scholarly journals Nitrogen metabolism in the isolated perfused rat liver. Nitrogen balance, redox state and rates of proteolysis

1974 ◽  
Vol 138 (3) ◽  
pp. 341-348 ◽  
Author(s):  
R. Parrilla ◽  
M. N. Goodman
Keyword(s):  
Glutamate Dehydrogenase ◽  
Nitrogen Metabolism ◽  
Redox State ◽  
Isolated Perfused Rat Liver ◽  
Experimental Conditions ◽  
Nitrogen Donor ◽  
Nadh Ratio ◽  
Metabolic Conditions ◽  
Rat Livers

Nitrogen balances were measured in isolated perfused rat livers in the presence and absence of nitrogen donors. In all instances the balance apparently was incomplete. The expression [alanine][α-oxoglutarate]/[pyruvate][glutamate] remained fairly constant under the metabolic conditions studied, indicating that it may be at near-equilibrium. The source of the extra nitrogen seems to be derived from increased hepatic proteolysis. The addition of a nitrogen donor to the perfusate arrested proteolysis, as did the addition of pyruvate. The free mitochondrial [NAD+]/[NADH] ratio, calculated from the glutamate dehydrogenase and β-hydroxybutyrate dehydrogenase reactants, showed similar values and exhibited parallel changes under most metabolic situations studied. These results suggest that, under the reported experimental conditions, both dehydrogenases share a common mitochondrial NAD pool. Glutamate dehydrogenase plays an important role in hepatic nitrogen metabolism in vivo.

scholarly journals The control of tricarboxylate-cycle oxidations in blowfly flight muscle. The steady-state concentrations of citrate, isocitrate 2-oxoglutarate and malate in flight muscle and isolated mitochondria

1975 ◽  
Vol 146 (3) ◽  
pp. 527-535 ◽  
Author(s):  
R N Johnson ◽  
R G Hansford
Keyword(s):  
Redox State ◽  
Flight Muscle ◽  
Total Content ◽  
Phormia Regina ◽  
Experimental Conditions ◽  
Main Determinant ◽  
Muscle Mitochondria ◽  
Isolated Mitochondria ◽  
Tricarboxylate Cycle

1. Blowfly (Phormia regina) flight-muscle mitochondria were allowed to oxidize pyruvate under a variety of experimental conditions, and determinations of the citrate, isocitrate, 2-oxoglutarate and malate contents of both the mitochondria and the incubation medium were made. For each intermediate a substantial portion of the total was present within the mitochondria. 2. Activation of respiration by either ADP or uncoupling agent resulted in a decreased content of citrate and isocitrate and an increased content of 2-oxoglutarate and malate when the substrate was pyruvate, APT and HCO3 minus. Such a decrease in citrate content was obscured when the substrate was pyruvate and proline owing to a large rise in the total content of tricarboxylate-cycle intermediates in the presence of proline and ADP. 3. An experiment involving oligomycin and uncoupling agent demonstrated that the ATP/ADP ratio is the main determinant of flux through the tricarboxylate cycle, with the redox state of nicotinamide nucleotide being of lesser importance. 4. Addition of ADP and Ca-2+ to activate the oxidation of both glycerol 3-phosphate and pyruvate, simulating conditions on initiation of flight, gave a decrease in citrate and isocitrate and an increase in 2-oxoglutarate and malate content. 5. There was a good correlation between these results with isolated flight-muscle mitochondria and the changes found in fly thoraces after 30s and 2 mihorax. 6. It is concluded that NAD-isocitrate dehydrogenase (EC 1.1.1.41) controls the rate of pyruvate oxidation in both resting fly flight muscle in vivo and isolated mitochondria in state 4 (nomenclature of Change & Williams, 1955).

scholarly journals Pyruvate dehydrogenase activity in hamster small intestine during development

1977 ◽  
Vol 164 (3) ◽  
pp. 693-697 ◽  
Author(s):  
C M Schiller
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Redox State ◽  
Direct Relationship ◽  
Active Form ◽  
Total Activity ◽  
Complete Conversion ◽  
Mitochondrial Redox State ◽  
Nadh Ratio

