SEARCH · PubMed Health
Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.
Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
A taxonomic study of specimens of Bothriocephalus from eels ( Anguilla spp.) in Japan has demonstrated the occurrence of two species, B. claviceps (Goeze, 1782) and B. japonicus Yamaguti, 1934. The former species is a parasite of eels ( A. anguilla and A. rostrata ) in the Holarctic Region and was recently reported from A. marmorata in Japan. The conspecificity of tapeworms newly found in an eel ( A. ? japonica ) from Lake Biwa, central Japan, with B. claviceps has been confirmed by the great similarity of their ITS-2 gene sequences (similarity 95.3% and 95.2%). However, the sequences of worms identified as B. claviceps from A. marmorata differed considerably from those of B. claviceps from two populations of A. anguilla from Europe and the above-mentioned one from Japan (similarity 66.3%, 67.1% and 65.1 %, respectively), thus indicating that the former cestodes may have been misidentified. This assumption was confirmed by morphological evaluation of a voucher specimen from A. marmorata. The morphology of this cestode, as well as those from A. japonica from two localities in Japan (Lakes Biwa and Suwa), indicates their conspecificity with B. japonicus. The validity of this taxon has been confirmed on the basis of a re-examination of the type-specimens. The two taxa, B. japonicus and B. claviceps, differ from each other in the shape and length of the scolex (619-730 microm in B. japonicus versus 1,180-2,100 microm in B. claviceps ), the relative position of the cirro-vaginal and uterine pores (opposite each other in relation to the median line of the body in B. japonicus versus tandem or slightly offset along the median line in the latter species), and the size of the eggs (41-52 x 28-35 microm in B. japonicus versus 50-70 x 31-43 microm in B. claviceps ).
The production of toxic ergopeptine alkaloids by the fungi Claviceps purpurea and Neotyphodium coenophialum involves the activity of one or more nonribosomal peptide synthetases. Claviceps purpurea and N. coenophialum each have several different peptide synthetase genes, fragments of which have been cloned previously. An additional Claviceps purpurea peptide synthetase gene was cloned by hydridization with one of the N. coenophialum peptide synthetase gene fragments. We detected the presence of mRNA from the peptide synthetase genes in cultures of different ages grown under conditions favorable or unfavorable for ergopeptine production. All four peptide synthetase genes from Claviceps purpurea were transcribed under at least some of the experimental conditions. Transcripts from three of the four genes were detected under conditions consistent with their potential involvement in ergopeptine biosynthesis. All three peptide synthetase genes previously identified in N. coenophialum were transcribed during symbiotic growth of this fungus with tall fescue, as well as ergopeptine-producing cultures. The data show that all of the peptide synthetase genes are transcribed, that one of the peptide synthetase genes is dissociated from ergopeptine biosynthesis, and, as a result, prioritize the remaining genes for functional analyses by transformation-mediated gene disruption.
Metabolic pattern of mycelial Claviceps paspali seed cultures during the submerged cultivation was established. By comparing it with conidial and mycelial Claviceps purpurea strains it was found that the biosynthesis of RNA, DNA, and proteins followed a similar course in all Claviceps strains, so the fall of RNA content in mycelium may be considered a general biochemical indicator for optimally developed inoculum. But, two different patterns of carbohydrate and lipid metabolism were observed one for conidial and one for mycelial strains.
A homologous transformation system was developed for the phytopathogenic fungus Claviceps purpurea. Orotidine-5'-monophosphate decarboxylase (OMPD)-deficient mutants were obtained by UV mutagenesis and selection for resistance against 5-fluoroorotate. These mutants could be complemented well by the corresponding genes of Aspergillus niger (pyrA) and Neurospora crassa (pyr4), yielding significantly higher transformation rates (and lower copy numbers per transformant) than the phleomycin resistance system. The homologous OMPD gene was isolated from a lambda genomic library by heterologous hybridization with the pyr4 gene of N. crassa, identified by complementation of Aspergillus and Claviceps mutants, and used to confirm homologous integration in Claviceps. The pyr transformation system also proved to be very efficient in cotransformation experiments using the bacterial beta-glucuronidase gene (uidA) as a reporter gene, which was also efficiently expressed during the parasitic cycle: honeydew produced by plants infected with pyr/uidA cotransformants was shown to contain significant levels of beta-glucuronidase activity.
A homokaryotic, clavine alkaloid-producing strain of ergot, Claviceps sp. strain SD 58, was used in an attempt to demonstrate parasexuality. Genetically marked auxotrophic strains were produced by mutation with N-methyl-N'-nitro-N-nitrosoguanidine. Protoplast fusion of pairs of unlike doubly auxotrophic strains and isolation of stable prototrophic fusion products were carried out. By growth of the fusion products on complete medium, selective pressure for prototrophy was removed and auxotrophic segregants were allowed to form. Analysis of these and recovery of segregants with nonleaky, non-parent-type combinations of auxotrophic characteristics has provided strong evidence that a parasexual cycle can function in Claviceps sp. strain SD 58. Preliminary work suggests that the genetics of ergot might be studied by mitotic analysis and that protoplast fusion and selection procedures might be useful for the enhancement of favorable characteristics in Claviceps strains.
