Enzymic degradation of chemically modified extracellular polysaccharides from Rhizobia

Extracellular polysaccharides from Rhizobium trifolii, U226, Coryn and Bart A; Rhizobium phaseoli, U453; Rhizobium leguminosarum, U331; and Rhizobium meliloti, U27, after chemical modification, become substrates for certain β-d-glucan hydrolases. The Streptomyces (1 → 4)-β-d-glucan endohydrolase (EC 3.2.1.4) hydrolyses reduced and deacetylated rhizobial polysaccharides, both before and after removal of carboxyethylidene substituents, to produce a series of oligosaccharides. The Rhizopus arrhizus (1 → 3)-β-d-glucan endohydrolase (EC 3.2.1.6) hydrolyses only fully modified polysaccharides to yield, in the case of R. meliloti U27, laminarabiose, and, in all other instances, a disaccharide identified β-d-Gal-(1 → 3)-D-Glc. The same disaccharides are released by the Rhizopus enzyme from oligosaccharides produced by the action of the Streptomyces enzyme on fully modified polysaccharides. The results are discussed in relation to the available data for the structure of the polysaccharides and the specificity of the enzymes.     

The golden root, Rhodiola rosea, prolongs lifespan but decreases oxidative stress resistance in yeast Saccharomyces cerevisiae

The effect of aqueous extract from R. rosea root on lifespan and the activity of antioxidant enzymes in budding yeast Saccharomyces cerevisiae have been studied. The supplementation of the growth medium with R. rosea extract decreased survival of exponentially growing S. cerevisiae cells under H₂O₂-induced oxidative stress, but increased viability and reproduction success of yeast cells in stationary phase. The extract did not significantly affect catalase activity and decreased SOD activity in chronologically aged yeast population. These results suggest that R. rosea acts as a stressor for S. cerevisiae cells, what sensitizes yeast cells to oxidative stress at exponential phase, but induces adaptation in stationary phase cells demonstrating the positive effect on yeast survival without activation of major antioxidant enzymes.     

Extraction, preliminary structural characterization, and antioxidant activities of polysaccharides from Salvia miltiorrhiza Bunge

Four polysaccharides were extracted from Salvia miltiorrhiza Bunge using hot water, ultrasonic, alkali, and enzyme methods. Preliminary structural characterization was conducted by physicochemical property, Fourier transform infrared spectroscopy (FTIR), and scanning electron microscopy (SEM) analyses. Antioxidant activities against 2,2-diphenyl-1-picryl-hydrazyl (DPPH), hydroxyl, and superoxide radicals were also evaluated. The physicochemical property analysis indicated the identicalness of the polysaccharide indices obtained by the hot water and ultrasonic methods. The indices obtained by the alkali and enzyme methods were significantly different. The FTIR spectra revealed the general characteristic absorption peaks of the four polysaccharides. The SEM images demonstrated significant differences in the surface features of the different polysaccharides. The antioxidant activity assay revealed the significant antioxidant activities of three polysaccharides. Overall, the polysaccharides from S. miltiorrhiza Bunge may have potential applications in the medical and food industries.     

Spectroscopic characterisation of order-disorder transitions for extracellular polysaccharides of arthrobacter species

The conformational behaviour of the extracellular polysaccharides from Arthrobacter species of soil-borne bacteria has been investigated by nuclear magnetic resonance relaxation and optical rotation. Polysaccharides from A. stabilis, A. viscosus, and A. viscosus sp. n, in solution at room temperature, all show evidence of an ordered conformation which can be melted out on heating. The temperature course of this transition, however, shows considerable variation with bacterial species. Thus A. stabilis polysaccharide shows a very sharp conformational transition centred around 60°, whereas the transitions of the polysaccharides from both strains of A. viscosus occur over a much broader temperature-range. The transition for the polysaccharide of A. viscosus sp. n is again centred close to 60°, whereas, for A. viscosus, melting of the tertiary structure of the polysaccharide is incomplete at 100°. O-Deacetylation destroys the ordered conformation of both A. viscosus polysaccharides. The ordered structure of A. stabilis polysaccharide, by contrast, is stabilised by removal of acyl substituents (which here include succinic half-ester). Understanding of the conformational state of these materials affords considerable insight into their gelation behaviour and unusual solution rheology. The known solution interactions with certain plant polysaccharides suggest a possible biological role for Arthrobacter polysaccharides in relationships with components of plant root-systems.     

