A chlorohydrin amino acid from Amanita abrupta

A new amino acid, (2S,4Z)-2-amino-5-chloro-6-hydroxy-4-hexenoic acid, has been isolated from Amanita abrupta. Three other unusual amino acids were also isolated from the same fungus.     

The sterol composition of mushrooms

Sterols were isolated from ten mushrooms, Hygrocybe punicea, Lampteromyces japonicus, Leucopaxillus giganteus, Lentinus edodes, Flammulina velutipes, Amanita caesarea, Coprinus atramentarius, Russula foetens, R. nigricans and R. senecis. The compositions of the sterol fractions were determined by GLC, combined GC/MS, and ¹H NMR. Ergosterol was present in all the mushrooms. Other sterols found were 5α-cholest-7-en-3β-ol and ergosta-5,7-dien-3β-ol. Ergosta-5,8,22-trien-3β-ol was isolated from F. velutipes.     

The effect of aluminum on cytokinins in the mycelia of Amanita muscaria

High performance liquid chromatography analysis of immunoaffinity-purified extracts of mycelia of Amanita muscaria, and the Amaranthus bioassay of the eluted fractions, revealed the following seven cytokinins: zeatin, zeatin riboside, zeatin N-9-glucoside, dihydrozeatin, dihydrozeatin riboside, isopentenyl adenine, and isopentenyl adenosine. The decreased growth of aluminum-treated mycelia correlated with a 35% decrease in the total amount of the cytokinins. Among individual cytokinins, zeatin was the most affected, exhibiting a reduction of about 90%. The results are compared with previous investigations of aluminum effects on cytokinins in the mycelia of Lactarius piperatus, whose growth is stimulated by aluminum.Abbreviations ZR zeatin riboside - iPA isopentenyl adenosine - Z zeatin - DHZ dihydrozeatin - iP isopentenyl adenine - DHZR dihydrozeatin riboside - Z-9G zeatin N-9-glucoside - iP-9G isopentenyl N-9-glucoside - HPLC high performance liquid chromatography - DHZRMP dihydrozeatin riboside monophosphate - ZRMP zeatin riboside monophosphate     

Investigations on Antioxidant,Antiproliferative and COX‐2 Inhibitory Potential of Alkaloids from Anthocephalus cadamba (Roxb.) Miq. Leaves

In the present study, an ayurvedic medicinal plant, Anthocephalus cadamba (Roxb .) Miq . commonly known as ‘Kadamb’ was explored for its potential against oxidative stress and cancer. The fractions namely AC‐4 and ACALK (alkaloid rich fraction) were isolated from A. cadamba leaves by employing two different isolation methods and evaluated for their in vitro antioxidant and antiproliferative activity. The structure of the isolated AC‐4 was characterized tentatively as dihydrocadambine by using various spectroscopic techniques such as ESI‐QTOF‐MS, ¹H‐ and ¹³C‐NMR, DEPT, COSY, HMQC, and HMBC. Results of various antioxidant assays viz. 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH), ABTS cation radical, superoxide anion radical scavenging, and plasmid nicking assay demonstrated that both the fractions viz. AC‐4 and ACALK possess ability to scavenge DPPH, ABTS radicals and effectively protected plasmid pBR322 DNA from damage caused by hydroxyl radicals. Further, when both fractions were evaluated for their potential to suppress growth of HeLa and COLO 205 cells, only ACALK fraction showed antiproliferative effects. ACALK exhibited GI₅₀ of 205.98 and 99.54 μg/ml in HeLa and COLO 205 cell lines, respectively. Results of Hoechst staining in cervical carcinoma (HeLa) cells confirmed that ACALK induced cell death in HeLa cells via apoptotic mode. Both the fractions also inhibited COX‐2 enzyme activity.     

PRIMER NOTE. Using the incomplete genome of the ectomycorrhizal fungus Amanita bisporigera to identify molecular polymorphisms in the related Amanita phalloides

We describe the cross‐genomic isolation of 13 single nucleotide polymorphisms (SNPs) and one variable microsatellite from five loci for the death cap mushroom Amanita phalloides. Microsatellite repeats were identified by searching the partial Amanita bisporigera genome. Flanking primers were designed for 25 of these microsatellite loci and tested for cross‐amplification in A. phalloides. One locus contained an interrupted, compound microsatellite, and four loci contained one to six SNPs. These results demonstrate the usefulness of even an incomplete genome to identify molecular markers for population studies in nonmodel organisms.     

