Depolymerization of legume seed galactomannan by Celloviridin G20x

The depolymerization of legume galactomannans by the commercial preparation Celloviridin G20x was studied with the aim of obtaining macromolecular fragments of a constant composition. Four galactomannans, representative of the entire range of monomer ratios characteristic of this class of phytopolysaccharides, were hydrolyzed. The galactomannans were isolated from oriental goat’s rue (Galega orientalis Lam., Man: Gal 1.1), guar (Cyamopsis tetragonoloba L., Man: Gal 1.6), honeylocust (Gleditsia triacanthos f. inermis L., Man: Gal 2.4), and sophora (Styphnolobium japonicum (L.) Schott., Man: Gal 5.1). Fragments with a monomer ratio close to that in the original polysaccharides were obtained with a high yield (75–80%). The degree of substitution of the 1,4-β-D-mannopyranose chain at position C-6 with α-galactose residues influenced the molecular weight of the final reaction product.     

Diverse patterns of cell wall mannan/galactomannan occurrence in seeds of the Leguminosae

Endosperms from seeds of different subfamilies of Leguminosae were submitted to sequential aqueous and alkaline aqueous extractions. The extractions from species belonging to the Mimosoideae and Faboideae subfamilies yielded galactomannans with constant Man:Gal ratios, whereas the extractions from Caesalpinioideae seeds gave rise to galactomannans with increasing values of the Man:Gal ratio. The presence of a family of galactomannans within the same species may be a trait found only in Caesalpinioideae subfamily. The final insoluble residues that were obtained after the removal of galactomannans from the Caesalpinioideae and Faboideae subfamilies are composed of pure mannans and do not contain cellulose, while those from the Mimosoideae subfamily are composed of cellulose. A mannan was isolated from the unripe endosperm of Caesalpinia pulcherrima, suggesting no developmental relationship between galactomannan and mannan. These results are consistent with the presence of a distinctive cell wall pattern in the endosperms of Leguminosae species.     

13C-n.m.r. studies of monomeric composition and sequence in alginate

High-resolution, ¹³C-n.m.r. spectra of slightly depolymerised alginates have been interpreted. The sequence of monomer units, l-guluronate (G) and d-mannuronate (M), markedly influenced the chemical shifts. At 50 MHz, some of the individual carbon resonances of both units were resolved into four lines, in evident dependence upon the identities of the units immediately preceding and following them in the chains. The relative intensifies of the signals permitted rapid computation of (1) monomeric composition (M/G ratio), (2) monomeric sequence in terms of a complete set of four diad and eight triad frequencies, and (3) the composition (M/G ratio) of end units and of the units adjacent to M-residues at the non-reducing end. The diad frequencies indicated that alginate was a block co-polymer containing number-average, co-monomer block-lengths of ～2?8. The triad frequencies indicated average lengths of ～4?8 for blocks containing two or more units, these being somewhat longer for G- than for M-blocks. Regions of the chains having a strictly alternating sequence of M- and G-residues were short. The relative occurrence of G-centred triads deviated significantly from those predicted by first-order Markovian statistics.     

Associations of Alleles of the Esterase-1 Locus with Gene Arrangements of the Left Arm of the Second Chromosome in DROSOPHILA ROBUSTA

Evidence of strong associations of Est-1 alleles with the 2L, 2L1 and 2L3 gene arrangements of the left arm of the second chromosome in D. robusta is presented. Each gene arrangement is polymorphic for three to four Est-1 alleles. The allele frequencies differ in the 2L3 and 2L arrangements; the allele Est-1^.92 is 8% in the 2L3 arrangement (n=203)—this allele is 82% in the 2L arrangement (n=203); the allele Est-1^1.0 is 66% and 14.8% in the 2L3 and 2L arrangements, respectively. There are no differences in allele frequencies in 2L3 arrangements from any of the widely separated seven different populations; similarly the allele frequencies in the 2L arrangement are alike in all five widely separated populations studied. The allele frequencies in the 2L1 arrangement are intermediate to those observed in the 2L3 and the 2L arrangements and show north-south clinal change. These associations between Est-1 alleles and gene arrangements of the left arm of the second chromosome are due to natural selection favoring different allele frequencies in different gene arrangements, as a result of epistatic interactions between the Est-1 locus and the loci on the gene arrangements. As expected, we observe that the proportion of heterozygotes is greater in the inversion heterokaryotypes than in the homokaryotypes.     

