Biological Activity and Food Analysis ofCyttaria spp. (Discomycetes)

The biological activity and nutritional composition of Chilean collections ofCyttaria berteroi C. darwinii, C. espinosae, C. harioti andC. johowii have been determined. The crude protein, lipid, ash, and carbohydrate content of the samples examined were similar to that of other edible fungi. Amino acid analysis of ChileanCyttaria showed that proteins of all species are deficient in methionine and cysteine and excepting oneC. espinosae collection, all samples proved to be below the WHO values for the essential amino acids valine, isoleucine, leucine, and lysine. The acute oral toxicity test ofC. espinosae in rats showed that doses up to 2.5 g extract/kg body weight (corresponding to 25.7–38.7 g fresh weight/kg) did not produce mortality or macroscopic damage in the organs examined of the test animals. Cyttaria extracts assayed at 50 fig/ml were inactive or moderately active as inhibitors towards the enzymes xanthine oxidase (0-31%) and β-glucuronidase (0-65%), and lacked antifungal and antibacterial effects in a battery of antimicrobial assays. When administered intravenously at 2.5 mg/kg, the water-soluble extract ofCyttaria produced a hypotensive response in rats (-16 to -21%). Furthermore, most of the aqueous extracts ofC. espinosae andC. harioti showed DNA binding activity. The main sterols fromCyttaria espinosae andC. berteroi were identified as dihydrobrassicasterol derivatives. Our study suggests that edibleCyttaria species do not represent an acute toxicity risk for consumers and that their nutritional value is similar to that of other edible, cultivated mushrooms. The preservation ofCyttaria spp. as a food resource is linked to the protection of the temperateNothofagus forests.     

A gel-forming (1→3)-β-D-glucan isolated from cyttaria harioti fischer

An alkali-soluble glucan, [α]_D + 11° (M potassium hydroxide) having a degree of polymerization of 220, has been isolated from the fruit bodies of the tree fungus Cyttaria harioti Fischer. Periodate oxidation and methylation analysis show that it consists of a highly branched β-D-(1→3)-linked backbone. Hydrolysis of the methylated polysaccharide yielded 2,3,4,6-tetra-O-methyl- (24.5 mol%), 2,4,6-tri-O-methyl-(39.4 mol%), 2,3,4-tri-O-methyl- (8.6 mol%), and 2,4-di-O-methyl-D-glucose (27.5 mol%). Periodate-oxidation results substantiate the methylation studies. The general structural features of the glucan are discussed.     

An unusual Xanthophyllomyces strain from leaves of Eucalyptus globulus in Chile

Xanthophyllomyces sp. was isolated as an epiphytic red yeast from leaves of Eucalyptus glo-bulus in Concepción, Chile. Sexual reproduction was by basidiospores produced from one or rarely two metabasidia arising from a yeast cell without preceding paedogamy. The main carotenoid pigment was astaxanthin. This isolate did not cluster with the X. dendrorhous complex (including Phaffia rhodozyma) in ITS and 26S rDNA-based phylogenetic analyses. The phylloplane may be a further habitat for Xanthophyllomyces, in addition to the well-known spring sap-flows of deciduous trees and the recently-characterised ascostromata of Cyttaria hariotii.     

Structural studies on a glycopeptide from the tree fungus Cyttaria harioti Fischer

A glycopeptide (In₁) was isolated by phenol-water extraction from Cyttaria harioti Fischer, parasite of Nothofagus sps. Neutral sugars account for 89% of In₁ and were characterized as glucose, mannose, and galactose. Glucosamine, identified by g.l.c., was colorimetrically estimated (5.8%). The molar ratio of Glc:Man:Gal:GlcNAc was 17:11:3:2. The linkages between the various monosaccharide residues were established through methylation analysis and periodate oxidation studies. The anomeric configurations of the various glycosyl groups were determined by chromium trioxide oxidation of the acetylated polysaccharide. The results were confirmed by ¹³C-n.m.r. spectroscopy. The sugar chain is N-glycosyl-linked to the peptide. Structural features of the carbohydrate moiety of glycopeptide In₁ are described.     