Marine antifoulants from bifurcaria bifurcata (phaeophyceae,cystoseiraceae) and other brown macroalgae

In this study, the antifouling activity of a series of extracts and linear diterpenes isolated from Bifurcaria bifurcata (Velley) Ross, a common brown alga of the Atlantic shores of Europe, and derivatives of these compounds was investigated. Antifouling assays with crude extracts from other brown algae, found abundantly along the coast of South Africa (Bifurcaria brassicaeformis, Bifurcariopsis capensis), the Atlantic shores of Europe (Halidrys siliquosa) and the coast of Mediterranean sea (Cladostephus verticillatus, Halopteris scoparia), are also reported. The fractions were tested in laboratory assays against representative species of the major groups of fouling organisms, viz. bacteria, fungi, diatoms, spores and zygotes of macroalgae and the blue mussel Mytilus edulis. Several components showed promising levels of activity. The high, albeit variable, level of antifouling activity suggests a potential for novel active ingredients in antifouling preparations.     

致命鹅膏菌毒素对DMBA/巴豆油诱导小鼠皮肤肿瘤的治疗作用

研究了致命鹅膏中的毒素在安全剂量下抑制小鼠皮肤肿瘤的效果,并对此毒素的药用安全性进行初步评价。采用经典的DMBA/巴豆油诱导小鼠皮肤乳头状瘤形成的二阶段致癌模型,观察和评价致命鹅膏毒素在不同剂量下抑制肿瘤的效果,并且通过测定血清中氨基己糖的含量来判断对肝损伤的情况。结果表明,致命鹅膏毒素在某一低剂量下对DMBA/巴豆油诱导小鼠皮肤乳头状瘤有较好的治愈效果,且对小鼠肝脏无损伤,证明了致命鹅膏毒素可以较好地治疗皮肤癌。     

Elaidic and trans-vaccenic acids in plasma phospholipids as indicators of dietary intake of 18:1 trans-fatty acids

Octadecenoic (18:1) trans-fatty acid fractions from margarine, butter and plasma phospholipids (PL) were isolated by silver ion TLC, and nine positional isomers (n-11-n-3) were identified by GC-MS based on their ozonolysis products. The GC analysis of the isolated fractions gave similar peak profiles and separated seven trans-isomers (n-11-n-6 and n-3). Without a preceding isolation step, the reproducibility of the Gc method for plasma PL elaidic (18:1 n-9 trans) and trans-vaccenic acids (n-7) was 3.4 and 2.7% (R.S.D.), respectively. These trans-isomers were rapidly incorporated and cleared in plasma PL and they closely reflected both increased and decreased intake of 18:1 trans-fatty acids during moderate fat substitutions. Significant associations between high-density lipoprotein cholesterol (HDL-C) and PL elaidic and trans-vaccenic acids appeared in habitual margarine users only.     

Plasma membranes from intestinal microvilli and erythrocytes contain cytochromes b5 and P-420

The presence of cytochromes b₅, P-450 and P-420 and activities of NADH- and NADPH-cytochrome c reductases were determined in plasma membranes isolated from microvilli of the chick and rat intestinal epithelium and erythrocyte membranes from chick, rat and man. The results are compared with the amounts of these components found in microsomal fractions from intestinal epithelium and in nuclear membranes from chick erythrocytes. Plasma membranes from intestinal microvilli and from erythrocytes contained significant amounts of NADH-cytochrome c reductase activity and of a pigment spectrophotometrically indistinguishable from rat liver microsomal cytochrome b₅. In addition, cytochrome b₅ fragments were prepared from the membranes by limited trypsin digestion and consisted of two to four components with M_r values in the range 10 000–13 500. In low-temperature difference spectra, the presence of a second cytochrome was noted which was similar to cytochrome P-420. Cytochrome P-450 and NADPH-cytochrome c reductase activities were not detected in plasma membrane fractions in significant concentrations but were present in the corresponding endomembrane fractions. These findings in highly purified, well defined plasma membrane fractions, in which contamination by endomembranes is minimal, strengthen the evidence for the existence of cytochrome-containing redox systems in plasma membranes of various cells and suggest that such redox components are general components of the cell surface. Possible functions and origins of these redox components in plasma membranes are discussed.     

Isolation and characterization of Perkinsus marinus proteases using bacitracin–sepharose affinity chromatography

Extracellular proteases were isolated from the cell-free culture supernatant of the oyster-pathogenic protozoan, Perkinsus marinus, by bacitracin–sepharose affinity chromatography. The purified protease fractions contained >75% of the protease activity initially loaded onto the column with very high specific activity that corresponded to 8–11-fold level of protease enrichment. The isolated proteases hydrolysed a variety of protein substrates including oyster plasma. All of the isolated P. marinus proteases belonged to the serine class of proteases. Inhibitor studies involving spectrophotometric assay and gelatin gel electrophoresis showed high levels of inhibition in the presence of the serine protease inhibitors PMSF, benzamidine and chymostatin, whereas inhibitors of cysteine, aspartic, and metalloproteases showed little or no inhibition. Spectrophotometric assays involving serine-specific peptide substrates further revealed that the isolated proteases belong to the class of chymotrypsin-like serine proteases. A 41.7 kDa monomeric, N-glycosylated, serine protease (designated Perkinsin) has been identified as the major P. marinus extracellular protease.     