Extraction,purification and characterization of galactomannans from non-traditional sources

This work presents a methodology for the extraction of galactomannans from seeds of four different species of Leguminosae (Adenanthera pavonina, Caesalpinia pulcherrima, Gleditsia triacanthos and Sophora japonica) to be used e.g. in the food and biomedical industries. The galactomannans were obtained by aqueous extraction followed by a precipitation with ethanol. This methodology is simpler and easier to perform than other existing extraction and purification methodologies, and because it avoids the use of organic solvents (other than ethanol), it is able to generate food grade substances and is environmentally friendlier. The yield of extraction in different stages of the process, monosaccharide composition, as well as physical and chemical parameters of the isolated galactomannans were determined and compared with previously published results. The mannose/galactose ratio of the extracted galactomannans ranged from 1.35 (A. pavonina) to 5.75 (S. japonica). The intrinsic viscosity ranged from 11.34 dL/g (C. pulcherrima) to 8.74 dL/g (S. japonica), while the viscosity average molecular mass ranged between 1.81 × 10⁶ Da and 1.17 × 10⁶ Da (A. pavonina > C. pulcherrima > G. triacanthos > S. japonica). The results confirm the suitability of the extraction and purification procedure to obtain galactomannans from non-traditional sources.     

Exopolysaccharide from surface-liquid culture of <Emphasis Type="Italic">Clonostachys</Emphasis><Emphasis Type="Italic">rosea</Emphasis> originates from autolysis of the biomass

We describe the purification and chemical characterization of galactomannans that appear both in the biomass and the culture broth during surface-liquid culture of the fungus Clonostachys rosea, a common facultative saprophyte that has potential to be used as a biological control agent against several plant pathogenic fungi, insects and nematodes. The galactomannans from both sources had comparable ratios of Man, Gal and Glc and the similarity were confirmed by ¹H, ¹³C NMR, HMQC, and COSY spectra. We propose that the galactomannan in the culture broth originates from autolysis of the biomass, based not only on the similarity that it has with the galactomannan extracted from the biomass but also on the fact that its concentration increased rapidly after glucose depletion from the medium, when biomass concentration was falling. Polysaccharides from C. rosea have not previously been characterized; we show that the characteristics of the galactomannans are consistent with those that have been reported for other members of the Bionectriaceae, the family to which C. rosea belongs.     

Intrinsic protein-lipid interactions. Infrared spectroscopic studies of gramicidin A, bacteriorhodopsin and Ca2+-ATPase in biomembranes and reconstituted systems

Infrared spectroscopy has been applied to the study of a number of aqueous systems of model and natural biomembranes. The absorption bands arising from water and buffer solutions were eliminated by means of an infrared spectrometer data station. Spectra were examined using H₂O and ²H₂O aqueous buffer systems. Pure lecithin-water systems, and various model biomembranes containing cholesterol, gramicidin A, bacteriorhodopsin or Ca²⁺-ATPase were examined. The infrared spectra of the reconstituted biomembranes were compared with those of the corresponding natural biomembranes, i.e. the purple membrane of Halobacterium halobium and also sarcoplasmic reticulum membranes, respectively.Changes in lipid chain conformation caused by the various intrinsic molecules incorporated within the model lipid bilayer structures were monitored by studying the shifts in frequency (cm^?1) of the CH₂ symmetric and asymmetric absorption bands arising from the lipid chains. The effect of gramicidin A and also the intrinsic proteins, as indicated by the shift of band frequencies, are quite different from that of cholesterol at temperatures above the main lipid transition temperature t_c. Cholesterol causes a reduction in gauche isomers which increases with concentration of cholesterol within the lipid bilayer. Whilst gramicidin A and the intrinsic proteins at low concentration cause a reduction of gauche isomers, at higher concentrations of these molecules, however, there is little difference in gauche isomer content when the intrinsic molecule is present compared with that of the fluid lipid alone. These results are considered and compared with previously published studies using deuterium nuclear magnetic resonance spectroscopy on similar model biomembrane systems. Below the lipid t_c value, all the intrinsic molecules produce an increase in gauche isomers presumably by disturbing the lipid chain packing in the crystalline lipid arrangement.Information about the polypeptide structure within gramicidin A. the reconstituted proteins and also the proteins in the natural biomembranes was obtained by examining the region of the infrared spectrum between 1600 and 1700 cm^?1 associated with the amide I and amide II bands. An examination of the infrared band frequencies of the different systems in this region leads to the conclusions: (1) that gramicidin A within a phospholipid bilayer structure probably has a single helix rather than a double helix structure; (2) that there are differences in band widths of the reconstituted Ca²⁺-ATPase and bacteriorhodopsin compared with the spectra of the corresponding sarcoplasmic reticulum and purple membrane; (3) different membrane proteins adopt different conformations as evinced by a comparison of the spectra of the sarcoplasmic reticulum and purple membrane; (4) the polypeptide arrangement in the purple membrane is mainly helical but the abnormal frequency of the amide I band suggests that some distortion of the helix occurs: and (5) the sarcoplasmic reticulum membrane contains unordered as well as α-helix polypeptide arrangements.     

