema (type Meige) with distichiasis was observed in father and son. The association with uvula bifida and submucous cleft of the palate is described for the first time.     

Water-soluble (1→3), (1→4)-β-D-glucans from barley (Hordeum vulgare) endosperm. II. Fine structure

Water-soluble (1→3),(1→4)-β-d-glucans isolated from barleys grown in Australia and the UK were depolymerised using a purified (1→3),(1→4)-β-d-glucan 4-glucanohydrolase (EC 3.2.1.73). Oligomeric products were quantitatively separated by high resolution gel filtration chromatography and their structures defined by methylation analysis. Approximately 90% (w/w) of each polysaccharide consists of cellotriosyl and cellotetraosyl residues separated by single (1→3)-linkages but blocks of 5–11 (1→4)-linked glucosyl residues are also present in significant proportions. Periodate oxidation followed by Smith degradation suggested that contiguous (1→3)-linked β-glucosyl residues are either absent, or present in very low frequency. The potential for misinterpretation of data due to incomplete Smith degradation was noted.The irregularly-spaced (1→3)-linkages interrupt the relatively rigid, ribbon-like (1→4)-β-glucan conformation and confer a flexibility and ‘irregular’ shape on the barley (1→3),(1→4)-β-d-glucan, consistent with its solubility in water. Molecular models incorporating the major structural features confirm that the polysaccharide is likely to assume a worm-like conformation in solution. Non-covalent interactions between long blocks of (1→4)-linkages in (1→3),(1→4)-β-d-glucans, or between these blocks and other polysaccharides, offer a possible explanation for the organisation of polysaccharides in the framework of the cell wall.     

Synthesis of two purple-membrane glycolipids and the glycolipid sulfate O-(β-d-glucopyranosyl 3-sulfate)-(1→6)-O-α-d-mannopyranosyl-(1→2)-O-α-d-glucopyranosyl-(1→1)-2, 3-di-O-phytanyl-sn-glycerol

The two purple-membrane glycolipids O-β-d-glucopyranosyl- and O-β-d-galactopyranosyl-(1→6)-O-α-d-mannopyranosyl-(1→2)-O-α-d-glucopyranosyl-(1→1)-2, 3-di-O-phytanyl-sn-glycerol were prepared by coupling O-(2,3,4-tri-O-acetyl-α-d-mannopyranosyl)-(1→2)-O-(3,4,6-tri-O-acetyl-α-d-glucopyranosyl)-(1→1)-2, 3-di-O-phytanyl-sn-glycerol (9) with 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide or 2,3,4,6-tetra-O-acetyl-α-d-mannopyranosyl bromide, respectively, followed by deacetylation. The glycolipid sulfate O-(β-d-glucopyranosyl 3-sulfate)-(1→6)-O-α-d-mannopyranosyl-(1→2)-O-α-d-glucopyranosyl-(1→1)-2,3-di-O-phytanyl-sn-glycerol was prepared by coupling of 9 with 2,4,6-tri-O-acetyl-3-O-trichloroethyloxycarbonyl-α-d-glucopyranosyl bromide in the presence of Hg(CN)₂/HgBr₂ followed by selective removal of the 3?-trichloroethyloxycarbonyl group, sulfation of HO-3?, and deacetylation. The suitably protected key-intermediate 9 could be prepared by two distinct approaches.     

Substrate specificities and kinetic properties of two (1→3), (1→4)-β-d-glucan endo-hydrolases from germinating barley (Hordeum vulgare)

Two β-d-glucan endo-hydrolases purified from germinating barley (Hordeum vulgare) hydrolyse (1→4)-β linkages in (1→3),(1→4)-β-d-glucans where the d-glucosyl residue is substituted at O-3, but will not hydrolyse (1→3)-β-d-glucans or (1→4)-β-d-glucans. Methylation analysis of hydrolytic products released from barley (1→3),(1→4)-β-d-glucan indicates that 3-O-β-cellobiosyl-d-glucose and 3-O-β-cellotriosyl-d-glucose are the major oligomers formed. The enzymes exhibit characteristic endo-hydrolase action-patterns on this substrate. Both enzyme can therefore be classified as (1→3),(1→4)-β-d-glucan 4-glucanohydrolases (EC 3.2.1.73). The reduced, pneumococcal polysaccharide RS III, which consists of alternating (1→3)- and (1→4)-linked β-d-glucosyl residues, is hydrolysed by the enzymes to release laminaribiose as a major oligomeric product. Although the kinetic parameters of the two enzymes are similar, one hydrolyses barley (1→3),(1→4)-β-d-glucan at a significantly higher rate than the other and is more stable at elevated temperatures.     