The structure of an α-D-glucan from Cyttaria harioti Fischer

A homogeneous glucan has been isolated from the fruiting bodies of Cyttaria harioti Fischer. Partial acid hydrolysis produced major amounts of isomaltose, whereas acetolysis gave maltose and maltotriose. Enzymic hydrolysis with amylo-glucosidase and pullulanase indicated a structure based on maltotriose residues connected by (1→6)-α-D linkages. This conclusion was supported by periodate-oxidation data which also showed that 3–7% of the glucose resisted oxidation. Methylation analysis confirmed the presence of (1→6) and (1→4) linkages in the ratio 1:2.4.     

Cophylogeny and biogeography of the fungal parasite Cyttaria and its host Nothofagus, southern beech

The obligate, biotrophic association among species of the fungal genus Cyttaria and their hosts in the plant genus Nothofagus often is cited as a classic example of cophylogeny and is one of the few cases in which the biogeography of a fungus is commonly mentioned or included in biogeographic analyses. In this study molecular and morphological data are used to examine hypotheses regarding the cophylogeny and biogeography of the 12 species of Cyttaria and their hosts, the 11 species of Nothofagus subgenera Lophozonia and Nothofagus. Our results indicate highly significant overall cophylogenetic structure, despite the fact that the associations between species of Cyttaria and Nothofagus usually do not correspond in a simple one to one relationship. Two major lineages of Cyttaria are confined to a single Nothofagus subgenus, a specificity that might account for a minimum of two codivergences. We hypothesize other major codivergences. Numerous extinction also are assumed, as are an independent parasite divergence followed by host switching to account for C. berteroi. Considering the historical association of Cyttaria and Nothofagus, our hypothesis may support the vicariance hypothesis for the trans-Antarctic distribution between Australasian and South American species of Cyttaria species hosted by subgenus Lophozonia. It also supports the hypothesis of transoceanic long distance dispersal to account for the relatively recent relationship between Australian and New Zealand Cyttaria species, which we estimate to have occurred 44.6-28.5 mya. Thus the history of these organisms is not only a reflection of the breakup of Gondwana but also of other events that have contributed to the distributions of many other southern hemisphere plants and fungi.     

Phylogeny of Cyttaria inferred from nuclear and mitochondrial sequence and morphological data

Cyttaria species (Leotiomycetes, Cyttariales) are obligate, biotrophic associates of Nothofagus (Hamamelididae, Nothofagaceae), the southern beech. As such Cyttaria species are restricted to the southern hemisphere, inhabiting southern South America (Argentina and Chile) and southeastern Australasia (southeastern Australia including Tasmania, and New Zealand). The relationship of Cyttaria to other Leotiomycetes and the relationships among species of Cyttaria were investigated with newly generated sequences of partial nucSSU, nucLSU and mitSSU rRNA, as well as TEF1 sequence data and morphological data. Results found Cyttaria to be defined as a strongly supported clade. There is evidence for a close relationship between Cyttaria and these members of the Helotiales: Cordierites, certain Encoelia spp., Ionomidotis and to a lesser extent Chlorociboria. Order Cyttariales is supported by molecular data, as well as by the unique endostromatic apothecia, lack of chitin and highly specific habit of Cyttaria species. Twelve Cyttaria species are hypothesized, including all 11 currently accepted species plus an undescribed species that accommodates specimens known in New Zealand by the misapplied name C. gunnii, as revealed by molecular data. Thus the name C. gunnii sensu stricto is reserved for specimens occurring on N. cunninghamii in Australia, including Tasmania. Morphological data now support the continued recognition of C. septentrionalis as a species separate from C. gunnii. Three major clades are identified within Cyttaria: one in South America hosted by subgenus Nothofagus, another in South America hosted by subgenera Nothofagus and Lophozonia, and a third in South America and Australasia hosted by subgenus Lophozonia, thus producing a non-monophyletic grade of South American species and a monophyletic clade of Australasian species, including monophyletic Australian and New Zealand clades. Cyttaria species do not sort into clades according to their associations with subgenera Lophozonia and Nothofagus.     

Chemical composition of the cell wall of the tree fungusCyttaria harioti Fischer

Isolated cell walls of stromata ofCyttaria harioti Fischer, collected in the field, accounted for ca. 67% of the fungus dry weight and were composed of neutral sugars (81%), 2-amino-2-deoxyglucose (0.2%), aminoacids (3.4%), and lipids (6.8%). Alkaline treatment gave a major soluble β-(1 → 3)-glucan and a minor insoluble one, structurally related, which in total accounted for 89% of the cell wall preparation. The absence of chitin is a remarkable feature since it is present in almost all the Ascomycetes previously studied.     

