The α-d-mannan core of a complex cell-wall heteroglycan of Trichoderma reesei is responsible for β-glucosidase activation

A heteroglycan responsible for the binding of the enzyme β-1,4-d-glucosidase (EC 3.2.1.21) to fungal cell walls was isolated from cell walls of the filamentous fungusTrichoderma reesei. The heteroglycan, composed of mannose, galactose, glucose, and glucuronic acid, also activated β-1,4-d-glucosidase, β-1,4-d-xylosidase andN-acetyl-β-1,4-d-glucosaminidase activity in vitro. The structural backbone of this heteroglycan was prepared by acid hydrolysis and gel filtration. The molecular structure of the core of the heteroglycan was determined by NMR studies as a linear α-1,6-d-mannan. The mannan core obtained by acid degradation stimulated the β-glucosidase activity by 90%. Several glycosidases fromAspergillus niger were also activated by theT. reesei heteroglycan. The β-glucosidase ofTrichoderma was activated by mannan fromSaccharomyces cerevisiae to a comparable extent.     

Modification of cambial cell wall architecture during cambium periodicity in Populus tomentosa Carr.

Cambium periodicity is correlated with changes in the cambial cell wall, but the heterogeneity of cell wall structure and composition makes it difficult to give an accurate interpretation, especially for complex secondary vascular tissues. A combination of different methods is necessary to reveal the structure of this complex cell wall. In this study, the cell wall architecture and composition of active and dormant cambial cells in Populus tomentosa were investigated by a combination of light microscopy, rapid-freezing and deep-etching electron microscopy, Fourier-transform infrared microspectroscopy and immuno-histochemistry. The results showed that the architecture of dormant cambial cell walls displayed a multi-layered structure, denser fibril network, smaller pore size, and fewer crosslinks between microfibrils than active cambial cell walls. The FTIR spectra of cell walls from active and dormant cambium showed differences in the intensity of bands near 1,740, 1,629, 1,537, 1,240, and 830 cm⁻¹, which reflected differences in cell wall composition. Immuno-labeling indicated that high methyl-esterified homogalacturonan and (1 → 4)-β-d-galactan epitopes were abundant and distributed in active cambial cell walls, and relatively de-esterified homogalacturonan and (1 → 5)-α-l-arabinan epitopes had weak labeling in the active cambium, while almost no labeling or very weak labeling for high methyl-esterified homogalacturonan, (1 → 4)-β-d-galactan and (1 → 5)-α-l-arabinan epitopes occurred in dormant cambial cells, except for the de-esterified homogalacturonan epitope, which was abundant in dormant cambial cells. These results demonstrate that there are great differences, both in structure and composition, between active and dormant cambial cell walls, which reflect their dynamic changes during cambium activity.     

Comparison of cell wall regeneration on maize protoplasts isolated from leaf tissue and suspension cultured cells

Summary The cell wall regeneration on protoplasts derived from maize mesophyll cells was compared with wall regeneration on protoplasts derived from suspension cultured cells using light microscopy, transmission electron microscopy, and mass spectrometry. The time course of cell wall regeneration has shown that the mesophyll protoplasts regenerated walls much slower than the protoplasts derived from cultured cells. Moreover, cell wall materials on the mesophyll protoplasts were often unevenly distributed. Electron microscopy has further demonstrated that the mesophyll protoplasts have less organized and compact walls than the protoplasts from cultured cells. Chemical analysis revealed that the mesophyll protoplasts had a lower ratio ofβ-(1–3)-glucan toβ-(1–4)-glucan than protoplasts from cultured cells. The significance of these results for the viability and development of protoplasts in culture is discussed. National Research Council of Canada paper no. 32458.     