Cell wall (1 → 3)- and (1 → 3, 1 → 4)-β-glucans during early grain development in rice (Oryza sativa L.)

Immunogold labeling was used to study the distribution of (1 → 3)-β-glucans and (1 → 3, 1 → 4)-β-glucans in the rice grain during cellularization of the endosperm. At approximately 3–5 d after pollination the syncytial endosperm is converted into a cellular tissue by three developmentally distinct types of wall. The initial free-growing anticlinal walls, which compartmentalize the syncytium into open-ended alveoli, are formed in the absence of mitosis and phragmoplasts. This stage is followed by unidirectional (centripetal) growth of the anticlinal walls mediated by adventitious phragmoplasts that form between adjacent interphase nuclei. Finally, the periclinal walls that divide the alveoli are formed in association with centripetally expanding interzonal phragmoplasts following karyokinesis. The second and third types of wall are formed alternately until the endosperm is cellular throughout. All three types of wall that cellularize the endosperm contain (1 → 3)-β-glucans but not (1 → 3, 1 → 4)-β-glucans, whereas cell walls in the surrounding maternal tissues contain considerable amounts of (1 → 3, 1 → 4)-β-glucans with (1 → 3)-β-glucans present only around plasmodesmata. The callosic endosperm walls remain thin and cell plate-like throughout the cellularization process, appearing to exhibit a prolonged juvenile state. Received: 7 January 1997 / Accepted: 11 February 1997     

One-pot enzymatic synthesis of the Galα1→3Galβ1→4GlcNAc sequence within situ UDP-Gal regeneration

The trisaccharide Gal13Gal14GlcNAc1O-(CH₂)₈COOCH₃ was enzymatically synthesized, within situ UDP-Gal regeneration. By combination in one pot of only four enzymes, namely, sucrose synthase, UDP-Glc 4-epimerase, UDP-Gal:GlcNAc 4-galactosyltransferase and UDP-Gal:Gal14GlcNAc 3-galactosyltransferase, Gal13Gal14GlcNAc1O-(CH₂)₈COOCH₃ was formed in a 2.2 µmol ml^–1 yield starting from the acceptor GlcNAc1O-(CH₂)₈COOCH₃. This is an efficient and convenient method for the synthesis of the Gal13Gal14GlcNAc epitope which plays an important role in various biological and immunological processes.     

Arabinoxylan and (1→3),(1→4)-β-glucan deposition in cell walls during wheat endosperm development

Arabinoxylans (AX) and (1→3),(1→4)-β-glucans are major components of wheat endosperm cell walls. Their chemical heterogeneity has been described but little is known about the sequence of their deposition in cell walls during endosperm development. The time course and pattern of deposition of the (1→3) and (1→3),(1→4)-β-glucans and AX in the endosperm cell walls of wheat (Triticum aestivum L. cv. Recital) during grain development was studied using specific antibodies. At approximately 45°D (degree-days) after anthesis the developing walls contained (1→3)-β-glucans but not (1→3),(1→4)-β-glucans. In contrast, (1→3),(1→4)-β-glucans occurred widely in the walls of maternal tissues. At the end of the cellularization stage (72°D), (1→3)-β-glucan epitopes disappeared and (1→3),(1→4)-β-glucans were found equally distributed in all thin walls of wheat endosperm. The AX were detected at the beginning of differentiation (245°D) in wheat endosperm, but were missing in previous stages. However, epitopes related to AX were present in nucellar epidermis and cross cells surrounding endosperm at all stages but not detected in the maternal outer tissues. As soon as the differentiation was apparent, the cell walls exhibited a strong heterogeneity in the distribution of polysaccharides within the endosperm.     

