scholarly journals Comparison of D0870, a new triazole antifungal agent, to fluconazole for inhibition of Candida albicans cytochrome P-450 by using in vitro assays.

1996 ◽  
Vol 40 (6) ◽  
pp. 1382-1386 ◽  
Author(s):  
K Venkateswarlu ◽  
D W Denning ◽  
N J Manning ◽  
S L Kelly
Keyword(s):  
Candida Albicans ◽  
Spectrophotometric Assay ◽  
In Vitro Assays ◽  
Type Ii ◽  
Intracellular Accumulation ◽  
Cell Extracts ◽  
Biochemical Comparison ◽  
Cytochrome P 450 ◽  
Differential Affinity

D0870 was 12 to 15 times more active than fluconazole in experiments to determine the MIC for growth arrest for two isolates of Candida albicans. A biochemical comparison of in vitro sterol biosynthesis in cell extracts showed only a twofold superiority of D0870 over fluconazole. A large differentiation (10-fold) in 50% saturating concentrations obtained by examining the binding of the azoles to microsomal P-450 was observed in a type II binding spectrophotometric assay, possibly reflecting the differential affinity for more than one P-450 enzyme. Additional mechanisms besides affinity for the target enzyme sterol 14 alpha-demethylase, such as differential intracellular accumulation of drug, may contribute to the differences in antifungal activity.

Ligand-dependent occupancy of the retinoic acid receptor beta 2 promoter in vivo

1994 ◽  
Vol 14 (12) ◽  
pp. 8191-8201
Author(s):  
A Dey ◽  
S Minucci ◽  
K Ozato
Keyword(s):  
Retinoic Acid ◽  
Dimethyl Sulfate ◽  
Dominant Negative ◽  
In Vitro Assays ◽  
Cell Extracts ◽  
Ec Cells ◽  
Beta 2 ◽  
Responsive Element ◽  
Receptor Beta

Retinoic acid (RA) activates transcription of the RA receptor beta 2 (RAR beta 2) gene in embryonal carcinoma (EC) cells. This activation involves binding of the RAR/retinoid X receptor (RAR/RXR) heterodimer to the RA-responsive element (beta RARE). Dimethyl sulfate-based genomic footprinting was performed to examine occupancy of this promoter in P19 EC cells. No footprint was detected at the beta RARE prior to RA treatment, but a footprint was detected within the first hour of RA treatment. Concomitantly, other elements in the promoter, the cyclic AMP-responsive element and tetradecanoyl phorbol acetate-like-responsive element became footprinted. Footprints at these elements were induced by RA without requiring new protein synthesis and remained for the entire duration of RA treatment but rapidly reversed upon withdrawal of RA. A delayed protection observed at the initiator site was also reversed upon RA withdrawal. The RA-inducible footprint was not due to induction of factors that bind to these element, since in vitro assays showed that these factors are present in P19 cell extracts before RA treatment. Significantly, no RA-induced footprint was observed at any of these elements in P19 cells expressing a dominant negative RXR beta, in which RXR heterodimers are unable to bind to the beta RARE. Results indicate that binding of a liganded heterodimer receptor to the beta RARE is the initial event that allows other elements to gain access to the factors. In accordance, reporter analyses showed that a mutation in the beta RARE, but not those in other elements, abrogates RA activation of the promoter. It is likely that the RAR beta 2 promoter opens in a hierarchically ordered manner, signalled by the occupancy of liganded heterodimers.

scholarly journals Identification and Characterization of the Endocytic Transmembrane Glycoprotein Endo180 as a Novel Collagen Receptor

2003 ◽  
Vol 14 (9) ◽  
pp. 3592-3604 ◽  
Author(s):  
Dirk Wienke ◽  
John R. MacFadyen ◽  
Clare M. Isacke
Keyword(s):  
Physiological Role ◽  
Collagen Matrix ◽  
Mannose Receptor ◽  
In Vitro Assays ◽  
Type Ii ◽  
Urokinase Plasminogen Activator Receptor ◽  
Coated Pits ◽  
Rich Domain ◽  
Fibronectin Type

