Fluorophore toxicity in mouse eggs and zygotes

Zygote ◽  
1998 ◽  
Vol 6 (2) ◽  
pp. 113-123 ◽  
Author(s):  
Karen P. Phillips ◽  
Wen-liang Zhou ◽  
Jay M. Baltz
Keyword(s):  
Membrane Damage ◽  
Fluorescence Excitation ◽  
Ion Concentrations ◽  
Fura 2 ◽  
Quantitative Measurements ◽  
Whole Cell Patch Clamp ◽  
Significant Toxicity ◽  
Intracellular Ion Concentrations ◽  
Mouse Eggs

Ion-sensitive fluorophores are commonly used for quantitative measurements of intracellular ion concentrations. However, both the method of intracellular loading — which for many fluorophores involves endogenous esterase-mediated removal of hydrophobic groups such as acetoxymethyl esters (AM) — and fluorescence excitation of fluorophores in the cell, can produce toxic metabolites and reactive species. Techniques used to measure intracellular ion concentrations in mammalian eggs and embryos are being increasingly employed, yet little information is available about any detrimental effects of the use of fluorophores. We have therefore used in vitro fertilisation (IVF) to assess potential fluorophore toxicity in mouse eggs, and whole cell patch-clamp recordings to detect fluorophore-associated membrane damage in zygotes. Four fluorophores were examined: SNARF-1 and BCECF (pH indicators), Fura-2 (Ca2+) and MQAE (Cl−). Cleavage of AM groups alone had no effect either on the success of IVF or on membrane electrical properties of mouse zygotes. Intracellularly loaded BCECF, SNARF-1 and Fura-2 followed by fluorescence excitation were not cell-toxic under the conditions examined. In contrast, MQAE demonstrated significant toxicity both alone and in combination with fluorescence excitation.

Characterization in vitro of H+ secretion and H+:Na+ exchange by an organism normally inhabiting a CO2-rich environment: Hymenolepis diminuta (Cestoda) in the rat intestine

1978 ◽  
Vol 56 (11) ◽  
pp. 2344-2354 ◽  
Author(s):  
R. B. Podesta
Keyword(s):  
Metabolic Pathways ◽  
Intestinal Parasite ◽  
Hymenolepis Diminuta ◽  
Ambient Fluid ◽  
Rat Intestine ◽  
Ion Concentrations ◽  
Intracellular Ion Concentrations ◽  
Metabolic Reactions ◽  
Stimulation Of

H+ and Na+ transport by the intestinal parasite Hymenolepis diminuta were studied in vitro. The flatworms acidified the ambient fluid by secreting H+ and the acidification could not be correlated with organic acid excretion. Ambient CO2-independent H+ secretion was attributed to protons of metabolic origin: dephosphorylation reactions and ionization of organic acids within the tissues. Ambient CO2-dependent H+ secretion was attributed to protons produced as a result of the hydration of CO2 within the tissue and to the stimulation of anaerobic metabolic pathways by CO2 acting as a cosubstrate in energy metabolism. Studies in which Na+ uptake was stimulated by CO2 or glucose and inhibited by ouabain, amiloride, or Na+ replacement suggested a partial direct coupling of Na+ absorption and H+ secretion but the different activation energies and the effect of buffer anions other than HCO3− suggested an indirect interaction. Various interactions were considered, including the effect of CO2 and intracellular ion concentrations on metabolic reactions leading to the supply of protons for H+ secretion and energy for ion transport.

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.

IMPACT OF SORBITOL- INDUCED OSMOTIC STRESS ON SOME BIOCHEMICAL TRAITS OF POTATO IN VITRO

2020 ◽  
Vol 51 (4) ◽  
pp. 1038-1047
Author(s):  
Mawia & et al.
Keyword(s):  
Ascorbic Acid ◽  
Osmotic Stress ◽  
Cytoplasmic Membrane ◽  
Genotypic Variation ◽  
Membrane Damage ◽  
Membrane Integrity ◽  
Culture Collection ◽  
Nutritive Medium ◽  
Starting Point

