Structural characterization and study of immunoenhancing properties of heteroglycan isolated from a somatic hybrid mushroom (PfloVv1aFB) of Pleurotus florida and Volvariella volvacea

A water soluble polysaccharide isolated from the hot aqueous extract of the fruit bodies of the somatic hybrid mushroom (PfloVv1aFB), raised through protoplast fusion between the strains of Pleurotus florida and Volvariella volvacea was found to consist of d-glucose, d-galactose, and d-mannose in a molar ratio of nearly 4:1:1 and showed macrophage, splenocyte, and thymocyte activation. On the basis of sugar analysis, methylation analysis, periodate oxidation, and NMR studies (¹H, ¹³C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HMQC and HMBC), the structure of the repeating unit of the polysaccharide was established as:     

Glucans from the alkaline extract of an edible mushroom, Pleurotus florida, cv Assam Florida: isolation, purification, and characterization

Three different glucans (PS-I, PS-II, and PS-III) were isolated from the alkaline extract of the fruiting bodies of an edible mushroom Pleurotus florida, cultivar Assam Florida. On the basis of total acid hydrolysis, methylation analysis, periodate oxidation, Smith degradation, and NMR experiments (¹H, ¹³C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating unit of these polysaccharides was established as follows:     

A test of the binary chloroplast membrane hypothesis by using a nonpenetrating chemical probe,p-(diazonium)-benzenesulfonic acid

Summary Spinach chloroplasts were exposed to³⁵S-labeledp-(diazonium)-benzenesulfonic acid (DABS), a water soluble compound which does not penetrate lipophilie regions of membranes, and which is highly reactive toward amino acid functionagroups such as -amino, sulfhydryl, histidine, and tyrosine groups. Amino groups inl lipids can also form similar, stable covalent bonds by diazo coupling. Both chloroplast lipids and proteins were labeled with DABS, the total binding being about 1 DABS per 10 chlorophylls, depending on the reaction conditions.After diazo coupling and subsequent digitonin fractionation into photosystems I and II enriched fractions, it was observed that PS-I was more highly labeled than PS-III usually by a factor of 10 to 24 times (on a per chlorophyll basis). After digitonin isolation, however, the PS-II portion bound an amount of DABS similar to the PS-I binding, We interpret these data as consistent with the binary membrane hypothesis (Arntzen. Dilley and Crane (1969),J. Cell Biol. 43:16), which visualizes PS-I on the externa, half of a 90 Å grana membrane, and PS-II occurring on the interior half of thel membrane. The alternative explanation that PS-II and PS-I are arranged as a mosaic, and that the low DABS binding in PS-II is caused by burial of the diazo reactive groups in the interior of the proteins (and only exposed through the denaturing effect of digitonin) is not directly ruled out. However, this alternative is not consistent with the facts that: (a) most of the membrane proteins in PS-I and PS-II are identical in electrophoretic properties and therefore probably have similar overall structures; and (b) digitonin does not lead to appreciable denaturation of proteins, evidenced by the retention of PS-II electron transport activity.     

Glucans from alkaline extract of a hybrid mushroom (backcross mating between PfloVv12 and Volvariella volvacea): structural characterization and study of immunoenhancing and antioxidant properties

Two different glucans (water-soluble PS-I, water-insoluble PS-II) were isolated from the alkaline extract of the fruit bodies of hybrid mushroom. PS-I was found to consist of only (1→6)-linked β-D-glucopyranose. PS-II was composed of terminal, (1→3,4)-linked, and (1→3)-linked β-D-glucopyranosyl moieties in a molar ratio of nearly 1:1:1. PS-I showed macrophages, splenocytes, and thymocytes activation as well as antioxidant property. On the basis of sugar analysis, methylation analysis, and NMR studies ((1)H, (13)C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating unit of these glucans were established as:     

Structural studies of water-soluble polysaccharides of an edible mushroom, Termitomyces eurhizus. A reinvestigation

The structure of two polysaccharides isolated from the hot aqueous extract of fruiting bodies of the mushroom, Termitomyces eurhizus, have been reinvestigated. These consist of two homogeneous fractions PS-I and PS-II. PS-I contains only D-glucose as the monosaccharide constituent. From methylation analysis and periodate oxidation studies, followed by GLC-MS analysis the linkages, the sugar units in PS-I were identified as (1-->3)-D-Glcp and (1-->6)-D-Glcp. PS-II contains D-glucose, and the mode of linkage of d-glucose was identified as (1-->6)-D-Glcp. Finally, the following possible structures of the polysaccharides were assigned using 1H, 2D-COSY, TOCSY, NOESY and 13C NMR spectral analysis: [carbohydrate structure: see text].     