Endo180, a member of the mannose receptor family, is constitutively recycled between clathrin-coated pits on the cell surface and intracellular endosomes. Its large extracellular domain contains an N-terminal cysteine-rich domain, a single fibronectin type II domain and eight C-type lectin-like domains. The second of these lectin-like domains has been shown to mediate Ca2+-dependent mannose binding. In addition, cross-linking studies have identified Endo180 as a urokinase plasminogen activator receptor–associated protein and this interaction can be blocked by collagen V. Here we demonstrate directly using in vitro assays, cell-based studies and tissue immunohistochemistry that Endo180 binds both to native and denatured collagens and provide evidence that this is mediated by the fibronectin type II domain. In cell culture systems, expression of Endo180 results in the rapid uptake of soluble collagens for delivery to lysosomal degradative compartments. Together with the observed restricted expression of Endo180 in both embryonic and adult tissue, we propose that Endo180 plays a physiological role in mediating collagen matrix remodelling during tissue development and homeostasis and that the observed receptor upregulation in pathological conditions may contribute to disease progression.

scholarly journals Prothioconazole and Prothioconazole-Desthio Activities against Candida albicans Sterol 14-α-Demethylase

2012 ◽  
Vol 79 (5) ◽  
pp. 1639-1645 ◽  
Author(s):  
Josie E. Parker ◽  
Andrew G. S. Warrilow ◽  
Hans J. Cools ◽  
Bart A. Fraaije ◽  
John A. Lucas ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Type Ii ◽  
Inhibitor Binding ◽  
Binding Properties ◽  
Content Type ◽  
Azole Antifungals ◽  
A Cell ◽  
Antifungal Action ◽  
Binding Spectra

ABSTRACTProthioconazole is a new triazolinthione fungicide used in agriculture. We have usedCandida albicansCYP51 (CaCYP51) to investigate thein vitroactivity of prothioconazole and to consider the use of such compounds in the medical arena. Treatment ofC. albicanscells with prothioconazole, prothioconazole-desthio, and voriconazole resulted in CYP51 inhibition, as evidenced by the accumulation of 14α-methylated sterol substrates (lanosterol and eburicol) and the depletion of ergosterol. We then compared the inhibitor binding properties of prothioconazole, prothioconazole-desthio, and voriconazole with CaCYP51. We observed that prothioconazole-desthio and voriconazole bind noncompetitively to CaCYP51 in the expected manner of azole antifungals (with type II inhibitors binding to heme as the sixth ligand), while prothioconazole binds competitively and does not exhibit classic inhibitor binding spectra. Inhibition of CaCYP51 activity in a cell-free assay demonstrated that prothioconazole-desthio is active, whereas prothioconazole does not inhibit CYP51 activity. Extracts fromC. albicansgrown in the presence of prothioconazole were found to contain prothioconazole-desthio. We conclude that the antifungal action of prothioconazole can be attributed to prothioconazole-desthio.

Comparison of Vibrio and Firefly Luciferases as Reporter Gene Systems for Use in Bacteria and Plants

1996 ◽  
Vol 23 (1) ◽  
pp. 75 ◽  
Author(s):  
SR Mudge ◽  
WR Lewis-Henderson ◽  
RG Birch
Keyword(s):  
Gene Expression ◽  
Reporter Gene ◽  
Ccd Camera ◽  
Reporter Gene Expression ◽  
In Vitro Assays ◽  
E Coli ◽  
Cell Extracts ◽  
Cooled Ccd

Luciferase genes from Vibrio harveyi (luxAB) and firefly (luc) were introduced into E. coli, Agrobacteriurn, Arabidopsis and tobacco. Transformed bacteria and plants were quantitatively assayed for luciferase activity using a range of in vitro and in vivo assay conditions. Both lux and luc proved efficient reporter genes in bacteria, although it is important to be aware that the sensitive assays may detect expression due to readthrough from distant promoters. LUX activity was undetectable by liquid nitrogen-cooled CCD camera assays on intact tissues of plants which showed strong luxAB expression by in vitro assays. The decanal substrate for the lux assay was toxic to many plant tissues, and caused chemiluminescence in untransformed Arabidopsis leaves. These are serious limitations to application of the lux system for sensitive, non-toxic assays of reporter gene expression in plants. In contrast, LUC activity was readily detectable in intact tissues of all plants with luc expression detectable by luminometer assays on cell extracts. Image intensities of luc-expressing leaves were commonly two to four orders of magnitude above controls under the CCD camera. Provided adequate penetration of the substrate luciferin is obtained, luc is suitable for applications requiring sensitive, non-toxic assays of reporter gene expression in plants.

