Reductive Activation of Carbon Tetrachloride by Human Haemoglobin

1993 ◽  
Vol 21 (1) ◽  
pp. 57-64
Author(s):  
Roberta Ferrara ◽  
Michela Rezzadore ◽  
Stefano Cazzaro ◽  
Roberto Tolando ◽  
Maurizio Manno
Keyword(s):  
Carbon Monoxide ◽  
Carbon Tetrachloride ◽  
Anaerobic Incubation ◽  
Reductive Activation ◽  
Anaerobic Conditions ◽  
Prosthetic Group ◽  
Experimental Conditions ◽  
Human Haemoglobin ◽  
Reductive Metabolism

The reductive metabolism of carbon tetrachloride (CC14) by human haemoglobin (Hb) was observed in vitro by absolute absorption spectra recorded under anaerobic conditions. The following results were obtained: 1) a decrease of the 430nm peak typical of free reduced Hb (Hb2+); 2) the formation of a shoulder of absorbance, attributable to the production of a complex between Hb2+ and a metabolite of CC14 carbon monoxide (Hb-CO); and 3) the oxidation of some Hb2+ to methaemoglobin (Hb3+). The concentration of these three forms — Hb2+, Hb-CO and Hb3+ — during anaerobic incubation of Hb with CC14 was calculated algebraically from the absolute spectra. CO production was then calculated from the concentration of Hb-CO, using a suitable calibration curve. Interestingly, under identical experimental conditions, a substrate-dependent loss of Hb-derived haem, but not of Hb itself nor of haem-derived porphyrin fluorescence, was measured. Preliminary HPLC studies to clarify the discrepancy and, in particular, the role and fate of the haem group, showed two substrate-dependent modified haem products. The results indicate that human Hb is able to catalyse the reductive activation of CCl4, and suggest that, during the process, its prosthetic group haem may be modified by CC14 metabolites to products which maintain a tetrapyrrolic structure but are unable to react with pyridine.

Observations of Frozen-Hydrated Microtubules

1990 ◽  
Vol 48 (1) ◽  
pp. 504-505
Author(s):  
D. Chrétien ◽  
D. Job ◽  
R.H. Wade
Keyword(s):  
Fourier Transforms ◽  
Structural Complexity ◽  
Image Contrast ◽  
Phase Behaviour ◽  
Experimental Conditions ◽  
Thin Sections ◽  
Surface Lattice ◽  
Cilia And Flagella ◽  
Outstanding Question

Microtubules are filamentary structures found in the cytoplasm of eukaryotic cells, where, together with actin and intermediate filaments, they form the components of the cytoskeleton. They have many functions and show various levels of structural complexity as witnessed by the singlet, doublet and triplet structures involved in the architecture of centrioles, basal bodies, cilia and flagella. The accepted microtubule model consists of a 25 nm diameter hollow tube with a wall made up of 13 paraxial protofilaments (pf). Each pf is a string of aligned tubulin dimers. Some results have suggested that the pfs follow a superhelix. To understand how microtubules function in the cell an accurate model of the surface lattice is one of the requirements. For example the 9x2 architecture of the axoneme will depend on the organisation of its component microtubules. We should also note that microtubules with different numbers of pfs have been observed in thin sections of cellular and of in-vitro material. An outstanding question is how does the surface lattice adjust to these different pf numbers?We have been using cryo-electron microscopy of frozen-hydrated samples to study in-vitro assembled microtubules. The experimental conditions are described in detail in this reference. The results obtained in conjunction with thin sections of similar specimens and with axoneme outer doublet fragments have already allowed us to characterise the image contrast of 13, 14 and 15 pf microtubules on the basis of the measured image widths, of the the image contrast symmetry and of the amplitude and phase behaviour along the equator in the computed Fourier transforms. The contrast variations along individual microtubule images can be interpreted in terms of the geometry of the microtubule surface lattice. We can extend these results and make some reasonable predictions about the probable surface lattices in the case of other pf numbers, see Table 1. Figure 1 shows observed images with which these predictions can be compared.