Temporal and spatial appearance of wall polysaccharides during cellularization of barley (Hordeum vulgare) endosperm

Barley endosperm begins development as a syncytium where numerous nuclei line the perimeter of a large vacuolated central cell. Between 3 and 6 days after pollination (DAP) the multinucleate syncytium is cellularized by the centripetal synthesis of cell walls at the interfaces of nuclear cytoplasmic domains between individual nuclei. Here we report the temporal and spatial appearance of key polysaccharides in the cell walls of early developing endosperm of barley, prior to aleurone differentiation. Flowering spikes of barley plants grown under controlled glasshouse conditions were hand-pollinated and the developing grains collected from 3 to 8 DAP. Barley endosperm development was followed at the light and electron microscope levels with monoclonal antibodies specific for (1→3)-β-d-glucan (callose), (1→3,1→4)-β-d-glucan, hetero-(1→4)-β-d-mannans, arabino-(1→4)-β-d-xylans, arabinogalactan-proteins (AGPs) and with the enzyme, cellobiohydrolase II, to detect (1→4)-β-d-glucan (cellulose). Callose and cellulose were present in the first formed cell walls between 3 and 4 DAP. However, the presence of callose in the endosperm walls was transient and at 6 DAP was only detected in collars surrounding plasmodesmata. (1→3,1→4)-β-d-Glucan was not deposited in the developing cell walls until approximately 5 DAP and hetero-(1→4)-β-d-mannans followed at 6 DAP. Deposition of AGPs and arabinoxylan in the wall began at 7 and 8 DAP, respectively. For arabinoxylans, there is a possibility that they are deposited earlier in a highly substituted form that is inaccessible to the antibody. Arabinoxylan and heteromannan were also detected in Golgi and associated vesicles in the cytoplasm. In contrast, (1→3,1→4)-β-d-glucan was not detected in the cytoplasm in endosperm cells; similar results were obtained for coleoptile and suspension cultured cells.     

Effects of Yariv phenylglycoside on cell wall assembly in the lily pollen tube

Arabinogalactan-proteins (AGPs) are proteoglycans with a high level of galactose and arabinose. Their current functions in plant development remain speculative. In this study, (β-D-glucosyl)₃ Yariv phenylglycoside [(β-D-Glc)₃] was used to perturb AGPs at the plasmalemma-cell wall interface in order to understand their functional significance in cell wall assembly during pollen tube growth. Lily (Lilium longiflorum Thunb.) pollen tubes, in which AGPs are deposited at the tip, were used as a model. Yariv phenylglycoside destabilizes the normal intercalation of new cell wall subunits, while exocytosis of the secretory vesicles still occurs. The accumulated components at the tip are segregated between fibrillar areas of homogalacturonans and translucent domains containing callose and AGPs. We propose that the formation of AGP/(β-D-Glc)₃ complexes is responsible for the lack of proper cell wall assembly. Pectin accumulation and callose synthesis at the tip may also change the molecular architecture of the cell wall and explain the lack of proper cell wall assembly. The data confirm the importance of AGPs in pollen tube growth and emphasize their role in the deposition of cell wall subunits within the previously synthesized cell wall. Received: 14 August 1997 / Accepted: 9 September 1997     

Production and characteristics of the exocellular polysaccharide in mutant strains ofXanthomonas fuscans

Production of the exocellular polysaccharide of the phytopathogenic bacteriumXanthomonas fuscans was investigated with respect to its possible use in utilization of industrial wastes containing lactose. Six stablelac ⁺ mutants were obtained after the treatment withN-methyl-N′-nitroso-N′-nitroguanidine. The mutants were compared with the parent strain. Morphological and cultivation characteristics, as well as production of the exooellular polysaccharide were compared. The production was found to be maximal during the stationary phase of growth in strains cultivated under submerged conditions. Gas chromatography revealed that the polysaccharide of the parent strain is formed by α- and β-D-glucose and α- and β-d-mannose with a small amount ofd-ribose and 6-deoxy-l-mannose. Composition of the polysaccharides produced by the mutant strains (lac ⁺) does not qualitatively differ from that of the parent strain. However, they were found to contain a higher quantity ofd-mannose, which is favourable for their industrial utilization.     

Different affinities of the α- and β-anomers ofD-glucose,D-mannose andD-xylose for the glucose uptake system of baker’s yeast

A recording technique for measuring the sugar uptake by cell suspensions using a polarimeter is described. The method makes it possible to calculate the uptake rates of the α-and β-anomers. The constitutive monosaccharide transport system ofSaccharomyces cerevisiae andSaccharomyces fragilis exhibits a higher affinity for the α-anomers ofd-glucose,d-manose andd-xylose than for the corresponding β-anomers, this resulting in a preferential uptake of the α-anomers from a mixture. The α-anomer ofd-xylose is preferred both during influx and efflux. The membrane transport ofd-xylose inSaccharomyces cerevisiae is not associated with a change of the anomer configuration. The facilitated diffusion system appears to possess a regulatory role for the utilization ofd-glucose andd-mannose in both yeast species investigated.     

