A convenient method for permeabilizing the fungus Cephalosporium acremonium

Felix, Hansruedi; Nüesch, J.; Wehrli, W.

doi:10.1016/0003-2697(80)90240-7

Cited by 14 publications

(10 citation statements)

References 21 publications

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“…Kuhberger et are not viable and allow the assessment of macromolecular synthesis in the absence of a barrier to low-molecular-weight compounds. This system has been developed to study DNA synthesis (41) and has more recently been used to study DNA transcription (18). We …”

Section: Discussionmentioning

confidence: 99%

“…The use of supernatants derived from 30,000-x -g centrifugation of disrupted bacteria (S30 fractions) as a cell-free protein synthesis system yielded very low incorporation of [14C]valine into trichloroacetic acid (TCA)-precipitable material in H. influenzae. Bacteria made permeable to small macromolecules by brief treatment with ether have been used previously to study DNA and RNA synthesis (18,41). We Media.…”

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confidence: 99%

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Effect of tobramycin on protein synthesis in 2-deoxystreptamine aminoglycoside-resistant clinical isolates of Haemophilus influenzae

Levy¹,

Burns²,

Mendelman³

et al. 1986

Antimicrob Agents Chemother

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Clinical isolates of Haemophilus influenzae resistant to a broad range of 2-deoxystreptamine aminoglycosides (2-DAM) were studied. The gene responsible for resistance could be mobilized by transformation into a 2-DAM susceptible laboratory strain of H. influenzae, enabling isogenic comparisons. The transformants had the same resistance phenotype as the parental strains. There was close linkage between 2-DAM resistance and streptomycin resistance, a chromosomal marker, but weak linkage between 2-DAM and erythromycin resistance. Resistant transformants exhibited a decreased accumulation of gentamicin due to the absence of the rapid, energy-dependent phase of uptake. Resistance was not through metabolic inactivation of the antibiotic; no aminoglycoside-acetylating, -adenylylating, or -phosphorylating activity was detected in the wild-type strains or in the 2-DAM-resistant transformants. Protein synthesis in 2-DAM-susceptible H. influenzae strains increased in the presence of low (1 ,ug/ml) and moderate (50 ,ug/ml) concentrations of tobramycin. With higher concentrations (100 and 500 ,ug/ml), protein synthesis was progressively inhibited. In contrast, protein synthesis in 2-DAM-resistant clinical isolates and transformants was inhibited by 1 ,ug of tobramycin per ml, and inhibition increased with higher drug concentrations. Since the stimulating effect of low concentrations of tobramycin in susceptible H. influenzae strains is probably due to misreading, these findings suggest that 2-DAM-resistant strains of H. influenzae have reduced sensitivity to misreading, indicating that altered ribosomes are responsible for the resistance.Although its pathogenic role has not been defined with certainty, nontypable Haemophilus influenzae can be cultured in high densities from the bronchial secretions of patients with cystic fibrosis (CF) or chronic bronchitis (21,26,33,38). With the use of selective culture media, this organism frequently can be recovered in the presence of other pathogenic organisms, such as members of the Enterobacteriaceae, Pseudomonas aeruginosa, Streptococcus pneumoniae, and Staphylococcus aureus (33,45). When patients with CF harbor P. aeruginosa and H. influenzae in similar densities in their bronchial secretions, the former organism is generally considered the main pathogen, and antimicrobial therapy that is effective against P. aeruginosa (i.e., an antipseudomonal penicillin and a 2-deoxystreptamine aminoglycoside [2-DAM]) is administered. The diffusion of these antibiotics into the bronchial secretions is poor, and their bioactivity in this environment is reduced; antipseudomonal penicillins are inactivated by P-lactamases (15), and aminoglycosides are bound to DNA from lysed cells (24,40) and antagonized by the low pH (3) and the ionic content of purulent bronchial secretions (24). Thus, high densities of H. influenzae in the bronchial secretions may be exposed for prolonged periods to subinhibitory concentrations of 2-DAM.We hypothesized that this situation could favor the emergence of resistance. ...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Effect of tobramycin on protein synthesis in 2-deoxystreptamine aminoglycoside-resistant clinical isolates of Haemophilus influenzae

