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Sorogen elongation and side branching during fruiting body development in Polysphondylium pallidum.

We report a cinematographic analysis of culmination in fruiting bodies of Polysphondylium pallidum. During the final stages of aggregation, sorogen elongation is slow. An increase in the rate of sorogen elongation occurs when the growing stalk contacts the substratum, lifting the cell mass into the air. The elongation process slows and finally stops as differentation of the terminal spore mass occurs. During the rapid elongation phase, variations in the rate of elongation of the main cell mass are correlated with the process of side branching. Side-branch elongation resembles elongation of the main sorogen with the rate of elongation increasing as the side-branch stalk contacts the main stalk and slowing as spore differentiation occurs. The morphological events of branching have been studied in detail and models to explain this process are discussed.

Morphogenesis↗

A novel ribonuclease from fruiting bodies of the common edible mushroom Pleurotus eryngii.

A ribonuclease with a temperature optimum of about 70 degrees C and a pH optimum of 6.5 was isolated from fruiting bodies of the mushroom Pleurotus eryngii. The ribonuclease was unadsorbed on DEAE-cellulose and adsorbed on Affi-gel blue gel and S-Sepharose. It possessed a molecular mass of 16 kDa, and exhibited higher ribonucleolytic activity toward poly A and poly G and lower ribonucleolytic activity toward poly C and poly U. Its N-terminal sequence was distinctly different from those of other mushroom ribonucleases, and resembled that of Pleurotus tuber-regium only by 40%. Furthermore, its thermostability characteristics, polyhomoribonucleotide specificity and molecular mass were dissimilar to those of other mushroom ribonucleases.

Amino Acid Sequence↗

DNA replication during sporulation in Myxococcus xanthus fruiting bodies.

During the developmental process of the Gram-negative soil bacterium Myxococcus xanthus, vegetatively growing rod cells differentiate to ultimately become metabolically quiescent and environmentally resistant myxospores encased within fruiting bodies. This program, initiated by nutrient deprivation, is propagated by both cell-autonomous and cell-nonautonomous signals. Our goal was to determine whether M. xanthus, like many other developmental systems, uses cell-cycle cues to regulate and control its developmental program. To address this question, the DNA replication cycle was used as a marker to monitor progression through the cell cycle in vegetative, stationary, and developing M. xanthus populations. Using flow cytometry, quantitative fluorescence microscopy, and FISH to establish the chromosome copy number of myxospores, it was determined that vegetatively growing cells contain one to two copies of the genome, but upon entry into stationary phase, the chromosome copy number drops to a single copy. Of particular interest, fruiting body-derived myxospores contain a specific two-chromosome DNA complement with both origin and terminus regions localized to the periphery of the myxospore. We speculate that this duplication of genetic information in the myxospore would help assure viability during germination by providing a second copy of each gene. The results of this study imply that not only is DNA replication tightly regulated during the developmental process of M. xanthus, but that there are also regulatory mechanisms to ensure that all myxospores acquire two copies of the chromosome.

Cell Cycle↗

Molecular species of cerebrosides in fruiting bodies of Lentinus edodes and their biological activity.

Cerebroside fraction was obtained from fresh fruiting bodies of Lentinus edodes and separated into ten molecular species by reverse-phase high-performance liquid chromatography. The species were identified by GLC, GC-MS and NMR. Their component glycosides and sphingoids were uniformly glucose and (4E,8E)-9-methyl-4,8-sphingadienine, respectively. The component fatty acids were 2-hydroxy acids with the carbon chain length of 16, 15, 14, 18, 24, 17, 25, 26, 22 and 23 (from major to minor). The cerebrosides with the C14-18 fatty acids showed strong fruiting-inducing activity in Schizophyllum commune. Those with the C22 and C23 ones had one-eighth and one-sixteenth of the activity, respectively, and those with C24-26 had no detectable activity. 22 and 23 must be the carbon chain lengths of the component fatty acid of the sphingolipids critical for expression of biological activity.

Basidiomycota↗

Analysis of recQ gene transcript in fruiting bodies of basidiomycetous mushroom Lentinula edodes.

Quantitative reverse transcription PCR analysis and total RNA staining demonstrated that Lentinula edodes recQ gene (Le.recQ) transcript is present in all the parts of the fruiting body, but in hymenophore at the highest density. Results of in situ RNA-RNA hybridization showed that the Le.recQ transcript level within the hymenophore is higher in the hymenium, subhymenium, and the outer region of the trama. Trama cells themselves contain a lower level of the transcript.

Adenosine Triphosphatases↗

A new perylenequinone from the fruit bodies of Bulgaria inquinans.