Structural features of the gum exudates from some Acacia species of the series Phyllodineae and Botryocephalae

Smith degradation of each of the polydisperse, gum polysaccharides from Acacia pycnantha, A. difformis, A. filicifolia, and A. podalyriaefolia, for which molecular-weight distributions have been measured by gel-permeation chromatography, gives, in good yield, a methanol-insoluble polysaccharide that shows a single peak on examination by this technique. The molecular weights of the Smith-degraded polysaccharides are close to the values expected if the respective gum polysaccharides, on losing periodate-vulnerable, peripheral sugar residues were to be split also at regular intervals between the otherwise (1→3)-linked d-galactose chains. The structures of these gums, which are similar in many respects, conform to the pattern shown recently to occur in the gum of A. baileyana.     

Polysaccharide structure of tetrasporic red seaweed Tichocarpus crinitus

Sulfated polysaccharide isolated from tetrasporic plants of Tichocarpus crinitus was investigated. The polysaccharide was isolated by two methods: with water extraction at 80 °C (HT) and with a mild alkaline extraction (AE). The extracted polysaccharides were presented by non-gelling ones only, while galactose and 3,6-AG were the main monosaccharides, at the same time amount of 3,6-AG in AE polysaccharides was the similar to that of HT. According to methods of spectroscopy and mass spectrometry, the polysaccharide from tetrasporic T. crinitus contains main blocks of 1,3-linked β-d-galactopyranosyl-2,4-disulfates and 1,4-linked 3,6-anhydro-α-d-galactopyranosyl while 6-sulfated 4-linked galactopyranosyl resudies are randomly distributed along the polysaccharide chain. The alkaline treatment of HT polysaccharide results in obtaining polysaccharide with regular structure that composed of alternating 1,3-linked β-d-galactopyranosyl-2,4-disulfates and 1,4-linked 3,6-anhydro-α-d-galactopyranosyl residues. Native polysaccharide (HT) possessed both high anticoagulant and antiplatelet activity measured by fibrin clotting and platelet aggregation induced by collagen. This activity could be connected with peculiar chemical structure of HT polysaccharide which has high sulfation degree and contains also 3,6-anhydrogalactose in the polymer chain.     

Mucilage from a fresh-water red alga of the genus Batrachospermum

The gelatinous polysaccharides of a Batrachospermum species have been extracted from the alga. The major polysaccharide is acidic and has been separated from neutral polysaccharides by chromatography on DEAE-cellulose. The constituent sugars of the acidic polysaccharide include d- and l-galactose, d-mannose, d-xylose, l-rhamnose, d-glucuronic acid, and two O-methyl sugars, which have been characterized as 3-O-methyl-l-rhamnose (l-acofriose and 3-O-methyl-d-galactose. Partial acid hydrolysis of this polysaccharide has given a complex mixture of neutral and acidic oligosaccharides. The two preponderant acidic oligosaccharides contained galactose and glucuronic acid in 1:1 ratio, suggesting the presence of a repeating sequence of these two residues as a major structural feature of the polysaccharide.     

The discernible,structural features of the acidic polysaccharides secreted by different Rhizobium species are the same

Octasaccharide repeating-units have been isolated from the acidic polysaccharides secreted by Rhizobium trifolii strain NA30, R. trifolii strain LPR5, R. leguminosarum strain LPR1, and R. phaseoli strain LPR49. (R. trifolii is the symbiont of clover, R. leguminosarum, of peas, and R. phaseoli, of beans). The repeating units were formed by treating the polysaccharides with an enzyme produced by a bacteriophage. The glycosyl sequence and the structures and locations of the non-glycosyl substituents were shown to be identical for repeating units derived from all of these polysaccharides, except for that derived from the polysaccharide produced by R. trifolii NA30. Therefore, the discernible structural features of the acidic polysaccharides secreted by Rhizobium species cannot be the determinant of host specificity. In support of this conclusion is the observation that R. trifolii LPR5045, produced by curing R. trifolii LPR5 of its Sym plasmid (the Sym plasmid is required for symbiosis and host specificity), secreted a polysaccharide having the same structure (including identities and locations of nonglycosyl substituents) as that of the polysaccharide secreted by its plasmid-containing parent. Thus, the structural genes that encode for synthesis of the acidic polysaccharide secreted by R. trifolii LPR5045 are not located on the Sym plasmid, and neither are the genes that encode for synthesis and attachment of non-glycosyl substituents of the polysaccharide. The possibility remains that a quantitatively minor component of the acidic polysaccharide could be a host-specific determinant.     