<Emphasis Type="Italic">QUASIMODO1</Emphasis> is expressed in vascular tissue of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> inflorescence stems,and affects homogalacturonan and xylan biosynthesis

An insertion in the promoter of the Arabidopsis thaliana QUA1 gene (qua1-1 allele) leads to a dwarf plant phenotype and a reduction in cell adhesion, particularly between epidermal cells in seedlings and young leaves. This coincides with a reduction in the level of homogalacturonan epitopes and the amount of GalA in isolated cell walls (Bouton et al., Plant Cell 14: 2577 2002). The present study was undertaken in order to investigate further the link between QUA1 and cell wall biosynthesis. We have used rapidly elongating inflorescence stems to compare cell wall biosynthesis in wild type and qua1-1 mutant tissue. Relative to the wild type, homogalacturonan α-1-4-D-galacturonosyltransferase activity was consistently reduced in qua1-1 stems (by about 23% in microsomal and 33% in detergent-solubilized membrane preparations). Activities of β-1-4-D-xylan synthase, β-1-4-D-galactan synthase and β-glucan synthase II activities were also measured in microsomal membranes. Of these, only β-1-4-D-xylan synthase was affected, and was reduced by about 40% in qua1-1 stems relative to wild type. The mutant phenotype was apparent in inflorescence stems, and was investigated in detail using microscopy and cell wall composition analyses. Using in situ PCR techniques, QUA1 mRNA was localized to discrete cells of the vascular tissue and subepidermal layers. In mutant stems, the organization of these tissues was disrupted and there was a modest reduction in homogalacturonan (JIM5) epitopes. This study demonstrates a specific role for QUA1 in the development of vascular tissue in rapidly elongating inflorescence stems and supports a role of QUA1 in pectin and hemicellulose cell wall synthesis through affects on α-1,4-D-galacturonosyltransferase and β-1,4-D-xylan synthase activities.     

Papulacandin B: Inhibitor of biogenesis of (1→3)-β-d-glucan fibrillar component of the cell wall ofSaccharomyces cerevisiae protoplasts

The effect of papulacandin B on regenerating protoplasts ofSaccharomyces cerevisiae was studied by light and electron microscopy. In liquid media it inhibited the biogenesis of (1→3)-β-d-glucan fibrillar nets; as a result, the protoplasts did not grow polarly but only spherically. The effect was reversible. Instead of the nets the inhibited protoplasts synthesized only individual microfibrils soluble in hydroxide; these were not joined in the nets and were partially masked by amorphous material. The microfibrils disintegrated after lysis and did not maintain the shape of protoplasts. Protoplasts inhibited in solid media grew spherically up to 25 μm but they did not divide or revert or revert, in spite of forming cell walls. These walls were amorphous and fragile and they disintegrated during preparation. Papulacandin B did not decrease the viability of protoplasts and did not interfere with their growth, biogenesis of alkali-soluble glucan microfibrils or amorphous wall matrix. It inhibited specifically the synthesis of alkali-insoluble branched (1→3)-β-d-glucan, a necessary building unit required for the formation of the fibrillar component of the cell wall responsible for the cell wall shape, its rigidity and tensile strength.     

Localization of cell wall polysaccharides in nonarticulated laticifers ofAsclepias speciosa Torr.

Summary Asclepias speciosa Torr, has latex-containing cells known as nonarticulated laticifers. In stem sections of this species, we have analyzed the cell walls of nonarticulated laticifers and surrounding cells with various stains, lectins, and monoclonal antibodies. These analyses revealed that laticifer walls are rich in (1→4) β-D-glucans and pectin polymers. Immunolocalization of pectic epitopes with the antihomogalacturonan antibodies JIM5 and JIM7 produced distinct labeling patterns. JIM7 labeled all cells including laticifers, while JIM5 only labeled mature epidermal cells and xylem elements. Two antibodies, LM5 and LM6, which recognize rhamnogalacturonan I epitopes distinctly labeled laticifer walls. LM6, which binds to a (l→5) α-arabinan epitope, labeled laticifer walls more intensely than walls of other cells. LM5, which recognizes a (1→4) β-D-galac-tan epitope, did not label laticifer segments at the shoot apex but labeled more mature portions of laticifers. Also the LM5 antibody did not label cells at the shoot apical meristem, but as cells grew and matured the LM5 epitope was expressed in all cells. LM2, a monoclonal antibody that binds to β-D-glucuronic acid residues in arabinogalactan proteins, did not label laticifers but specifically labeled sieve tubes. Sieve tubes were also specifically labeled byRicinus communis agglutinin, a lectin that binds to terminal β-D-galactosyl residues. Taken together, the analyses conducted showed that laticifer walls have distinctive cytochemical properties and that these properties change along the length of laticifers. In addition, this study revealed differences in the expression of pectin and arabinogalactan protein epitopes during shoot development or among different cell types.     

