Structure and anticoagulant activity of a sulfated galactan from the red alga, Gelidium crinale. Is there a specific structural requirement for the anticoagulant action?

Marine red algae are an abundant source of sulfated galactans with potent anticoagulant activity. However, the specific structural motifs that confer biological activity remain to be elucidated. We have now isolated and purified a sulfated galactan from the marine red alga, Gellidium crinale. The structure of this polysaccharide was determined using NMR spectroscopy. It is composed of the repeating structure -4-alpha-Galp-(1-->3)-beta-Galp1--> but with a variable sulfation pattern. Clearly 15% of the total alpha-units are 2,3-di-sulfated and another 55% are 2-sulfated. No evidence for the occurrence of 3,6-anhydro alpha-galactose units was observed in the NMR spectra. We also compared the anticoagulant activity of this sulfated galactan with a polysaccharide from the species, Botryocladia occidentalis, with a similar saccharide chain but with higher amounts of 2,3-di-sulfated alpha-units. The sulfated galactan from G. crinale has a lower anticoagulant activity on a clotting assay when compared with the polysaccharide from B. occidentalis. When tested in assays using specific proteases and coagulation inhibitors, these two galactans showed significant differences in their activity. They do not differ in thrombin inhibition mediated by antithrombin, but in assays where heparin cofactor II replaces antithrombin, the sulfated galactan from G. crinale requires a significantly higher concentration to achieve the same inhibitory effect as the polysaccharide from B. occidentalis. In contrast, when factor Xa instead of thrombin is used as the target protease, the sulfated galactan from G. crinale is a more potent anticoagulant. These observations suggest that the proportion and/or the distribution of 2,3-di-sulfated alpha-units along the galactan chain may be a critical structural motif to promote the interaction of the protease with specific protease and coagulation inhibitors.     

Induction of phlorotannin production in a brown alga: defense or resource dynamics?

Increase of phenolic secondary metabolites, phlorotannins, in brown algae due to gastropod grazing has been interpreted as an anti-herbivore adaptation. Here we tested whether such a response could be due to changes in truly available resources for the alga, not by the grazing activity of snails as such. We allowed two species of snails, Theodoxus fluviatilis and Physa fontinalis to graze on Fucus vesiculosus . These species feed on epibiota and particulate matter on the thallus but do not eat the thallus of F. vesiculosus . We further simulated snail grazing by nutrient enhancement, removal of epibiota and by a combination of the two. Manipulations of nutrient and light availability revealed the crucial role of epibiota in mediating resource availability for F. vesiculosus . Nutrient enhancement alone increased epibiota and decreased phlorotannins. Cleaning the thallus resulted in increased growth, and together with nutrient enhancement also in a trade-off with phlorotannins. Presence of T. fluviatilis on the thallus induced phlorotannin production, a response differing from the simulations of snail grazing. However, we suggest that the increase in phlorotannins may not be an induced defense but rather a consequence of a specific way of resource manipulation by this snail species. T. fluviatilis removes hyaline hairs that facilitate nutrient uptake. P. fontinalis did not remove hyaline hairs and the response of the alga to its grazing was similar to the treatment where we mechanically removed epibiota suggesting that cleaning of the thallus is the major mechanism how this snail species affects F. vesiculosus . Genetic variation in phlorotannin concentrations highly exceeded the induced responses of simulated or real snail grazing. This casts doubt for the efficiency of induced phlorotannin production to act as a defense, but is not contradictory with the interpretation of phlorotannins responding to variation in resource availability.     

Is there a correlation between structure and anticoagulant action of sulfated galactans and sulfated fucans?

We attempted to identify the specific structural features in sulfated galactans and sulfated fucans that confer anticoagulant activity. For this study we employed a variety of invertebrate polysaccharides with simple structures composed of well-defined units of oligosaccharides. Our results indicate that a 2-O-sulfated, 3-linked alpha-L-galactan, but not a alpha-L-fucan with a similar molecular size, is a potent thrombin inhibitor mediated by antithrombin or heparin cofactor II. The difference between the activities of these two polysaccharides is not very pronounced when factor Xa replaced thrombin. The occurrence of 2,4-di-O-sulfated units is an amplifying motif for 3-linked alpha-fucan-enhanced thrombin inhibition by antithrombin. If we replace antithrombin by heparin cofactor II, then the major structural requirement for the activity becomes single 4-O-sulfated fucose units. The presence of 2-O-sulfated fucose residues always had a deleterious effect on anticoagulant activity. Overall, our results indicate that the structural requirements for interaction of sulfated galactans and sulfated fucans with coagulation cofactors and their target proteases are stereospecific and not merely a consequence of their charge density and sulfate content.     

