scholarly journals Characterization and Optimization of In Vitro Assay Conditions for (1,3).BETA.-Glucan Synthase Activity from Aspergillus fumigatus and Candida albicans for Enzyme Inhibition Screening.

1998 ◽  
Vol 51 (7) ◽  
pp. 665-675 ◽  
Author(s):  
REBECCA L. WOOD ◽  
TAMARA K. MILLER ◽  
AMY WRIGHT ◽  
PETER MCCARTHY ◽  
CATHY S. TAFT ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Aspergillus Fumigatus ◽  
Enzyme Inhibition ◽  
In Vitro Assay ◽  
Beta Glucan ◽  
Vitro Assay ◽  
Glucan Synthase ◽  
Assay Conditions

scholarly journals Cryptococcus neoformans Resistance to Echinocandins: (1,3)β-Glucan Synthase Activity Is Sensitive to Echinocandins

2005 ◽  
Vol 49 (7) ◽  
pp. 2851-2856 ◽  
Author(s):  
Marybeth A. Maligie ◽  
Claude P. Selitrennikoff
Keyword(s):  
Cryptococcus Neoformans ◽  
Specific Activity ◽  
In Vitro Assay ◽  
Carbohydrate Component ◽  
Phenylmethylsulfonyl Fluoride ◽  
Fungal Cell ◽  
Vitro Assay ◽  
Glucan Synthase ◽  
Assay Conditions

ABSTRACT (1,3)β-d-Glucan synthase (EC 2.4.1.34. UDP-glucose: 1,3-β-d-glucan 3-β-glucosyltransferase) uses UDP-glucose as substrate and catalyzes the polymerization of glucose ([1,3]-β-linkages) to form the major carbohydrate component of the fungal cell wall. We have optimized in vitro assay conditions for (1,3)β-glucan synthase activity from Cryptococcus neoformans. Cells lysed in 50 mM Tris, pH 7.75, containing 20% glycerol, 2 mM NaF, 1 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, 5 mM MgCl2, 0.1% protease and phosphatase inhibitor cocktails, and 60 μM GTPγS produced maximum specific activity in vitro. We tested in vitro C. neoformans (1,3)β-glucan synthase activity against the (1,3)β-glucan synthase inhibitors, caspofungin and cilofungin, and have determined that (1,3)β-glucan synthase activity is very sensitive (apparent Ki of 0.17 ± 0.02 μM and 22 ± 5.7 μM, respectively) to these echinocandins. Taken together with high MICs for C. neoformans (caspofungin MIC, 16 μg/ml; cilofungin MIC, 64 μg/ml), our results indicate that C. neoformans is resistant to caspofungin and cilofungin by a mechanism(s) unrelated to (1,3)β-glucan synthase resistance.

scholarly journals A high throughput in vitro assay for fungal (1,3).BETA.-glucan synthase inhibitors.

1994 ◽  
Vol 47 (9) ◽  
pp. 1001-1009 ◽  
Author(s):  
CATHY S. TAFT ◽  
CAROL S. ENDERLIN ◽  
CLAUDE P. SELITRENNIKOFF
Keyword(s):  
High Throughput ◽  
In Vitro Assay ◽  
Beta Glucan ◽  
Vitro Assay ◽  
Glucan Synthase

Glucuronidation of thyroid hormone in rat liver: effects of in vivo treatment with microsomal enzyme inducers and in vitro assay conditions.

Endocrinology ◽  
1993 ◽  
Vol 133 (5) ◽  
pp. 2177-2186 ◽  
Author(s):  
T J Visser ◽  
E Kaptein ◽  
H van Toor ◽  
J A van Raaij ◽  
K J van den Berg ◽  
...  
Keyword(s):  
Thyroid Hormone ◽  
Rat Liver ◽  
In Vitro Assay ◽  
Microsomal Enzyme ◽  
Vitro Assay ◽  
In Vivo Treatment ◽  
Microsomal Enzyme Inducers ◽  
Assay Conditions

Phosphorylation of the neuronal protein kinase C substrate B-50: In vitro assay conditions alter sensitivity to ACTH

1988 ◽  
Vol 13 (4) ◽  
pp. 343-348 ◽  
Author(s):  
Vincent J. Aloyo ◽  
Hindrik Zwiers ◽  
Pierre N. E. De Graan ◽  
Willem Hendrik Gispen
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
In Vitro Assay ◽  
Vitro Assay ◽  
Neuronal Protein ◽  
Kinase C ◽  
Assay Conditions

In vitro binding of Candida albicans yeast cells to human fibronectin

1984 ◽  
Vol 30 (2) ◽  
pp. 221-227 ◽  
Author(s):  
Katherine G. Skerl ◽  
Richard A. Calderone ◽  
Esther Segal ◽  
T. Sreevalsan ◽  
W. Michael Scheld
Keyword(s):  
Candida Albicans ◽  
Epithelial Cells ◽  
Mammalian Cells ◽  
Fluorescent Antibody ◽  
In Vitro Assay ◽  
Yeast Cells ◽  
Vitro Assay ◽  
Vaginal Epithelial Cells ◽  
Human Fibronectin

The binding of Candida albicans yeast cells to human fibronectin (Fn), a major glycoprotein of mammalian cells, was studied using an in vitro assay. Adherence was quantitated in microtiter dishes coated with Fn to which radiolabeled yeast cells were added. Under optimum conditions of the assay, i.e., 1 mM CaCl2 and 70 μg Fn protein, approximately 40% of the radiolabeled yeast cells adhered to the Fn. Adherence to Fn was greater at 30 °C than at 4 °C and was greater with viable yeast cells than with heat-killed cells. Candida albicans (two strains) and C. tropicalis adhered to Fn to a greater extent than C. pseudotropicalis, C. krusei, or Saccharomyces cerevisiae. Pretreatment of C. albicans with chymotrypsin, pronase, or papain, but not pepsin, decreased adherence to Fn. Blocking experiments using mannan, sugars, or amino sugars were carried out by preabsorbing the Fn with each of the above-mentioned compounds. Candida mannan blocked adherence of C. albicans to Fn. The mannan effect was dose dependent. However, adherence of C. albicans to Fn was not significantly reduced by mannose, glucose, or several other sugars. The role of FN as a receptor for the binding of C. albicans yeast cells to buccal and vaginal epithelial cells was investigated also using an in vitro assay. We determined, using indirect fluorescent antibody techniques, that both buccal and vaginal epithelial cells possessed Fn. In addition, yeast cells, when pretreated with Fn, showed reduced adherence with buccal and vaginal cells when compared with nontreated cells. These studies may indicate a role for Fn in the adherence of C. albicans to buccal and vaginal epithelial cells.

A novel in vitro assay for assessing efficacy and toxicity of antifungals using human leukaemic cells infected with Candida albicans

2015 ◽  
Vol 119 (1) ◽  
pp. 177-187 ◽  
Author(s):  
J. Boros-Majewska ◽  
Ł. Turczyk ◽  
X. Wei ◽  
S. Milewski ◽  
D.W. Williams
Keyword(s):  
Candida Albicans ◽  
In Vitro Assay ◽  
Vitro Assay ◽  
Leukaemic Cells
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