Simultaneous measurement of intracellular pH and Ca2+ using the fluorescence of SNARF-1 and fura-2

1991 ◽  
Vol 260 (2) ◽  
pp. C297-C307 ◽  
Author(s):  
R. Martinez-Zaguilan ◽  
G. M. Martinez ◽  
F. Lattanzio ◽  
R. J. Gillies
Keyword(s):  
Intracellular Ph ◽  
Simultaneous Measurement ◽  
Physiological Response ◽  
Second Messengers ◽  
Physiological Responses ◽  
High Time Resolution ◽  
Experimental Conditions ◽  
Fura 2 ◽  
Fluorescence Characteristics

Upon cell stimulation with hormones and other mitogens, a variety of biochemical and physiological responses occur within the first few minutes. Changes in both intracellular pH (pHin) and intracellular Ca2+ concentration ([Ca2+]in) are prominent and play a major role in the signal transduction mechanism leading to the physiological response, i.e., secretion, neurotransmission, proliferation, or differentiation. However, it is not clear whether these ions work independently in the activation pathway leading to a particular physiological response. The fluorescence characteristics of most Ca2+ indicators are pH sensitive, and quantitative estimates of [Ca2+]in cannot be made without knowledge of pHin. Thus it is desirable to have a technique to simultaneously monitor these two ions with relatively high time resolution. Here we have developed experimental conditions that allow us to use optimum emission conditions for a pH fluorescent indicator SNARF-1 and optimum excitation conditions for the Ca2+ indicator fura-2. The fluorescence spectra of these compounds are sufficiently different to allow simultaneous measurement of pH and Ca2+ both in vitro and in situ. We have observed simultaneous changes in both pHin and [Ca2+]in in BALB/c 3T3 cells on treatment with the nonfluorescent Ca2+ ionophore 4-bromo-A23187. This temporal relationship between pHin and Ca2+ gives further credence to the interrelationship between these two second messengers in the expression of physiological responses.

pH of isolated resting skeletal muscle and its relation to potassium content

1963 ◽  
Vol 204 (6) ◽  
pp. 1048-1054 ◽  
Author(s):  
Ronald B. Miller ◽  
Ian Tyson ◽  
Arnold S. Relman
Keyword(s):  
Skeletal Muscle ◽  
Intracellular Ph ◽  
Potassium Content ◽  
Ion Concentration ◽  
Detectable Effect ◽  
Rat Diaphragm ◽  
Intracellular Acidosis ◽  
Experimental Conditions ◽  
Hydrogen Ion Concentration

Intracellular pH of isolated rat diaphragm was measured with both a C14-DMO method and a tissue CO2 technique. The values for intracellular pH by each method, although slightly different, changed in parallel under most experimental conditions. Acute, severe potassium depletion in vitro had no detectable effect on intracellular pH, nor did prior depletion in vivo followed by incubation in a potassium-free bath. This was true whether or not the potassium-depleted muscle was exposed to normal or elevated extracellular levels of bicarbonate, and was unaffected by the presence of cationic amino acids in the bath. Acute repletion of previously potassium-depleted muscle resulted in a small rise in cell pH, but this was no greater than that produced by loading normal tissues with potassium. It is concluded that under the conditions of these experiments there is no evidence of intracellular acidosis in potassium-depleted skeletal muscle. Rat diaphragm can lose up to half its potassium content in vitro without detectable increase in hydrogen ion concentration.

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.

Enhanced Increases in Cytosolic Ca2+ in ADP-Stimulated Platelets from Patients with Delta-Storage Pool Deficiency – A Possible Indicator of Interactions between Granule-Bound ADP and the Membrane ADP Receptor

1997 ◽  
Vol 77 (02) ◽  
pp. 376-382 ◽  
Author(s):  
Bruce Lages ◽  
Harvey J Weiss
Keyword(s):  
Platelet Rich Plasma ◽  
Limited Range ◽  
Dense Granule ◽  
Receptor Desensitization ◽  
Fura 2 ◽  
Storage Pool ◽  
Low Levels ◽  
The Right

