(1/240) Contaminations occurring in fungal PCR assays.

Successful in vitro amplification of fungal DNA in clinical specimens has been reported recently. In a collaboration among five European centers, the frequency and risk of contamination due to airborne spore inoculation or carryover contamination in fungal PCR were analyzed. The identities of all contaminants were specified by cycle sequencing and GenBank analysis. Twelve of 150 PCR assays that together included over 2,800 samples were found to be contaminated (3.3% of the negative controls were contaminated during the DNA extraction, and 4.7% of the PCR mixtures were contaminated during the amplification process). Contaminants were specified as Aspergillus fumigatus, Saccharomyces cerevisiae, and Acremonium spp. Further analysis showed that commercially available products like zymolyase powder or 10x PCR buffer may contain fungal DNA. In conclusion, the risk of contamination is not higher in fungal PCR assays than in other diagnostic PCR-based assays if general precautions are taken.  (+info)

(2/240) Chemical modification of NADP-isocitrate dehydrogenase from Cephalosporium acremonium evidence of essential histidine and lysine groups at the active site.

NADP-isocitrate dehydrogenase from Cephalosporium acremonium CW-19 has been inactivated by diethyl pyrocarbonate following a first-order process giving a second-order rate constant of 3.0 m-1. s-1 at pH 6.5 and 25 degrees C. The pH-inactivation rate data indicated the participation of a group with a pK value of 6.9. Quantifying the increase in absorbance at 240 nm showed that six histidine residues per subunit were modified during total inactivation, only one of which was essential for catalysis, and substrate protection analysis would seem to indicate its location at the substrate binding site. The enzyme was not inactivated by 5, 5'-dithiobis(2-nitrobenzoate), N-ethylmaleimide or iodoacetate, which would point to the absence of an essential reactive cysteine residue at the active site. Pyridoxal 5'-phosphate reversibly inactivated the enzyme at pH 7.7 and 5 degrees C, with enzyme activity declining to an equilibrium value within 15 min. The remaining activity depended on the modifier concentration up to about 2 mm. The kinetic analysis of inactivation and reactivation rate data is consistent with a reversible two-step inactivation mechanism with formation of a noncovalent enzyme-pyridoxal 5'-phosphate complex prior to Schiff base formation with a probable lysyl residue of the enzyme. The analysis of substrate protection shows the essential residue(s) to be at the active site of the enzyme and probably to be involved in catalysis.  (+info)

(3/240) High performance liquid chromatography of natural products. I. Separation of cephalosporin C derivatives and cephalosporin antibiotics;isolation of cephalosporin C from fermentation broth.

Microbonded propylamine silica with a solvent system containing acetic acid, methanol, acetonitrile and water (2:4:7.5:86.5) is suitable for an efficient separation of mixtures containing several closely related cephem derivatives. The same system with preparative columns was used for the isolation of cephalosporin C directly from the filtered broth of C. acremonium fermentation.  (+info)

(4/240) RIT 2214, a new biosynthetic penicillin produced by a mutant of Cephalosporium acremonium.

A number of lysine-requiring auxotrophs of Cephalosporium acremonium were investigated for incorporation of side-chain precursors and for accumulation of beta-lactam compounds. One of the auxotrophs, Acremonium chrysogenum ATCC 20389, producing cephalosporin C and penicillin N only if grown in media supplemented with DL-alpha-amino-adipic acid (DL-alpha-AAA), was found to use L-S-carboxymethylcysteine (L-CMC) as a side-chain precursor for the synthesis of a new penicillin (RIT 2214). No corresponding cephalosporin was detected. The penicillin present in the culture filtrate, was concentrated by adsorption on activated carbon and successive column chromatography on Amberlite IRA-68 and Amberlite XAD-4. Final purification was achieved by cellulose column chromatography. RIT 2214 was identified as 6-(D)-[(2-amino-2-carboxy)-ethylthio]-acetamido]-penicillanic acid by spectral analysis, bioactivity spectrum, elucidation of side-chain structure and finally by semisynthesis. Its biological properties were also evaluated.  (+info)

(5/240) Fungi from geothermal soils in Yellowstone National Park.

Geothermal soils near Amphitheater Springs in Yellowstone National Park were characterized by high temperatures (up to 70 degrees C), high heavy metal content, low pH values (down to pH 2.7), sparse vegetation, and limited organic carbon. From these soils we cultured 16 fungal species. Two of these species were thermophilic, and six were thermotolerant. We cultured only three of these species from nearby cool (0 to 22 degrees C) soils. Transect studies revealed that higher numbers of CFUs occurred in and below the root zone of the perennial plant Dichanthelium lanuginosum (hot springs panic grass). The dynamics of fungal CFUs in geothermal soil and nearby nongeothermal soil were investigated for 12 months by examining soil cores and in situ mesocosms. For all of the fungal species studied, the temperature of the soil from which the organisms were cultured corresponded with their optimum axenic growth temperature.  (+info)

(6/240) Quantitative assessment of in planta distribution of metabolic activity and gene expression of an endophytic fungus.