Seed galactomannans: an overview

Seed galactomannans are vegetable, heterogeneous polysaccharides widely distributed in nature. Generally, they possess (1-->4)-linked D-mannopyranose (Man) main chains to which are attached (1-->6)-linked D-galactopyranosyl (Gal) units. The Man/Gal ratios differ from gum to gum, resulting in a change in structure, which, in turn, determines the various industrial applications of seed galactomannans. These materials are important in paper, textile, petroleum-drilling, pharmaceutics, food, cosmaceutics, and explosives industries. In this review, the biodiversified applicability of galactomannan gums is discussed, particularly with respect to structural aspects, properties, human consumption, and technical applications. Especially important is that the solution properties (rheological behaviour, viscosity, emulsifying tendency, etc.) of natural and chemically modified galactomannans can be tuned by interaction with other (carbohydrate-based) monomers or polymers.     

Agar/galactomannan gels applied to shoot regeneration from tobacco leaves

This study concerns the efficacy of partial agar substitution by galactomannans as support in plant regeneration media for Nicotiana tabacum. The production of multiple shoots from leaf-derived callus and their rooting were evaluated. The galactomannans applied were obtained from Cassia fastuosa (cassia) and Cyamopsis tetragonolobus (guar gum — a commercial galactomannan) seeds. The results obtained on media solidified with mixtures of agar/galactomannan (3 g dm⁻³ each) gels were compared with those on media gelled with a standard concentration of agar (6 g dm⁻³). The in vitro performance allowed to conclude that the use of galactomannans raised the number of shoots and improved their quality. Furthermore, the length of roots and the size of leaves were significantly higher in the media solidified with agar/guar galactomannan mixtures.     

STRUCTURAL STUDIES ON THE GALACTOMANNANS OF LICHENS OF THE GENUSCLADONIA

Galactomannans were isolated fromCladonia signata,C. furcata,C. imperialis, andC. clathratavia successive alkaline extraction and precipitation with Fehling solution and Cetavlon. They were investigated using¹³C-NMR spectroscopy, methylation analysis, and Smith degradation, and their specific rotations and monosaccharide compositions determined. As with galactomannans of otherCladoniaspecies, they contained (1→6)-linked main chains of α-mannopyranose, which were non-substituted (structure4in Fig. 2), mono-substituted at O-2 with α-mannopyranose (structure6) or α-galactopyranose (structure1), O-4 with β-galactopyranose (structure2), and disubstituted at O-2 and O-4 with α-mannopyranosyl and β-galactopyranosyl units, respectively (structure5). Disubstitution was present to a greater extent in the galactomannans ofC. clathrataandC. imperialisthan in those ofC. signataandC. furcata. In the case of the galactomannans ofC. furcata,C. clathrata, andC. imperialis, substitution also occurred at O-2 withO-β-galactofuranosyl-(1→6)-O-α-mannopyranosyl units (structure7). As observed in previous investigations, the C-1 portion of the¹³C-NMR of mannose-containing polysaccharides is typical of the lichen species. However, those of galactomannans ofC. imperialisandC. clathrataare almost identical and, although other chemical data showed many structures in common, some differences were evident.     

The galactomannan-like oligosaccharides from the endosperm of the seed of Gleditsia triacanthos

The endosperm of the seed of Gleditsia triacanthos contains 4.8% of 85% ethanol-soluble, galactomannan-like oligosaccharides having Man:Gal ratios of 1.5–2.6:1 and an average degree of polymerization of 15. They have a narrow distribution of molecular weights and of ratio of components. The oligosaccharides have the gross structure accepted for the galactomannans, namely, a β-(1→4)-linked d-mannopyranosyl backbone having single stubs of α-(1→6)-linked d-galactopyranosyl groups. Some of the lateral chains contain more than one unit, and a minor proportion of the branches are ended by arabinofuranose or fucopyranose residues. Unusual branching points formed by 3,4-linked d-mannosyl, or 3,6-linked d-galactosyl units, or both, were also found. Despite their low molecular weight, the oligosaccharides form aggregates with a structure similar to that of the aggregates of the related galactomannans, but having a lower association energy. This fact, together with the difficulty of combining with more than one partner (due to the short, central chain), results in an increased solubility and in nonviscous solutions. The ¹³C-n.m.r. spectrum differentiated clearly the five structural units of the oligosaccharides, namely, the reducing and nonreducing end-chains of the d-mannosyl backbone; substituted and nonsubstitued, internal β-(1→4)-linked mannopyranosyl units of the backbone; and the galactosyl nonreducing end-chain of the lateral chains. The C-4 signal of the (1→4)-linked d-mannose and the C-6 signal of the same, but substituted, units showed splitting into three lines. The first has been attributed to sequence-related heterogeneity, whereas the latter is tentatively explained by assuming that this resonance is sensitive to whether the mannosyl units linked to that residue are also branched, or not.     

