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C M Douglas

Publications and source records attributed to C M Douglas.

At least 19 recordsLinked to original sources
PubMed2006

Nasal insertion of St John's wort: an unusual cause of epistaxis.

We report a case of severe unilateral epistaxis requiring surgical arrest of bleeding, via endoscopic sphenopalatine artery ligation and anterior ethmoidal artery ligation. This followed recreational nasal insertion of St John's wort (Hypericum perforatum). Interactions between this substance and prescribed drugs have been described following oral ingestion. However, this unusual case highlights a further, worrying potential rhinological side effect of this substance.

Administration, Intranasal↗
PubMed2005

Anthrax lethal factor inhibition.

The primary virulence factor of Bacillus anthracis is a secreted zinc-dependent metalloprotease toxin known as lethal factor (LF) that is lethal to the host through disruption of signaling pathways, cell destruction, and circulatory shock. Inhibition of this proteolytic-based LF toxemia could be expected to provide therapeutic value in combination with an antibiotic during and immediately after an active anthrax infection. Herein is shown the crystal structure of an intimate complex between a hydroxamate, (2R)-2-[(4-fluoro-3-methylphenyl)sulfonylamino]-N-hydroxy-2-(tetrahydro-2H-pyran-4-yl)acetamide, and LF at the LF-active site. Most importantly, this molecular interaction between the hydroxamate and the LF active site resulted in (i) inhibited LF protease activity in an enzyme assay and protected macrophages against recombinant LF and protective antigen in a cell-based assay, (ii) 100% protection in a lethal mouse toxemia model against recombinant LF and protective antigen, (iii) approximately 50% survival advantage to mice given a lethal challenge of B. anthracis Sterne vegetative cells and to rabbits given a lethal challenge of B. anthracis Ames spores and doubled the mean time to death in those that died in both species, and (iv) 100% protection against B. anthracis spore challenge when used in combination therapy with ciprofloxacin in a rabbit "point of no return" model for which ciprofloxacin alone provided 50% protection. These results indicate that a small molecule, hydroxamate LF inhibitor, as revealed herein, can ameliorate the toxemia characteristic of an active B. anthracis infection and could be a vital adjunct to our ability to combat anthrax.

Animals↗
PubMed2005

Specific substitutions in the echinocandin target Fks1p account for reduced susceptibility of rare laboratory and clinical Candida sp. isolates.

An association between reduced susceptibility to echinocandins and changes in the 1,3-beta-d-glucan synthase (GS) subunit Fks1p was investigated. Specific mutations in fks1 genes from Saccharomyces cerevisiae and Candida albicans mutants are described that are necessary and sufficient for reduced susceptibility to the echinocandin drug caspofungin. One group of amino acid changes in ScFks1p, ScFks2p, and CaFks1p defines a conserved region (Phe 641 to Asp 648 of CaFks1p) in the Fks1 family of proteins. The relationship between several of these fks1 mutations and the phenotype of reduced caspofungin susceptibility was confirmed using site-directed mutagenesis or integrative transformation. Glucan synthase activity from these mutants was less susceptible to caspofungin inhibition, and heterozygous and homozygous Cafks1 C. albicans mutants could be distinguished based on the shape of inhibition curves. The C. albicans mutants were less susceptible to caspofungin than wild-type strains in a murine model of disseminated candidiasis. Five Candida isolates with reduced susceptibility to caspofungin were recovered from three patients enrolled in a clinical trial. Four C. albicans strains showed amino acid changes at Ser 645 of CaFks1p, while a single Candida krusei isolate had a deduced R1361G substitution. The clinical C. albicans mutants were less susceptible to caspofungin in the disseminated candidiasis model, and GS inhibition profiles and DNA sequence analyses were consistent with a homozygous fks1 mutation. Our results indicate that substitutions in the Fks1p subunit of GS are sufficient to confer reduced susceptibility to echinocandins in S. cerevisiae and the pathogens C. albicans and C. krusei.