Probe design and synthesis of Galβ(1→3)[NeuAcα(2→6)]GlcNAcβ(1→2)Man motif of N-glycan

Synthesis and clusterization of Galβ(1→3)[NeuAcα(2→6)]GlcNAcβ(1→2)Man motif of the N-glycan, as the molecular probes for their biological evaluation, are reported. Key step is the quantitative and the completely α-selective sialylation of the C5-azide N-phenyltrifluoroacetimidate with the disaccharide acceptor, Galβ(1→3)GlcNTroc. Clusterization of the 16 molecules of trisaccharide motif was also achieved by the ‘self-activating click reaction’. These probes could efficiently be labeled by biotin and/or other fluorescence- or radioactive reporter groups through either cross metathesis, acylation, Cu(I)-mediated Huisgen [2+3]-cycloaddition, or the azaelectrocyclization to utilize the various biological techniques.     

The Maize Mixed-Linkage (1→3),(1→4)-β-d-Glucan Polysaccharide Is Synthesized at the Golgi Membrane

With the exception of cellulose and callose, the cell wall polysaccharides are synthesized in Golgi membranes, packaged into vesicles, and exported to the plasma membrane where they are integrated into the microfibrillar structure. Consistent with this paradigm, several published reports have shown that the maize (Zea mays) mixed-linkage (1→3),(1→4)-β-d-glucan, a polysaccharide that among angiosperms is unique to the grasses and related Poales species, is synthesized in vitro with isolated maize coleoptile Golgi membranes and the nucleotide-sugar substrate, UDP-glucose. However, a recent study reported the inability to detect the β-glucan immunocytochemically at the Golgi, resulting in a hypothesis that the mixed-linkage β-glucan oligomers may be initiated at the Golgi but are polymerized at the plasma membrane surface. Here, we demonstrate that (1→3),(1→4)-β-d-glucans are detected immunocytochemically at the Golgi of the developing maize coleoptiles. Further, when maize seedlings at the third-leaf stage were pulse labeled with [¹⁴C]O₂ and Golgi membranes were isolated from elongating cells at the base of the developing leaves, (1→3),(1→4)-β-d-glucans of an average molecular mass of 250 kD and higher were detected in isolated Golgi membranes. When the pulse was followed by a chase period, the labeled polysaccharides of the Golgi membrane diminished with subsequent transfer to the cell wall. (1→3),(1→4)-β-d-Glucans of at least 250 kD were isolated from cell walls, but much larger aggregates were also detected, indicating a potential for intermolecular interactions with glucuronoarabinoxylans or intermolecular grafting in muro.An overwhelming body of evidence accumulated has established that the (1→4)-β-d-glucan chains of cellulose microfibrils are synthesized and assembled at the plasma membrane surface (Delmer, 1999; Saxena and Brown, 2005), whereas, with the lone exception of the (1→3)-β-d-glucan, callose, all noncellulosic pectin and cross-linking glycan polysaccharides are synthesized in Golgi membranes (Northcote and Pickett-Heaps, 1966; Ray et al., 1969, 1976; Harris and Northcote, 1971; Zhang and Staehelin, 1992). Using several plant systems, including grass species, autoradiography and membrane fractionation showed that monosaccharides from ¹⁴C-labeled substrates accumulated in cell wall polysaccharides in Golgi vesicles during a pulse were subsequently transferred to the cell wall when chased with unlabeled substrates (Northcote and Pickett-Heaps, 1966; Pickett-Heaps, 1967; Jilka et al., 1972). Early studies showed that labeled sugars from nucleotide-sugar substrates could be incorporated into alcohol-insoluble polysaccharides using microsomal membranes, and later refined by isolation of Golgi membranes and the synthesis of defined polysaccharides with combinations of nucleotide sugars (Bailey and Hassid, 1966; Ray et al., 1969, 1976; Smith and Stone, 1973; Ray, 1980; Hayashi and Matsuda, 1981a; Gordon and Maclachlan, 1989; Gibeaut and Carpita, 1993).When micromolar concentrations of substrates were used, only small chains of the glycan products were typically made in vitro. For example, xyloglucan oligomers with the characteristic α-d-Xyl-(1→6)-d-glucosyl unit, isoprimeverose, were synthesized with isolated microsomal membranes and low concentrations of UDP-Glc and UDP-Xyl (Ray et al., 1976; Hayashi and Matsuda, 1981b). When concentrations of each nucleotide sugar were increased to millimolar concentrations, then polysaccharides of about 250 kD were synthesized containing the characteristic XXXG heptasaccharide unit structure (Gordon and Maclachlan, 1989). Immunocytochemical evidence with antibodies directed against the terminal nonreducing xylosyl and fucosyl residues confirm that synthesis of the xyloglucan backbone begins in the cis-Golgi membrane and culminates with