Applicability of Instability Index for In vitro Protein Stability Prediction

Background: The dipeptide composition-based Instability Index (II) is one of the protein primary structure-dependent methods available for in vivo protein stability predictions. As per this method, proteins with II value below 40 are stable proteins. Intracellular protein stability principles guided the original development of the II method. However, the use of the II method for in vitro protein stability predictions raises questions about the validity of applying the II method under experimental conditions that are different from the in vivo setting. Objective: The aim of this study is to experimentally test the validity of the use of II as an in vitro protein stability predictor. Methods: A representative protein CCM (CCM - Caulobacter crescentus metalloprotein) that rapidly degrades under in vitro conditions was used to probe the dipeptide sequence-dependent degradation properties of CCM by generating CCM mutants to represent stable and unstable II values. A comparative degradation analysis was carried out under in vitro conditions using wildtype CCM, CCM mutants and two other candidate proteins: metallo-β-lactamase L1 and α -S1- casein representing stable, borderline stable/unstable, and unstable proteins as per the II predictions. The effect of temperature and a protein stabilizing agent on CCM degradation was also tested. Results: Data support the dipeptide composition-dependent protein stability/instability in wt-CCM and mutants as predicted by the II method under in vitro conditions. However, the II failed to accurately represent the stability of other tested proteins. Data indicate the influence of protein environmental factors on the autoproteolysis of proteins. Conclusion: Broader application of the II method for the prediction of protein stability under in vitro conditions is questionable as the stability of the protein may be dependent not only on the intrinsic nature of the protein but also on the conditions of the protein milieu.

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METABOLIC CHANGES IN TESTICULAR CELLS FROM RATS AFTER LONG-TERM EXPOSURE TO 37 °C IN VIVO OR IN VITRO

Journal of Endocrinology ◽

10.1677/joe.0.0850471 ◽

1980 ◽

Vol 85 (3) ◽

pp. 471-479 ◽

Cited By ~ 20

Author(s):

F. F. G. ROMMERTS ◽

F. H. DE JONG ◽

J. A. GROOTEGOED ◽

H. J. VAN DER MOLEN

Keyword(s):

Sertoli Cells ◽

Leydig Cells ◽

Biochemical Properties ◽

Effect Of Temperature ◽

Seminiferous Tubules ◽

Experimental Conditions ◽

Testicular Cells ◽

Old Rats

Biochemical properties of isolated Leydig cells, Sertoli cells and spermatocytes from rat testes have been investigated after in-vivo or in-vitro exposure of these cells to abdominal temperature (37 °C). The rate of production of testosterone and pregnenolone by isolated Leydig cells from cryptorchid and normal testes from mature rats was not different. Production of pregnenolone by mitochondria prepared from cryptorchid testes was 6·7 times higher than production by mitochondria from normal testes. Sertoli cells prepared from immature rats and incubated in vitro at 32 or 37 °C showed, on day 1 of the culture period, an initial twofold increase in the secretion of androgen-binding protein which was absent after 6 days in culture. In contrast, incorporation of [3H]leucine into secreted proteins was stimulated twofold on day 1 as well as by day 6 of culture. Secretion of oestradiol was increased 30-fold by day 6 when compared with the level found on day 1 when cells had been cultured at 37 °C and the increased secretion of oestradiol was maintained for approximately 2 days when the temperature of incubation was decreased to 32 °C Spermatocytes isolated from seminiferous tubules incubated for 20 h at 37 °C were active in the synthesis of RNA. No degeneration of these cells was observed in testes of 25-day-old rats 5 days after experimental cryptorchidism, whereas under similar conditions massive degeneration of spermatocytes was shown in the testes of mature rats. These results suggest that the effects of temperature on the different testicular cells greatly depend on the experimental conditions used to study the effect of temperature.

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Studies on the interaction of Escherichia coli endotoxin with erythrocyte membranes

Canadian Journal of Physiology and Pharmacology ◽

10.1139/y82-137 ◽

1982 ◽

Vol 60 (7) ◽

pp. 977-985 ◽

Cited By ~ 9

Author(s):

David V. Godin ◽

John M. Tuchek ◽

Maureen E. Garnett

Keyword(s):