A three-enzyme complex containing anthranilate synthetase, phosphoribosyl anthranilate isomerase and indole-3-glycerol phosphate synthetase was partially purified from Claviceps spec., strain SD 58. The anthranilate synthetase activity of the enzyme complex was quite unstable unless glutamine, MgCl2, TRIS and, most importantly, glycerol were included in the extraction buffer. The three-enzyme complex showed a molecular weight of 400,000 when estimated using Sephadex gel filtration, and a molecular weight of 200,000 when using sucrose density gradient centrifugation. At least two bands of anthranilate synthetase were detected on disc gel electrophoresis. An enzyme complex containing phosphoribosyl anthranilate synthetase and indoleglycerol phosphate synthetase, but no anthranilate synthetase, was isolated from Claviceps. This enzyme complex had an apparent molecular weight of 165,000 as estimated by sucrose gradient centrifugation. Anthranilate synthetase is inhibited by L-tryptophan and elymoclavine, the terminal ergot alkaloid produced by this strain of Claviceps. No differences could be detected between the enzyme complexes isolated from 2-day-old growing mycelia and from 6-day-old alkaloid-producing mycelia of the organism.
Tryptophan serves as a precursor for the biosynthesis of alkaloids in the ergot fungus, Claviceps purpurea (Fries) Tulasne, and also is believed to act as an inducer of the enzymes necessary for alkaloid production. The characteristics of the transport system responsible for the accumulation of tryptophan in ergot mycelium were investigated, with the goal of clarifying the complex relationships among tryptophan uptake, size of the free intracellular pool of tryptophan, and alkaloid production. The characteristics of tryptophan uptake were studied by pulse feeding radioactively labeled tryptophan to cultures of Claviceps species, strain SD-58, which represented a variety of ages and nutritional states. Tryptophan accumulation in strain SD-58 is mediated by an energy-requiring system which exhibits specificity for neutral aromatic and aliphatic l-amino acids, is pH and temperature dependent, and shows saturation at high substrate concentrations. Tryptophan transport is a function of the intracellular concentration of free tryptophan, the nitrogen deficiency of the mycelium, the rate of growth, and alkaloid production, which were measured in Claviceps strain SD-58 growth in several culture media, some of which promoted alkaloid production and some of which did not. The results indicate that the initial velocity of tryptophan transport is not directly related to alkaloid production.
Two strains (pepty 695 and pur 221) of Claviceps purpurea have been used to study the dependence of proline turnover on incubation time (1, 3, 6, and 12 hours) under different culture conditions (synthetic saccharose-citrate medium NL 720 and complex wort medium M 107) by means of tracer technique. In the saprophytically ergotoxine producing strain pepty 695 the proline is utilized in the protein and alkaloid biosyntheses. On the other hand, under non-producing conditions (M 10) the radioactivity of proline-14C is incorporated into many fractions and finally into CO2. In submerged culture (NL 720) the Claviceps strain pur 221 which does not produce ergolines uses proline only to a small extent in the protein biosynthesis, while most of this amino acid is not metabolized. The extent of uptake of proline is not correlated with the alkaloid synthesis. For comparison the turnover of L-glutamate-14C and D,L-ornithine-1-14C by strain pepty 695 cultivated in the NL 720 medium has been studied.
The mutant strain Claviceps purpurea 244 forming hyphae composed mainly from sclerotium-like cells was found to sporulate both in liquid and solid media, particularly in the form of terminal chlamydospores (4.0 x 6.5 micrometer). Chlamydospores produced during submerged cultivation germinated, new chlamydospores being formed directly from germ tubes, or, occasionally, conidia (the so-called microcycle) or new vegetative mycelium were formed. The ultrastructures of the chlamydospores and vegetative cells was identical. The cytoplasm was filled with ribosomes and contained lipid inclusions and vacuoles with membrane invaginations. Strain 244 cultivated under submerged conditions produced 150 microgram/ml clavins, with elymoclavin predominating (82%). The parent strain Claviceps pururea 129 only produced chlamydospores on the vegetative mycelium, whereas no microcycle was detected; under submerged conditions it produced mainly agroclavin (85%) at a concentration of 4 mg/ml.
Panicum repens and P. antidotale were found to be infected with Claviceps sp. This is the first report of ergot on P. repens. The pyrenomycete produced abundant sclerotia on the host plants. The sclerotia contained 0.71 and 0.68% alkaloids, respectively, which predominantly consisted of chanoclavine, festuclavine and agroclavine. The infected grasses were possibly mycotoxic. Submerged cultures of Claviceps strain isolated from Panicum spp. produced significant amount of chanoclavine, festuclavine and agroclavine. No pharmaceutically important alkaloid was found in sclerotia or in submerged culture.