This study had as principal objective identification of osmotic-tolerant potato genotypes by using "in vitro" tissue culture and sorbitol as a stimulating agent, to induce water stress, which was added to the  culture nutritive medium in different concentration (0,50, 110, 220, 330 and 440 mM).  The starting point was represented by plantlets culture collection, belonging to eleven potato genotypes: Barcelona, Nectar, Alison, Jelly, Malice, Nazca, Toronto, Farida, Fabulla, Colomba and Spunta. Plantlets were multiplied between two internodes to obtain microcuttings (in sterile condition), which were inoculated on medium. Sorbitol-induced osmotic stress caused a significant reduction in the ascorbic acid, while the concentration of proline, H2O2 and solutes leakage increased compared with the control. Increased the proline content prevented lipid peroxidation, which played a pivotal role in the maintenance of membrane integrity under osmotic stress conditions. The extent of the cytoplasmic membrane damage depends on osmotic stress severity and the genotypic variation in the maintenance of membranes stability was highly associated with the ability of producing more amounts of osmoprotectants (proline) and the non-enzymic antioxidant ascorbic acid in response to osmotic stress level. The results showed that the genotypes Jelly, Nectar, Allison, Toronto, and Colomba are classified as highly osmotic stress tolerant genotypes, while the genotypes Nazca and Farida are classified as osmotic stress susceptible ones.

X-ray microanalysis of intracellular ions in the anaerobic halophilic eubacterium Haloanaerobium praevalens

1997 ◽  
Vol 43 (6) ◽  
pp. 588-592 ◽  
Author(s):  
Aharon Oren ◽  
Mikal Heldal ◽  
Svein Norland
Keyword(s):  
Electron Microscope ◽  
High Variability ◽  
Sodium Potassium ◽  
X Ray ◽  
Ion Concentrations ◽  
Major Cation ◽  
Transmission Electron ◽  
Intracellular Ion Concentrations ◽  
Intracellular Ions ◽  
Chloride Concentrations

The intracellular concentrations of Na+, K+, and Cl− of the anaerobic halophilic eubacterium Haloanaerobium praevalens were assayed by means of X-ray microanalysis with the transmission electron microscope. Apparent intracellular cation concentrations between 1.22 and 1.91 M and chloride concentrations of 0.93–1.57 M were measured in cells growing exponentially in 2.6 M total salts. In exponentially growing cells, K+ was the major cation (70% of the cation sum). Stationary phase cells showed a high variability among individual cells, some of the cells containing higher Na+ than K+ concentrations.Key words: Haloanaerobium praevalens, intracellular ion concentrations, sodium, potassium, X-ray microanalysis.

scholarly journals Needle-Free Delivery of Acetalated Dextran-Encapsulated AR-12 Protects Mice from Francisella tularensis Lethal Challenge

2016 ◽  
Vol 60 (4) ◽  
pp. 2052-2062 ◽  
Author(s):  
Ky V. Hoang ◽  
Heather Curry ◽  
Michael A. Collier ◽  
Hassan Borteh ◽  
Eric M. Bachelder ◽  
...  
Keyword(s):  
Francisella Tularensis ◽  
Lethal Challenge ◽  
Content Type ◽  
Human Macrophages ◽  
Gentamicin Treatment ◽  
Significant Toxicity ◽  
Serovar Typhimurium ◽  
Spectrum Activity

ABSTRACTFrancisella tularensiscauses tularemia and is a potential biothreat. Given the limited antibiotics for treating tularemia and the possible use of antibiotic-resistant strains as a biowarfare agent, new antibacterial agents are needed. AR-12 is an FDA-approved investigational new drug (IND) compound that induces autophagy and has shown host-directed, broad-spectrum activityin vitroagainstSalmonella entericaserovar Typhimurium andF. tularensis. We have shown that AR-12 encapsulated within acetalated dextran (Ace-DEX) microparticles (AR-12/MPs) significantly reduces host cell cytotoxicity compared to that with free AR-12, while retaining the ability to controlS.Typhimurium within infected human macrophages. In the present study, the toxicity and efficacy of AR-12/MPs in controlling virulent type AF. tularensisSchuS4 infection were examinedin vitroandin vivo. No significant toxicity of blank MPs or AR-12/MPs was observed in lung histology sections when the formulations were given intranasally to uninfected mice. In histology sections from the lungs of intranasally infected mice treated with the formulations, increased macrophage infiltration was observed for AR-12/MPs, with or without suboptimal gentamicin treatment, but not for blank MPs, soluble AR-12, or suboptimal gentamicin alone. AR-12/MPs dramatically reduced the burden ofF. tularensisin infected human macrophages, in a manner similar to that of free AR-12. However,in vivo, AR-12/MPs significantly enhanced the survival ofF. tularensisSchuS4-infected mice compared to that seen with free AR-12. In combination with suboptimal gentamicin treatment, AR-12/MPs further improved the survival ofF. tularensisSchuS4-infected mice. These studies provide support for Ace-DEX-encapsulated AR-12 as a promising new therapeutic agent for tularemia.

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