A new perylenequinone, 4,9-dihydroxy-1,2,11,12-tetrahydroperylene-3,10-quinone (1), together with three known compounds, have been isolated from the ethanolic extract of the fruit bodies of Bulgaria inquinans, and their structures elucidated on the basis of the spectral data and comparison with the literature.

Ascomycota↗

Fruiting body morphogenesis in submerged cultures of Myxococcus xanthus.

Induced by starvation, the development of fruiting bodies by Myxococcus xanthus on glass and plastic surfaces under a layer of liquid was followed microscopically. Calcium ions and a neutral pH were required for development of a Myxococcus strain that grew dispersed in liquid culture. Initially asymmetric aggregates later became round, and sporulation followed aggregation.

Calcium↗

Cell differentiation during fruiting body formation in polysphondylium pallidum.

The spatial pattern of cellular differentiation was studied during fruiting body formation in Polysphondylium pallidum using 3 different staining methods: Calcofluor fluorescence (cellulose accumulation), neutral red (prestalk cells) and immunofluorescence (prespore cells). Neutral-red staining revealed the existence of a clear prestalk region which becomes evident during aggregation and continues throughout culmination. Immunofluorescent staining demonstrated that cells in the prestalk region gradually lose their presporeness (fluorescence) as they are transformed into differentiated stalk cells. Calcofluor staining revealed that stalk cell differentiation begins during the mid-aggregation phase and that the mode of formation of the main stalk and the side branches differs slightly in morphology. Calcofluor staining also demonstrated the development, during aggregation, of a thick cellulosic girdle with lateral tubular extensions which surround the aggregation streams. The above results are discussed in terms of our present knowledge about differentiation and morphogenesis in cellular slime moulds.

Dictyostelium↗

Oligonucleotidase activity of phosphodiesterase from the fruit body of Flammulina velutipes.

A phosphodiesterase (EC 3.1.4.1) was purified to homogeneity from the fruit body of Flammulina velutipes. The enzyme had considerable activity toward oligonucleotides. The Km values were 0.66 mM for ApA, 2.47 mM for (Ap)2A, and 3.03 mM for (Ap)3A. The enzyme hydrolyzed oligodeoxyribonucleotides as well as oligoribonucleotides. The oligoribonucleotides bearing a phosphate residue at the 3' end were not hydrolyzed by the enzyme. The enzyme hydrolyzed the oligoribonucleotides exonucleolytically from the 3' to 5' end. Thus the PDase of F. velutipes is considered to function in vivo as an oligonucleotidase (EC 3.1.13.3), which efficiently converts oligonucleotides to 5'-mononucleotides in the cell.

Chromatography↗

Evaluation of the anti-inflammatory activity of zhankuic acids isolated from the fruiting bodies of Antrodia camphorata.

We have previously shown that a concentrated ethanol extract of the fruiting bodies of Antrodia camphorata exhibited immunomodulating effects in human leukocytes and fourteen compounds including zhankuic acids A, B, C, and antcin K were identified in the extract. In this study, an acute cellular model in isolated peripheral human neutrophils was established to elucidate the anti-inflammatory effects of these compounds. Reactive oxygen species (ROS) production and firm adhesion by neutrophils display two important responses during inflammation. To evaluate whether these compounds could prevent inflammatory responses by neutrophils, their effects on N-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol 12-myristate-13-acetate (PMA)-activated peripheral human neutrophils were examined. Pretreatment with 1 - 25 microM of zhankuic acids A, B, C, or antcin K concentration-dependently diminished fMLP- or PMA-induced ROS production, as measured by a lucigenin-amplified chemiluminescence, with IC (50) (microM) around 5 - 20 microM. Zhankuic acids A, B, C, or antcin K also effectively inhibited the fMLP- or PMA-induced firm adhesion without interfering with the up-expression of surface Mac-1 (CD11b/CD18), a beta2 integrin mediating the firm adhesion of neutrophils to endothelium. The anti-inflammatory actions of these drugs were not due to cytotoxic effects because no significant difference in cell viability was observed compared to vehicle control. These data suggest that inhibition of both ROS production and firm adhesion by neutrophils has no significant cytotoxic effect that could give these drugs the potential to be anti-inflammatory agents for the clinical treatment.

Anti-Inflammatory Agents, Non-Steroidal↗

Antibacterial constituents from fruit bodies of ascomyce Bulgaria inquinans.

Two ergosterins and two triterpenoids were isolated from the dried fruit bodies of Ascomyce Bulgaria inquinans. By means of chemical (hydrolysis) and spectroscopic methods (NMR, El-MS), their structures were established as betuinic acid (1), cerevisterol (2), (24R)ergosta-7, 22E-diene-3beta, 5alpha, 6beta-triol-3-O-palmitate (3) and ursolic acid (4). Compound 3 is a new compound.