Hyphal wall composition inApodachlyella completa

Mechanically isolated hyphal walls of the oomyceteApodachlyella completa (Humphrey) Indoh. (Leptomitales) were investigated by means of biochemical, cytochemical, and X-ray analyses. Microscopic examination of the cell wall preparations revealed them to be free of cytoplasmic contaminants β-Glucans account for 88% of wall weight and contain predominantly I→3 and I→6 linkages. Cellulose was demonstrated cytochemically and by X-ray diffraction and accounts for approximately 6% of wall dry weight. Additionally, walls contain 5% insoluble hexosamine, 4.8% protein, 1.1% lipid, and 0.5% ash. Chitin was qualitatively detected by means of cytochemical and X-ray analysis. The cell wall composition ofA. completa is similar to that reported for other oomycetes, in that β-glucans are the primary wall constituents. However, this fungus shows the unusual feature of chitin occurring simultaneously with cellulose, as do the closely related speciesApodachlya sp. andLeptomitus lacteus. *** DIRECT SUPPORT *** A01R4022 00007     

Changes in cell wall composition of deformedras1 − cells ofSchizosaccharomyces pombe

Disruption of theSchizosaccharomyces pombe ras1 gene results in a morphological transformation to large spheres, in contrast to wild-type cells which grow as rods. Chemical analysis of isolated cell walls showed no significant changes in saccharide content but an increase in protein and phosphate contents inras1 ⁻ walls relative to parent walls. Polymers tightly bound to the cell wall were solubilized by SDS treatment. Several compounds with molar mass ranging from 22 to 130 kDa and more were resolved by gel filtration and SDS-PAGE. Among low-molar-mass species, a component moving as a band at 31 kDa was conspicuous inras1 ⁻ cell walls. It was solubilized by heating in Tris-HCl buffer and shown to have a β-1,3-glucanase activity against laminarin. The level of the enzyme was by 30% higher in theras1 ⁻ cell wall than in the wild-type cell wall. This enzyme may participate in the remodelling of the rigid glucan network and account (at least partially) for the aberrant cell shape. Theras1 ⁻ cell wall contained a high level of charged polymers, especially phosphoproteins, raising the appealing possibility thatras1 ⁻ is involved in a putative kinase cascade required to sense and respond to external stimuli destined for the cell wall. Although the present study shows thatras1 loss of function and altered cell wall composition are closely linked defects, it has still to be shown that theras1 protein is directly involved in alterations found in the mutant cell walls.     

Production and structure elucidation of di- and oligosaccharide lipids (biosurfactants) from Tsukamurella sp. nov.

The bacterium Tsukamurella sp. nov., isolated from soil, was found to produce novel glycolipids when grown on sunflower oil as the sole carbon source. The glycolipids were isolated by chromatography on silica columns and their structures elucidated using a combination of multidimensional NMR and MS techniques. The three main components are 2,3-di-O-acyl-α-d-glucopyranosyl-(1-1)-α-d-glucopyranose, 2,3-di-O-acyl-β-d-glucopyranosyl-(1-2)-4,6-di-O-acyl-α-d-glucopyranosyl-(1-1)-α-d-glucopyranose and 2,3-di-O-acyl-β-d-glucopyranosyl-(1-2)-β-d-galactopyranosyl-(1-6)-4,6-di-O-acyl-α-d-glucopyranosyl-(1-1)-α-d-glucopyranosl which are linked to fatty acids varying in chain length from C₄ to C₁₈. The glycolipids are mainly extracellular but are also found attached to the cell walls. During the cultivation the composition of the glycolipids changed from disaccharide- to tri- and tetrasaccharide lipids. The glycolipids show good surface-active behaviour and have antimicrobial properties. Received: 22 May 1998 / Received revision: 24 August 1998 / Accepted: 26 August 1998     