NONDIALYZABLE SULFATED SIALOGLYCOPEPTIDE FRACTIONS DERIVED FROM BOVINE HEIFER BRAIN GLYCOPROTEINS. ISOLATION,CHARACTERIZATTON AND CARBOHYDRATE-PEPTIDE LINKAGE STUDIES1

The nondialyzable sialoglycopeptides dcrived from defatted, protease digested whole boyine heifer brain tissue were isolated and characterized. The partially purified bovine heifer brain tissue were isolated and characterized. The partially purified sialoglycopeptide mixtures contained 7-8% ester sulfate. The amount of nonsulfated material present was of the order of 15%. The total mixture was fractionated by anion exchange chromatography on AG1-X2(CI^-) using a sodium chloride gradient to yield twelve fractions all of which contained ester sulfate to varying degrees (5.6-21.9%). The molecular size of the fractions ranged from molecular weights of 10,400-12,500. The molar sulfate/galactose plus glucosamine ratios ranged from 0.3 to 1.1. Carbohydrate peptide linkage studies of the fractions by treatment with 0.2 M-NaOH-O.3 M-NaBH₄ led to the destruction of a portion of the threonine, serine and galactosamine and the appearance in acid hydrolysates of α-amino-n-butyric acid and galactosaminitol with an increase in alanine. Partial acid hydrolysis of the most abundant sialoglycopeptide liberated detectable amounts of 2-acetarnido-1-(L-4-aspartamido-)-1,2-dideoxy-β-D- glucose. These results indicated that the GalNac at the reducing end of the alkali labile carbohydrate prosthetic groups were linked O-glycosidically to thc hydroxyl groups of threonine and serine and the alkali-stable region of the principal sialoglycopeptide involves N-acetylglycosaminyl-asparagine linkages. Mild acid hydrolysis of the alkali-stable portion of the sulfated fractions indicated that the ester sulfate was confined to the alkali-stable region and exists as galactose 6-O-sulfate and N-acetylglucosa- mine 6-O-sulfate, structural fcatures similar to those present in rat brain glycopcptides (Markgolis & Margolis , 1970; Margolis et al.; 1972). Fucose and ester sulfate were confined to the alkali-stable region while mannose was located in both the alkali-labilc and alkali-stable regions of the sialoglyco-peptide fractions.     

The House Fly Attractants in Mushrooms

Several active concentrates responsible for the long known house fly attracting property of Amanita muscaria (L.) Fr. were obtained through steam distillation and exhaustive solvent extraction of the filtered juice of the fruiting body. Among the active concentrates, one fraction referred to as D₃ was isolated as colorless crystals, m.p. 22~23°C, with a chromatographycally proved homogeneity and exhibited a marked attractiveness to house flies, especially to mature females.     

Cell-Cycle-Specific Activity of Cyclic Nucleotide Phosphodiesterase In Physarum polycephalum

The cell-cycle-related activities of the cAMP- and cGMP-dependent phosphodiesterases of Physarum polycephalum were assayed. the activities of plasmodial homogenate and of selected subcellular fractions were measured. the results suggested the presence of both cAMP- and cGMP-dependent phosphodiesterase in the isolated nuclei of P. polycephalum. In addition, they reveal that the cAMP- and cGMP-dependent phosphodiesterase activities of the subcellular fractions fluctuate throughout the cell cycle. the whole-cell homogenates exhibit no cell-cycle-related changes in the presence of 5 × 10^-4 m cGMP. Kinetic data suggest the presence of multiple phosphodiesterase activities in the homogenate and its particulate fractions for the cGMP-dependent enzyme. Multiple cAMP activities are also suggested for the particulate fractions. the K_m values indicate that the substrate affinities of the phosphodiesterases from P. polycephalum are similar to those found previously in mammalian systems.     