Amino Acid Substitution↗
PubMed2002

The antifungal echinocandin caspofungin acetate kills growing cells of Aspergillus fumigatus in vitro.

Caspofungin acetate is an antifungal antibiotic that inhibits synthesis of 1,3-beta-D-glucan, an essential component of the fungal cell wall. While caspofungin causes cell death in yeasts and dimorphic fungi such as Candida albicans, its effect on Aspergillus fumigatus is less well understood. We used the fluorescent dyes 5,(6)-carboxyfluorescein diacetate (CFDA) and bis-(1,3-dibutylbarbituric acid) trimethine oxonol (DiBAC), which stain live and dead cells, respectively, to further characterize the antifungal activity of caspofungin. For comparison, compounds whose mode of action was either fungistatic (fluconazole, itraconazole) or fungicidal (amphotericin B) were also evaluated. A correlation between caspofungin-induced loss of viability, decreased CFDA staining, and increased DiBAC staining was established first with C. albicans. For A. fumigatus, caspofungin caused similar dye-staining changes, which were quantified by fluorimetric analysis of stained hyphae grown in a medium that promoted dispersed growth. The minimum concentration of caspofungin required to produce these changes also decreased the level of growth-dependent reduction of the indicator dye Alamar Blue. We observed a differential effect of caspofungin as a function of cell position: 88% of apical cells and 61% of subapical branching cells failed to stain with the viable dye CFDA, but only 24% of subapical cells were unstained. Complementary results were seen with germlings from DiBAC-stained, caspofungin-treated cultures. Extended incubation of A. fumigatus with a single dose of caspofungin affected the same proportion of apical and subapical branching cells for up to 72 h. The dye-staining patterns illustrate that the cells at the active centers for new cell wall synthesis within A. fumigatus hyphae are killed when they are exposed to caspofungin.

Anti-Bacterial Agents↗
PubMed2001

Fungal beta(1,3)-D-glucan synthesis.

The polysaccharide beta(1,3)-D-glucan is a component of the cell wall of many fungi. Synthesis of the linear polymer is catalysed by UDP-glucose beta(1,3)-D-glucan beta(3)-D-glucosyltransferase. Because this enzyme has a key role in fungal cell-wall synthesis, and because many organisms that are responsible for human mycoses, including Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans, produce walls that are rich in beta(1,3)-glucan, it has been and remains the focus of intensive study. From early characterization of the enzymatic activity in Saccharomyces cerevisiae, advances have been made in purification of the enzyme, identification of essential subunits and description of regulatory circuitry that controls expression and localization of different components of the multisubunit enzyme complex. Progress in each of these areas has been enhanced dramatically by the availability of specific inhibitors of the enzymatic reaction that produces beta(1,3)-glucan. These natural product inhibitors have utility both as tools to dissect the biology of beta(1,3)-glucan synthase and as sources for development of semisynthetic derivatives with clinical utility in treatment of human fungal disease. This review will focus on the biochemistry, genetics and regulation of the enzyme.

Echinocandins↗
PubMed2001

Quantitative PCR assay to measure Aspergillus fumigatus burden in a murine model of disseminated aspergillosis: demonstration of efficacy of caspofungin acetate.