Contribution of peptidoglycan to the binding of metal ions by the cell wall ofBacillus subtilis

Equilibrium dialysis techniques were utilized in assessing the interaction between the cell walls ofBacillus subtilis and metal ions. The results show that cell wall teichoic acid molecules are not required for wall-metal ion complex formation. The numbers of combining sites for metals on unmodified cell walls were 0.7–0.9 μmol metal bound per mg wall. Thus, it is likely that the metals are combining with identical sites in the cell walls. The geometry of the sites may be responsible for the changes in affinity of the walls for various metals.     

Fracture mechanics of the cell wall of Chara corallina

Previous mechanical studies using algae have concentrated on cell extension and growth using creep-type experiments, but there appears to be no published study of their failure properties. The mechanical strength of single large internode cell walls (up to 2 mm diameter and 100 mm in length) of the charophyte (giant alga) Chara corallina was determined by dissecting cells to give sheets of cell wall, which were then notched and fractured under tension. Tensile tests, using a range of notch sizes, were conducted on cell walls of varying age and maturity to establish their notch sensitivity and to investigate the propagation of cracks in plant cell walls. The thickness and stiffness of the walls increased with age whereas their strength was little affected. The strength of unnotched walls was estimated as 47 ± 13 MPa, comparable to that of some grasses but an order of magnitude higher than that published for model bacterial cellulose composite walls. The strength was notch-sensitive and the critical stress intensity factor K _1c was estimated to be 0.63 ± 0.19 MNm^−3/2, comparable to published values for grasses. Received: 4 April 2000 / Accepted: 21 July 2000     

Isolation of a new flavonol glycoside and its effects on the blue color of seed coats ofOphiopogon jaburan

From the blue seed coats ofOphiopogon jaburan, a new flavonol glycoside was isolated as needles and determined to be kaempferol 3-O-β-d-galactoside-4′-O-β-d-glucoside (OK-2) by UV and NMR spectral analyses. OK-2 and kaempfrol 3, 4′-di-O-β-d-glucoside (OK-1), which was detected previously, in the blue seed coat were present in a molar ratio of about 13:7. OK-2 was newly found as a factor causing the blueing effects on ophionin which is a main anthocyanin in the blue seed coats. The mixture of 4.8×10⁻³ M OK-2 and 2.5×10⁻³ M ophionin in Mcllvaine's buffer solution (pH 5.6) showed stable blue color, and the absorption spectrum of the mixture showed two absorption peaks and a shoulder in visible reasion, coinciding with that of the fresh blue seed coat. The effect of ophionin and OK-2 co-pigmentation on the blue color of seed coat ofO. jaburan was discussed.     

A role for arabinogalactan-proteins in root epidermal cell expansion

Arabinogalactan-proteins (AGPs) are abundant plant proteoglycans that react with (β-d-Glc)₃ but not (β-d-Man)₃ Yariv reagent. We report here that treatment with (β-d-Glc)₃ Yariv reagent caused inhibition of root growth of Arabidopsis thaliana (L.) Heynh. seedlings. Moreover, the treated roots exhibited numerous bulging epidermal cells. Treatment with (β-d-Man)₃ Yariv reagent did not have any such effects. These results indicate a role for AGPs in root growth and control of epidermal cell expansion. Because treatment with (β-d-Glc)₃ Yariv reagent phenocopies the reb1 (root epidermal cell bulging) mutant of Arabidopsis, AGPs were extracted from the reb1-1 mutant and compared with those of the wild type. The reb1-1 roots contained an approximately 30% lower level of AGPs than the wild type. More importantly, while the profile of AGPs from wild-type roots showed two major peaks upon crossed electrophoresis, the profile of AGPs from reb1-1 roots exhibited only one of the major peaks. Therefore, the reb1 phenotype appears to be a result of defective or missing root AGPs. Taken together, this pharmacological and genetic evidence strongly indicates a function of AGPs in the control of root epidermal cell expansion. Received: 13 February 1997 / Accepted: 1 April 1997     

Differential effects of amino acid analogs on growth and heterocyst differentiation in two nitrogen-fixing blue-green algae

