scholarly journals Functions of FKBP12 and Mitochondrial Cyclophilin Active Site Residues In Vitro and In Vivo inSaccharomyces cerevisiae

1997 ◽  
Vol 8 (11) ◽  
pp. 2267-2280 ◽  
Author(s):  
Kara Dolinski ◽  
Christian Scholz ◽  
R. Scott Muir ◽  
Sabine Rospert ◽  
Franz X. Schmid ◽  
...  
Keyword(s):  
Active Site ◽  
Active Sites ◽  
In Vitro Assay ◽  
Biologically Active ◽  
Wild Type ◽  
Prolyl Isomerase ◽  
Vitro Assay ◽  
Isomerase Activity

Cyclophilin and FK506 binding protein (FKBP) acceleratecis–trans peptidyl-prolyl isomerization and bind to and mediate the effects of the immunosuppressants cyclosporin A and FK506. The normal cellular functions of these proteins, however, are unknown. We altered the active sites of FKBP12 and mitochondrial cyclophilin from the yeast Saccharomyces cerevisiae by introducing mutations previously reported to inactivate these enzymes. Surprisingly, most of these mutant enzymes were biologically active in vivo. In accord with previous reports, all of the mutant enzymes had little or no detectable prolyl isomerase activity in the standard peptide substrate-chymotrypsin coupled in vitro assay. However, in a variation of this assay in which the protease is omitted, the mutant enzymes exhibited substantial levels of prolyl isomerase activity (5–20% of wild-type), revealing that these mutations confer sensitivity to protease digestion and that the classic in vitro assay for prolyl isomerase activity may be misleading. In addition, the mutant enzymes exhibited near wild-type activity with two protein substrates, dihydrofolate reductase and ribonuclease T1, whose folding is accelerated by prolyl isomerases. Thus, a number of cyclophilin and FKBP12 “active-site” mutants previously identified are largely active but protease sensitive, in accord with our findings that these mutants display wild-type functions in vivo. One mitochondrial cyclophilin mutant (R73A), and also the wild-type human FKBP12 enzyme, catalyze protein folding in vitro but lack biological activity in vivo in yeast. Our findings provide evidence that both prolyl isomerase activity and other structural features are linked to FKBP and cyclophilin in vivo functions and suggest caution in the use of these active-site mutations to study FKBP and cyclophilin functions.

scholarly journals Endogenous Synthesis of Erythritol, a Novel Biomarker of Weight Gain (P15-016-19)

2019 ◽  
Vol 3 (Supplement_1) ◽  
Author(s):  
Semira Ortiz ◽  
Doletha Szebenyi ◽  
Martha Field
Keyword(s):  
Molecular Modeling ◽  
Active Site ◽  
Active Sites ◽  
A549 Cells ◽  
Site Directed Mutagenesis ◽  
Central Adiposity ◽  
Wild Type ◽  
Reduction Activity

Abstract Objectives Serum erythritol is associated with central adiposity gain in young adults. Erythritol, a 4-carbon polyol, is synthesized endogenously from erythrose through the pentose phosphate pathway. We have identified two enzymes which catalyze this reaction: alcohol dehydrogenase 1 (ADH1) and sorbitol dehydrogenase (SORD). Interestingly, ADH1 isoforms ADH1B1 and ADH1C2 catalyze NADPH-dependent synthesis of erythritol in vitro, but ADH1B1 does not. In A549 cells, siRNA knockdown of SORD levels to less than 15% of control levels reduced erythritol by 50%. A549 cells also have low levels of ADH1 expression. This indicates that other enzymes may be capable of endogenous erythritol production. Based on its high degree of homology to ADH1, we hypothesize that ADH4 also catalyzes erythritol synthesis. The objective of this study was to further elucidate the mechanism of erythritol synthesis by: determining key differences between the active site of ADH1B2 compared to ADH1B1 and SORD, and screening a new candidate enzyme, ADH4. Methods This study used molecular modeling to evaluate the enzyme, erythrose, NADPH complex. Site-directed mutagenesis was performed to replace Arg208 (R208) of recombinant human SORD with histidine. Recombinant SORD R208H mutant and ADH4 were then expressed, purified, and their erythrose reduction activity was kinetically characterized. Results Molecular modeling of the active sites of ADH1B1 and ADH1B2 revealed that the Arg48 residue of ADH1B1 makes three contacts with NADPH, whereas His48 of ADH1B2 only makes one contact. Mutation of the SORD active-site Arg208 to histidine reduced the kcat by more than 80% compared to wild-type (95 ± 21 min−1 wild-type, 16 ± 2 min−1 R208H, P < 0.05). ADH4 erythrose reduction activity reduced by more than 95% compared to ADH1 variant proteins in vitro (kcat 2 ± 1 min−1). Conclusions Mutation of arginine 208 to histidine in the active site of SORD significantly reduced the kcat, indicating that the presence of an arginine residue is essential for binding NADPH, the cofactor required in vivo for erythritol synthesis. The low erythrose reduction activity of ADH4 suggests that ADH4 does not contribute to erythritol synthesis in humans. Funding Sources None.

