In vitro characterisation of calcium influx across skin and gut epithelia of the Pacific hagfish, Eptatretus stoutii

2020 ◽  
Vol 190 (2) ◽  
pp. 149-160
Author(s):  
Chris N. Glover ◽  
Greg G. Goss
Keyword(s):  
Calcium Influx ◽  
The Pacific ◽  
Eptatretus Stoutii

scholarly journals A sand fly salivary protein acts as a neutrophil chemoattractant

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Anderson B. Guimaraes-Costa ◽  
John P. Shannon ◽  
Ingrid Waclawiak ◽  
Jullyanna Oliveira ◽  
Claudio Meneses ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Calcium Influx ◽  
Parasite Burden ◽  
Salivary Protein ◽  
Sand Fly ◽  
Human Neutrophils ◽  
Chemotactic Protein ◽  
The Impact

AbstractApart from bacterial formyl peptides or viral chemokine mimicry, a non-vertebrate or insect protein that directly attracts mammalian innate cells such as neutrophils has not been molecularly characterized. Here, we show that members of sand fly yellow salivary proteins induce in vitro chemotaxis of mouse, canine and human neutrophils in transwell migration or EZ-TAXIScan assays. We demonstrate murine neutrophil recruitment in vivo using flow cytometry and two-photon intravital microscopy in Lysozyme-M-eGFP transgenic mice. We establish that the structure of this ~ 45 kDa neutrophil chemotactic protein does not resemble that of known chemokines. This chemoattractant acts through a G-protein-coupled receptor and is dependent on calcium influx. Of significance, this chemoattractant protein enhances lesion pathology (P < 0.0001) and increases parasite burden (P < 0.001) in mice upon co-injection with Leishmania parasites, underlining the impact of the sand fly salivary yellow proteins on disease outcome. These findings show that some arthropod vector-derived factors, such as this chemotactic salivary protein, activate rather than inhibit the host innate immune response, and that pathogens take advantage of these inflammatory responses to establish in the host.

scholarly journals Contractile and Endothelium-Dependent Dilatory Responses of Cerebral Arteries at Various Extracellular Magnesium Concentrations

1991 ◽  
Vol 11 (1) ◽  
pp. 161-164 ◽  
Author(s):  
Mária Faragó ◽  
Csaba Szabó ◽  
Eörs Dóra ◽  
Ildikó Horváth ◽  
Arisztid G. B. Kovách
Keyword(s):  
Smooth Muscle ◽  
Vascular Reactivity ◽  
Calcium Influx ◽  
Cerebral Arteries ◽  
Inhibitory Effect ◽  
Contractile Responses ◽  
Middle Cerebral Arteries ◽  
The Right ◽  
Endothelial Receptor

To clarify the effect of extracellular magnesium (Mg2+) on the vascular reactivity of feline isolated middle cerebral arteries, the effects of slight alterations in the Mg2+ concentration on the contractile and endothelium-dependent dilatory responses were investigated in vitro. The contractions, induced by 10−8-10−5 M norepinephrine, were significantly potentiated at low Mg2+ (0.8 m M v. the normal, 1.2 m M). High (1.6 and 2.0 m M) Mg2+ exhibited an inhibitory effect on the contractile responses. No significant changes, however, in the EC50 values for norepinephrine were found. The endothelium-dependent relaxations induced by 108–10−5 M acetylcholine were inhibited by high (1.6 and 2.0 m M) Mg2+. Lowering of the Mg2+ concentration to 0.8 m M or total withdrawal of this ion from the medium failed to alter the dilatory potency of acetylcholine. The changes in the dilatory responses also shifted the EC50 values for acetylcholine to the right. The present results show that the contractile responses of the cerebral arteries are extremely susceptible to the changes of Mg2+ concentrations. In response to contractile and endothelium-dependent dilatory agonists, Mg2+ probably affects both the calcium influx into the endothelial and smooth muscle cells as well as the binding of acetylcholine to its endothelial receptor. Since Mg2+ deficiency might facilitate the contractile but not the endothelium-dependent relaxant responses, the present study supports a role for Mg2+ deficiency in the development of the cerebral vasospasm.