Effects of a few amino acid analogs on growth and heterocyst differentiation have been studied in two nitrogen-fixing species ofAnabaena. All the analogs except α-methyl-dl-aspartic acid inhibited growth. Exposure ofAnabaena doliolum, todl-5-fluorotryptophan anddl-p-fluorophenylalanine caused pronounced fragmentation of filaments into single cells. At low concentrations (0.01 mM), α-methyl-dl-aspartic acid stimulated growth of the strain ofA. doliolum as well as the strain of the second (unidentified)Anabaena species. Ethionine,dl-p-fluorophenylalanine,dl-5-fluorotryptophan, and canavanine blocked heterocyst differentiation, whereas α-methyl-dl-aspartic acid, α-methyl-dl-methionine,N-o-nitrophenylsulfenyl-l-tryptophan, norleucine, andS-2-aminoethyl-l-cysteine did not show any significant effect. Treatment with 7-azatryptophan,dl-β-hydroxynorvaline,l-methionine-dl-sulfoximine,l-methionine sulfone, and β-2-thienyl-dl-alanine led to a twofold increase in heterocyst frequency. Possible modes of action of the analogs in growth inhibition and changes in heterocyst frequency are discussed.     

Agar degradation by microorganisms and agar-degrading enzymes

Agar is a mixture of heterogeneous galactans, mainly composed of 3,6-anhydro-l-galactoses (or l-galactose-6-sulfates) d-galactoses and l-galactoses (routinely in the forms of 3,6-anhydro-l-galactoses or l-galactose-6-sulfates) alternately linked by β-(1,4) and α-(1,3) linkages. It is a major component of the cell walls of red algae and has been used in a variety of laboratory and industrial applications, owing to its jellifying properties. Many microorganisms that can hydrolyze and metabolize agar as a carbon and energy source have been identified in seawater and marine sediments. Agarolytic microorganisms commonly produce agarases, which catalyze the hydrolysis of agar. Numerous agarases have been identified in microorganisms of various genera. They are classified according to their cleavage pattern into three types—α-agarase, β-agarase, and β-porphyranase. Although, in a broad sense, many other agarases are involved in complete hydrolysis of agar, most of those identified are β-agarases. In this article we review agarolytic microorganisms and their agar-hydrolyzing systems, covering β-agarases as well as α-agarases, α-neoagarobiose hydrolases, and β-porphyranases, with emphasis on the recent discoveries. We also present an overview of the biochemical and structural characteristics of the various types of agarases. Further, we summarize and compare the agar-hydrolyzing systems of two specific microorganisms: Gram-negative Saccharophagus degradans 2–40 and Gram-positive Streptomyces coelicolor A3(2). We conclude with a brief discussion of the importance of agarases and their possible future application in producing oligosaccharides with various nutraceutical activities and in sustainably generating stock chemicals for biorefinement and bioenergy.     

The zygote cell wall of Chlamydomonas reinhardii: a structural,chemical and immunological approach

The zygote cell wall of Chlamydomonas reinhardii has been studied using structural, chemical and immunological methods. Monoclonal antibodies and polyclonal antisera that were originally raised to the major hydroxyproline-rich glycoproteins of the vegetative cell wall were used to probe the zygote wall for common antigenic components. These antibodies cross-reacted strongly and specifically with components of the zygote cell wall, and were used to show the origin, route of transport, and the location of these antigens within the zygote cell wall. The zygote cell wall contained about 10% protein, with hydroxyproline accounting for 22.5 mol % of the total amino acids present. Glucose was the most abundant sugar residue, and accounted for 56% of the total sugar present. Gas liquid chromatography-mass spectrometry showed the presence of a (1-3)-d-glucan as the major structural polysaccharide within the zygote cell wall. The (1-3)-d-glucan was detected and localised within the zygote cell wall by immunogold labelling of thin sections. Using an antiserum directed against (1-3)-d-linked glucose units, this polysaccharide was found to be consistently present within the non-staining layer of both young and mature zygote cell walls. (1-3)-d-Glucan was also detected in other wall layer using higher concentrations of antiserum. No intracellular labelling was found, indicating that the plasmamembrane is the site for the synthesis of this polysaccharide within the Chlamydomonas zygote.Abbreviations DGP antiserum to deglycosylated 2-BII glycoprotein - GLC-MS gas liquid chromatography-mass spectrometry - MAC monoclonal antibody centre