Escherichia Coli ◽

Membrane Lipids ◽

Effect Of Temperature ◽

Erythrocyte Membranes ◽

Simple Relationship ◽

In Vitro Effects ◽

The Stability ◽

Erythrocyte Stability

Escherichia coli lipopolysaccharide (endotoxin) alters the stability of erythrocytes to hypotonic lysis although the nature and magnitude of the effect varied with temperature and with the type of red blood cell examined. Evidence has been obtained suggesting a possible modulatory role of membrane lipids in governing the molecular consequences of membrane–endotoxin interaction. The marked effect of temperature on the stabilization of red cells by endotoxin was not attributable to variations in toxin binding and was not observed with more conventional structurally unrelated antihemolytic agents. Although chemical modifications which alter the toxicity of endotoxin in vivo also modify its ability to stabilize erythrocytes in vitro, no simple relationship between in vivo endotoxin toxicity and in vitro effects on erythrocyte stability was apparent. The critical dependence of endotoxin antihemolytic effects in vitro on molecular structure may offer a convenient means of assessing the homogeneity of these preparations before performing experiments in vivo.

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Formation and Structure of Casein Micelles in Lactating Mammary Tissue

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100067741 ◽

1970 ◽

Vol 28 ◽

pp. 150-151

Author(s):

Robert J. Carroll ◽

Marvin P. Thompson ◽

Harold M. Farrell

Keyword(s):

Size Distribution ◽

G Protein ◽

Milk Proteins ◽

Mammary Tissue ◽

Colloidal System ◽

Casein Micelles ◽

The Stability

Milk is an unusually stable colloidal system; the stability of this system is due primarily to the formation of micelles by the major milk proteins, the caseins. Numerous models for the structure of casein micelles have been proposed; these models have been formulated on the basis of in vitro studies. Synthetic casein micelles (i.e., those formed by mixing the purified αsl- and k-caseins with Ca2+ in appropriate ratios) are dissimilar to those from freshly-drawn milks in (i) size distribution, (ii) ratio of Ca/P, and (iii) solvation (g. water/g. protein). Evidently, in vivo organization of the caseins into the micellar form occurs in-a manner which is not identical to the in vitro mode of formation.

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Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

Nuklearmedizin ◽

10.1055/s-0037-1620623 ◽

1977 ◽

Vol 16 (04) ◽

pp. 157-162 ◽

Cited By ~ 1

Author(s):

C. Schümichen ◽

B. Mackenbrock ◽

G. Hoffmann

Keyword(s):

Normal Saline ◽

Half Life ◽

Compound A ◽

Short Half Life ◽

Low Concentrations ◽

The Stability ◽

Kinetics Of ◽

In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

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Superoxide, Xanthine Oxidase and Platelet Reactions: Further Studies on Mechanisms by which Oxidants Influence Platelets

Thrombosis and Haemostasis ◽

10.1055/s-0038-1650190 ◽

1981 ◽

Vol 45 (03) ◽

pp. 290-293 ◽

Cited By ~ 9

Author(s):

Peter H Levine ◽

Danielle G Sladdin ◽

Norman I Krinsky

Keyword(s):

Superoxide Dismutase ◽

Cytochrome C ◽

Xanthine Oxidase ◽

Direct Effect ◽

Platelet Function ◽

Experimental Conditions ◽

Release Reaction

SummaryIn the course of studying the effects on platelets of the oxidant species superoxide (O- 2), Of was generated by the interaction of xanthine oxidase plus xanthine. Surprisingly, gel-filtered platelets, when exposed to xanthine oxidase in the absence of xanthine substrate, were found to generate superoxide (O- 2), as determined by the reduction of added cytochrome c and by the inhibition of this reduction in the presence of superoxide dismutase.In addition to generating Of, the xanthine oxidase-treated platelets display both aggregation and evidence of the release reaction. This xanthine oxidase induced aggreagtion is not inhibited by the addition of either superoxide dismutase or cytochrome c, suggesting that it is due to either a further metabolite of O- 2, or that O- 2 itself exerts no important direct effect on platelet function under these experimental conditions. The ability of Of to modulate platelet reactions in vivo or in vitro remains in doubt, and xanthine oxidase is an unsuitable source of O- 2 in platelet studies because of its own effects on platelets.

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Desmopressin (DDAVP) Enhances Platelet Adhesion to the Extracellular Matrix of Cultured Human Endothelial Cells through Increased Expression of Tissue Factor

Thrombosis and Haemostasis ◽

10.1055/s-0038-1656088 ◽

1997 ◽

Vol 77 (05) ◽

pp. 0975-0980 ◽

Cited By ~ 23

Author(s):

Angel Gálvez ◽

Goretti Gómez-Ortiz ◽

Maribel Díaz-Ricart ◽

Ginés Escolar ◽

Rogelio González-Sarmiento ◽

...