Anti-Bacterial Agents↗

Clitocypin, a new type of cysteine proteinase inhibitor from fruit bodies of mushroom clitocybe nebularis.

A novel inhibitor of cysteine proteinases has been isolated from fruit bodies of a mushroom Clitocybe nebularis. The inhibitor was purified to homogeneity by affinity chromatography and gel filtration, followed by reverse-phase high pressure liquid chromatography. The active inhibitor has an apparent molecular mass of about 34 kDa by gel filtration and by SDS-polyacrylamide gel electrophoresis without prior boiling of the sample. Boiling in 2.5% SDS or incubation in 6 m guanidine hydrochloride resulted in a single band of 17 kDa, indicating homodimer composition with no intersubunit disulfide bonds. The inhibitor in nondenaturing buffer is resistant to boiling in water, retaining its activity and dimer composition. The mushroom protein is a tight binding inhibitor of papain (K(i) = 0.59 nm), cathepsin L (K(i) = 0.41 nm), cathepsin B (K(i) = 0.48 micrometer), and bromelain (K(i) = 0.16 micrometer) but is inactive toward cathepsin H, trypsin, and pepsin. Its isoelectric point is 4.4, and sugar analysis indicates the absence of carbohydrate. A single protein sequence of 150 amino acids, containing no cysteine or methionine residues, was obtained by amino acid sequencing. The calculated molecular mass of 16854 Da corresponds well with the value obtained by mass spectrometry. A major part of this sequence was verified by molecular cloning. The monomer sequence is clearly devoid of typical cystatin structure elements and has no similarity to any other known cysteine proteinase inhibitors but bears some similarity to a lectin-like family of proteins from mushrooms. The inhibitor, which is present in at least two other members of the Clitocybe genus, has been named clitocypin (Clitocybe cysteine proteinase inhibitor).

Agaricus↗

Important role of fungal intracellular laccase for melanin synthesis: purification and characterization of an intracellular laccase from Lentinula edodes fruit bodies.

A laccase (EC 1.10.3.2) was isolated from the fully browned gills of Lentinula edodes fruit bodies. The enzyme was purified to a homogeneous preparation using hydrophobic, cation-exchange and size-exclusion chromatography. SDS-PAGE analysis showed the purified laccase, Lcc 2, to be a monomeric protein of 58.0 kDa. The enzyme had an isoelectric point of around pH 6.9. The optimum pH for enzyme activity was around 3.0 against 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)diammonium salt (ABTS), and it was most active at 40 degrees C and stable up to 50 degrees C. The enzyme contained 8.6 % carbohydrate and some copper atoms. The enzyme oxidized ABTS, p-phenylenediamine, pyrogallol, guaiacol, 2,6-dimethoxyphenol, catechol and ferulic acid, but not veratryl alcohol and tyrosine. Beta-(3,4-dihydroxyphenyl)alanine (L-DOPA), which was not oxidized by a laccase previously reported from the culture filtrate of L. edodes, was also oxidized by Lcc 2, and the oxidative product of L-dopa was identified as L-DOPA quinone by HPLC analysis. Lcc 2 was able to oxidize phenolic compounds extracted from fresh gills to brown-coloured products, suggesting a role for laccase in melanin synthesis in this strain.

Hydrogen-Ion Concentration↗

Antitumor activity of a polysaccharide fraction extracted from cultured fruiting bodies of Grifola frondosa.

Antitumor activity of a polysaccharide fraction (GF-1) extracted from cultured fruiting bodies of a fungus, Grifola frondosa, was examined on allogeneic and syngeneic tumors in mice. GF-1 had a marked inhibitory activity against the growth of subcutaneously (s.c.) inoculated Sarcoma 180 by the intraperitoneally (i.p.) injection at 0.5-5.0 mg/mouse for 10 successive days. A significant antitumor activity was also observed when GF-1 at 4.0 mg/mouse was i.p. injected successively on days +1-+5, +7-+11, +14-+18 or +21-+25 if the tumor cells were inoculated s.c. on day 0. Similar results were obtained by a single i.p. injection of GF-1 at 2.0 mg/mouse on day +1, +7, +14 or +21. When GF-1 was injected i.p., intravenously (i.v.) or intratumorally (i.t.), all of them showed an equivalent level of higher inhibitory activity (inhibition ratio; over 90%). However, the oral (p.o.) administration was not effective. The pretreatment of mice with GF-1 at 2.0 or 4.0 mg/mouse for 5 times before tumor inoculation did not show a significant antitumor activity. In addition, GF-1 administered i.p. at varying times before and/or after the tumor inoculation showed no antitumor activity against ascites form of Sarcoma 180. In the syngeneic systems, GF-1 exhibited an antitumor activity against solid form of Meth A fibrosarcoma in BALB/c mice and MM46 carcinoma in C3H/He mice.

Animals↗