The extracellular polysaccharides of Rhizobium japonicum: Compositional studies

The extracellular polysaccharides of seven strains of Rhizobium japonicum were investigated by using a gas-chromatographic scheme developed for determination of the various sugars present. These polysaccharides were more heterogeneous in their composition than those of any other species of Rhizobium yet examined. Five strains (1809, 110, 123, 127, and 709) produced polysaccharides containing the same constituents, although in varying relative amounts: glucose (36–44%), galactose (7–25%), mannose (18–20%), 4-O-methylgalactose (5–13%), galacturonic acid (12–16%), and acetyl groups (4–8%). The sugars of the polysaccharide of strain 1809 were all of the d series. These are the first bacterial polysaccharides reported to contain 4-O-methylgalactose and the first Rhizobium polysaccharides in which galacturonic acid has been found. In contrast to this, the polysaccharide of strain 129 consisted of glucose (7%), galactose (51%), mannose (5%), xylose (5%), glucuronic acid (5%), and pyruvic acid (2%). The polysaccharide of strain 711 contained glucose (34%), galactose (13%), mannose (27%), and pyruvic acid (6%).     

Mechanism of action of novel synthetic dodecapeptides against Candida albicans

Background

Three de novo designed low molecular weight cationic peptides (IJ2, IJ3 and IJ4) containing an unnatural amino acid α,β-didehydrophenylalanine (?Phe) exhibited potent antifungal activity against fluconazole (FLC) sensitive and resistant clinical isolates of Candida albicans as well as non-albicans and other yeast and filamentous pathogenic fungi. In the present study, their synthesis, susceptibility of different fungi and the mechanism of anti-candidal action have been elucidated.

Methods

The antimicrobial peptides (AMPs) were synthesized by solid-phase method and checked for antifungal activity against different yeasts and fungi by broth microdilution method. Anti-candidal mode of action of the peptides was investigated through detecting membrane permeabilization by confocal microscopy, Reactive Oxygen Species (ROS) generation by fluorometry, apoptosis and necrosis by flow cytometry and cell wall damage using Scanning and Transmission Electron Microscopy.

Results and conclusions

The MIC of the peptides against C. albicans and other yeast and filamentous fungal pathogens ranged between 3.91 and 250 μM. All three peptides exhibited effect on multiple targets in C. albicans including disruption of cell wall structures, compromised cell membrane permeability leading to their enhanced entry into the cells, accumulation of ROS and induction of apoptosis. The peptides also showed synergistic effect when used in combination with fluconazole (FLC) and caspofungin (CAS) against C. albicans.

General significance

The study suggests that the AMPs alone or in combination with conventional antifungals hold promise for the control of fungal pathogens, and need to be further explored for treatment of fungal infections.     

Identification and properties of a sterol-binding polysaccharide isolated from Saccharomyces Cerevesiae

A sterol-solubilizing polysaccharide isolated from yeast has been described (Adams, B. G. and Parks L. W. (1967) Biochem. Biophys. Res. Commun. 28,490–494) which greatly simplifies the addition of sterolsto aqueous media. The polysaccharide has now been identified by gas-liquid chromatography and carbazole reactions as yeast cell wall mannan. The cell wall mannan has been purified and binding constants for several sterols have been determined. Binding of sterols is a biphasic function of mannan concentration and is independent of pH over a range of 5 to 8.     

Binding Hot Spot in the Weak Protein Complex of Physiological Redox Partners Yeast Cytochrome c and Cytochrome c Peroxidase

Transient protein interactions mediate many vital cellular processes such as signal transduction or intermolecular electron transfer. However, due to difficulties associated with their structural characterization, little is known about the principles governing recognition and binding in weak transient protein complexes. In particular, it has not been well established whether binding hot spots, which are frequently found in strong static complexes, also govern transient protein interactions. To address this issue, we have investigated an electron transfer complex of physiological partners from yeast: yeast iso-1-cytochrome c (Cc) and yeast cytochrome c peroxidase (CcP). Using isothermal titration calorimetry and NMR spectroscopy, we show that Cc R13 is a hot-spot residue, as R13A mutation has a strong destabilizing effect on binding. Furthermore, we employ a double-mutant cycle to illustrate that Cc R13 interacts with CcP Y39. The present results, in combination with those of earlier mutational studies, have enabled us to outline the extent of the energetically important Cc-CcP binding region. Based on our analysis, we propose that binding energy hot spots, which are prevalent in static protein complexes, could also govern transient protein interactions.     