scholarly journals Stepwise Reconstitution of Interphase Microtubule Dynamics in Permeabilized Cells and Comparison to Dynamic Mechanisms in Intact Cells

1998 ◽  
Vol 142 (6) ◽  
pp. 1519-1532 ◽  
Author(s):  
Yasmina Saoudi ◽  
Rati Fotedar ◽  
Ariane Abrieu ◽  
Marcel Dorée ◽  
Jürgen Wehland ◽  
...  
Keyword(s):  
Model System ◽  
Microtubule Dynamics ◽  
In Vitro Assays ◽  
Microtubule Depolymerization ◽  
Dynamic Activity ◽  
Intact Cells ◽  
Permeabilized Cells ◽  
Cell Extracts

Microtubules in permeabilized cells are devoid of dynamic activity and are insensitive to depolymerizing drugs such as nocodazole. Using this model system we have established conditions for stepwise reconstitution of microtubule dynamics in permeabilized interphase cells when supplemented with various cell extracts. When permeabilized cells are supplemented with mammalian cell extracts in the presence of protein phosphatase inhibitors, microtubules become sensitive to nocodazole. Depolymerization induced by nocodazole proceeds from microtubule plus ends, whereas microtubule minus ends remain inactive. Such nocodazole-sensitive microtubules do not exhibit subunit turnover. By contrast, when permeabilized cells are supplemented with Xenopus egg extracts, microtubules actively turn over. This involves continuous creation of free microtubule minus ends through microtubule fragmentation. Newly created minus ends apparently serve as sites of microtubule depolymerization, while net microtubule polymerization occurs at microtubule plus ends. We provide evidence that similar microtubule fragmentation and minus end–directed disassembly occur at the whole-cell level in intact cells. These data suggest that microtubule dynamics resembling dynamics observed in vivo can be reconstituted in permeabilized cells. This model system should provide means for in vitro assays to identify molecules important in regulating microtubule dynamics. Furthermore, our data support recent work suggesting that microtubule treadmilling is an important mechanism of microtubule turnover.

Virulent bacteriophage infection of Rhizobium leguminosarum

1972 ◽  
Vol 18 (4) ◽  
pp. 375-384 ◽  
Author(s):  
A. N. Ley ◽  
H. R. Warner ◽  
Phyllis L. Kahn
Keyword(s):  
Molecular Weight ◽  
Rna Synthesis ◽  
Rhizobium Leguminosarum ◽  
Deoxyribonucleic Acid ◽  
In Vitro Assays ◽  
Tail Fiber ◽  
Bacteriophage Infection ◽  
Cell Extracts ◽  
High Level

Bacteriophage 317, which virulently infects Rhizobium leguminosarum, has an eclipse period of 60–70 min and a latent period of 100 min at 30°. Electron micrographs of the phage indicated head, tail, and tail-fiber structural components.Base analysis of phage 317 deoxyribonucleic acid (DNA) indicated the presence of equimolar amounts of adenine and thymine and of guanine and cytosine, which suggests that the DNA is double stranded. The DNA has a molecular weight of 41 × 106 daltons as determined from electron micrographs.The results of 14C-uracil incorporation studies showed that net ribonucleic acid (RNA) synthesis was markedly inhibited after infection. There was a slight stimulation in DNA synthesis after infection as indicated by 14C-thymidine incorporation.The results of in vitro assays of enzymes involved in the biosynthesis of DNA showed that deoxyuridine 5′-triphosphate nucleotidohydrolase (dUTPase) and deoxythymidine 5′-monophosphate kinase (dTMP kinase) increased 50- and 30-fold respectively, after infection. The reason for the increased dUTPase activity is not readily apparent. Phage 317 DNA contains only the standard bases, unlike the DNA of other phages that induce an increase in this enzyme after infection. A high level of deoxythymidine 5′-monophosphatase (dTMPase) was observed in both uninfected and infected crude cell extracts. Further work is necessary to see if similar changes occur in Rhizobium during the establishment of symbiosis with legumes.

scholarly journals A Dictyostelium mutant lacking an F-actin cross-linking protein, the 120-kD gelation factor.