Levobupivacaine-induced contraction of isolated rat aorta is calcium dependent

2011 ◽  
Vol 89 (7) ◽  
pp. 467-476 ◽  
Author(s):  
Ji Seok Baik ◽  
Ju-Tae Sohn ◽  
Seong-Ho Ok ◽  
Jae-Gak Kim ◽  
Hui-Jin Sung ◽  
...  
Keyword(s):  
Methylene Blue ◽  
Smooth Muscle ◽  
Calcium Influx ◽  
Rat Aorta ◽  
Isometric Tension ◽  
Guanosine Monophosphate ◽  
Response Curves ◽  
Calcium Dependent ◽  
Isolated Rat

Levobupivacaine is a long-acting local anesthetic that intrinsically produces vasoconstriction in isolated vessels. The goals of this study were to investigate the calcium-dependent mechanism underlying levobupivacaine-induced contraction of isolated rat aorta in vitro and to elucidate the pathway responsible for the endothelium-dependent attenuation of levobupivacaine-induced contraction. Isolated rat aortic rings were suspended to record isometric tension. Cumulative levobupivacaine concentration–response curves were generated in either the presence or absence of the antagonists verapamil, nifedipine, SKF-96365, 2-aminoethoxydiphenylborate, Gd3+, NW-nitro-l-arginine methyl ester (L-NAME), 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), and methylene blue, either alone or in combination. Verapamil, nifedipine, SKF-96365, 2-aminoethoxydiphenylborate, low calcium concentrations, and calcium-free Krebs solution attenuated levobupivacaine-induced contraction. Gd3+ had no effect on levobupivacaine-induced contraction. Levobupivacaine increased intracellular calcium levels in vascular smooth muscle cells. L-NAME, ODQ, and methylene blue increased levobupivacaine-induced contraction in endothelium-intact aorta. SKF-96365 attenuated calcium-induced contraction in a previously calcium-free isotonic depolarizing solution containing 100 mmol/L KCl. Levobupivacaine-induced contraction of rat aortic smooth muscle is mediated primarily by calcium influx from the extracellular space mainly via voltage-operated calcium channels and, in part, by inositol 1,4,5-trisphosphate receptor-mediated release of calcium from the sarcoplasmic reticulum. The nitric oxide – cyclic guanosine monophosphate pathway is involved in the endothelium-dependent attenuation of levobupivacaine-induced contraction.

Evaluation of the phytotoxicity of 2,4-diacetylphloroglucinol and Pseudomonas brassicacearum Q8r1-96 on different wheat cultivars

2021 ◽  
Author(s):  
Mingming Yang ◽  
Linda S Thomashow ◽  
David M Weller
Keyword(s):  
Seed Germination ◽  
Pacific Northwest ◽  
Root Colonization ◽  
The United States ◽  
Wheat Cultivars ◽  
The Pacific ◽  
Root Necrosis ◽  
Growth Promoting ◽  
Pseudomonas Brassicacearum

Pseudomonas brassicacearum Q8r1-96 and other 2,4-diacetylphloroglucinol (DAPG)-producing pseudomonads of the Pseudomonas fluorescens complex possess both biocontrol and growth-promoting properties and play an important role in suppression of take-all of wheat in the Pacific Northwest (PNW) of the United States. However, P. brassicacearum can also reduce seed germination and cause root necrosis on some wheat cultivars. We evaluated the effect of Q8r1-96 and DAPG on the germination of 69 wheat cultivars that have been or currently are grown in the PNW. Cultivars varied widely in their ability to tolerate P. brassicacearum or DAPG. The frequency of germination of the cultivars ranged from 0 to 0.87 and from 0.47 to 0.90 when treated with Q8r1-96 and DAPG, respectively. There was a significant positive correlation between the frequency of germination of cultivars treated with Q8r1-96 in assays conducted in vitro and in the greenhouse. The correlation was greater for spring than for winter cultivars. In contrast, the effect of Q8r1-96 on seed germination was not correlated with that of DAPG alone, suggesting that DAPG is not the only factor responsible for the phytotoxicity of Q8r1-96. Three wheat cultivars with the greatest tolerance and three cultivars with the least tolerance to Q8r1-96 were tested for their ability to support root colonization by strain Q8r1-96. Cultivars with the greatest tolerance supported significantly greater populations of strain Q8r1-96 than those with the least tolerance to the bacteria. Our results show that wheat cultivars differ widely in their interaction with P. brassicacearum and the biocontrol antibiotic DAPG.

scholarly journals Signal transduction mechanisms involved in in vitro ram sperm capacitation

Reproduction ◽  
2006 ◽  
Vol 132 (5) ◽  
pp. 721-732 ◽  
Author(s):  
Patricia Grasa ◽  
José Álvaro Cebrián-Pérez ◽  
Teresa Muiño-Blanco
Keyword(s):  
Tyrosine Phosphorylation ◽  
Calcium Influx ◽  
Calcium Entry ◽  
Calcium Entry Blocker ◽  
Protein Tyrosine Phosphorylation ◽  
Sperm Capacitation ◽  
Protein Tyrosine ◽  
Entry Blocker ◽  
Ram Sperm