Isolation and Structural Determination of a Glucan from the Spores of Ganoderma lucidum

A water soluble, (1→6)-branched, (1→4)linked D-glucan (LB-B1),［α］21D=+174.2° (c 0.87, H2O), was obtained from a hot-water extract of the sporoderm-broken spores of Ganoderma lucidum (Fr.) Karst by HPSEC, with 0.001 mol/L sodium hydroxide as the eluant, the molecular weight ( Mw ) of LB-B1 was estimated to be 9.3×103. From the results of total hydrolysis, methylation analysis, acetolysis and 1D, 2D NMR experimentation, it was concluded that LB-B1 was composed of repeating units with the following structure:α-D-Glcp-(1→6)-α-D -Glcp-(1→6)-α-D-Glcp １↓6→4)-α- D -Glcp-(1→4)-α- D -Glcp-(1→4)-α- D -Glcp-(1→4)-α-D-Glcp-(1→4-)-α-D-Glcp-(1→)n     

Structural studies of β-carbonic anhydrase from the green alga Coccomyxa: inhibitor complexes with anions and acetazolamide

The β-class carbonic anhydrases (β-CAs) are widely distributed among lower eukaryotes, prokaryotes, archaea, and plants. Like all CAs, the β-enzymes catalyze an important physiological reaction, namely the interconversion between carbon dioxide and bicarbonate. In plants the enzyme plays an important role in carbon fixation and metabolism. To further explore the structure-function relationship of β-CA, we have determined the crystal structures of the photoautotroph unicellular green alga Coccomyxa β-CA in complex with five different inhibitors: acetazolamide, thiocyanate, azide, iodide, and phosphate ions. The tetrameric Coccomyxa β-CA structure is similar to other β-CAs but it has a 15 amino acid extension in the C-terminal end, which stabilizes the tetramer by strengthening the interface. Four of the five inhibitors bind in a manner similar to what is found in complexes with α-type CAs. Iodide ions, however, make contact to the zinc ion via a zinc-bound water molecule or hydroxide ion — a type of binding mode not previously observed in any CA. Binding of inhibitors to Coccomyxa β-CA is mediated by side-chain movements of the conserved residue Tyr-88, extending the width of the active site cavity with 1.5-1.8 Å. Structural analysis and comparisons with other α- and β-class members suggest a catalytic mechanism in which the movements of Tyr-88 are important for the CO₂-HCO₃ ^- interconversion, whereas a structurally conserved water molecule that bridges residues Tyr-88 and Gln-38, seems important for proton transfer, linking water molecules from the zinc-bound water to His-92 and buffer molecules.     

Interspecific hybridization, a matter of pioneering? Insights from Atlantic salmon and brown trout

Interspecific hybridization may occur in situations of recent contact between a colonizer and a resident species, being more intense in the colonization front. Atlantic salmon Salmo salar and brown trout S. trutta have been sympatric species since their origin and they share spatial and temporal spawning niches, exhibiting low levels of bidirectional interspecific hybridization and introgression throughout their distribution range. Different causes have been identified for increased hybridization, from escapes or deliberate releases of domesticated fish to sneaking male behavior. We have examined hybridization rates and direction in different situations of advance of one of these species into a territory formerly inhabited by the other (247 samples were analyzed in northern Spain and 487 in Kerguelen Islands). In all cases, hybrids found in the colonization front were offspring of colonizer females and resident males. We hypothesize that these findings are the result of adaptive relaxed mate choice of colonizing females, regardless of the relative abundance of each species.     