Caspofungin acetate (MK-0991) is an antifungal antibiotic that inhibits the synthesis of 1,3-beta-D-glucan, an essential component of the cell wall of several pathogenic fungi. Caspofungin acetate was recently approved for the treatment of invasive aspergillosis in patients who are refractory to or intolerant of other therapies. The activity of 1,3-beta-D-glucan synthesis inhibitors against Aspergillus fumigatus has been evaluated in animal models of pulmonary or disseminated disease by using prolongation of survival or reduction in tissue CFU as assay endpoints. Because these methods suffer from limited sensitivity or poor correlation with fungal growth, we have developed a quantitative PCR-based (qPCR) (TaqMan) assay to monitor disease progression and measure drug efficacy. A. fumigatus added to naïve, uninfected kidneys as either ungerminated conidia or small germlings yielded a linear qPCR response over at least 4 orders of magnitude. In a murine model of disseminated aspergillosis, a burden of A. fumigatus was detected in each of five different organs at 4 days postinfection by the qPCR assay, and the mean fungal load in these organs was 1.2 to 3.5 log(10) units greater than mean values determined by CFU measurement. When used to monitor disease progression in infected mice, the qPCR assay detected an increase of nearly 4 log(10) conidial equivalents/g of kidney between days 1 and 4 following infection, with a peak fungal burden that coincided with the onset of significant mortality. Traditional CFU methodology detected only a marginal increase in fungal load in the same tissues. In contrast, when mice were infected with Candida albicans, which does not form true mycelia in tissues, quantitation of kidney burden by both qPCR and CFU assays was strongly correlated as the infection progressed. Finally, treatment of mice with induced disseminated aspergillosis with either caspofungin or amphotericin B reduced the A. fumigatus burden in infected kidneys to the limit of detection for the qPCR assay. Because of its much larger dynamic range, the qPCR assay is superior to traditional CFU determination for monitoring the progression of disseminated aspergillosis and evaluating the activity of antifungal antibiotics against A. fumigatus.

Amphotericin B↗
PubMed1999

A glucan synthase FKS1 homolog in cryptococcus neoformans is single copy and encodes an essential function.

Cryptococcal meningitis is a fungal infection, caused by Cryptococcus neoformans, which is prevalent in immunocompromised patient populations. Treatment failures of this disease are emerging in the clinic, usually associated with long-term treatment with existing antifungal agents. The fungal cell wall is an attractive target for drug therapy because the syntheses of cell wall glucan and chitin are processes that are absent in mammalian cells. Echinocandins comprise a class of lipopeptide compounds known to inhibit 1,3-beta-glucan synthesis, and at least two compounds belonging to this class are currently in clinical trials as therapy for life-threatening fungal infections. Studies of Saccharomyces cerevisiae and Candida albicans mutants identify the membrane-spanning subunit of glucan synthase, encoded by the FKS genes, as the molecular target of echinocandins. In vitro, the echinocandins show potent antifungal activity against Candida and Aspergillus species but are much less potent against C. neoformans. In order to examine why C. neoformans cells are less susceptible to echinocandin treatment, we have cloned a homolog of S. cerevisiae FKS1 from C. neoformans. We have developed a generalized method to evaluate the essentiality of genes in Cryptococcus and applied it to the FKS1 gene. The method relies on homologous integrative transformation with a plasmid that can integrate in two orientations, only one of which will disrupt the target gene function. The results of this analysis suggest that the C. neoformans FKS1 gene is essential for viability. The C. neoformans FKS1 sequence is closely related to the FKS1 sequences from other fungal species and appears to be single copy in C. neoformans. Furthermore, amino acid residues known to be critical for echinocandin susceptibility in Saccharomyces are conserved in the C. neoformans FKS1 sequence.

Amino Acid Sequence↗
PubMed1997

Identification of the FKS1 gene of Candida albicans as the essential target of 1,3-beta-D-glucan synthase inhibitors.