Evolution of photosynthetic reaction centers

The common ancestor of all photosynthetic prokaryotes and organelles contained chlorophyll (Chl) a. All green and purple photosynthetic bacteria descended from a common bacteriochlorophyll (Bchl) a-containing ancestor which diverged from the Chl a line. Separate PS-I and PS-II reaction centers may have evolved before the appearance of Bchl a. When the transition to Bchl a occurred, the resultant organism contained two types of reaction center, “PS-I” and “PS-II.” One line of development eliminated “PS-II” and evolved into the green bacteria. The other line eliminated “PS-I” and became the purple bacteria. In the Chl a-containing organisms the evolution of PS-II continued until oxygen evolution was achieved.     

Effect of lignin-derived phenolic monomers on the growth of the edible mushrooms Lentinus edodes,Pleurotus sajor-caju and Volvariella volvacea

Pleurotus sajor-caju was generally more tolerant to lignin-related phenolic monomers and tannin derivatives than Lentinus edodes and the straw mushroom, Volvariella volvacea. Several phenols, at up to 5 mM, enhanced mycelial growth of P. sajor-caju. No clear pattern was evident when the effects of phenols and tannins on the growth of V. volvacea and L. edodes were compared, but the lower concentrations of 4-hydroxybenzaldehyde and vanillin which were tested were markedly more toxic to the straw mushroom. The distribution of phenolic monomers and tannin derivatives in the agricultural wastes used for mushroom cultivation may be an important growth determinant. However, the differences in the growth inhibition profiles of L. edodes, P. sajor-caju and V. volvacea suggest that, alone, the effect of these compounds on fungal growth is unlikely to account for the varying abilities of the three mushroom species to grow and fruit on a particular lignocellulosic substrate.     

Chemical analysis of new water-soluble (1→6)-, (1→4)-α, β-glucan and water-insoluble (1→3)-, (1→4)-β-glucan (Calocyban) from alkaline extract of an edible mushroom, Calocybe indica (Dudh Chattu)

Two different glucans (PS-I, water-soluble; and PS-II, water-insoluble) were isolated from the alkaline extract of fruit bodies of an edible mushroom Calocybe indica. On the basis of acid hydrolysis, methylation analysis, periodate oxidation, and NMR analysis ((1)H, (13)C, DEPT-135, TOCSY, DQF-COSY, NOESY, ROESY, HMQC, and HMBC), the structure of the repeating unit of these polysaccharides were established as: PS-I: →6)-β-D-Glcp-(1→6)-β-D-glcp-(1→6)-)-β-D-Glcp-(1→ α-D=Glcp (Water-soluble glucan). PS-II: →3)-β-D-Glcp-(1→3)-β-D-glcp-(1→3)-)-β-D-Glcp-(1→3)-β-D-Glcp-(1→ β-D-Glcp (Water-insoluble glucan, Calocyban).     

Bioconversion of agricultural lignocellulosic wastes through the cultivation of the edible mushrooms Agrocybe aegerita,Volvariella volvacea and Pleurotus spp.

Ten selected wild and commercial strains of Pleurotus ostreatus,Pleurotus eryngii,Pleurotus pulmonarius, Agrocybe aegerita andVolvariella volvacea were cultivated on three agricultural wastes, i.e. wheat straw (WS), cotton waste (CW) and peanut shells (PS). All species demonstrated significantly higher colonization rates on WS and CW than on PS. WS supported faster growth of A. aegerita and Pleurotus spp., whereas V. volvacea performed better on CW. Comparison of growth rates on composted and non-composted WS and CW substrates revealed that in the latter case faster colonization was achieved, particularly for Pleurotus spp. However, one commercial strain of V. volvacea presented higher growth rates when the composted CW medium was used. Furthermore, earliness in the fructification of P. ostreatus, P. pulmonarius and V. volvacea strains was promoted in CW substrates, while WS favoured earliness of P. eryngii and A. aegerita. Similarly, high sporophore yields were obtained by P. ostreatus and P. pulmonarius on both wastes, whereas WS enhanced yield and basidioma size of P. eryngii and A. aegerita strains and CW production of V. volvacea. The substrates cellulose:lignin ratios were found to be positively correlated to mycelial growth rates and to mushroom yield of P. ostreatus and P. pulmonarius; in addition, positive correlation was also detected for carbon:nitrogen ratio and mushroom yield in P. eryngii and A. aegerita and between cellulose content and mushroom yield for V. volvacea strains.     