SummaryThe possible involvement of secreted platelet substances in agonist- induced [Ca2+]i increases was investigated by comparing these increases in aspirin-treated, fura-2-loaded normal platelets and platelets from patients with storage pool deficiencies (SPD). In the presence and absence of extracellular calcium, the [Ca2+]i response induced by 10 µM ADP, but not those induced by 0.1 unit/ml thrombin, 3.3 µM U46619, or 20 µM serotonin, was significantly greater in SPD platelets than in normal platelets, and was increased to the greatest extent in SPD patients with Hermansky-Pudlak syndrome (HPS), in whom the dense granule deficiencies are the most severe. Pre-incubation of SPD-HPS and normal platelets with 0.005-5 µM ADP produced a dose-dependent inhibition of the [Ca2+]i response induced by 10 µ M ADP, but did not alter the [Ca2+]i increases induced by thrombin or U46619. Within a limited range of ADP concentrations, the dose-inhibition curve of the [Ca2+]i response to 10 µM ADP was significantly shifted to the right in SPD-HPS platelets, indicating that pre-incubation with greater amounts of ADP were required to achieve the same extent of inhibition as in normal platelets. These results are consistent with a hypothesis that the smaller ADP-induced [Ca2+]i increases seen in normal platelets may result from prior interactions of dense granule ADP, released via leakage or low levels of activation, with membrane ADP receptors, causing receptor desensitization. Addition of apyrase to platelet-rich plasma prior to fura-2 loading increased the ADP-induced [Ca2+]i response in both normal and SPD-HPS platelets, suggesting that some release of ADP derived from both dense granule and non-granular sources occurs during in vitro fura-2 loading and platelet washing procedures. However, this [Ca2+]i response was also greater in SPD-HPS platelets when blood was collected with minimal manipulation directly into anticoagulant containing apyrase, raising the possibility that release of dense granule ADP resulting in receptor desensitization may also occur in vivo. Thus, in addition to enhancing platelet activation, dense granule ADP could also act to limit the ADP-mediated reactivity of platelets exposed in vivo to low levels of stimulation.

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 In vitro Osmotic Stress Memory in Chrisanthemum hybridum: Structural and Physiological Responses

2017 ◽  
Vol 27 (2) ◽  
pp. 161-169 ◽  
Author(s):  
Lidiia Samarina ◽  
Valentina Malyarovskaya ◽  
Yulija Abilfazova ◽  
Natalia Platonova ◽  
Kristina Klemeshova ◽  
...  
Keyword(s):  
Osmotic Stress ◽  
Relative Water Content ◽  
Soluble Protein ◽  
Growth Parameters ◽  
Physiological Responses ◽  
Proline Content ◽  
Stress Exposure ◽  
Stress Memory ◽  
Relative Water

Structural and physiological responses of chrysanthemum to repeated osmotic stress were studied. Plants were cultured for 2 weeks (for each stress1 and stress 2) on half MS supplemented with mannitol 100 mM (Treatment I) and 200 mM (Treatment II). First stress inhibited growth parameters stronger than second stress in treatment I. In treatment II both stress events strongly inhibited growth parameters of micro‐shoots. Proline content exceeded control 6 ‐ 8 times after 1st stress, and 2 ‐ 5 times after the 2nd stress in treatments I and II, respectively. Soluble protein was accumulated in leaves during both stress exposures, and 2 ‐ 2.5 times exceeded control after the 2nd stress. Relative water content in both treatments increased after the 2nd stress exposure. In treatment II chlorophyll а and carotenoids contents were 8.78 and 4.62 mg/g comparing to control (4.21 and 2.25 mg/g, respectively) after the 1st stress. But after the 2nd stress there was no difference with control.Plant Tissue Cult. & Biotech. 27(2): 161-169, 2017 (December)

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 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.

scholarly journals Link between Lipid Second Messengers and Osmotic Stress in Plants

2021 ◽  
Vol 22 (5) ◽  
pp. 2658
Author(s):  
Beatriz A. Rodas-Junco ◽  
Graciela E. Racagni-Di-Palma ◽  
Michel Canul-Chan ◽  
Javier Usorach ◽  
S. M. Teresa Hernández-Sotomayor
Keyword(s):  
Osmotic Stress ◽  
Protein Conformation ◽  
Membrane Lipids ◽  
Second Messengers ◽  
Physiological Responses ◽  
Plant Cells ◽  
Transient Molecules ◽  
Molecular Approaches ◽  
Extracellular Signal ◽  
Different Types

Plants are subject to different types of stress, which consequently affect their growth and development. They have developed mechanisms for recognizing and processing an extracellular signal. Second messengers are transient molecules that modulate the physiological responses in plant cells under stress conditions. In this sense, it has been shown in various plant models that membrane lipids are substrates for the generation of second lipid messengers such as phosphoinositide, phosphatidic acid, sphingolipids, and lysophospholipids. In recent years, research on lipid second messengers has been moving toward using genetic and molecular approaches to reveal the molecular setting in which these molecules act in response to osmotic stress. In this sense, these studies have established that second messengers can transiently recruit target proteins to the membrane and, therefore, affect protein conformation, activity, and gene expression. This review summarizes recent advances in responses related to the link between lipid second messengers and osmotic stress in plant cells.

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