M?ssbauer spectroscopy of the nitrogenase proteins from Klebsiella pneumoniae. Structural assignments and mechanistic conclusions

The Mo–Fe protein and the Fe protein which together constitute the nitrogenase of Klebsiella pneumoniae were prepared from bacteria grown in ⁵⁷Fe-enriched medium. The Mössbauer spectrum of the Mo–Fe protein, as isolated in the presence of Na₂S₂O₄, showed that the protein contained three iron species, called M4, M5 and M6. The area of the spectrum associated with species M4, with δ=0.65mm/s and ΔE=3.05mm/s at 4.2°K, corresponded to two iron atoms/molecule of protein and it is interpreted as being due to a high-spin ferrous, spin-coupled pair of iron atoms. The iron atoms of species M4 may be involved in the quaternary structure of the protein. Species M5, with δ=0.61mm/s and ΔE=0.83mm/s at 77°K, corresponded to eight iron atoms/molecule of protein and is interpreted as being due to Fe₄S₄ or Fe₂S₂ low-spin ferrous iron clusters. Species M6, with δ=0.37mm/s and ΔE=0.71mm/s at 77°K, also corresponded to eight iron atoms/molecule of protein and, at 4.2°K, became a broad shallow absorption, characteristic of magnetic hyperfine interaction. Oxidation of the Mo–Fe protein with the redox dye Lauth''s Violet did not affect the activity of the protein but changed species M4, M5 and M6 into the species M1 (δ=0.37mm/s, ΔE=0.75mm/s at 77°K, broad magnetic component at 4.2°K) and M2 (δ=0.35mm/s, ΔE=0.9mm/s at 4.2°K). In the presence of the Fe protein, Na₂S₂O₄, ATP and Mg²⁺, the M6 component of the Mo–Fe protein was replaced by species M7 with δ=0.46mm/s, ΔE=1.04mm/s at 4.2°K. The change in Mössbauer parameters associated with the M6 → M7 transformation was very similar to the change observed on reduction of the high-potential Fe protein from Chromatium vinosum. In contrast, Na₂S₂O₄-reduced Fe protein contained only one type of iron cluster (F4). Species F4 had δ=0.50mm/s, ΔE=0.9mm/s at 195°K, and at 4.2°K broadened in a manner characteristic of a magnetic hyperfine interaction, associated with half-integral spin, equally distributed over all four atoms of the Fe protein. The Mössbauer spectra of the Mo–Fe and the Fe protein under argon were unaffected by the reducible substrates N₂ and C₂H₂ and the inhibitor CO in the presence of ATP, Mg²⁺ and Na₂S₂O₄. A number of Mössbauer spectral species associated with inactivated Mo–Fe and Fe proteins are described and discussed.     

Nucleotide sequence and phylogenetic implication of the ATPase subunits β and ε encoded in the chloroplast genome of the brown alga Dictyota dichotoma

We have cloned and sequenced the genes atpB and atpE, coding for CF₁ subunits and , respectively, of the chloroplast genome of the brown alga Dictyota dichotoma. Although the coding site of atpE cannot be demonstrated by heterologous Southern hybridizations, a 417 bp reading frame 3 to atpB was identified as the gene atpE by sequence similarities with atpE genes from other sources. A maximum sequence identity of 30% is found between the predicted amino acid sequence of the Dictyota subunit and the corresponding cyanobacterial subunits. Including conserved amino acid replacements, the Dictyota subunit exhibits about 70% sequence similarity with the cyanobacterial and land plant subunits. As in cyanobacteria, the atpE gene does not overlap the preceding gene atpB. The deduced amino acid sequence of atpB is 74–79% identical to the corresponding cyanobacterial and chloroplast subunits. Entirely conserved are regions referred to as the catalytic and/or regulatory sites of ATP formation, including interacting regions between subunits and . A phylogram predicted from F₁/CF₁- subunits of eleven different organisms suggests a common evolutionary origin of plastids from chlorophytes and brown algae.     

Structural analysis of carrageenans from the red alga, Callophyllis hombroniana Mont. Kütz (Kallymeniaceae, Rhodophyta)

The use of a range of modern analytical techniques has facilitated the structural characterisation of the polysaccharide from the New Zealand endemic red alga, Callophyllis hombroniana. The native polysaccharide contains a number of structural units with the largest proportion consisting of 3-linked beta-D-galactopyranosyl 2-sulfate units, alternating with 4-linked 3,6-anhydro-alpha-D-galactopyranosyl 2-sulfate units, that is, theta-carrageenan (36 mol%). C. hombroniana is the first red seaweed reported to naturally contain such a large proportion of theta-carrageenan.     