Total pyruvate dehydrogenase activities in hamster intestine increase from 40 nmol/min (munits) per g of intestine in the foetal animals to 460 munits/g in the adult, whereas the fraction of the enzyme in the active form increases from 34 to 42% of the total activity over the same period. However, a complete conversion of the enzyme into the active form is observed in the neonatal animal immediately after birth. Results from experiments in vitro suggested that the activation of pyruvate dehydrogenase is controlled, in part, by the [NAD+]/[NADH] ratio. This proposal was tested in vivo by examining the proportion of the enzyme in the active form during conditions when the [NAD+]/[NADH] ratio was markedly altered, and the data show a direct relationship between the mitochondrial redox state and activity of the active form.

scholarly journals hSirT1-Dependent Regulation of the PCAF-E2F1-p73 Apoptotic Pathway in Response to DNA Damage

2009 ◽  
Vol 29 (8) ◽  
pp. 1989-1998 ◽  
Author(s):  
Natalia Pediconi ◽  
Francesca Guerrieri ◽  
Stefania Vossio ◽  
Tiziana Bruno ◽  
Laura Belloni ◽  
...  
Keyword(s):  
Dna Damage ◽  
Cell Survival ◽  
Stress Responses ◽  
Promoter Activity ◽  
Redox State ◽  
Apoptotic Pathway ◽  
Nadh Ratio ◽  
P53 Status

ABSTRACT The NAD+-dependent histone deacetylase hSirT1 regulates cell survival and stress responses by inhibiting p53-, NF-κB-, and E2F1-dependent transcription. Here we show that the hSirT1/PCAF interaction controls the E2F1/p73 apoptotic pathway. hSirT1 represses E2F1-dependent P1p73 promoter activity in untreated cells and inhibits its activation in response to DNA damage. hSirT1, PCAF, and E2F1 are corecruited in vivo on theP1p73 promoter. hSirT1 deacetylates PCAF in vitro and modulates PCAF acetylation in vivo. In cells exposed to apoptotic DNA damage, nuclear NAD+ levels decrease and inactivate hSirT1 without altering the hSirT1 interaction with PCAF and hSirT1 binding to the P1p73 promoter. The reactivation of hSirT1 by pyruvate that increases the [NAD+]/[NADH] ratio completely abolished the DNA damage-induced activation of TAp73 expression, thus linking the modulation of chromatin-bound hSirT1 deacetylase activity by the intracellular redox state with P1p73 promoter activity. The release of PCAF from hSirT1 repression favors the assembly of transcriptionally active PCAF/E2F1 complexes onto the P1p73 promoter and p53-independent apoptosis. Our results identify hSirT1 and PCAF as potential targets to modulate tumor cell survival and chemoresistance irrespective of p53 status.

Kinetics of Distribution of D2O and Antipyrine in Isolated Perfused Rat Liver

1958 ◽  
Vol 192 (3) ◽  
pp. 531-537 ◽  
Author(s):  
Alan M. Thompson ◽  
H. Mead Cavert ◽  
Nathan Lifson
Keyword(s):  
Rat Liver ◽  
Early Period ◽  
Tissue Perfusion ◽  
Isolated Perfused Rat Liver ◽  
Perfused Rat Liver ◽  
Rate Of Increase ◽  
The Difference ◽  
Rat Livers ◽  
Kinetics Of

Isolated rat livers were perfused via the portal vein with a blood-Ringer mixture containing a constant inflow concentration of D2O and, in some cases, antipyrine. The rate of increase of outflow concentration was studied, comparisons being made between D2O, antipyrine and a theoretical outflow curve based on completely flow-limited distribution. The effect of flow rate on the deviation of D2O from theoretical was also studied. The results indicate that exchange of D2O and antipyrine between blood and tissue in the perfused rat liver is extremely rapid relative to the rate of blood supply of these substances to the organ, even at flows several times that occurring in vivo. In the average experiment the average D2O concentration in the liver during the early period of the perfusion was about 90% of the mixed venous concentration. The factors responsible for the failure of D2O to distribute maximally, particularly tissue perfusion heterogeneity and diffusion limitation, are discussed. Although quantitation of the difference is difficult, antipyrine appears to distribute somewhat more rapidly than D2O due to its greater solubility in cell membranes.