Inhibitory effect of the compounds from the water extract of Curcuma longa on the production of PGE2 and NO in a macrophage cell line stimulated by LPS

ABSTRACT

We wished to search for the compounds contributing to the anti-inflammatory effects of the water extract of Curcuma longa (WEC). WEC was fractioned and the fractions were evaluated with regard to their inhibitory effect on the production of nitric oxide (NO) from the macrophage cell line stimulated by lipopolysaccharide. Compounds in the active fractions were isolated and identified. One isolated compound was identified as new: (6S)-2-methyl-5-hydroxy-6-(3-hydroxy-4-methylphenyl)-2-heptene-4-one (1). Four isolated compounds were identified as known: (6S)-2-methyl-6-(4-hydroxyphenyl)-2-heptene-4-one (4), bisabolone-4-one (5), curcumenone (6), and turmeronol A (8). Three isolated compounds were not identified their stereostructures but their planar structures: 2-methyl-6-(4-hydroxymethyl-phenyl)-2-heptene-4-one (2), 2-methyl-6-(2,3-epoxy-4-methyl-4-cyclohexene)-2-heptene (3), and 4-methylene-5-hydroxybisabola-2,10-diene-9-one (7). Compounds 1, 4, 7 and 8 inhibited production of prostaglandin E2 and NO. Others inhibited NO production only. These results (at least in part) show the active compounds contributing to the anti-inflammatory effects of WEC, and may be useful for elucidating its various beneficial physiologic effects.     

Sterol glucosylation by the plasma membrane from cotyledons of Phaseolus aureus

Membrane fractions were isolated from dark grown cotyledons of Phaseolus auneus by differential and sucrose density gradient centrifugation. Endoplasmic reticulum-, Golgi apparatus- and plasma membrane-rich fractions were identified by their respective enzymic activities and tested for their ability to transfer glucose from UDP-glucose to endogenous sterols to form steryl glucosides. The glucosyltransferase activity was shown to be located mainly at the plasma membrane.ABBREVIATIONS SG steryl glucoside - ASG acylated steryl glucoside - UDP-glc Uridine diphosphoglucose     

Design of bacterial defined mixed cultures for biodegradation of specific crude oil fractions, using population dynamics analysis by DGGE

Hydrocarbon-degrading bacteria isolated from oil-polluted soils, were used to design three defined mixed cultures (DMC) for biodegradation of Maya crude oil fractions. The first degrading culture, DMC A was made up with 10 strains. Design of DMC B (six strains) and DMC C (three strains) was based on DGGE profiles obtained throughout biodegradation assays of different petroleum fractions. Biodegradation of the aliphatic fraction (10 000 mg l⁻¹) and an aromatic–polar mixture (5000 mg l⁻¹) was evaluated for the DMC B. Biodegradation of total hydrocarbons (10 000 mg l⁻¹) and its fractions was evaluated for DMC B and DMC C. During biodegradation assays, O₂ consumption and CO₂ production were assessed by respirometry, while population dynamics of predominant strains was based on PCR-DGGE profiles of partial 16S rDNA. Aliphatic fraction was completely biodegraded by DMC B, while degradation of the aromatic–polar mixture was 12.5% and for total hydrocarbons 40.5%. DMC B was able to degrade the aromatic fraction (31%) and even the polar fraction (19.6%) present in total hydrocarbons. DMC C degraded the aromatic and polar fractions (5.6% and 2%, respectively) present in total hydrocarbons. DGGE profiles of the DMCs indicated that Pseudomonas sp., Gordonia rubripertincta and a non-identified strain were predominant and probably responsible of the hydrocarbons biodegradation. The use of DGGE-fingerprinting to track microbial populations, allowed selecting strains to design efficient oil-degrading defined mixed cultures.     

Amanita xenokommosis (Agaricales,Basidiomycota), a striking new species from sand dune forest of northeastern Brazil,with a key for phenetically similar species*

Abstract

Amanita is a well established genus corresponding one to the major groups of Agaricales having with a rich systematic story. Several species belonging to this genus were recently reported from Brazil. Recent studies changed drastically its infrageneric classification, recognizing the subgenera Amanita, Amanitina and Lepidella. Within the subg. Amanitina, the ectomycorrhizal species with amyloid basidiospores, appendiculate pileus and stipe base with more globose to subglobose bulb belong to sect. Roanokenses. Amanita xenokommosis belongs to this section and is described here a new species from the Brazilian Coastal Sand Dune. It is characterized by elongate to cylindrical amyloid basidiospores, brown pileus with sharp floccose appendiculate remnants and bulb covered by a large limbate/saccate universal veil. Amanita caojizong, A. manginiana, A. modesta, A. pseudomanginiana and A. pseudoporphyria are similar species, but differ in many aspects, such as basidiospores size, shape of bulb and characteristics of the universal veil.     