Pneumocandins and echinocandins are fungicidal antibiotics, currently in clinical development, that inhibit 1,3-beta-D-glucan synthase (GS) in several human fungal pathogens. We have identified a gene from the diploid organism Candida albicans that encodes a target of these inhibitors. A 2.1-kb portion of this gene, designated CaFKS1, has significant homology to the Saccharomyces cerevisiae FKS1 and FKS2 genes, which encode partially functionally redundant subunits of GS. To evaluate the role of CaFkslp in susceptibility to echinocandins, we disrupted CaFKS1 on one homolog each of the spontaneous pneumocandin-resistant C. albicans mutants CAI4R1, NR2, NR3, and NR4. These mutants had been selected previously on agar plates containing the pneumocandin L-733,560. The clones derived from this transformation were either resistant (Ech[r]) or fully sensitive (Ech[s]) to inhibition by L-733,560 in both liquid broth microdilution and in vitro GS assays. The site of plasmid insertion in the transformants was mapped by Southern blot analysis, using restriction site polymorphisms in the CaFKS1 gene to distinguish between the two alleles (designated CaFKS1h and CaFKS1b). For strains CAI4R1 and NR2, the CaFKS1b allele was disrupted in each Ech(r) transformant; for strain NR4, CaFKS1h was disrupted in each Ech(r) transformant. We conclude that (i) strains CAI4R1, NR2, and NR4 are heterozygous for a dominant or semidominant pneumocandin resistance mutation at CaFKS1, (ii) drug resistance mutations can occur in either CaFKS1 allele, and (iii) CaFks1p is a target of the echinocandins. For transformants of strain NR3, all the clones we analyzed were uniformly Ech(r), and only the CaFKS1h allele, either in disrupted or wild-type form, was detected on genomic Southern blots. We believe gene conversion at the CaFKS1 locus may have produced two Cafks1h alleles that each contain an Ech(r) mutation. Transformants derived from the mutants were analyzed for susceptibility to pneumocandin treatment in a mouse model of disseminated candidiasis. Strains heterozygous for the resistant allele (i.e., C. albicans CAI4R1, NR2, and NR4) were moderately resistant to treatment, while strains without a functional Ech(s) allele (i.e., strain NR3 and derivatives of strain CAI4R1 with the disruption plasmid integrated in the Ech[s] allele) displayed strong in vivo echinocandin resistance. Finally, we were unable to inactivate both alleles at CaFKS1 by two-step integrative disruption, suggesting that CaFks1p is likely to be an essential protein in C. albicans.

Amino Acid Sequence↗
PubMed1996

Characterization of echinocandin-resistant mutants of Candida albicans: genetic, biochemical, and virulence studies.

The pneumocandins are potent antifungal agents of the echinocandin class which are under development for use as broad-spectrum antimycotic therapy. One important consideration for any new therapeutic class for treating serious fungal infections is the potential for drug resistance development. In this study we have isolated and characterized four independent spontaneous Candida albicans mutants resistant to the potent semisynthetic pneumocandin L-733,560. These mutants have many of the properties of FKS1/ETG1 echinocandin-resistant mutants of Saccharomyces cerevisiae, including (i) cross-resistance to other 1,3-beta-D-glucan synthase inhibitors, such as papulacandin and echinocandins, but no change in sensitivity to other antifungal agents; (ii) in vitro glucan synthase activity that is more resistant to pneumocandins than the wild-type parent enzyme; and (iii) semidominant drug resistance in spheroplast fusion strains. The mutants were compared with C. albicans echinocandin-resistant mutants isolated by mutagenesis by L. Beckford and D. Kerridge (mutant M-2) (abstr. PS3.11, in Proceedings of the XI Congress of the International Society for Human and Animal Mycology, Montreal, Canada, 1992) and by A. Cassone, R. E. Mason, and D. Kerridge (mutant CA-2) (Sabouraudia 19:97-110, 1981). All of the strains had resistant enzyme activity in vitro. M-2 grew poorly and had low levels of enzyme activity. In contrast, CA-2 and the spontaneous mutants grew as well as the parents and had normal levels of glucan synthase activity. These results suggest that these resistant mutants may have alterations in glucan synthase. CA-2 was unable to form germ tubes, an ability retained by the spontaneous mutants. The virulence of the spontaneous mutants was unimpaired in a mouse model of disseminated candidiasis, while M-2 and CA-2 were 2 orders of magnitude less virulent than their parent strains. Significantly, mice challenged with the spontaneous mutant CAI4R1 responded therapeutically to lower levels of L-733,560 than would he predicted by the increase in in vitro susceptibility.