scholarly journals Validation of a Gas Chromatography-Mass Spectrometry Method for the Measurement of the Redox State Metabolic Ratios Lactate/Pyruvate and β-Hydroxybutyrate/Acetoacetate in Biological Samples

2021 ◽  
Vol 22 (9) ◽  
pp. 4752
Author(s):  
Robin Wijngaard ◽  
Meritxell Perramón ◽  
Marina Parra-Robert ◽  
Susana Hidalgo ◽  
Gina Butrico ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Gas Chromatography ◽  
Redox State ◽  
Gas Chromatography Mass Spectrometry ◽  
Spectrometry Method ◽  
Limit Of Quantification ◽  
Experimental Conditions ◽  
Mass Spectrometry Method ◽  
Chromatography Mass Spectrometry

The metabolic ratios lactate/pyruvate and β-hydroxybutyrate/acetoacetate are considered valuable tools to evaluate the in vivo redox cellular state by estimating the free NAD+/NADH in cytoplasm and mitochondria, respectively. The aim of the current study was to validate a gas-chromatography mass spectrometry method for simultaneous determination of the four metabolites in plasma and liver tissue. The procedure included an o-phenylenediamine microwave-assisted derivatization, followed by liquid-liquid extraction with ethyl acetate and silylation with bis(trimethylsilyl)trifluoroacetamide:trimethylchlorosilane 99:1. The calibration curves presented acceptable linearity, with a limit of quantification of 0.001 mM for pyruvate, β-hydroxybutyrate and acetoacetate and of 0.01 mM for lactate. The intra-day and inter-day accuracy and precision were within the European Medicines Agency’s Guideline specifications. No significant differences were observed in the slope coefficient of three-point standard metabolite-spiked curves in plasma or liver and water, and acceptable recoveries were obtained in the metabolite-spiked samples. Applicability of the method was tested in precision-cut liver rat slices and also in HepG2 cells incubated under different experimental conditions challenging the redox state. In conclusion, the validated method presented good sensitivity, specificity and reproducibility in the quantification of lactate/pyruvate and β-hydroxybutyrate/acetate metabolites and may be useful in the evaluation of in vivo redox states.

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.

In vivo Production of Soluble Inorganic Pyrophosphatases in Two Strains of Vibrio cholerae

1970 ◽  
Vol 24 (1) ◽  
pp. 38-41
Author(s):  
Taslima Taher Lina ◽  
Mohammad Ilias
Keyword(s):  
Vibrio Cholerae ◽  
Specific Activity ◽  
Experimental Conditions ◽  
Environmental Strain ◽  
Cell Extracts ◽  
Atcc Strain ◽  
The Family ◽  
Effects Of Ph ◽  
Vibrio Cholerae O1

The in vivo production of soluble inorganic pyrophosphatases (PPases) was investigated in two strains, namely, Vibrio cholerae EM 004 (environmental strain) and Vibrio cholerae O1 757 (ATCC strain). V. cholerae is known to contain both family I and family II PPase coding sequences. The production of family I and family II PPases were determined by measuring the enzyme activity in cell extracts. The effects of pH, temperature, salinity of the growth medium on the production of soluble PPases were studied. In case of family I PPase, V. cholerae EM 004 gave the highest specific activity at pH 9.0, with 2% NaCl + 0.011% NaF and at 37°C. The strain V. cholerae O1 757 gave the highest specific activity at pH 9.0, with media containing 0% NaCl and at 37°C. On the other hand, under all the conditions family II PPase did not give any significant specific activity, suggesting that the family II PPase was not produced in vivo in either strains of V. cholerae under different experimental conditions. Keywords: Vibrio cholerae, Pyrophosphatases (PPases), Specific activityDOI: http://dx.doi.org/10.3329/bjm.v24i1.1235 Bangladesh J Microbiol, Volume 24, Number 1, June 2007, pp 38-41

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.

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