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

1981 ◽  
Vol 45 (03) ◽  
pp. 290-293 ◽  
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.

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

1997 ◽  
Vol 77 (05) ◽  
pp. 0975-0980 ◽  
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.

Oxygenation of 18-hydroxycorticosterone as the final reaction for aldosterone biosynthesis

1984 ◽  
Vol 107 (3) ◽  
pp. 395-400 ◽  
Author(s):  
Itaru Kojima ◽  
Etsuro Ogata ◽  
Hiroshi Inano ◽  
Bun-ichi Tamaoki
Keyword(s):  
Carbon Monoxide ◽  
Hydroxysteroid Dehydrogenase ◽  
Dependent Manner ◽  
In Vitro Production ◽  
Mitochondrial Preparation ◽  
Final Reaction ◽  
Vitro Production ◽  
Dose Dependent ◽  
Cytochrome P 450

Abstract. Incubation of 18-hydroxycorticosterone with the sonicated mitochondrial preparation of bovine adrenal glomerulosa tissue leads to the production of aldosterone, as measured by radioimmunoassay. The in vitro production of aldosterone from 18-hydroxycorticosterone requires both molecular oxygen and NADPH, and is inhibited by carbon monoxide. Cytochrome P-450 inhibitors such as metyrapone, SU 8000. SU 10603, SKF 525A, amphenone B and spironolactone decrease the biosynthesis of aldosterone from 18-hydroxycorticosterone. These results support the conclusion that the final reaction in aldosterone synthesis from 18-hydroxycorticosterone is catalyzed by an oxygenase, but not by 18-hydroxysteroid dehydrogenase. By the same preparation, the production of [3H]aldosterone but not [3H]18-hydroxycorticosterone from [1,2-3H ]corticosterone is decreased in a dose-dependent manner by addition of non-radioactive 18-hydroxycorticosterone.

Hepato- and Nephroprotective Effects of the Polyphenol Concentrate From Cabernet Sauvignon Grape Varieties Cultivated in Kazakhstan in the Experimental Model Of CCL4-Induced Toxicity

2017 ◽  
Vol 9 (03) ◽  
Author(s):  
Nurgozhin T. ◽  
Sergazy S. H. ◽  
Adilgozhina G. ◽  
Gulyayev A. ◽  
Shulgau Z. ◽  
...  
Keyword(s):  
Carbon Tetrachloride ◽  
Experimental Model ◽  
Lethal Dose ◽  
Radical Scavenging ◽  
Cabernet Sauvignon ◽  
Intragastric Administration ◽  
Liver And Kidney ◽  
Antioxidant Stress

Objective:This study investigates the hepatoprotective effect and the antioxidant role of polyphenol concentrate in the experimental model of carbon tetrachloride (CCl4) induced toxicity. Methods: Antioxidant activity of Cabernet Sauvignon grape polyphenol were evaluated by radical scavenging of 1,1-diphenyl-2-picryl hydrazyl radical (DPPH), 2,2’-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS.+). In addition, the effects of polyphenol concentrate on the survival of Wistar rats in the toxicity model, was also investigated. The polyphenol concentrate was administered for 5 five days prior to injection of carbon tetrachloride in a sub-lethal dose of 300 mg/kg of animal body weight in order to perform histological examinations of the liver and kidney, and detect the levels of AST, ALT and bilirubin. Results: Administration of polyphenol concentrate increased animal survival in the experimental model. Moreover, the intragastric administration of polyphenol concentrate prior to the initiation of the experimental model of toxicity, which was caused by a sub-lethal CCl4 dose, reduced morphological injuries in the liver and kidney, decreased the AST and ALT levels of the blood serum. Discussion and conclusion: Our data demonstrate that polyphenol concentrate possesses an antioxidant potential both in vitro and in vivo by reducing antioxidant stress that was caused by CCl4 administration into rats.