1990 ◽  
Vol 111 (4) ◽  
pp. 1477-1489 ◽  
Author(s):  
M Brink ◽  
G Gerisch ◽  
G Isenberg ◽  
A A Noegel ◽  
J E Segall ◽  
...  
Keyword(s):  
Binding Proteins ◽  
Mutant Cell ◽  
Actin Binding ◽  
In Vitro Assays ◽  
Cell Cortex ◽  
Cross Linking ◽  
Actin Binding Proteins ◽  
Cell Extracts ◽  
Nonmuscle Cells

Actin-binding proteins are known to regulate in vitro the assembly of actin into supramolecular structures, but evidence for their activities in living nonmuscle cells is scarce. Amebae of Dictyostelium discoideum are nonmuscle cells in which mutants defective in several actin-binding proteins have been described. Here we characterize a mutant deficient in the 120-kD gelation factor, one of the most abundant F-actin cross-linking proteins of D. discoideum cells. No F-actin cross-linking activity attributable to the 120-kD protein was detected in mutant cell extracts, and antibodies recognizing different epitopes on the polypeptide showed the entire protein was lacking. Under the conditions used, elimination of the gelation factor did not substantially alter growth, shape, motility, or chemotactic orientation of the cells towards a cAMP source. Aggregates of the mutant developed into fruiting bodies consisting of normally differentiated spores and stalk cells. In cytoskeleton preparations a dense network of actin filaments as typical of the cell cortex, and bundles as they extend along the axis of filopods, were recognized. A significant alteration found was an enhanced accumulation of actin in cytoskeletons of the mutant when cells were stimulated with cyclic AMP. Our results indicate that control of cell shape and motility does not require the fine-tuned interactions of all proteins that have been identified as actin-binding proteins by in vitro assays.

Cellular Alterations Induced by Candida albicans RC Nosodes: an in vitro Study

2021 ◽  
Vol 11 (40) ◽  
pp. 209-210
Author(s):  
Fortune Homsani ◽  
Gleyce Moreno ◽  
Camila Siqueira ◽  
Juliana Grechi ◽  
André Luis Santos ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Cell Viability ◽  
Cell Model ◽  
Etiologic Agent ◽  
Yeast Cells ◽  
In Vitro Assays ◽  
Mitochondrial Activity ◽  
No Production ◽  
Cellular Alterations

Introduction: Candidiasis is an opportunist infection, caused by yeast of the genus Candida, which emerges as one of the main causes of systemic infections in hospitalized patients. Candida albicans is the most common causing agent of these infections. According to the Brazilian Homeopathic Pharmacopeia[1], nosodes are medicines compounded from chemically undefined biological products. Living nosodes are prepared using the etiologic agent of an illness in its infective form, were first developed by Brazilian physician Roberto Costa (RC). Roberto Costa’s research indicated that living nosodes present a higher capability to stimulate the host’s immunological system [2]. Aim: This study aims to evaluate cellular alterations induced in C. albicans yeasts and RAW 264-7 macrophages by Candida albicans RC. Methodology: To prepare Candida albicans RC, one part of C. albicans infective yeast suspension (108 cell/ml) was diluted in 9 parts of sterile distilled water and submitted to 100 mechanical succussions. This process was successively repeated to the potencies of 12x and 30x1. Water 30x was prepared by the same technique, as control. The cell viability of C. albicans previously treated with nosodes in both potencies and respective controls was evaluated using the samples at the concentration of 10% (V/V), in a volume of 1ml, distributed in 1-3 days. The viability of the yeast cells was analyzed by MTT (3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazolic) (5mg/ml) assay [3] and by Propidium Iodide (PI) incorporation methods. Additionally, using macrophages RAW 264-7 as a cell model, Nitric Oxide (NO) production and cell viability were also evaluated. For this, the following protocol of cell treatment was employed: on each experimental day, RAW 264-7 cells were treated 4 times (4 stimuli) with RC nosode 30x at the concentration of 10% (V/V). Results: The nosodes (12x and 30x) did not present cytotoxic effects on macrophage cells (n=1), or on C. albicans yeasts (n=2), as detected by MTT and PI methods. Moreover, no statistically significant differences on NO production were detected among the experimental groups (n=6). Conclusion: Preliminary results of in vitro assays indicate that nosodes (12x and 30x) do not alter mitochondrial activity or cell viability of C. albicans. Similarly, treatment by RC nosodes does not seem to alter NO release and mitochondrial activity of RAW macrophages. New experiments are being performed to confirm these preliminary data.