Cephalosporinase and penicillinase activities of a β-lactamase from Pseudomonas pyocyanea

1. Pseudomonas pyocyanea N.C.T.C. 8203 produces a beta-lactamase that is inducible by high concentrations of benzylpenicillin or cephalosporin C. Methicillin appeared to be a relatively poor inducer, but this could be attributed in part to its ability to mask the enzyme produced. Much of the enzyme is normally cell-bound. 2. No evidence was obtained that the crude enzyme preparation consisted of more than one beta-lactamase and the preparation appeared to contain no significant amount of benzylpenicillin amidase or of an acetyl esterase. 3. The maximum rate of hydrolysis of cephalosporin C and several other derivatives of 7-aminocephalosporanic acid by the crude enzyme was more than five times that of benzylpenicillin. Methicillin, cloxacillin, 6-aminopenicillanic acid and 7-aminocephalosporanic acid were resistant to hydrolysis, and methicillin and cloxacillin were powerful competitive inhibitors of the action of the enzyme on easily hydrolysable substrates. 4. Cephalosporin C, cephalothin and cephaloridine yielded 2 equiv. of acid/mole on enzymic hydrolysis, and deacetylcephalorsporin C yielded 1 equiv./mole. Evidence was obtained that the opening of the beta-lactam ring of cephalosporin C and cephalothin is accompanied by the spontaneous expulsion of an acetoxy group and that of cephaloridine by the expulsion of pyridine. 5. A marked decrease in the minimum inhibitory concentration of benzylpenicillin and several hydrolysable derivatives of 7-aminocephalosporanic acid was observed when the size of the inoculum was decreased. This suggested that the production of a beta-lactamase contributed to the factors responsible for the very high resistance of Ps. pyocyanea to these substances. It was therefore concluded that the latter might show synergism with the enzyme inhibitors, methicillin and cloxacillin, against this organism.     

Arvoredol—An unusual chlorinated and biofilm inhibiting polyketide from a marine Penicillium sp. of the Brazilian coast

Penicillium sp. F37 has been isolated from the marine sponge Axinella corrugata and shown to be closely related to Penicillium maximae. From the culture of Penicillium sp. F37 arvoredol, a novel chlorinated polyketide with 6,7-dihydro-4(5H)-benzofuranone moiety has been isolated and characterized by spectroscopic methods Arvoredol prevented biofilm formation of the human pathogen Staphylococcus epidermidis at a concentration of 125 μg mL⁻¹ by 40%. It was also active against colorectal carcinoma HCT116 cells with a MIC of 7.9 μg mL⁻¹.     

Estimating the Probability of a Major Outbreak from the Timing of Early Cases: An Indeterminate Problem?

Conservation biologists, as well as veterinary and public health officials, would benefit greatly from being able to forecast whether outbreaks of infectious disease will be major. For values of the basic reproductive number (R ₀) between one and two, infectious disease outbreaks have a reasonable chance of either fading out at an early stage or, in the absence of intervention, spreading widely within the population. If it were possible to predict when fadeout was likely to occur, the need for costly precautionary control strategies could be minimized. However, the predictability of even simple epidemic processes remains largely unexplored. Here we conduct an examination of simulated data from the early stages of a fatal disease outbreak and explore how observable information might be useful for predicting major outbreaks. Specifically, would knowing the time of deaths for the first few cases allow us to predict whether an outbreak will be major? Using two approaches, trajectory matching and discriminant function analysis, we find that even in our best-case scenario (with accurate knowledge of epidemiological parameters, and precise times of death), it was not possible to reliably predict the outcome of a stochastic Susceptible-Exposed–Infectious-Recovered (SEIR) process.     