Studies of oxidative phosphorylation in Saccharomyces cerevisiae and Saccharomyces carlsbergensis

Mitochondria isolated from cultures of Saccharomyces cerevisiae and Saccharomyces carlsbergensis, which were harvested late in the stationary phase of growth at a time when full development of the electron transport and associated phosphorylation systems had occurred, carried out oxidative phosphorylation with P:O ratios of approximately 3 and 2 when pyruvate-malate and succinate, respectively, were used as substrates.     

Polysaccharide component in the stigmatic exudate from Lilium longiflorum

The polysaccharide component of the stigmatic exudate from Lilium longiflorum has the composition, arabinose (26%), rhamnose (6%), galactose (57%) and glucuronic acid (11%). The highly branched polysaccharide bears a striking resemblance to the acidic polysaccharide exudate from Araucaria bidwillii in belonging to the galactan group and in carrying outer chains terminated by arabinofuranose, rhamnopyranose, galactopyranose and glucuronic acid residues. Both polysaccharides contain the sequence O-rhamnopyranosyl-(1→4)-glucopyranosyluronic acid-(1→6)-galactopyranose in some of the outer chains.     

Regulation of hyphal morphogenesis by Ras and Rho small GTPases

The fungal kingdom is extremely diverse – comprised of over 1.5 million species including yeasts, molds and mushrooms. Essentially, all fungi have cell walls that contain chitin and the cells of most fungi grow as tube-like filaments called hyphae. These filamentous fungi, such as the mold Neurospora crassa, develop branched radial networks of hyphae referred to as mycelium. In contrast, non-filamentous fungi do not form radial mycelia, but grow as single cells, which reproduce by either budding or fission such as Saccharomyces cerevisiae or Schizosaccharomyces pombe, respectively. Finally, there are fungi that are capable of switching between single cell, yeast form growth and filamentous growth such as Candida albicans. The switch from yeast to filamentous growth in these so-called dimorphic fungi is a virulence trait in many human and plant pathogens. Highly conserved master regulators of all three fungal growth modes – filamentous, non-filamentous and dimorphic – are the Ras and Rho small GTPases, which spatially and temporally control cell polarity establishment and maintenance. This review summarizes the key roles of the Ras and Rho GTPases during hyphal morphogenesis in a range of fungi.     

Receptor Site on Clover and Alfalfa Roots for Rhizobium

Sites on white clover and alfalfa roots that bind Rhizobium trifolii and R. meliloti capsular polysaccharides, respectively, were examined by fluorescence microscopy. Fluorescein isothiocyanate-labeled capsular material from R. trifolii bound specifically to root hairs of clover but not alfalfa. Binding was most intense at the root hair tips. Treatment of clover roots with 2-deoxyglucose (2-dG) prevented binding of R. trifolii capsular material to the roots. The sugar 2-dG enhanced the elution of clover root protein, which could bind to and specifically agglutinate R. trifolii but not R. meliloti or R. japonicum. The mild elution procedure left the roots intact. Agglutination of R. trifolii and passive hemagglutination of rabbit erythrocytes coated with the capsular material of R. trifolii were specifically inhibited by 2-dG. These results suggest that clover roots contain proteins that cross-link complementary polysaccharides on the surface of clover root hairs and infective R. trifolii through 2-dG-sensitive binding sites. Alfalfa root hairs were shown to specifically bind to a surface polysaccharide from R. meliloti.     

Profiling Carbohydrate-Receptor Interaction with Recombinant Innate Immunity Receptor-Fc Fusion Proteins

The recognition of bacteria, viruses, fungi, and other microbes is controlled by host immune cells, which are equipped with many innate immunity receptors, such as Toll-like receptors, C-type lectin receptors, and immunoglobulin-like receptors. Our studies indicate that the immune modulating properties of many herbal drugs, for instance, the medicinal fungus Reishi (Ganoderma lucidum) and Cordyceps sinensis, could be attributed to their polysaccharide components. These polysaccharides specifically interact with and activate surface receptors involved in innate immunity. However, due to the complexity of polysaccharides and their various sources from medicinal fungi, quantitative analysis of medicinal polysaccharide extracts with regard to their functions represents a major challenge. To profile carbohydrate-immune receptor interactions, the extracellular domains of 17 receptors were cloned as Fc-fusion proteins, such that their interactions with immobilized polysaccharides could be probed in an enzyme-linked immunosorbent assay. The results show that several innate immune receptors, including Dectin-1, DC-SIGN, Langerin, Kupffer cell receptor, macrophage mannose receptor, TLR2, and TLR4, interact with the polysaccharide extracts from G. lucidum (GLPS). This analysis revealed distinct polysaccharide profiles from different sources of medicinal fungi, and the innate immune receptor-based enzyme-linked immunosorbent assay described here can serve as a high-throughput profiling method for the characterization and quality control of medicinal polysaccharides. It also provides a means to dissect the molecular mechanism of medicinal polysaccharide-induced immunomodulation events.     