Levy¹,

Burns²,

Mendelman³

et al. 1986

Antimicrob Agents Chemother

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“…Two enzyme systems, hexokinase/glucose 6-phosphate dehydrogenase and RNA polymerases, have been assayed in ether-treated cells of the fungus Cephalosporium acremonium. Furthermore, in a crude cell extract of C. acremonium no RNA synthesis was detected [1]. Furthermore, in a crude cell extract of C. acremonium no RNA synthesis was detected [1].…”

Section: Introductionmentioning

confidence: 91%

“…The advantages of using a permeabilised system over cell extracts/lysates include (a) quick preparation (necessary for the measurement of RNA synthesis); (b) some reactions are not easily measured in crude extracts [1]; (c) warming up of the sample during homogenisation or sonication, leading to enzyme destruction is avoided; (d) activity of degrading enzymes such as proteases or phosphatases is reduced [2]. The advantages of using a permeabilised system over cell extracts/lysates include (a) quick preparation (necessary for the measurement of RNA synthesis); (b) some reactions are not easily measured in crude extracts [1]; (c) warming up of the sample during homogenisation or sonication, leading to enzyme destruction is avoided; (d) activity of degrading enzymes such as proteases or phosphatases is reduced [2].…”

Section: Introductionmentioning

confidence: 99%

Ultrastructure of permeabilised cells of Escherichia coli and Cephalosporium acremonium

Hobot¹

1982

FEMS Microbiology Letters

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“…Permeability issues in gram-negative bacteria have been recently discussed by Chen [16]. Apart from that, a number of chemical and physical treatment methods have been described to permeabilize yeast cells [17].…”

Section: Introductionmentioning

confidence: 99%

Effect of Cultural Conditions and Media Constituents on Production of Penicillin V Acylase and CTAB Treatment to Enhance Whole-Cell Enzyme Activity of Rhodotorula aurantiaca (NCIM 3425)

Kumar

Singh

Poddar

et al. 2008

Appl Biochem Biotechnol

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Penicillin V acylase (PVA) is a pharmaceutically important enzyme as it plays a vital role in the manufacture of semi-synthetic beta-lactam antibiotics. Rhodotorula aurantiaca (NCIM 3425) produced high levels of intracellular penicillin V acylase after 18 h at pH 8.0 and temperature 27 degrees C. Fructose was the best carbon source for PVA production, whereas tryptone was the best nitrogen source to produce the enzyme up to 170 and 1,088 IU/l of culture, respectively. Additionally, the cell-bound PVA activity was enhanced on treatment with cationic detergent. Whole-cell activity was found to be doubled (204%) on treatment of 0.01 g dry weight of cells with 50 microg/ml solution of N-cetyl-N,N,N-trimethylammoniumbromide at pH 8.0 for 1 h at room temperature. Atomic force microscopy images of permeabilized cells show perturbation in the cell wall and offer first-ever visual illustration of surface structure modifications that occur during permeabilization of R. aurantiaca cells leading to enhancement in activity of intracellular enzyme.

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A convenient method for permeabilizing the fungus Cephalosporium acremonium

Cited by 14 publications

References 21 publications

Effect of tobramycin on protein synthesis in 2-deoxystreptamine aminoglycoside-resistant clinical isolates of Haemophilus influenzae

Effect of tobramycin on protein synthesis in 2-deoxystreptamine aminoglycoside-resistant clinical isolates of Haemophilus influenzae

Ultrastructure of permeabilised cells of Escherichia coli and Cephalosporium acremonium

Effect of Cultural Conditions and Media Constituents on Production of Penicillin V Acylase and CTAB Treatment to Enhance Whole-Cell Enzyme Activity of Rhodotorula aurantiaca (NCIM 3425)

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