Glucans of Pleurotus florida blue variant: isolation, purification, characterization and immunological studies

Two different glucans (PS-I and PS-II) were isolated from the alkaline extract of the fruiting bodies of an edible mushroom, Pleurotus florida blue variant and the PS-I showed macrophage, splenocyte and thymocyte activations. On the basis of sugar analysis, methylation analysis, periodate oxidation, and NMR studies ((1)H, (13)C, DEPT-135, DQF-COSY, TOCSY, NOESY, ROESY, HMQC and HMBC), the structure of the repeating unit of these polysaccharides were established.     

Molecular Cloning,Sequencing, and Expression of a <Emphasis Type="SmallCaps">l</Emphasis>-Glutamine <Emphasis Type="SmallCaps">d</Emphasis>-Fructose 6-Phosphate Amidotransferase Gene from <Emphasis Type="Italic">Volvariella volvacea</Emphasis>

Using 3′-RACE and 5′-RACE, we have cloned and sequenced the genomic gene and complete cDNA encoding l-glutamine d-fructose 6-phosphate amidotransferase (GFAT) from the edible straw mushroom, Volvariella volvacea. Gfat contains five introns, and encodes a predicted protein of 697 amino acids that is homologous to other reported GFAT sequences. Southern hybridization indicated that a single gfat gene locus exists in the V. volvacea genome. Recombinant native V. volvacea GFAT enzyme, over-expressed using Escherichia coli and partially purified, had an estimated molecular mass of 306 kDa and consisted of four equal-sized subunits of 77 kD. Reciprocal plots revealed K _m values of 0.55 and 0.75 mM for fructose 6-phosphate and l-glutamine, respectively. V. volvacea GFAT activity was inhibited by the end-product of the hexosamine pathway, UDP-GlcNAc, and by the glutamine analogues N ³-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid and 2-amino-2-deoxy-d-glucitol-6-phosphate.     

Immunogold localization of photosystem I and photosystem II light-harvesting complexes in cryptomonad thylakoids

Summary— The molecular organization of the thylakoids of Cryptomonas rufescens was studied by immunoelectron microscopy employing antibodies against photosystem (PS)-I and two PS-II antenna proteins. The PS-I complex and the 19-kDa chlorophyll a/c light-harvesting (LH) protein are both localized along the length of the thylakoid membranes. The external membranes of the paired thylakoids are enriched in PS-I whereas the chlorophyll a/c LH protein is more concentrated in the internal or appressed membranes. However, unlike the situation in higher plants and Chlamydomonas, there is not a marked asymmetry in the concentration of PS-I and chorophyll a/c LH protein in the two types of membranes. Double labelling studies of sections and isolated PE-PS-II particles with anti-phycoerythrin and anti-LH confirmed that phycoerythrin is localized in the thylakoid lumen and that this pigment exists in two forms, a fraction closely associated with the thylakoid membranes and another fraction free in the lumen. These results confirm the uniqueness of cryptomonad thylakoids.     

Effect of Growth Regulators and Herbicides on Photosynthetic Partial Reactions in Isolated Leaf Cells

The effect of indoleacetic acid, gibberellic acid, 2,4-dichloro-phenoxyacetic acid and amitrole at various concentration levels ranging from 0.05 to 1.0 mM on the photosynthetic evolution of O₂ and ¹⁴CO₂ fixation by isolated leaf cells was studied. The plant growth regulators enhanced O₂ evolution and ¹⁴CO₂ fixation at low concentrations and were inhibitory beyond a critical level. The amitrole had an inhibitory effect at all the concentration levels used. All the substances exhibited similar patterns of effect on the ferricyanide reduction by isolated chloroplasts and on the electron transport rates of sub-chloro-plast particles containing PS-I and PS-II independently, under non-phosphorylating conditions. As was seen from the response in all the three electron transport systems of the chloroplast studied, the electron transport chain connecting PS-II and PS-I could be considered as a possible site of action at least for the growth regulating substances as it is the only part that is common to all the three reactions. The phosphorylation associated with this part of the electron transport was “inhibited” by the substances even at the lowest concentration used. The stimulation of non-phosphorylating electron flow, with a simultaneous reduction in the rate of ATP synthesis, at low concentration levels indicated that these substances played a possible uncoupling role. The amitrole on the other hand appeared to have a generalized non-specific inhibitory action on all the partial reactions of photosynthesis.     