Using perennial ryegrass infected with an Acremonium transformant carrying the Escherichia coli beta-D-glucuronidase gene (gusA) (GUS system) under control of a constitutive promoter, we have developed methods for the quantitative extraction of endophyte-associated GUS activity from plant material. Fluorometric assays of these extracts allow quantitative assessment of the distribution of endophyte-associated GUS activity within single plants (tillers) with high resolution. Fluorescence microscopy with the dye Imagene Green can in addition visualize individual GUS-expressing hyphae. Since the transformant expresses the GUS gene constitutively, GUS activity can be used as an indicator of in planta endophyte metabolic activity. Using this approach we found that (i) the concentration of endophyte metabolic activity in plant tissue decreases with increasing plant size, (ii) approximately 70% of endophyte metabolic activity present in a plant is located in the leaf sheaths, (iii) basal-apical gradients and lateral (younger to older tissue) gradients of endophyte metabolic activity exist and (iv) basal-apical gradients are established early in leaf development. Our data suggest that the concentration of endophyte in each part of the plant is regulated so that a predetermined threshold of total endophyte activity per plant is not exceeded and a consistent distribution pattern is maintained.  (+info)

(7/240) Influence of consumption of endophyte-infested tall fescue hay on performance of heifers and lambs.

Two experiments were conducted to evaluate performance and physiological responses of heifers and lambs to Neotyphodium coenophialum-infested tall fescue hay fed under European rearing conditions. Endophyte-free (E-) or 100% endophyte-infested (E+) hay was derived from the same cultivar (cv. Clarine) so that the effect of the endophytic fungus could be clearly separated from a possible cultivar effect. In Exp. 1, starting in June 1996, 20 age- and body weight-paired Holstein dairy heifers were assigned for 97 d to one of two treatments consisting of ad libitum access to either E- or E+ hay, corresponding to 0 and .41 mg/kg ergovaline, respectively. During the experimental period, no significant difference (P>.20) in forage consumption, rectal temperature, or behavioral status of the animals was observed between the two treatments. The E+ diet induced a 10% apparent decrease in ADG and a clear reduction in prolactin (PRL) plasma concentration compared to the E- diet. When animals were all reassigned to a common endophyte-free diet, the E+ group recovered body weight and PRL to levels similar to those in animals fed E- after 7 wk. In Exp. 2, 30 Texel ram lambs were assigned to two treatments consisting of dietary E- or E+ tall fescue hay. The E- and E+ hays were harvested from the same plots as used in Exp. 1 and contained 0 and .96 mg/kg ergovaline, respectively. No effect of the endophyte was found on intake or carcass or testicle weight (P>.20) after the 95-d feeding period. The E+ treatment resulted in a slight reduction in BW at slaughter, mainly explained by a lower ruminal fill (P<.01). In E+ treated animals, prolactin concentrations dropped significantly (P<.001) from d 27. Hay assessment in both experiments showed no difference in chemical composition and IVDMD. The endophytic fungus strongly lowered the palatability of the E+ hay, although there was no effect on intake with heifers (Exp. 1) or with lambs (Exp. 2). The potential of severe heat stress, as expressed by the temperature humidity index, was not high in our experimental conditions, although they were considered rather unusually stressful for the western part of northern Europe. Yet, no economic effect on cattle was observed, in disagreement with results obtained in many previous U.S. studies.  (+info)

(8/240) The fungal CPCR1 protein, which binds specifically to beta-lactam biosynthesis genes, is related to human regulatory factor X transcription factors.

Here we report the isolation and characterization of a novel transcription factor from the cephalosporin C-producing fungus Acremonium chrysogenum. We have identified a protein binding site in the promoter of the beta-lactam biosynthesis gene pcbC, located 418 nucleotides upstream of the translational start. Using the yeast one-hybrid system, we succeeded in isolating a cDNA clone encoding a polypeptide, which binds specifically to the pcbC promoter. The polypeptid shows significant sequence homology to human transcription factors of the regulatory factor X (RFX) family and was designated CPCR1. A high degree of CPCR1 binding specificity was observed in in vivo and in vitro experiments using mutated versions of the DNA binding site. The A. chrysogenum RFX protein CPCR1 recognizes an imperfect palindrome, which resembles binding sites of human RFX transcription factors. One- and two-hybrid experiments with truncated versions of CPCR1 showed that the protein forms a DNA binding homodimer. Nondenaturing electrophoresis revealed that the CPCR1 protein exists in vitro solely in a multimeric, probably dimeric, state. Finally, we isolated a homologue of the cpcR1 gene from the penicillin-producing fungus Penicillium chrysogenum and determined about 60% identical amino acid residues in the DNA binding domain of both fungal RFX proteins, which show an overall amino acid sequence identity of 29%.  (+info)

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