Animals↗
PubMed1995

Aerosol and parenteral pneumocandins are effective in a rat model of pulmonary aspergillosis.

The pneumocandins are semisynthetic analogs of echinocandin-like compounds that have shown efficacy in animal models of systemic candidiasis, disseminated aspergillosis, and pneumocystis pneumonia. However, the most common form of Aspergillus infection in susceptible patients is pulmonary aspergillosis, which was not directly tested in the mouse models used in the past. We have evaluated three pneumocandins, L-693,989, L-731,373, and L-733,560, in a rat model of pulmonary aspergillosis. Male Sprague-Dawley rats were treated for 2 weeks with cortisone and tetracycline and fed a low-protein diet before being inoculated via the trachea with 10(6) conidia of Aspergillus fumigatus H11-20. In the absence of drug treatment, the animals developed a progressive, rapidly fatal bronchopneumonia. All three pneumocandins at doses of 5 mg/kg (intraperitoneally [i.p.] every 12 h [q12h]) were effective in delaying mortality in this model. Survival at day 7 postinfection was 20% among controls (n = 10 for all groups), while it was 60, 80, and 90% in groups that were treated with L-693,989, L-731,373, and L-733,560, respectively. In another trial, survival at day 7 postinfection was 25% among controls (n = 8 for all groups); it was 87.5% in a group treated with amphotericin B (0.5 mg/kg i.p. q12h) and was 100% in a group treated with L-733,560 (0.625 mg/kg i.p. q12h). In a separate trial, aerosol L-693,989 administered 2 h before infection (5 mg/kg) delayed mortality. Eight of the 10 animals treated with aerosol L-693,989 survived for 7 days, whereas only 2 of 10 control animals survived. We conclude that the pneumocandins we tested were highly effective in an animal model of pulmonary aspergillosis.

Aerosols↗
PubMed1994

The Saccharomyces cerevisiae FKS1 (ETG1) gene encodes an integral membrane protein which is a subunit of 1,3-beta-D-glucan synthase.

In Saccharomyces cerevisiae, mutations in FKS1 confer hypersensitivity to the immunosuppressants FK506 and cyclosporin A, while mutations in ETG1 confer resistance to the cell-wall-active echinocandins (inhibitors of 1,3-beta-D-glucan synthase) and, in some cases, concomitant hypersensitivity to the chitin synthase inhibitor nikkomycin Z. The FKS1 and ETG1 genes were cloned by complementation of these phenotypes and were found to be identical. Disruption of the gene results in (i) a pronounced slow-growth phenotype, (ii) hypersensitivity to FK506 and cyclosporin A, (iii) a slight increase in sensitivity to echinocandin, and (iv) a significant reduction in 1,3-beta-D-glucan synthase activity in vitro. The nucleotide sequence encodes a 215-kDa polypeptide predicted to be an integral membrane protein with 16 transmembrane helices, consistent with previous observations that the etg1-1 mutation results in echinocandin-resistant glucan synthase activity associated with the nonextractable membrane fraction of the enzyme. These results suggest that FKS1 encodes a subunit of 1,3-beta-D-glucan synthase. The residual activity present in the disruption mutant, the nonessential nature of the gene, and results of Southern blot hybridization analysis point to the existence of a glucan synthase isozyme.

Base Sequence↗
PubMed1994

A Saccharomyces cerevisiae mutant with echinocandin-resistant 1,3-beta-D-glucan synthase.