Structural studies of urinary oliogosaccharides from patients with mannosidosis

Eight neutral oligosaccharide fractions were obtained from the pooled urine of two patients with mannosidosis by Bio-Gel P2 and Bio-Gel P4 column chromatography. The structures of seventeen oligosaccharides were determined by monosaccharide composition analysis, methylation studies, acetolysis, Smith degradation, and ¹³C NMR analysis. Three of the proposed structures, Manα1-3Manβ1-4GlcNAc, Manα1-2Manα1-3Manβ1-4GlcNAc, and Manα1-2Manα1-2Manα1-3Manβ1-4GlcNAc are identical to those first published by Norden et al. (N. E. Norden, A. Lundblad, S. Svennson, P. A. Ockerman, and S. Autio, 1973. J. Biol. Chem.248, 6210–6215; N. E. Norden, A. Lundblad, S. Svennson, and S. Autio, 1974. Biochemistry13, 871–874). Thirteen of them, Manα1-3Manα1-6(Manα1-3)-Manβ1-4GlcNAc, Manα1-3Manα1-6(Manα1-2Manα1-3)Manβ1-4GlcNAc, and 11 isomers of (Manα1-2)_0–4[Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAc], are the same as those first published by Yamashita et al. (K. Yamashita, Y. Tachibana, K. Mihara, S. Okada, H. Yabuuchi, and A. Kobata, 1980, J. Biol. Chem.255, 5126–5133); a tetrasac-charide, Manα1-6(Manα1-3)Manβ1-4GlcNAc, is newly reported and several other structural possibilities are proposed.     

Structural analysis of galactomannans by NMR spectroscopy

The structure of naturally occurring galactomannans was characterized by high resolution NMR spectroscopy involving two-dimensional (2D) NMR measurements of the field gradient DQF-COSY, HMQC, HMBC, and ROESY experiments. Four galactomannans with different proportions of galactose (G) and mannose (M), from fenugreek gum (FG), guar gum (GG), tara gum (TG), and locust bean gum (LG), were investigated. Because these galactomannans had very high molecular weights, hydrolysis by dilute H₂SO₄ was carried out to give the corresponding low molecular weight galactomannans, the structural identities of which were established by comparison of the specific rotations, shape of the GPC profiles, and NMR spectra with those of higher molecular weight galactomannans. The correlation signals GH1-GC4, -GC5, and -MC6 in HMBC and GH1-GH6 in ROESY spectra of FG showed that more than two galactopyranose units with the 1 → 4 linkage were connected at C6 of the mannopyranose main chain. The coupling constant (J_H1,2) of galactose was 3.4 Hz, indicating that galactose has an α-linkage. The main chain mannose was found to connect through the 1 → 4 linkage, because of the appearance of the correlation signals MH1-MC4, and MC1-MH4 in the HMBC spectrum due to the long-range correlation signals between two neighboring mannopyranose residues through the M4-O-M1 bond. Although the main chain mannose J_H1,2 was not observed, probably because of the high molecular weight, the specific rotation of LG with a higher proportion of mannose was low, [α]_D²⁵ = +10.8°, compared with that of FG with a lower proportion of mannose, [α]_D²⁵ = +90.5°, suggesting that the mannose in the main chain had a α-linkage. These results suggest that the galactomannans comprise a (1 → 4)-β-mannopyranosidic main chain connected with more than two (1 → 4)-α-galactopyranosidic side chains, in addition to the single galactopyranose side chain, at C6 of the mannopyranose main chain.     