Biogeography, Host Specificity, and Molecular Phylogeny of the Basidiomycetous Yeast Phaffia rhodozyma and Its Sexual Form, Xanthophyllomyces dendrorhous

Phaffia rhodozyma (sexual form, Xanthophyllomyces dendrorhous) is a basidiomycetous yeast that has been found in tree exudates in the Northern Hemisphere at high altitudes and latitudes. This yeast produces astaxanthin, a carotenoid pigment with biotechnological importance because it is used in aquaculture for fish pigmentation. We isolated X. dendrorhous from the Southern Hemisphere (Patagonia, Argentina), where it was associated with fruiting bodies of Cyttaria hariotii, an ascomycetous parasite of Nothofagus trees. We compared internal transcribed spacer (ITS)-based phylogenies of P. rhodozyma and its tree host (Betulaceae, Corneaceae, Fagaceae, and Nothofagaceae) and found them to be generally concordant, suggesting that different yeast lineages colonize different trees and providing an explanation for the phylogenetic distance observed between the type strains of P. rhodozyma and X. dendrorhous. We hypothesize that the association of Xanthophyllomyces with Cyttaria derives from a previous association of the yeast with Nothofagus, and the sister relationship between Nothofagaceae and Betulaceae plus Fagaceae correlates with the phylogeny of X. dendrorhous strains originating from these three plant families. The two most basal strains of X. dendrorhous are those isolated from Cornus, an ancestral genus in the phylogenetic analysis of the host trees. Thus, we question previous conclusions that P. rhodozyma and X. dendrorhous represent different species since the polymorphisms detected in the ITS and intergenic spacer sequences can be attributed to intraspecific variation associated with host specificity. Our study provides a deeper understanding of Phaffia biogeography, ecology, and molecular phylogeny. Such knowledge is essential for the comprehension of many aspects of the biology of this organism and will facilitate the study of astaxanthin production within an evolutionary and ecological framework.     

Two-Electron Reactions S2QB → S0QB and S3QB → S1QB are Involved in Deactivation of Higher S States of the Oxygen-Evolving Complex of Photosystem II

The oxygen-evolving complex of Photosystem II cycles through five oxidation states (S₀-S₄), and dark incubation leads to 25% S₀ and 75% S₁. This distribution cannot be reached with charge recombination reactions between the higher S states and the electron acceptor Q_B⁻. We measured flash-induced oxygen evolution to understand how S₃ and S₂ are converted to lower S states when the electron required to reduce the manganese cluster does not come from Q_B⁻. Thylakoid samples preconditioned to make the concentration of the S₁ state 100% and to oxidize tyrosine Y_D were illuminated by one or two laser preflashes, and flash-induced oxygen evolution sequences were recorded at various time intervals after the preflashes. The distribution of the S states was calculated from the flash-induced oxygen evolution pattern using an extended Kok model. The results suggest that S₂ and S₃ are converted to lower S states via recombination from S₂Q_B⁻ and S₃Q_B⁻ and by a slow change of the state of oxygen-evolving complex from S₃ and S₂ to S₁ and S₀ in reactions with unspecified electron donors. The slow pathway appears to contain two-electron routes, S₂Q_B → S₀Q_B, and S₃Q_B → S₁Q_B. The two-electron reactions dominate in intact thylakoid preparations in the absence of chemical additives. The two-electron reaction was replaced by a one-electron-per-step pathway, S₃Q_B → S₂Q_B → S₁Q_B in PS II-enriched membrane fragments and in thylakoids measured in the presence of artificial electron acceptors. A catalase effect suggested that H₂O₂ acts as an electron donor for the reaction S₂Q_B → S₀Q_B but added H₂O₂ did not enhance this reaction.     