Improved fruiting of the straw mushroom (Volvariella volvacea) on cotton waste supplemented with sodium acetate

Compared with controls, treatment of the cultivation substrate for Volvariella volvacea with 0.02% NaAc at the “spraying water” stage increased the number of fruiting body primordia by 280, the mushroom yield by 16.25%, the number of fruiting bodies by 35.57%, and the biological efficiency by 16.28%. The average single mushroom weight increased by 19.33%, but there was no significant difference between treatments and controls. A correlation analysis revealed a significant (p < 0.05) positive correlation between the total yield and the number of fruiting bodies. Comparisons of the cost and profit values for the sodium acetate-treated and untreated groups revealed that the former generated a higher income for the grower. Our data indicate that sodium acetate treatment is a promising new method for increasing V. volvacea yields. A possible mechanism whereby sodium acetate increased the mushroom yield is discussed.

     

Analysis of photosystem II using particle II preparation. I. Experimental evidence supporting the idea of the involvement of two light reactions in photosystem II of green plant photosynthesis

(1) To analyze the photoelectron flow related to photosystemII, particle II preparation, i.e., the chloroplast fragmenthaving only photosystem II activity, proved to be far betterthan the generally used chloroplast preparations having activitiesof both PS-I and PS-II. (2) By simultaneous measurements ofthe activities of O₂ evolution and DPIP- and ferricyanide photoreductionusing variously-treated particle II preparations, it was foundthat a noticeable activity of ferricyanide photoreduction wasstill observed, though the former two activities were completelylost in the course of treatments such as Tris-treatment, pre-illuminationand aging. (3) Besides this, differences were found betweenferricyanide- and DPIP-photoreduction in respect to susceptibilityto CCCP, availability of artificial electron donor, and theeffect of chloride addition. However, both photo-reductionswere equally inactivated by heat-treatment and addition of DCMU.(4) To explain the observed distinctions between DPIP and ferricyanidein their mode of action as electron acceptor for PS-II, a schemesuggesting the involvement of two light reactions in PS-II isproposed and the electron flow near PS-II is discussed. ¹ This work has been supported by Grants from the Ministry ofEducation (Nos. 8425- 70-'71; 4970l4-'69-'71), which are gratefullyacknowledged here. (Received January 12, 1972; )     

五种矿质元素对草菇菌丝生长和活性氧清除能力的影响

【背景】菌种退化是草菇生产中面临的难题,探究一种简单高效的复壮方法是草菇产业亟须解决的问题。【目的】探讨外源添加5种矿质元素对草菇退化菌株的菌丝性状和活性氧(reactive oxygen species, ROS)清除能力的影响。【方法】筛选出5种矿质元素的最佳浓度并添加于PDA培养基中,测定草菇菌落形态、菌丝生长速度、菌丝生物量、ROS含量、抗氧化酶活性及抗氧化物质含量。【结果】MnSO4、Na2SeO3、CaSO4、FeSO4可有效提高草菇退化菌株D1和D2的菌丝生长速度和生物量;降低O2·-、H2O2等ROS的含量,并增加还原型谷胱甘肽(glutathione, GSH)、氧化型谷胱甘肽(glutathionedisulfide,GSSG)的含量及超氧化物歧化酶(superoxidedismutase,SOD)、过氧化氢酶(catalase, CAT)、谷胱甘肽...     