A novel, potent, semisynthetic pneumocandin, L-733,560, was used to isolate a resistant mutant in Saccharomyces cerevisiae. This compound, like other pneumocandins and echinocandins, inhibits 1,3-beta-D-glucan synthase from Candida albicans (F.A. Bouffard, R.A. Zambias, J. F. Dropinski, J.M. Balkovec, M.L. Hammond, G.K. Abruzzo, K.F. Bartizal, J.A. Marrinan, M. B. Kurtz, D.C. McFadden, K.H. Nollstadt, M.A. Powles, and D.M. Schmatz, J. Med. Chem. 37:222-225, 1994). Glucan synthesis catalyzed by a crude membrane fraction prepared from the S. cerevisiae mutant R560-1C was resistant to inhibition by L-733,560. The nearly 50-fold increase in the 50% inhibitory concentration against glucan synthase was commensurate with the increase in whole-cell resistance. R560-1C was cross-resistant to other inhibitors of C. albicans 1,3-beta-D-glucan synthase (aculeacin A, dihydropapulacandin, and others) but not to compounds with different modes of action. Genetic analysis revealed that enzyme and whole-cell pneumocandin resistance was due to a single mutant gene, designated etg1-1 (echinocandin target gene 1), which was semidominant in heterozygous diploids. The etg1-1 mutation did not confer enhanced ability to metabolize L-733,560 and had no effect on the membrane-bound enzymes chitin synthase I and squalene synthase. Alkali-soluble beta-glucan synthesized by crude microsomes from R560-1C was indistinguishable from the wild-type product. 1,3-beta-D-Glucan synthase activity from R560-1C was fractionated with NaCl and Tergitol NP-40; reconstitution with fractions from wild-type membranes revealed that drug resistance is associated with the insoluble membrane fraction. We propose that the etg1-1 mutant gene encodes a subunit of the 1,3-beta-D-glucan synthase complex.

Anti-Bacterial Agents↗
PubMed1990

Pseudomonas aeruginosa exotoxin A: alterations of biological and biochemical properties resulting from mutation of glutamic acid 553 to aspartic acid.

Glutamic acid 553 of Pseudomonas aeruginosa exotoxin A (ETA) was identified earlier as a putative active-site residue by photoaffinity labeling with NAD. Here ETA-E553D, a cloned form of the toxin in which Glu-553 has been replaced by aspartic acid, was purified from Escherichia coli extracts and characterized. Cytotoxicity of the mutant toxin for mouse L-M cells was less than 1/400,000 that of the wild type. The mutation caused a 3200-fold reduction in NAD:elongation factor 2 ADP-ribosyltransferase activity, as estimated by assays with an active fragment derived from the toxin by digestion with thermolysin. NAD glycohydrolase activity was reduced somewhat less, by a factor of 50, and photoaffinity labeling with NAD by a factor of 2. We detected less than 2-fold change in the values of KM for NAD or elongation factor 2 and no change in KD for NAD, as determined by quenching of protein fluorescence. The drastic reduction of ADP-ribosyltransferase activity therefore results primarily from an effect of the mutation on kcat, implying that Glu-553 plays an important and possibly direct role in catalyzing this reaction. The effects of the E553D mutation are similar to those of the E148D mutation in diphtheria toxin, supporting the notion that these two Glu residues perform the same function in their respective toxins.

ADP Ribose Transferases↗
PubMed1988

Role of protein processing, intracellular trafficking and endocytosis in production of and immunity to yeast killer toxin.

Yeast strains harboring M1-dsRNA and its packaging virus ScV-L secrete a disulfide-linked, heterodimeric toxin which kills sensitive yeast cells by disrupting plasma membrane function. The mature toxin is derived from a precursor (preprotoxin) which undergoes post-translational processing steps during export via the established yeast secretory pathway. Cleavage by both the KEX1 and KEX2 endopeptidases is required for expression of killing activity. The same 1.0 kb open reading frame on M1-dsRNA directs the expression of immunity to toxin. Differentially processed derivatives of protoxin, as well as protoxin itself, have been proposed to serve as mediators of immunity. To understand the mechanisms by which the killing and immunity phenotypes can be derived from a common precursor, we have: 1) studied cellular processes implicated in expression of the phenotypes; and 2) developed a system to produce mutants defective in immunity, killing, or both. In the first approach, the role played by both endocytosis and vesicular traffiking in expression of killing and immunity was examined. Strains defective in endocytosis (end1, end2) or vacuolar protein localization (vpl3, vpl6) were transformed with a plasmid encoding killer toxin under control of the pho5 promoter. When induced by phosphate starvation, both end mutants and all vpl mutants expressed killing activity. Immunity to exogenous toxin, however, was significantly decreased in strains carrying both vpl mutant alleles and in one of the endocytosis mutants (end1]. This suicidal phenotype (rex for resistance expression) has been described previously in M1-containing strains as a leaky phenocopy.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Membrane↗
PubMed1987