Applicability of Instability Index for In vitro Protein Stability Prediction

2019 ◽  
Vol 26 (5) ◽  
pp. 339-347 ◽  
Author(s):  
Dilani G. Gamage ◽  
Ajith Gunaratne ◽  
Gopal R. Periyannan ◽  
Timothy G. Russell
Keyword(s):  
Protein Stability ◽  
Caulobacter Crescentus ◽  
Effect Of Temperature ◽  
Instability Index ◽  
Dipeptide Composition ◽  
Experimental Conditions ◽  
The Stability ◽  
Vitro Protein

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.

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

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.

scholarly journals Effects of Substituents on Photophysical and CO-Photoreleasing Properties of 2,6-Substituted meso-Carboxy BODIPY Derivatives

Chemistry ◽  
2021 ◽  
Vol 3 (1) ◽  
pp. 238-255
Author(s):  
Esther M. Sánchez-Carnerero ◽  
Marina Russo ◽  
Andreas Jakob ◽  
Lucie Muchová ◽  
Libor Vítek ◽  
...  
Keyword(s):  
Carbon Monoxide ◽  
Absorption Spectra ◽  
Wavelength Range ◽  
Toxic Effects ◽  
Biological Research ◽  
In Vitro Experiments ◽  
Vast Array ◽  
Physiological Processes ◽  
Photochemical Properties

Carbon monoxide (CO) is an endogenously produced signaling molecule involved in the control of a vast array of physiological processes. One of the strategies to administer therapeutic amounts of CO is the precise spatial and temporal control over its release from photoactivatable CO-releasing molecules (photoCORMs). Here we present the synthesis and photophysical and photochemical properties of a small library of meso-carboxy BODIPY derivatives bearing different substituents at positions 2 and 6. We show that the nature of substituents has a major impact on both their photophysics and the efficiency of CO photorelease. CO was found to be efficiently released from π-extended 2,6-arylethynyl BODIPY derivatives possessing absorption spectra shifted to a more biologically desirable wavelength range. Selected photoCORMs were subjected to in vitro experiments that did not reveal any serious toxic effects, suggesting their potential for further biological research.

scholarly journals Evaluation of Indigenous Olive Biocontrol Rhizobacteria as Protectants against Drought and Salt Stress

2021 ◽  
Vol 9 (6) ◽  
pp. 1209
Author(s):  
Nuria Montes-Osuna ◽  
Carmen Gómez-Lama Cabanás ◽  
Antonio Valverde-Corredor ◽  
Garikoitz Legarda ◽  
Pilar Prieto ◽  
...  
Keyword(s):  
Salt Stress ◽  
Abiotic Stresses ◽  
Biochemical Parameters ◽  
Biocontrol Agent ◽  
Negative Effects ◽  
Experimental Conditions ◽  
Acds Gene ◽  
Drought And Salt Stress ◽  
Physiological And Biochemical

Stress caused by drought and salinity may compromise growth and productivity of olive (Olea europaea L.) tree crops. Several studies have reported the use of beneficial rhizobacteria to alleviate symptoms produced by these stresses, which is attributed in some cases to the activity of 1-aminocyclopropane-1-carboxylic acid deaminase (ACD). A collection of beneficial olive rhizobacteria was in vitro screened for ACD activity. Pseudomonas sp. PICF6 displayed this phenotype and sequencing of its genome confirmed the presence of an acdS gene. In contrast, the well-known root endophyte and biocontrol agent Pseudomonas simiae PICF7 was defective in ACD activity, even though the presence of an ACD-coding gene was earlier predicted in its genome. In this study, an unidentified deaminase was confirmed instead. Greenhouse experiments with olive ‘Picual’ plants inoculated either with PICF6 or PICF7, or co-inoculated with both strains, and subjected to drought or salt stress were carried out. Several physiological and biochemical parameters increased in stressed plants (i.e., stomatal conductance and flavonoids content), regardless of whether or not they were previously bacterized. Results showed that neither PICF6 (ACD positive) nor PICF7 (ACD negative) lessened the negative effects caused by the abiotic stresses tested, at least under our experimental conditions.

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