Distribution of D-galactosyl groups in guaran and locust-bean gum

Distribution of α-d-galactopyranosyl side-chain groups in two galactomannans, guaran and locust-bean gum, was determined by measurement of the O-acetyl-O-methyl-d-mannitol derivatives obtained from the corresponding primary C-p-tolylsulfonyl polysaccharide derivatives. The O-acetyl-O-methyl-d-mannitol derivatives were produced by β-elimination and methylation, with sodium (methylsulfinyl)methide and methyl iodide, of the primary C-p-toluenesulfinylated galactomannans, followed by sequential acid hydrolysis, reduction, and acetylation of the partially degraded p-tolyl sulfones. The results indicated that side-chain units of guaran are alternately disposed along the d-mannan backbone, whereas those of locust-bean gum are disposed in uniform blocks along the backbone.     

Cell-Wall Carbohydrates of the Endosperm of the Seed of Gleditsia triacanthos

The endosperm of the seed of Gleditsia triacanthos L. contains 18.55% of its dry weight as nonreserve, cell-wall carbohydrates. Of this carbohydrate material, comprising mainly mannose, galactose, and glucose, 76.1% was of low-molecular weight or highly hydrophilic. Mannose, galactose, and glucose were also the major sugar components of the polysaccharides extracted with alkali (23.1% of the cell-wall), while the same sugars, with minor amounts of arabinose, form the residues. Methylation analysis of the polysaccharides and the borate-sodium hydroxide residue indicate that the cell walls are built up on a network of galactomannans, with high Man/Gal ratios, reinforced with minor amounts of cellulose.     

Nuclear Magnetic Resonance of Tissue 23Na: II. Theoretical Line Shape

The theoretical line-shape function of the nuclear magnetic resonance (NMR) signal of ²³Na in biological tissue (and other unoriented systems) was obtained under the following conditions: (I) there occur two states of ²³Na in the system, (II) the exchange of ²³Na between the two states is rapid (but not too rapid), (III) in the absence of exchange, the ²³Na in one state is characterized by a single transverse relaxation time T₂ and a single Larmor frequency, and (IV) in the absence of exchange, the ²³Na in the other state possesses (a) two different values of T₂ and/or (b) more than one Larmor frequencies in the first order perturbation effect. The theoretical signal obtained consists of two Lorentzian components, which are centered at the same frequency, but characterized by different T₂. Only the narrower component, comprising 40% of the total intensity, is visible, when the fast T₂ is sufficiently short. The theoretical line-shape function of ²³Na signal was also calculated for oriented systems in which the above conditions are fulfilled.     

Genetic Coadaptation in the Chromosomal Polymorphism of DROSOPHILA SUBOBSCURA. I. Seasonal Changes of Gametic Disequilibrium in a Natural Population

Seasonal changes in gene arrangement and allozyme frequencies have been investigated in Drosophila subobscura for several years. Some arrangements (O_st and O₃₊₄₊₇) show seasonal variation, which suggests that chromosomal polymorphism is flexible in this species. Seasonal changes in allozyme frequencies for Lap and Pept-1 loci, both located within the same inversions of chromosome O, are significant only inside the O_st arrangement, but not inside O₃₊₄ arrangement. This arrangement-dependent response of allozyme generates variation in arrangement-allozyme disequilibrium. The historical hypothesis on the maintenance of disequilibria cannot explain these seasonal changes, and some kind of natural selection must be invoked. Association between Lap and Pept-1 is also seasonal inside O_st but not inside O₃₊₄. We propose that O_st probably consists of a finite array of supergenes that are differentially favored in each season by natural selection. The present evidence on this supergene selection and other genetic, biogeographic and phylogenetic data points to O₃₊₄ as the most primitive gene order among the present arrangements.     

Influence of domestic sewage on growth and nitrogen fixation of Azolla pinnata R. Br.

Biomass production of Azolla pinnata R.Br., cultured in sewage effluents under full sunlight, correlated closely with the changes of orthophosphate phosphorus (P) in the medium: it was highest (13.18 g m⁻²) in August as opposed to 6.34 g m⁻² in January, though the environmental influence was also remarkable. Phosphorus contents in different media, particularly in one receiving a higher quantity of sewage effluents, gradually increased to 20.30 μg 1⁻¹ in January, reaching a maximum in May (37.60 μg 1⁻¹), followed by a gradual decline with a further rise in August (32.70 μg 1⁻¹). The seasonal variation in phosphorus content of the medium was closely associated with the carbon, phosphorus and nitrogen contents of plant tissue, as well as the heterocyst frequency of the symbiont, Anabaena azollae Strasb. The respective increases in nitrogen, carbon and phosphorus contents of plant tissue in mg 1⁻¹ during August were 39.80, 280.00 and 2.20, compared with values of 28.20, 245.00 and 1.50 during January, while the heterocyst frequencies were 15.96 and 10.92, respectively.