Exotoxin A of Pseudomonas aeruginosa: active, cloned toxin is secreted into the periplasmic space of Escherichia coli.

We subcloned the structural gene for exotoxin A (ETA) of Pseudomonas aeruginosa in front of the tac promoter in an Escherichia coli expression vector and studied the intracellular location and properties of the protein product. The E. coli K-12 strain that carried this recombinant plasmid produced an immunoreactive protein that was identical to authentic ETA in size and in cytotoxic and ADP-ribosyl transferase activities per unit of immunoreactive material. The protein was predominantly in the periplasmic fraction; and a mutation in the secA gene blocked secretion, processing, and conversion of the protein to a fully toxic conformation. The results indicate that expression of the ETA gene in E. coli yields native ETA, which is localized within the periplasmic space. This organism may therefore serve as a useful host for studying structure and function in ETA.

ADP Ribose Transferases↗
PubMed1987

Exotoxin A of Pseudomonas aeruginosa: substitution of glutamic acid 553 with aspartic acid drastically reduces toxicity and enzymatic activity.

Glutamic acid 553 of Pseudomonas aeruginosa exotoxin A (ETA) has been identified by photoaffinity labeling as a residue within the NAD binding site (S.F. Carroll and R.J. Collier, J. Biol. Chem. 262:8707-8711, 1987). To explore the function of Glu-553 we used oligonucleotide-directed mutagenesis to replace this residue with Asp in cloned ETA and expressed the mutant gene in Escherichia coli K-12. ADP-ribosylation activity of Asp-553 ETA in cell extracts was about 1,800-fold lower and toxicity for mouse L-M929 fibroblasts was at least 10,000-fold lower than that of the wild-type toxin. Extracts containing Asp-553 ETA inhibited the cytotoxicity of authentic ETA on L-M929 fibroblasts, suggesting that the mutant toxin competes for ETA receptors. The results indicate that Glu-553 is crucial for ADP-ribosylation activity and, consequently, cytotoxicity of ETA. Substitution or deletion of this residue may be a route to new ETA vaccines.

ADP Ribose Transferases↗
PubMed1986

Comparison of histochemical and biochemical assays for oestrogen receptor in breast tumour tissue.

Oestrogen receptors were measured by a histochemical technique in tumour tissue from 45 patients with breast cancer, and the results compared with receptor levels measured by a standard biochemical dextran-charcoal assay. Histochemical measurements were made by incubating frozen sections of tumour tissue with 17-fluoresceinated oestrone, using dithiothreitol to inhibit uptake by low affinity binding sites, and estimating receptor content by the extent of fluorescence seen microscopically. Results from the two assays gave varying estimates of receptor concentration, with 66% of patients classed as receptor-positive by the biochemical assay, and 44% of patients classed as receptor-positive using the histochemical technique. Because of these discrepancies the histochemical assay cannot be considered a direct substitute for biochemical receptor measurements. Progesterone receptor was detected by biochemical measurement in 80% of the subgroup of patients who were positive for oestrogen receptor in both assays. The histochemical assay may thus identify a subset of those patients considered oestrogen receptor-positive by conventional receptor assays, and this group may be more likely to respond to hormonal therapy. However, whether the histochemical method provides a predictive measurement for endocrine responsiveness which is superior to standard progesterone receptor assay remains to be determined.

Adult↗