fucosylation in the trans-Golgi membrane and trans-Golgi network (Moore et al., 1991; Lynch and Staehelin, 1992; Zhang and Staehelin, 1992). The fucosyl transferase responsible for xyloglucan side chain decoration was also shown to be a Golgi-resident protein by in vitro synthesis of xyloglucan polymers (Camirand and Maclachlan, 1986).In Poales species, including all grasses, the mixed-linkage (1→3),(1→4)-β-d-glucan is a major cross-linking glycan that appears transiently during cell elongation in growing tissues and accumulates to large amounts in the cell walls of the endosperm of certain grains (Stone and Clarke, 1992; Trethewey et al., 2005). Bailey and Hassid (1966) demonstrated the synthesis in vitro of noncellulosic glucans with microsomal membranes from grasses. Henry and Stone (1982) used the Bacillus subtilis endoglucanase, an enzyme that generates diagnostic cellodextrin-(1→3)-β-Glc units from (1→3),(1→4)-β-d-glucan to show that the mixed-linkage β-glucan was made specifically with UDP-Glc and microsomal membranes. We used flotation centrifugation to obtain highly enriched Golgi membranes from which (1→3),(1→4)-β-d-glucans of an average of about 250 kD were synthesized (Gibeaut and Carpita, 1993).The BG1 monoclonal antibody recognizes the (1→3),(1→4)-β-d-glucan with high specificity (Meikle et al., 1994). This monoclonal antibody has been used to show dramatic changes in epitope abundance of (1→3),(1→4)-β-d-glucan in the cell walls of developing tissues (Meikle et al., 1994; Trethewey et al., 2005; McCann et al., 2007) and its appearance in the cell walls of Arabidopsis (Arabidopsis thaliana) following heterologous expression of genes thought to encode its synthases (Burton et al., 2006; Doblin et al., 2009). The failure to detect (1→3),(1→4)-β-d-glucan in Golgi membranes and only in the cell wall prompted Fincher (2009) to conclude that cellodextrin oligomers of the (1→3),(1→4)-β-d-glucan may be initiated in the Golgi membrane, but the actual polymerization of the polysaccharide occurs at the plasma membrane.While there is little question that synthesis of full-length polymers is possible in vitro with isolated Golgi membranes and UDP-Glc (Gibeaut and Carpita, 1993; Buckeridge et al., 1999, 2001; Urbanowicz et al., 2004), Fincher (2009) asserts correctly that there exists no experimental evidence that the polymer is made in vivo within the Golgi membrane in intact tissues. In fact, earlier work showing the paucity of immunolabeling of (1→3),(1→4)-β-d-glucan in Golgi membranes of developing wheat (Triticum aestivum) endosperm at a time of active deposition called to question the site of synthesis in vivo (Philippe et al., 2006). There is precedence for the synthesis of chitin in vitro with precociously activated chitisomes (Bracker et al., 1976), a vesicular package of chitin synthase that in vivo is quiescent until reaching the plasma membrane. No activity of chitin synthase from isolated plasma membranes could be demonstrated. In a similar way, the Golgi synthase activity of (1→3),(1→4)-β-d-glucan could be a precocious activation in vitro of a plasma membrane activity.As in vitro synthesis studies clearly show synthesis of full-length (1→3),(1→4)-β-d-glucan only at the Golgi, we reexamined the puzzling finding of its absence from Golgi bodies to determine the true site of synthesis in vivo. In contrast to Fincher (2009), our own immunocytochemistry shows (1→3),(1→4)-β-d-glucan is indeed in the Golgi membrane in 2-d-old coleoptiles, when rapid growth is just beginning. However, we are unable to detect the β-glucan in Golgi after the peak rate of elongation. We pulse labeled maize (Zea mays) seedlings with radiolabeled CO₂ and followed the fate of label captured by photosynthesis and translocated to elongating cells at the base of the seedling. We found by flotation centrifugation that Golgi membranes contain (1→3),(1→4)-β-d-glucan of at least 250 kD, similar to that of the product of in vitro synthesis at optimal UDP-Glc concentrations and commercial preparations of barley (Hordeum vulgare) endosperm (1→3),(1→4)-β-d-glucan (Gibeaut and Carpita, 1993; Buckeridge et al., 1999, 2001; Urbanowicz et al., 2004). When polysaccharides are extracted sequentially from the cell walls by hot ammonium oxalate, and increasing concentrations of NaOH to 4 m, the (1→3),(1→4)-β-d-glucans are found mostly in the higher concentrations of alkali fractions. While 250 kD polymers are observed, most of the (1→3),(1→4)-β-d-glucans eluted in fractions containing glucuronoarabinoxylans (GAXs), which are much larger, indicating either that an aggregation with GAXs increase the apparent size or that trans-glucosylation events increase the degree of polymerization of the (1→3),(1→4)-β-d-glucans.     