Development of photosystem-II activity during irradiance of etiolated Helianthus (Asteraceae) seedlings

Light-induced development of photosystem (PS)-II activity was followed during irradiance of etiolated Helianthus annuus (sunflower) cotyledons using chlorophyll a fluorescence. Cotyledons from seedlings grown in continuous darkness for 6 d were exposed to 100 μmol photons·m⁻²·s⁻¹ for time periods of 1, 3, 6, and 12 h. Associated with increased time of irradiance exposure were significant: (1) increases in concentration of PS II, (2) increases in quantum efficiency of PS II, (3) decreases in the ratio of PS-II quinone_B (Q_B)-nonreducing centers to total PS-II centers (PS-II Q_B-nonreducing centers + PS-II Q_B-reducing centers), and (4) decreases in the ratio of slow PS-II Q_B-reducing centers to total PS-II Q_B-reducing centers (fast PS-II Q_B-reducing centers + slow PS-II Q_B-reducing centers). The results support the hypotheses that development of PS II involves assembly of complexes which initially cannot reduce Q_B and that heterogeneous aspects of PS-II pools during chloroplast maturation may represent different developmental states.     

Pyruvic-acid-containing polysaccharide in the cell wall of Bacillus polymyxa AHU 1385

Three acidic polymer fractions with molecular masses of about 16 kDa, 35 kDa and 70 kDa were isolated from lysozyme digests of N-acetylated cell walls of Bacillus polymyxa AHU 1385 by ion-exchange chromatography and gel chromatography. These fractions, containing mannosamine, glucosamine and pyruvic acid in a molar ratio of about 1:1:1 together with glycopeptide components, were characterized as polysaccharide-linked glycopeptides with one, two and more polysaccharide chains. On the other hand, treatment of the cell walls with glycine/HC1 buffer, pH 2.5, at 100 degrees C for 10 min followed by separation of water-soluble products on ion-exchange chromatography gave three polysaccharide fractions, PS-I-III, which contained different amounts of pyruvic acid (0,0.6 and 0.9 residue/mannosamine residue) along with equimolar amounts of mannosamine and glucosamine. Pyruvate-free polysaccharides similar to PS-I were also obtained from PS-II, PS-III and polysaccharide-linked glycopeptides by treatment with 10 mM HC1 at 100 degrees C for 1 h. Results of analyses of these polysaccharide preparations by 1H-NMR and 13C-NMR measurement and methylation, together with data from characterization of fragments obtained by hydrogen fluoride hydrolysis, lead to the most likely structure, ----3)[4,6-O-(1-carboxyethylidene)]ManNAc(beta 1----4)GlcNac(beta 1----, for the acidic polysaccharide of this strain.     

Mutagenesis of Protoplasts and Regeneration of Mycelium in the Mushroom Volvariella volvacea

Regenerating protoplasts were obtained from mycelial culture of the mushroom Volvariella volvacea by the action of the lytic enzyme Novozym 234 in the presence of 0.01 M phosphate buffer (pH 6.0) containing 0.6 M NaCl. Regeneration was found to be poor in liquid medium, but more than 50% regeneration was achieved on solid 2% agar medium overlaid with 0.5% agar. Protoplasts of V. volvacea were found to be highly sensitive to the killing action of both UV irradiation and N-methyl-N′-nitro-N-nitrosoguanidine. However, no morphological or auxotrophic mutants could be obtained from protoplasts by chemical mutagenesis. Four types of morphological mutants and one auxotrophic (adenine-negative) mutant were obtained from UV-irradiated protoplasts. The adenine-negative mutant of V. volvacea was found to be stable, not losing auxotrophy on repeated subculture.     

大麦突变种Chlorina-f 2类囊体膜的叶绿素蛋白复合体

当突变种大麦Chlorina-f 2的类囊体膜在SDS/叶绿素的重量比为10:1,叶绿素的浓度为0.5mg/ml的条件下增溶,并在SDS-聚丙烯酰胺凝胶电泳中进行分离时,共出现4条含叶绿素的带。按电泳迁移率的增加,这些带分别是CP Ⅰ,CPa 1,CPa 2和FC。光谱测定表明CP Ⅰ为混有少量光系统Ⅱ??成分的光系统Ⅰ反应中心复合体,CPa 2为光系统Ⅱ反应中心复合体,CPa 2为光系统Ⅱ内周天线复合体。属于光系统Ⅰ的CP Ⅰ的叶绿素含量占总叶绿素的45.6%,而属于光系统Ⅱ的CPa Ⅰ和CPa 2的叶绿素之和则占总叶绿素的43.2%。可见在缺b大麦中,两个都失缺其外周天线的光系统的叶绿素含量是基本相等的。这和光合作用中两个光反应相互串联的理论是完全一致的。