Somatic variations on a yellow mutant in Nicotiana tabacum L. (a1+/a1a2+/a2) I. Non-reciprocal genetic events occurring in leaf cells

A greenish-yellow mutant was obtained after treatment of seeds of Nicotiana tabacum L. var. Xanthi n.c. with ethyl methanesulfonate (EMS). Two genetically independent mutations (a₁ and a₂) were isolated. The first mutation (a₁) antagonizes the function of its partially dominant a₁⁺ allele. The second mutation (a₂) is amorphous but strongly interacts with a₁.Among the nine possible genotypes at the two loci, five varied in somatic cells. The heterozygous state a₁⁺/a₁ strongly increased the frequency of both spontaneous and induced variations. However, two homozygotes also showed variations.Variants were isolated from induced and spontaneous non-reciprocal and reciprocal variations within paliside tissues by bud induction in vitro. They were genetically tested. In this first paper, only non-reciprocal variations are reported.Green variants from the greenish-yellow (J¹) dihybrid a₁⁺/a₁a₂⁺/a₂ clone had two genotypes: the first was due to true reversions of a₁ to a₁⁺, whereas the second was due to amorphous a₁⁰ mutations from a₁. These a₁⁰ mutations may well be deletions.The lightest yellow variants from J¹ were due to mutations either from a₁⁺ into a₁ or from a₂⁺ into a₂.Deletions at the a₁⁺?a₁ locus led to either yellow variations when a₁⁺ was lost, or to false reversions when the antagonistic allele a₁ was lost.Amorphous alleles at the a₁⁺?a₁ locus were also isolated from tissues other than J⁺. They gave zygotic lethality (s) that probably varied with the size of the deletions. Thus, true reversions and deletions at the a₁⁺?a₁ locus could be distinguished from one another by progeny tests.Other variants showed higher frequencies of spontaneous variations (instability). Somatic changes observed in these unstable systems were due to modifications at the marker loci. The genetic nature of this instability is not yet known.There is strong evidence that the genetic events involved in these non-reciprocal variations were deletions, conversions and point mutations. True reversions from a₁ into a₁⁺ and new mutations from a₁⁺ into a₁ were obtained only from a₁⁺/a₁. It was therefore supposed that the changes observed took place only in heterozygotes, and the conversion hypothesis was made. Attempts are being made to prove that conversions do exist in higher plants, and to find out if this process, as deletions, is induced by radiation.     

A nhaD Na/Hantiporter and a pcd homologues are among the Rhodothermus marinus complex I genes

The NADH:menaquinone oxidoreductase (Nqo) is one of the enzymes present in the respiratory chain of the thermohalophilic bacterium Rhodothermus marinus. The genes coding for the R. marinus Nqo subunits were isolated and sequenced, clustering in two operons [nqo₁ to nqo₇ (nqo_A) and nqo₁₀ to nqo₁₄ (nqo_B)] and two independent genes (nqo₈ and nqo₉). Unexpectedly, two genes encoding homologues of a NhaD Na⁺/H⁺ antiporter (NhaD) and of a pterin-4α-carbinolamine dehydratase (PCD) were identified within nqo_B, flanked by nqo₁₃ and nqo₁₄. Eight conserved motives to harbour iron-sulphur centres are identified in the deduced primary structures, as well as two consensus sequences to bind nucleotides, in this case NADH and FMN. Moreover, the open-reading-frames of the putative NhaD and PCD were shown to be co-transcribed with the other complex I genes encoded by nqo_B. The possible role of these two genes in R. marinus complex I is discussed.     

Engineered biosynthesis of bacteriochlorophyll gF in Rhodobacter sphaeroides

Engineering photosynthetic bacteria to utilize a heterologous reaction center that contains a different (bacterio) chlorophyll could improve solar energy conversion efficiency by allowing cells to absorb a broader range of the solar spectrum. One promising candidate is the homodimeric type I reaction center from Heliobacterium modesticaldum. It is the simplest known reaction center and uses bacteriochlorophyll (BChl) g, which absorbs in the near-infrared region of the spectrum. Like the more common BChls a and b, BChl g is a true bacteriochlorin. It carries characteristic C3-vinyl and C8-ethylidene groups, the latter shared with BChl b. The purple phototrophic bacterium Rhodobacter (Rba.) sphaeroides was chosen as the platform into which the engineered production of BChl g_F, where F is farnesyl, was attempted. Using a strain of Rba. sphaeroides that produces BChl b_P, where P is phytyl, rather than the native BChl a_P, we deleted bchF, a gene that encodes an enzyme responsible for the hydration of the C3-vinyl group of a precursor of BChls. This led to the production of BChl g_P. Next, the crtE gene was deleted, thereby producing BChl g carrying a THF (tetrahydrofarnesol) moiety. Additionally, the bchG^Rs gene from Rba. sphaeroides was replaced with bchG^Hm from Hba. modesticaldum. To prevent reduction of the tail, bchP was deleted, which yielded BChl g_F. The construction of a strain producing BChl g_F validates the biosynthetic pathway established for its synthesis and satisfies a precondition for assembling the simplest reaction center in a heterologous organism, namely the biosynthesis of its native pigment, BChl g_F.     