scholarly journals The metabolism in vitro and hepatic microsomal interactions of some enantiomeric drug substrates

1970 ◽  
Vol 117 (5) ◽  
pp. 833-841 ◽  
Author(s):  
David S. Hewick ◽  
James R. Fouts
Keyword(s):  
Type I ◽  
Type Ii ◽  
Difference Spectra ◽  
Microsomal Cytochrome ◽  
Hepatic Microsomes ◽  
Michaelis Constants ◽  
Nadph Oxidation ◽  
Identical Type ◽  
Cytochrome P 450

1. The metabolism in vitro and microsomal interactions of (+)-amphetamine, (−)-amphetamine, (+)-benzphetamine and (−)-benzphetamine were studied with hepatic microsomes from phenobarbitone-pretreated male rabbits. 2. (+)-Benzphetamine was N-demethylated 30–35% faster than (−)-benzphetamine, but the apparent Michaelis constants for the two enantiomers were similar. 3. (−)-Amphetamine was deaminated about 200% faster than (+)-amphetamine. 4. The benzphetamine enantiomers gave qualitatively and quantitatively identical type I microsomal difference spectra (peak, 390nm; trough, 425nm) indicating identical apparent binding affinities for microsomes and identical spectral changes at maxima (ΔEmax. values). 5. The amphetamine enantiomers gave qualitatively identical type II microsomal difference spectra (peak, 433nm; trough, 395nm). However, the type II spectral data indicated that (+)-amphetamine had a markedly higher apparent binding affinity than (−)-amphetamine for microsomes. The amphetamine enantiomers gave identical ΔEmax. values. 6. The benzphetamine enantiomers (0.5mm) enhanced the rate of microsomal cytochrome P-450 reduction by NADPH by 400–500%, (+)-benzphetamine enhancing the rate 20–25% more than (−)-benzphetamine. 7. The amphetamine enantiomers decreased the rate of microsomal cytochrome P-450 reduction by NADPH. At a concentration of 2mm, (+)-amphetamine decreased the rate more than (−)-amphetamine. 7. All four enantiomers enhanced microsomal NADPH oxidation.

Inhibition of nitric oxide synthesis improves detoxication in inflammatory liver dysfunction in vivo

1997 ◽  
Vol 273 (2) ◽  
pp. G530-G536 ◽  
Author(s):  
A. Veihelmann ◽  
T. Brill ◽  
M. Blobner ◽  
I. Scheller ◽  
B. Mayer ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
In Vitro Assays ◽  
Serum Concentrations ◽  
Aminopyrine Breath Test ◽  
Vitro Assay ◽  
Synthase Inhibitor ◽  
Cytochrome P 450 ◽  
Stimulation Of

Inflammatory stimulation of the liver induces nitric oxide (NO) biosynthesis and suppression of detoxication. In this study the effect of NO biosynthesis on cytochrome P-450 (CYP) enzyme activity was investigated by comparing in vivo and in vitro assays. To establish liver inflammation, CD rats were injected with Corynebacterium parvum (C. parvum) suspension. After 5 days NO biosynthesis was highly induced as indicated by increased NO2- plus NO3- serum concentrations. At the same time the aminopyrine breath test (ABT), measuring CYP activity in vivo, was reduced to 42% and the in vitro assay of aminopyrine turnover was suppressed to 12% of NaCl- injected controls. When C. parvum-injected animals were treated with the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA), CYP activities significantly improved with an ABT of 76% and an in vitro aminopyrine turnover of 47% of controls. Neither C. parvum injections nor L-NMMA treatment resulted in a significant change of CYP protein concentrations. These data indicate that suppression of xenobiotic metabolism can be attenuated by inhibition of NO biosynthesis during an ongoing process of inflammation.

Sign in / Sign up

Export Citation Format

Share Document