We validate the chlortetracycline (CTC) technique for the evaluation of capacitation and acrosome reaction-like changes in ram sperm, carrying out a double estimation of the acrosome status after treatment with lysophosphatidylcholine, using fluorescein isocyanate (FITC)-RCA/ethidium homodimer 1 (EthD-1) and CTC/EthD-1. Highly consistent results and a positive correlation between the results of acrosome-reacted sperm evaluated with both techniques were obtained. In this study, we evaluate the effects of ram sperm capacitation of BSA, Ca2+, NaHCO3and cAMP agonists and their influence on the associated protein tyrosine phosphorylation. We found a time-dependent increase in capacitation related to protein tyrosine phosphorylation, either in the absence or the presence of BSA. The addition of an increasing concentration of cholesterol to samples containing BSA did not influence results. The effect of bicarbonate was concentration-dependent, with a significantly lowered value of non-capacitated sperm in the presence 18 and 25 mM. The addition of extracellular calcium did not significantly increase either the proportion of capacitated sperm or the protein tyrosine phosphorylation signalling, although a significantly higher value of acrosome-reacted sperm was found in samples containing 4 mM Ca2+. cAMP agonists increased capacitated sperm and protein tyrosine phosphorylation signalling. The inhibition of protein kinase A by H-89 caused a decrease in sperm capacitation. Addition of a calcium-entry blocker (Verapamil; Sigma) did not influence results, which suggests that the calcium entry blocker was unable to inhibit the calcium influx associated with capacitation in ram sperm. Our findings might benefit our understanding of the biochemical mechanisms involved in mammalian sperm capacitation and ultimately, fertility.

scholarly journals Nanobody-Mediated Inhibition of P2X7 on Microglia Improves Stroke Outcome

2021 ◽  
Author(s):  
Maximilian Wilmes ◽  
Carolina Pinto Espinoza ◽  
Peter Ludewig ◽  
Arthur Liesz ◽  
Annette Nicke ◽  
...  
Keyword(s):  
Ischemic Stroke ◽  
Transgenic Mice ◽  
Tissue Damage ◽  
Calcium Influx ◽  
Atp Release ◽  
Artery Occlusion ◽  
Membrane Pores ◽  
Caspase 1 ◽  
Ischemic Tissue

Abstract BackgroundPrevious studies have demonstrated that purinergic receptors could be therapeutic targets to modulate the inflammatory response in multiple brain disease models. However, tools for the selective and efficient targeting of these receptors are scarce. The new development of P2X7-specific nanobodies (nbs) enables us to effectively block the P2X7-channel.MethodsTemporary middle cerebral artery occlusion (tMCAO) in wildtype and P2X7-transgenic mice was used as a model for ischemic stroke. ATP release was assessed in transgenic ATP sensor mice. Stroke size was measured without treatment and after injection of P2X7-specific nbs i.v. and i.c.v. directly before tMCAO-surgery. P2X7-GFP expressing transgenic mice were used to show immunhistochemically P2X7 distribution in the brain. In vitro cultured microglia were used to investigate calcium-influx, pore-formation via DAPI uptake, caspase 1 activation and IL-1b release after incubation with P2X7-specific nbs. ResultsATP sensor mice showed an increase of ATP-release in the ischemic hemisphere compared to the contralateral hemisphere or sham mice up to 24 h after stroke. We could further verify the role of the ATP-P2X7 axis in P2X7-overexpressing mice, which showed significantly greater stroke volumes after 24 h. In vitro experiments with primary microglia cells showed that P2X7-specific nanobodies were capable of dampening the ATP-trigged calcium-influx and formation of membrane pores measured by Fluo4 fluorescence or DAPI uptake. We found a lower caspase 1 activity and a subsequently lower IL-1b release. However, the intravenous (i.v.) injection of P2X7-specific nanobodies compared to isotype controls before the tMCAO-surgery did not result in smaller stroke size compared to isotype controls. As demonstrated by FACS, nbs had only reached brain infiltrating macrophages but not microglia. To reach microglia, we injected the P2X7-spezific nbs or the isotype directly intraventricularly (icv). 30 mg of P2X7-specific nbs proved efficient for microglial targeting, reducing post-stroke microglia activation and stroke size significantly.ConclusionHere, we demonstrate the importance of locally produced ATP for the tissue damage observed in ischemic stroke and we show the potential of icv injected P2X7-specific nbs to reduce ischemic tissue damage.

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