scholarly journals DIGESTIBLE ENERGY IN FORAGE BYIN VIVOANDIN VITROASSAYS

1970 ◽  
Vol 50 (3) ◽  
pp. 557-562 ◽  
Author(s):  
J. E. TROELSEN
Keyword(s):  
Energy Analysis ◽  
Energy Content ◽  
In Vitro Assay ◽  
Pure Species ◽  
Vitro Assay ◽  
Digestible Energy ◽  
The Relationship ◽  
Cattle And Sheep

Forage of six pure species was harvested for hay at several maturity stages during four years. The digestible energy content of 102 different lots of hay was determined by feeding to four groups of sheep during the same period, and by in vitro digestions and energy analysis of the undigested residues. The relationship between digestible energy content assayed by the two methods was highly significant (r = 0.85) and did not differ between years and species. Exclusion from regression of the hays containing less than 2 or more than 3 digestible kcal/g revealed that the in vitro assay could reproduce the in vivo digestible energy value with a standard deviation of 0.31 in over 70% of the hays. This represented the maturity and quality range of forage commonly fed to cattle and sheep. The in vitro assay therefore appeared promising for commercial quality determinations.

scholarly journals Mutagenicity evaluation of Anastatica hierochuntica L. aqueous extract in vitro and in vivo

2017 ◽  
Vol 243 (4) ◽  
pp. 375-385 ◽  
Author(s):  
Siti Rosmani Md Zin ◽  
Zahurin Mohamed ◽  
Mohammed A Alshawsh ◽  
Won F Wong ◽  
Normadiah M Kassim
Keyword(s):  
Aqueous Extract ◽  
Positive Control ◽  
In Vitro Assay ◽  
In Vivo Study ◽  
Sufficient Evidence ◽  
Aqueous Extracts ◽  
Mutagenic Potential ◽  
Vitro Assay

Anastatica hierochuntica L. ( A. hierochuntica), a folk medicinal plant, was evaluated for mutagenic potential via in vitro and in vivo assays. The in vitro assay was conducted according to modified Ames test, while the in vivo study was performed according to Organisation for Economic Co-operation and Development guideline for mammalian erythrocyte micronucleus assay. Four groups ( n= 5 males and 5 females per group) Sprague Dawley rats were randomly chosen as the negative control, positive control (received a single intramuscular injection of cyclophosphamide 50 mg/kg), 1000 and, 2000 mg/kg A. hierochuntica aqueous extracts. All groups except the positive control were treated orally for three days. Findings of the in vitro assay showed mutagenic potential of AHAE at 0.04 and 0.2 mg/ml. However, no mutagenic effect was demonstrated in the in vivo study up to 2000 mg/kg. No significant reduction in the polychromatic and normochromatic erythrocytes ratio was noted in any of the groups. Meanwhile, high micronucleated polychromatic erythrocytes frequency was seen in cyclophosphamide-treated group only. These findings could perhaps be due to insufficient dosage of A. hierochuntica aqueous extracts to cause genetic damage on the bone marrow target cells. Further acute and chronic in vivo toxicity studies may be required to draw pertinent conclusion on the safety aspect of A. hierochuntica aqueous extracts consumption. Impact statement In this paper, we report on the mutagenicity evaluation of Anastatica hierochuntica aqueous extract. This is a significant research in view of the popularity of this herb consumption by the people across the globe despite of limited scientific evidence on its toxicity potential. This study is intended to encourage more extensive related research in order to provide sufficient evidence and guidance for determining its safe dosage.

scholarly journals A Novel 3-Deoxy-d-manno-Octulosonic Acid (Kdo) Hydrolase That Removes the Outer Kdo Sugar of Helicobacter pylori Lipopolysaccharide

2005 ◽  
Vol 187 (10) ◽  
pp. 3374-3383 ◽  
Author(s):  
Christopher Stead ◽  
An Tran ◽  
Donald Ferguson ◽  
Sara McGrath ◽  
Robert Cotter ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Lipid A ◽  
In Vitro Assay ◽  
Spectrometric Analysis ◽  
Vitro Assay ◽  
The Core ◽  
H Pylori