Relative Contributions of Bacteria and Fungi to Rates of Degradation of Lignocellulosic Detritus in Salt-Marsh Sediments

Specifically radiolabeled [¹⁴C-lignin]lignocellulose and [¹⁴C-polysaccharide]lignocellulose from the salt-marsh cordgrass Spartina alterniflora were incubated with an intact salt-marsh sediment microbial assemblage, with a mixed (size-fractionated) bacterial assemblage, and with each of three marine fungi, Buergenerula spartinae, Phaeosphaeria typharum, and Leptosphaeria obiones, isolated from decaying S. alterniflora. The bacterial assemblage alone mineralized the lignin and polysaccharide components of S. alterniflora lignocellulose at approximately the same rate as did intact salt-marsh sediment inocula. The polysaccharide component was mineralized twice as fast as the lignin component; after 23 days of incubation, ca. 10% of the lignin component and 20% of the polysaccharide component of S. alterniflora lignocellulose were mineralized. Relative to the total sediment and bacterial inocula, the three species of fungi mediated only very slow mineralization of the lignin and polysaccharide components of S. alterniflora lignocellulose. Experiments with uniformly ¹⁴C-labeled S. alterniflora material indicated that the three fungi and the bacterial assemblage were capable of degrading the non-lignocellulosic fraction of S. alterniflora material, but only the bacterial assemblage significantly degraded the lignocellulosic fraction. Our results suggest that bacteria are the predominant degraders of lignocellulosic detritus in salt-marsh sediments.     

In vitro antioxidant and antitumor activities of polysaccharides extracted from Asparagus officinalis

Ultrasonic circulating extraction technology was applied for the polysaccharide extraction from Asparagus officinalis. The crude polysaccharides were deproteinized by Sevag method and three main polysaccharide fractions, AOP-4, AOP-6 and AOP-8 were obtained by fractional precipitation with gradient concentrations of ethanol (40%, 60% and 80%). The in vitro antitumor and antioxidant activities of the polysaccharide fractions were evaluated by MTT assay and free radical-scavenging assay, respectively. Deproteinized AOPs showed higher antioxidant and antitumor activities than crude AOP. AOP-4 with molecular weight 5.75 × 10⁴ Da showed significant function of scavenging hydroxyl radical. Three AOP fractions had significant antitumor activity against HeLa and BEL-7404 cells in a dose dependent manner. Furthermore, the inhibit activity of AOP-4 against HeLa cells was higher than those of other AOPs and the inhibition rate reached 83.96% at the concentration of 10 mg/mL. These results indicated that the AOP might be useful for developing natural safe antitumor drugs or health food.     

Antifungal properties of Canavalia ensiformis urease and derived peptides

Ureases (EC 3.5.1.5) are metalloenzymes that hydrolyze urea into ammonia and CO₂. These proteins have insecticidal and fungicidal effects not related to their enzymatic activity. The insecticidal activity of urease is mostly dependent on the release of internal peptides after hydrolysis by insect digestive cathepsins. Jaburetox is a recombinant version of one of these peptides, expressed in Escherichia coli. The antifungal activity of ureases in filamentous fungi occurs at submicromolar doses, with damage to the cell membranes. Here we evaluated the toxic effect of Canavalia ensiformis urease (JBU) on different yeast species and carried out studies aiming to identify antifungal domain(s) of JBU. Data showed that toxicity of JBU varied according to the genus and species of yeasts, causing inhibition of proliferation, induction of morphological alterations with formation of pseudohyphae, changes in the transport of H⁺ and carbohydrate metabolism, and permeabilization of membranes, which eventually lead to cell death. Hydrolysis of JBU with papain resulted in fungitoxic peptides (∼10 kDa), which analyzed by mass spectrometry, revealed the presence of a fragment containing the N-terminal sequence of the entomotoxic peptide Jaburetox. Tests with Jaburetox on yeasts and filamentous fungi indicated a fungitoxic activity similar to ureases. Plant ureases, such as JBU, and its derived peptides, may represent a new alternative to control medically important mycoses as well as phytopathogenic fungi, especially considering their potent activity in the range of 10⁻⁶–10⁻⁷ M.