S-allele diversity in Prunus L. Cerasus subgenus from Iran

In this study, S-allele diversity of eight wild and two commercial species of the Cerasus subgenus in Iran was investigated using two primer pairs. A high level of S-allele polymorphism was detected among and within the species evaluated. Furthermore, most of wild species showed 2–4 alleles based on S-allele primers and may be considered as tetraploid. Sweet cherry cultivars, Siah-Mashhad, Siah-Shabestar, Takdaneh-Mashhad, Siah-Daneshkadeh and Protiva showed S₃S₁₂, S₃S₁₂, S₃S₁₂, S₃S₅ and S₃S₄ combinations, respectively, allele S₃ showing the highest frequency. Three Iranian sweet cherry cultivars had the same allelic combination (S₃S₁₂) that the same ancestor in genealogy of these cultivars may explain the loss of diversity observed at the S-locus. Wild cherry (mazzard) accessions showed wide range of alleles such as S₁, S₂, S₇, S₁₄ and S₂₀ and unknown alleles, while sour cherries showed S₆, S₉, S₁₃ and S₂₇ alleles. In conclusion, the conservation of these highly diverse native species of Iranian wild Cerasus germplasm is recommended for future breeding activity.     

Oxidative addition of 3,6-di-tert-butyl-o-benzoquinone and 4,6-di-tert-butyl-N-(2,6-di-iso-propylphenyl)-o-iminobenzoquinone to SnCl2

Addition of 3,6-di-tert-butyl-o-benzoquinone (3,6-DBBQ) to SnCl₂ in THF leads to the oxidation of Sn(II) to Sn(IV) with formation of catecholate complex (3,6-DBCat)SnCl₂ · 2THF (1), where 3,6-DBCat is 3,6-di-tert-butyl-catecholate dianion. The reaction of 4,6-di-tert-butyl-N-(2,6-di-iso-propylphenyl)-o-iminobenzoquinone (IBQ-Prⁱ) also proceeds on the oxidative-addition mechanism yielding bis-iminosemiquinonato species (ISQ-Prⁱ)₂SnCl₂(2), where ISQ-Prⁱ is anion-radical 4,6-di-tert-butyl-N-(2,6-di-iso-propylphenyl)-o-iminobenzosemiquinolate. The complexes have been characterized by IR, X-band EPR, ¹H NMR (for 1) spectroscopy and magnetochemistry (for 2). X-ray analysis data show the distorted octahedral environment of tin(IV) for both complexes. Complex 1 is diamagnetic (ground state S = 0), while 2 has triplet ground state (S = 1, biradical). Catecholate complex 1 is able to be a spin trap for different organic radicals.     

13C magnetic resonance study of the ionization of N-acetyl-dl-proline in aqueous solution

NMR titration curves have been recorded for all the ¹³C resonances of cis and trans$\text{N-}\text{acetyl}\text{-}\text{dl}\text{-}\text{proline}$ in ²H₂O. the measured pK_2H values are 3.4 ± 0.8 and 4.13 ± 0.08 respectively; the free energy of ionization for the trans isomer being (3.8 kJ/mole) greater than for the cis. The ionization shifts of the two isomers differ significantly only at the acetyl carbonyl and C_γ positions. It is suggested that these are related to conformational changes which stabilize the trans form at low p²H.     