ABSTRACT The lipid A domain anchors lipopolysaccharide (LPS) to the outer membrane and is typically a disaccharide of glucosamine that is both acylated and phosphorylated. The core and O-antigen carbohydrate domains are linked to the lipid A moiety through the eight-carbon sugar 3-deoxy-d-manno-octulosonic acid known as Kdo. Helicobacter pylori LPS has been characterized as having a single Kdo residue attached to lipid A, predicting in vivo a monofunctional Kdo transferase (WaaA). However, using an in vitro assay system we demonstrate that H. pylori WaaA is a bifunctional enzyme transferring two Kdo sugars to the tetra-acylated lipid A precursor lipid IVA. In the present work we report the discovery of a Kdo hydrolase in membranes of H. pylori capable of removing the outer Kdo sugar from Kdo2-lipid A. Enzymatic removal of the Kdo group was dependent upon prior removal of the 1-phosphate group from the lipid A domain, and mass spectrometric analysis of the reaction product confirmed the enzymatic removal of a single Kdo residue by the Kdo-trimming enzyme. This is the first characterization of a Kdo hydrolase involved in the modification of gram-negative bacterial LPS.

Carcinogenic and co-carcinogenic potential of 2,3,7,8-tetrachlorodibenzodioxin in a host-mediated in vivo/in vitro assay

Chemosphere ◽  
1992 ◽  
Vol 25 (7-10) ◽  
pp. 1085-1090 ◽  
Author(s):  
T. Massa ◽  
A. Esmseili ◽  
H. Fortmeyer ◽  
B. Schlatterer ◽  
H. Hagenmaier ◽  
...  
Keyword(s):  
In Vitro Assay ◽  
Vitro Assay ◽  
Carcinogenic Potential

scholarly journals An in-vitro assay using human spermatozoa to detect toxicity of biologically active substances

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Tino Vollmer ◽  
Börje Ljungberg ◽  
Vera Jankowski ◽  
Joachim Jankowski ◽  
Griet Glorieux ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Toxicity Testing ◽  
Biologically Active ◽  
Human Spermatozoa ◽  
Vitro Assay ◽  
Cellular Behavior ◽  
Novel Method ◽  
Set Up

Abstract Identifying the key toxic players within an in-vivo toxic syndrome is crucial to develop targeted therapies. Here, we established a novel method that characterizes the effect of single substances by means of an ex-vivo incubation set-up. We found that primary human spermatozoa elicit a distinct motile response on a (uremic) toxic milieu. Specifically, this approach describes the influence of a bulk toxic environment (uremia) as well as single substances (uremic toxins) by real-time analyzing motile cellular behavior. We established the human spermatozoa-based toxicity testing (HSTT) for detecting single substance-induced toxicity to be used as a screening tool to identify in-vivo toxins. Further, we propose an application of the HSTT as a method of clinical use to evaluate toxin-removing interventions (hemodialysis).

scholarly journals Ruminal fermentation and digestion of cattle diets with total and partial replacement of soybean meal by a slow-release urea product

2019 ◽  
Vol 64 (No. 7) ◽  
pp. 294-301
Author(s):  
S Gonzalez-Munoz ◽  
J Sanchez ◽  
S Lopez-Aguirre ◽  
J Vicente ◽  
J Pinos-Rodriguez
Keyword(s):  
Soybean Meal ◽  
Degradation Rate ◽  
Slow Release ◽  
In Vitro Assay ◽  
Partial Replacement ◽  
Vitro Assay ◽  
Dietary Substitution ◽  
Slow Release Urea

One in vitro assay and one in vivo trial with ruminally cannulated Holstein steers were conducted to evaluate the effects of a dietary substitution of soybean meal by a urea and slow-release urea source of fermentation and degradation of diets for cattle. The experimental diets consisted of the total mixed rations defined as the control with soybean meal (SBM), U (urea), SRU (slow-release urea), and SRU+U+AA (0.42% + 0.42% + 1% amino acids methionine and lysine). The dietary substitution of SBM by U or SRU reduced (P &lt; 0.05) the total gas production (V), microbial mass and degradation at 72 h incubation under the in vitro conditions, as well as the degradation rate (c) and the total volatile fatty acids (VFA) in the rumen of the steers; however, when the dietary substitution of SBM was by U+SRU+AA, those values did not decrease. In the steers, the dietary substitution of SBM by U and SRU reduced the ruminal degradation rate and the total VFA, and increased the ammonia N, but when SBM was substituted by U+SRU+AA in the diets, these changes were not observed. No advantage of SRU over U was found. The dietary substitution of SBM by U, SRU, U+SRU+AA did not modify the molar proportion of the VFA in the rumen nor were there changes in the nutrient digestion or excretion. Both the in vitro assay and the in vivo trial indicated that replacing SBM with U or SRU increases the ruminal ammonia N concentrations and reduces the degradation rate in the rumen, although those undesirable findings were not found when the SBM was replaced by U+SRU+AA. Therefore, it is feasible to replace the SBM with a combination of urea, slow-release urea, lysine and methionine in the diet for the ruminants.

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