Synthesis of p-trifluoroacetamidophenyl O-α-D-galactopyranosyl-(1→2)-O-α-D-mannopyranosyl-(1→4)-α-L-rhamnopyranoside

The role of exposed tyrosine side-chains in enzyme-catalysed reactions has been studied for porcine-pancreatic alpha-amylase, sweet-potato beta-amylase, and Aspergillus niger glucamylase using N-acetylimidazole as the specific protein reagent. The changes in activity binding affinity (Δk_?1/k₊₁), and kinetic parameters (K_m,k₂) due to acetylation of the phenolic hydroxyl groups have been determined. Acetylation of each enzyme occurred by an “apparent” first-order reaction with a rate constant of 0.72–1.4 x 10^?1min^?1. Acetylation increased the apparent K_m (soluble starch as the substrate) for each enzyme (appreciably for alpha-amylase and glucamylase), whereas k² remained unchanged. Similarly, for each enzyme, the binding affinity for immobilised cyclohexa-amylose decreased appreciably, whereas the catalytic activity was reduced only to a small degree (and remained unchanged for beta-amylase). It is concluded that the tyrosine groups located in the active centre of each enzyme have a substrate-binding function.     

Water-soluble (1→3), (1→4)-β-d-glucans from barley (Hordeum vulgare) endosperm. I. Physicochemical properties

Physicochemical methods have been used to define molecular weight, molecular weight distribution, solution behaviour and shape of (1→3), (1→4)-β-d-glucans purified from the 40°C water-extract of barley endosperm by precipitation with 30% saturated ammonium sulphate. The molecular weight and solution properties of a (1→3), (1→4)-β-d-glucan from Australian grown barley (cv. Clipper) are compared with a commercially available preparation. Weight and number average molecular weights are 290 000 and 210 000 respectively for the Clipper (1→3), (1→4)-β-d-glucan and 160 000 and 150 000 respectively for the commercial preparation. The degree of polydispersity is small, but this probably results from the selection of a specific population of (1→3), (1→4)-β-d-glucan molecules during isolation. The higher molecular weight of the Clipper (1→3), (1→4)-β-d-glucan is reflected in higher sedimentation coefficient and intrinsic viscosity values. Viscosity and sedimentation data indicate that the molecules are highly asymmetric, with axial ratios of approximately 100 and 80 for the Clipper (1→3), (1→4)-β-d-glucan and the commercial preparation, respectively. Both polysaccharides appear to exist in solution as extended, worm-like chains.     