Preparation, properties and crystal structure of two isomers of R,R,S,S- and R,S,R,S-[chloro(1,3,10,12,16,19-hexaazatetracyclo[17,3,1,1,0]tetracosane)copper(II)]

Two isomers (R,S,R,S- and R,R,S,S-) of five coordinate complex [Cu(L)Cl]⁺ have been separated and characterised. These two isomers have significantly different spectrochemical and electrochemical properties. Absorption maximum of R,S,R,S-[Cu(L)Cl]⁺ shifts to longer wavelength and its reduction potential shifts to more positive direction comparing those of R,R,S,S-[Cu(L)Cl]⁺. R,S,R,S-[Cu(L)Cl]⁺ is significantly distorted to trigonal-bipyramidal structure, whereas R,R,S,S-[Cu(L)Cl]⁺ retains almost square-planar geometry. The average bond distance of Cu-N in basal plane of R,S,R,S-[Cu(L)Cl]⁺ is longer by 0.024 Å than that of R,R,S,S-[Cu(L)Cl]⁺, whereas the bond distance of Cu-Cl in former is shorter by 0.200 Å than that in latter. The isolated square-planar complexes of R,R,S,S- and R,S,R,S-[Cu(L)](ClO₄)₂ are converted to the R,R,S,S- and R,S,R,S-[Cu(L)Cl]⁺ by the addition of Cl⁻ in nitromethane solution with the rate constants, k=1.70 (±0.02) and 8.31 (±0.07) M⁻¹ s⁻¹, respectively.     

Pro-tRNA synthetase from Phaseolus aureus and Delonix regia

Pro-tRNA synthetase from Phaseolus aureus was photoinactivated in the presence of methylene blue or rose bengal. Pro and several imino acid analogues protected the enzyme against dye-mediated photoinactivation but ATP was ineffective. Together with kinetic data, this evidence suggested that a His-residue near the Pro-binding site was involved in the enzyme reaction. In the absence of methylene blue, Phaseolus enzyme was stable to light whilst that from Delonix was rapidly and reversibly photoinactivated. ATP as well as Pro, protected the Delonix enzyme against dye-independent photoinactivation. In the presence of methylene blue, the Delonix enzyme was more rapidly photoinactivated than in the absence of the dye. p-Chloromercuribenzoate (pCMB)-inhibited enzyme from both Phaseolus and Delonix was reactivated by sulphydryl reducing reagents. Reactivation of Delonix enzyme was markedly temperature-dependent whilst Phaseolus enzyme was reactivated equally efficiently at all temperatures tested. ATP, tRNA, Pro and several analogues of Pro, protected both the Phaseolus and Delonix enzymes against pCMB inhibition. The possible roles of the His-residue and SH group are discussed in relation to the known differences in substrate specificity between the Phaseolus and Delonix enzymes.     

Nδ-acetylornithine and S-methylcysteine in blood plasma

N^δ-Acetylornithine and S-methylcysteine have been identified as minor components of deproteinized blood plasma of human and bovine blood. Human blood plasma contains a variable amount of acetylornithine, averaging 1.1 ± 0.4 μmol/l (range 0.8–0.2 μmol/l). Urine contains a very small amount of acetylornithine, approximately 1 nmol/mg creatinine (1 μmol/day). Human blood plasma contains 3.9 ± 1.9 μmol/l (range 1.4–6.5 μmol/l) of S-methylcysteine. Urine contains approximately 5 nmol/mg creatinine; after acid hydrolysis the amount is increased to 20 nmol/mg creatinine.     

Association of the eukaryotic V1VO ATPase subunits a with d and d with A

Owing to the complex nature of V₁V_O ATPases, identification of neighboring subunits is essential for mechanistic understanding of this enzyme. Here, we describe the links between the V₁ headpiece and the V_O-domain of the yeast V₁V_O ATPase via subunit A and d as well as the V_O subunits a and d using surface plasmon resonance and fluorescence correlation spectroscopy. Binding constants of about 60 and 200 nM have been determined for the a-d and d-A assembly, respectively. The data are discussed in light of subunit a and d forming a peripheral stalk, connecting the catalytic A₃B₃ hexamer with V_O.

Structured summary

MINT-7012054: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by fluorescence correlation spectroscopy (MI:0052)MINT-7012041: d (uniprotkb:P32366) binds (MI:0407) to A (uniprotkb:P17255) by surface plasmon resonance (MI:0107)MINT-7012028: d (uniprotkb:P32366) binds (MI:0407) to a (uniprotkb:P32563) by surface plasmon resonance (MI:0107)