Among 18 tested strains of Claviceps spp., 7 produced significant amounts of exocellular polysaccharide (EPS). The maximum production of EPS was found in fermentation broth of Claviceps viridis. The kinetics of growth, substrate consumption, and EPS production in the batch, aerobic, submerged culture of this fungus were investigated in detail. The experimental data were processed by a simple mathematical model describing mass balance of growth, substrate consumption, formation of intermediates, and production of EPS. The parameters of the model were estimated from data obtained in cultivation performed in flasks and two laboratory fermentors of different size. Physiological similarity was obtained during process scale-up in volumetric ratio 1:100. The sugar consumption efficiency (52%) and observed EPS productivity (1.9 kg/m3 per d) were comparable with literature data.
The application of a two-phase fermentation system for the production of ergot peptide alkaloids by Claviceps purpurea is described. Perfluorocarbons (PFC) are used as oxygen vectors in Claviceps fermentation for the first time. In shake-flask cultivations, the inclusion of PFC in the medium brings about a five-fold increase in the total alkaloid production and a six-fold increase in the pharmaceutically important component, ergotamine. This rise cannot be correlated with the concentration of the added PFC and it is thought that the enhancement is due to a combination of factors, including the influence of PFC. Other oxygen vectors, such as several hydrocarbons, prove to be poor oxygen carriers in our study. Cultivations with PFC in a bioreactor are reproducible, the maximum total alkaloid and ergotamine production being attained on the 11th and 9th days, respectively. The relatively lower increase in the total alkaloid production in the bioreactor as compared to the shake-flasks is attributed to the unequal oxygen availability in the reactor. Processes with PFC offer the operational advantage of a five-fold reduction in aeration rate.
A gene (cpd1) coding for the dimethylallyltryptophan synthase (DMATS) that catalyzes the first specific step in the biosynthesis of ergot alkaloids, was cloned from a strain of Claviceps purpurea that produces alkaloids in axenic culture. The derived gene product (CPD1) shows only 70% similarity to the corresponding gene previously isolated from Claviceps strain ATCC 26245, which is likely to be an isolate of C. fusiformis. Therefore, the related cpd1 most probably represents the first C. purpurea gene coding for an enzymatic step of the alkaloid biosynthetic pathway to be cloned. Analysis of the 3'-flanking region of cpd1 revealed a second, closely linked ergot alkaloid biosynthetic gene named cpps1, which codes for a 356-kDa polypeptide showing significant similarity to fungal modular peptide synthetases. The protein contains three amino acid-activating modules, and in the second module a sequence is found which matches that of an internal peptide (17 amino acids in length) obtained from a tryptic digest of lysergyl peptide synthetase 1 (LPS1) of C. purpurea, thus confirming that cpps1 encodes LPS1. LPS1 activates the three amino acids of the peptide portion of ergot peptide alkaloids during D-lysergyl peptide assembly. Chromosome walking revealed the presence of additional genes upstream of cpd1 which are probably also involved in ergot alkaloid biosynthesis: cpox1 probably codes for an FAD-dependent oxidoreductase (which could represent the chanoclavine cyclase), and a second putative oxidoreductase gene, cpox2, is closely linked to it in inverse orientation. RT-PCR experiments confirm that all four genes are expressed under conditions of peptide alkaloid biosynthesis. These results strongly suggest that at least some genes of ergot alkaloid biosynthesis in C. purpurea are clustered, opening the way for a detailed molecular genetic analysis of the pathway.
Hydrophobins are unique fungal extracellular proteins that produce amphipathic films at interfaces, mediate contact to hydrophobic surfaces and are known to be important in phytopathogenicity. In the pathogenic ascomycete Claviceps purpurea, causing ergot disease in grasses and cereals and ergotism in livestock, a gene encoding an extraordinary type of hydrophobin has been detected, which appeared to be induced during alkaloid synthesis in axenic culture of an ergot-alkaloid producing strain of Claviceps (V. Garre and P. Tudzynski, pers. communication; Arntz and Tudzynski, 1997, Curr. Genet. 31, 357-360). To elucidate presence and function of this hydrophobin during infection of rye, the nonradioactive in situ hybridization technique was successfully adapted to the fungal organism and optimized in the pathogenic interaction system. Semithin cryosections proved to be suitable for microscopical gene expression analysis using immune-mediated alkaline-phosphatase staining for detection of digoxigenin-labeled cRNA probes. Specific hybridization of the prepared antisense riboprobe to hydrophobin mRNA was confirmed in nonradioactive Northern blots. While permeabilization by proteinase K had only a minor effect, the inclusion of detergent into the hybridization solutions enhanced specific RNA-RNA hybridization under maximum stringency. Hydrophobin mRNA was found in fungal cells, growing in axenic culture. In the disease cycle, hydrophobin transcripts were localized in abundance during vegetative fructification in conidiophores that actively produced conidia. No signals were observed in sclerotial hyphae during formation of the alkaloid-containing ergots, although they fluoresced intensely during total RNA detection using acridine orange. Notably, in situ hybridization experiments resulted in specific signals during early infection and colonization phases in the external mycelia and in hyphae penetrating the host epidermal layer. The presumed role of the hydrophobin gene product in ergot pathogenicity is discussed with respect to the described spatio-temporal distribution of the hydrophobin transcripts.