β-1-3- and β-1-4-glucan synthesis by membrane fractions from the fungus Saprolegnia

The -glucan synthetase activity of the fungus Saprolegnia monoica was assayed by supplying UDP-glucose to membrane fractions of mycelial homogenate. The analysis of glucan products by hydrolysis with various -glucanases and by chromatography show that both -1-3- and -1-4-linkages are formed at high substrate concentrations. In the absence of MgCl₂, -1-3-linked glucans are mainly produced. By increasing MgCl₂ concentrations the total synthesis activity and -1-3-linkages production are reduced. At low substrate concentrations in the presence of MgCl₂, -1-4-linked glucans are the only polysaccharide synthesized. Electron microscopy of radioactive products, synthesized by original membrane fractions or by membrane fractions isolated from continuous sucrose density gradients, shows microfibrils when the assays are conducted at high substrate concentrations in the absence of MgCl₂.Abbreviations G.S. I glucan synthetase I - G.S. II glucan synthetase II - Dol. P dolichol phosphate     

Enzyme immunoassays of N 6-benzyladenine and N 6-(meta-hydroxybenzyl)adenine cytokinins

Enzyme-linked immunosorbent assays (ELISAs) were developed for determination of N ⁶-benzyladenosine, N ⁶-(meta-hydroxybenzyl)adenosine, and structurally related cytokinins. The use of the ELISAs allowed detection over the range of 0.05–70 pmol for N ⁶-benzyladenine and 0.01–20 pmol for the N ⁶-(meta-hydroxybenzyl)adenine cytokinins. Polyclonal antibodies used in the assays were specific for N ⁶-benzyladenine and N ⁶-(meta-hydroxybenzyl)adenine and their corresponding N ⁹-substituted derivatives. By the use of internal standardization, dilution assays, authentic [2-³H]cytokinin recovery markers, and immunohistograms, the ELISAs have been shown to be applicable for the estimation of N ⁶-benzyladenine and N ⁶-(meta-hydroxybenzyl)adenine-type cytokinins in plant tissues. For the analysis of cytokinins in the tissues of young poplar leaves and Solarium teratoma shoot culture, the extracts were fractionated by high performance liquid chromatography (HPLC) and the fractions analyzed by ELISAs. Immunohistogram ELISA analysis of fractions from different HPLC systems indicated major peaks of immunoreactivity co-chromatographing with the labeled and unlabeled standards of N ⁶-benzyladenine, N ⁶-meta-hydroxybenzyl)adenine, and their N ⁹-glycosides in these tissues.Abbreviations ELISA enzyme-linked immunosorbent assay - FW fresh weight - (mOH)[9R]BAP N ⁶-(meta-hydroxybenzyl)adenosine - HPLC high performance liquid chromatography - TBS Tris-buffered saline - TEAA triethylammonium acetate - [9R]BAP N ⁶-benzyladenosine     

Regulatory properties of the ADPglucose pyrophosphorylase from Rhodopseudomonas sphaeroides and from Rhodopseudomonas gelatinosa

Highly purified fractions of chlorosomes and cytoplasmic membranes were isolated from Chloroflexus aurantiacus Ok-70-fl and Chlorobium limicola 6230. These fractions were comparatively analyzed for their pigmentation, phospholipid, glycolipid, and cytochrome c content as well as for their specific activities of succinate dehydrogenase and NADH-oxidase. The data showed that there are some differences in pigmentation and phospholipid content between the isolated fractions of Chloroflexus and Chlorobium. Chlorosomes of Chloroflexus contained a specific BChl a-complex with a characteristic absorption maximum at about 790 nm. This BChl a-complex could not be detected in spectra of chlorosomes from Chlorobium. The near infrared region of the spectra of the isolated cytoplasmic membranes of both organisms revealed considerable differences: The BChl a-complexes of Chloroflexus membranes exhibited peaks at 806 and 868 nm whereas the membranes of Chlorobium had a single BChl a-peak at 710 nm. In contrast to the findings with Chlorobium the chlorosomes of Chloroflexus contained at least twice as much phospholipids as did the cytoplasmic membranes. In Chlorobium the phospholipid content of cytoplasmic membranes is three times that of their chlorosomes. The distribution of all other components (carotenoid composition, enzyme activities, cytochrome c content, and glycolipids) was about the same in both strains. From the data it was concluded that differences in the organization of the photosynthetic apparatus are mainly based on differences of the organization of the photosynthetic units in the cytoplasmic membrane and probably the kind of linkage of the light harvesting system in the chlorosomes with the reaction center in the cytoplasmic membranes.Abbreviations BChl c bacteriochlorophyll c - BChl a bacteriochlorophyll a - DSM Deutsche Sammlung von Mikrorganismen