Synthetic Studies on Nephritogenic Glycosides. Synthesis of β-Glc-(1→6)-α-Glc-(l→6)-α-Glc-(1→Asn)

Gibberellins (GAs) A₉, A₁₅, A₁₉, A₂₀, A₂₉, A₃₅, A₄₄, A₅₀ and A₆₁ were identified by capillary gas chromatography/selected ion monitoring (GC/SIM) in immature seeds of loquat (Eriobotrya japonica Lindl). Furthermore, five unknown GA-like compounds with apparent parent ions of m/z 418, 504 or 506 (as methyl ester trimethylsilyl ether derivatives) were found by GC/mass spectrometry (GC/MS) in the biologically active fractions. The m/z 418 and 504 compounds may have been C-11β hydroxylated GA₉ and dehydro-GA₃₅, respectively. The bioassay and GC/MS results suggest that the major GAs were GA₅₀ and the five unknown GA-like compounds. In the immature seeds, at least two GA metabolic pathways may thus exist, one being the non-hydroxylation pathway of GA₁₅→GA₂₄→GA₉, and the other, the early C-13 hydroxylation pathway of GA₄₄→GA₁₉→GA₂₀→GA₂₉. A late C-11β hydroxylation pathway is also possible.     

TheX → Y → Z scheme after 23 years

Mathematical modelling of the course of the immune response is undoubtedly one of the most progressive and most promising areas of modern immunology. Mathematical models (along with computer programs) can be taken as "the only means of thoroughly testing and examining a large and intricate theory" (Partridge et al. 1984). The first phase of construction of mathematical models is the formulation of assumptions based on the knowledge of the facts to be modelled (manifested usually in a scheme of the presumed course of the modelled process). The first mathematical models of immune response were based on the hypothesis of a two-stage differentiation of cells participating in the humoral response, published in Prague 23 years ago (Sercarz and Coons 1962; Sterzl 1962) and illustrated by the X----Y----Z scheme. Many contemporary mathematical models still stem from this scheme which undoubtedly fits the fundamental data concerning the immune system.     

Synthesis of phenyl O-α-l-fucopyranosyl-(1→2)-O-β-d-galactopyranosyl-(1→3)-2-acetamido-2-deoxy-α-d-galactopyranoside

phenyl 2-acetamido-2-deoxy-4,6-O-(p-methoxybenzylidene)-3-O-[4,6-O-(p-methoxybenzylidene)-β-d-alactopyranosyl]-α-d-galactopyranoside (3) was prepared from phenyl 2-acetamido-2-deoxy-4,6-O-(p-methoxybenzylidene)-3-O-(2,3,4,6-tetra-O-acetyl-β-d-galactopyranosyl)-α-d-galactopyranoside by zemplén deacetylation, followed by reaction with p-methoxybenzaldehyde in the presence of anhydrous zinc chloride. The selective benzoylation of 3 gave the 3′-benzoate which, on condensation with 2,3,4-tri-O-benzyl-α- l-fucopyranosyl bromide under catalysis by halide ion, afforded a crystalline trisaccharide from which the title trisaccharide was obtained by debenzoylation followed by catalytic hydrogenolysis.     

Water-soluble (1→3), (1→4)-β-d-glucans from barley (Hordeum vulgare) endosperm. III. Distribution of cellotriosyl and cellotetraosyl residues