Structural studies of α-melanocyte-stimulating hormone and a novel β-melanocyte-stimulating hormone from the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias

A melanocyte-stimulating hormone (MSH) has been isolated from extracts of the neurointermediate lobe of the pituitary of the dogfish Squalus acanthias by gel-filtration and ion-exchange chromatography. It had approximately 1% of the potency of mammalian alpha-MSH on bioassays in vitro on frog skin and dogfish skin. Sequence analysis revealed it to be a hexadecapeptide with the following primary structure: Asp-Gly-Asp-Asp-Tyr-Lys-Phe-Gly-His-Phe-Arg-Trp-Ser-Val-Pro-Leu. It appears to be related to the beta-MSH species of mammalian species but has only the sequence -His-Phe-Arg-Trp- in common with the heptapeptide core -Met-Glu-His-Phe-Arg-Trp-Gly- which is characteristic not only of the MSH peptides but also of the adrenocorticotrophins and lipotrophins studied so far. An alpha-MSH was also isolated, 50% of which was amidated at the C-terminus group. Sequence data from this study taken in conjunction with those from a previous study (Lowry & Chadwick, 1970b) revealed it to be a tridecapeptide which is identical with the N-terminal sequence of dogfish adrenocorticotrophin.     

Water-soluble sulfated polysaccharides from the red seaweed Chaetangium fastigiatum. Analysis of the system and the structures of the α-d-(1 → 3)-linked mannans

The water-soluble polysaccharides from Chaetangium fastigiatum were fractionated with cetrimide. The complexed material was subjected to fractional solubilization in solutions of increasing sodium chloride concentration and seven fractions were separated and analyzed. Two of the fractions were subjected to methylation and desulfation-methylation analyses. The results indicate that this seaweed contains a system of sulfated polysaccharides consisting in part of a galactan and an α-d-(1 → 3)-linked mannan, 2- and 6-sulfated, and having single stubs of β-(1 → 2)-linked d-xylose. Composition dispersity of the mannan is produced by variation of the amount and disposition of the sulfate groups and of the content of the xylose side-chains.     

The polyphenolic content of fruit and vegetables and their antioxidant activities. What does a serving constitute?

Analysis of the major flavone, flavonol, anthocyanidin and hydroxycinnamic acid constituents (and their glycosides) of onion, tomato, egg plant and apple has been undertaken and the antioxidant activities of the phenolic extracts determined. The major phenolic antioxidant components of egg plant are chlorogenic acid in the flesh and a delphinidin conjugate in the skin. In the case of apple, the major phenolic antioxidants detected are chlorogenic acid, procyanidins/catechin compounds, rutin and phloridzin. Quercetin glycosides are well-known to be the major phenolic components of onion. Assessment of the antioxidant activities of a serving of 100 g fresh weight fruit, vegetable and comparison with previously reported findings for 150 ml beverage (500 ml portion in the case of beer), expressed in μmol Trolox equivalents show that the antioxidant activities of 1 glass (150 ml) red wine ≡ 12 glasses white wine ≡ 2 cups of tea ≡ 4 apples ≡ 5 portions of onion ≡ 5.5 portions egg plant ≡ 3.5 glasses of blackcurrant juice ≡ 3.5 (500 ml) glasses of beer ≡ 7 glasses of orange juice ≡ 20 glasses of apple juice (long life).     

Structural and Functional Characterization of the Redβ Recombinase from Bacteriophage λ

The Red system of bacteriophage λ is responsible for the genetic rearrangements that contribute to its rapid evolution and has been successfully harnessed as a research tool for genome manipulation. The key recombination component is Redβ, a ring-shaped protein that facilitates annealing of complementary DNA strands. Redβ shares functional similarities with the human Rad52 single-stranded DNA (ssDNA) annealing protein although their evolutionary relatedness is not well established. Alignment of Rad52 and Redβ sequences shows an overall low level of homology, with 15% identity in the N-terminal core domains as well as important similarities with the Rad52 homolog Sak from phage ul36. Key conserved residues were chosen for mutagenesis and their impact on oligomer formation, ssDNA binding and annealing was probed. Two conserved regions were identified as sites important for binding ssDNA; a surface basic cluster and an intersubunit hydrophobic patch, consistent with findings for Rad52. Surprisingly, mutation of Redβ residues in the basic cluster that in Rad52 are involved in ssDNA binding disrupted both oligomer formation and ssDNA binding. Mutations in the equivalent of the intersubunit hydrophobic patch in Rad52 did not affect Redβ oligomerization but did impair DNA binding and annealing. We also identified a single amino acid substitution which had little effect on oligomerization and DNA binding but which inhibited DNA annealing, indicating that these two functions of Redβ can be separated. Taken together, the results provide fresh insights into the structural basis for Redβ function and the important role of quaternary structure.