Keyword(s):

Extracellular Matrix ◽

Endothelial Cells ◽

Tissue Factor ◽

Platelet Adhesion ◽

Umbilical Vein ◽

Procoagulant Activity ◽

Experimental Conditions ◽

Chromogenic Assay

SummaryThe effect of desmopressin (DDAVP) on thrombogenicity, expression of tissue factor and procoagulant activity (PCA) of extracellular matrix (ECM) generated by human umbilical vein endothelial cells cultures (HUVEC), was studied under different experimental conditions. HUVEC were incubated with DDAVP (1, 5 and 30 ng/ml) and then detached from their ECM. The reactivity towards platelets of this ECM was tested in a perfusion system. Coverslips covered with DD A VP-treated ECMs were inserted in a parallel-plate chamber and exposed to normal blood anticoagulated with low molecular weight heparin (Fragmin®, 20 U/ml). Perfusions were run for 5 min at a shear rate of 800 s1. Deposition of platelets on ECMs was significantly increased with respect to control ECMs when DDAVP was used at 5 and 30 ng/ml (p <0.05 and p <0.01 respectively). The increase in platelet deposition was prevented by incubation of ECMs with an antibody against human tissue factor prior to perfusion. Immunofluorescence studies positively detected tissue factor antigen on DDAVP derived ECMs. A chromogenic assay performed under standardized conditions revealed a statistically significant increase in the procoagulant activity of the ECMs produced by ECs incubated with 30 ng/ml DDAVP (p <0.01 vs. control samples). Northern blot analysis revealed increased levels of tissue factor mRNA in extracts from ECs exposed to DDAVP. Our data indicate that DDAVP in vitro enhances platelet adhesion to the ECMs through increased expression of tissue factor. A similar increase in the expression of tissue factor might contribute to the in vivo hemostatic effect of DDAVP.

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Non-invasive skin sampling of tryptophan/kynurenine ratio in vitro towards a skin cancer biomarker

Scientific Reports ◽

10.1038/s41598-020-79903-w ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Skaidre Jankovskaja ◽

Johan Engblom ◽

Melinda Rezeli ◽

György Marko-Varga ◽

Tautgirdas Ruzgas ◽

...

Keyword(s):

Skin Cancer ◽

Relative Increase ◽

Cancer Biomarker ◽

Cancer Diagnostics ◽

Sampling Time ◽

Skin Permeability ◽

Experimental Conditions ◽

Non Invasive

AbstractThe tryptophan to kynurenine ratio (Trp/Kyn) has been proposed as a cancer biomarker. Non-invasive topical sampling of Trp/Kyn can therefore serve as a promising concept for skin cancer diagnostics. By performing in vitro pig skin permeability studies, we conclude that non-invasive topical sampling of Trp and Kyn is feasible. We explore the influence of different experimental conditions, which are relevant for the clinical in vivo setting, such as pH variations, sampling time, and microbial degradation of Trp and Kyn. The permeabilities of Trp and Kyn are overall similar. However, the permeated Trp/Kyn ratio is generally higher than unity due to endogenous Trp, which should be taken into account to obtain a non-biased Trp/Kyn ratio accurately reflecting systemic concentrations. Additionally, prolonged sampling time is associated with bacterial Trp and Kyn degradation and should be considered in a clinical setting. Finally, the experimental results are supported by the four permeation pathways model, predicting that the hydrophilic Trp and Kyn molecules mainly permeate through lipid defects (i.e., the porous pathway). However, the hydrophobic indole ring of Trp is suggested to result in a small but noticeable relative increase of Trp diffusion via pathways across the SC lipid lamellae, while the shunt pathway is proposed to slightly favor permeation of Kyn relative to Trp.

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Factors affecting in vitro and in vivo antioxidant effects. Experimental conditions and nature of oxidants determine antioxidant efficacy

Journal of Berry Research ◽

10.3233/jbr-200695 ◽

2021 ◽

pp. 1-9

Author(s):

Etsuo Niki

Keyword(s):

Biological Molecules ◽

Experimental Conditions ◽

Factors Affecting ◽

Beneficial Effects ◽

Multiple Oxidants ◽

Antioxidant Effects

Reactive oxygen and nitrogen species have been implicated in the onset and progression of various diseases and the role of antioxidants in the maintenance of health and prevention of diseases has received much attention. The action and effect of antioxidants have been studied extensively under different reaction conditions in multiple media. The antioxidant effects are determined by many factors. This review aims to discuss several important issues that should be considered for determination of experimental conditions and interpretation of experimental results in order to understand the beneficial effects and limit of antioxidants against detrimental oxidation of biological molecules. Emphasis was laid on cell culture experiments and effects of diversity of multiple oxidants on antioxidant efficacy.

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