Cellotriosyl and cellotetraosyl residues, linked by single (1→3)-β-linkages, account for more than 90% of the 40°C water-soluble (1→3), (1→4)-β-d-glucan from barley flour. We have analysed their sequence dependence by treating the polymer as a two-state Markov chain with stationary distribution. Quantitation of the penultimate oligosaccharides released during hydrolysis of the (1→3), (1→4)-β-d-glucan with (1→3), (1→4)-β-d-glucan 4-glucanohydrolase (EC 3.2.1.73) by analytical gel filtration chromatography enabled the relative abundance of two adjacent cellotriosyl, two adjacent cellotetraosyl and adjacent cellotetraosyl/cellotriosyl residues to be estimated and the sequence dependence to be evaluated.Within the theoretical and practical constraints of the method it is concluded that the cellotriosyl and cellotetraosyl residues are arranged in an essentially independent (random) fashion. Thus, any mechanism proposed for the biosynthesis of the molecule should explain this apparently random distribution of cellotriosyl and cellotetraosyl residues as well as the presence, in relatively low frequency, of blocks of up to 10 or more adjacent (1→4)-linkages.     

Purification and characterization of (1→3, 1→4)-β-glucan endohydrolases from germinated wheat (Triticum aestivum)

A (13, 14)--glucan 4-glucanohydrolase [(13, 14)--glucanase, EC 3.2.1.73] was purified to homogeneity from extracts of germinated wheat grain. The enzyme, which was identified as an endohydrolase on the basis of oligosaccharide products released from a (13, 14)--glucan substrate, has an apparent pI of 8.2 and an apparent molecular mass of 30 kDa. Western blot analyses with specific monoclonal antibodies indicated that the enzyme is related to (13, 14)--glucanase isoenzyme EI from barley. The complete primary structure of the wheat (13, 14)--glucanase has been deduced from nucleotide sequence analysis of cDNAs isolated from a library prepared using poly(A)⁺ RNA from gibberellic acid-treated wheat aleurone layers. One cDNA, designated LW2, is 1426 nucleotide pairs in length and encodes a 306 amino acid enzyme, together with a NH₂-terminal signal peptide of 28 amino acid residues. The mature polypeptide encoded by this cDNA has a molecular mass of 32085 and a predicted pI of 8.1. The other cDNA, designated LW1, carries a 109 nucleotide pair sequence at its 5 end that is characteristic of plant introns and therefore appears to have been synthesized from an incompletely processed mRNA. Comparison of the coding and 3-untranslated regions of the two cDNAs reveals 31 nucleotide substitutions, but none of these result in amino acid substitutions. Thus, the cDNAs encode enzymes with identical primary structures, but their corresponding mRNAs may have originated from homeologous chromosomes in the hexaploid wheat genome.     

(1→2) and (1→6)-linked β-d-galactofuranan of microalga Myrmecia biatorellae, symbiotic partner of Lobaria linita

A structural study of the cell wall polysaccharides of Myrmecia biatorellae, the symbiotic algal partner of the lichenized fungus Lobaria linita was carried out. It produced a rhamnogalactofuranan, with a (1→6)-β-d-galactofuranose in the main-chain, substituted at O-2 by single units of β-d-Galf, α-l-Rhap or by side chains of 2-O-linked β-d-Galf units. The structure of the polysaccharide was established by chemical and NMR spectroscopic analysis, and is new among natural polysaccharides. Moreover, in a preliminary study, this polysaccharide increased the lethality of mice submitted to polymicrobial sepsis induced by cecal ligation and puncture, probably due to the presence of galactofuranose, which have been shown to be highy immunogenic in mammals.     

Synthesis of O-β-d-mannopyranosyl-(1→4)-O-α-l-rhamnopyranosyl-(1→3)-d-galactopyranose,the trisaccharide repeating-unit of the O-specific polysaccharide from Salmonella anatum

Glycosylation of 1,2:5,6-di-O-isopropylidene-α-d-galactofuranose with 2,3-di-O-acetyl-4-O-(2,3,4,6-tetra-O-acetyl-β-d-mannopyranosyl)-α-l-rhamnopyranosyl bromide, followed by removal of the protecting groups, gave O-β-d-mannopyranosyl-(1→4)-O-α-l-rhamnopyranosyl-(1→3)-d-galactose, which is the trisaccharide repeating-unit of the O-specific polysaccharide chain of the lipopolysaccharide from Salmonella anatum. The formation of the β-d-mannopyranosyl linkage was achieved by a glucose-mannose conversion via stereoselective reduction of the corresponding oxo-disaccharide.