scholarly journals Development of an In Vitro Membrane Model to Study the Function of EsxAB Heterodimer and Establish the Role of EsxB in Membrane Permeabilizing Activity of Mycobacterium tuberculosis

Pathogens ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1015
Author(s):  
Salvador Vazquez Reyes ◽  
Supriyo Ray ◽  
Javier Aguilera ◽  
Jianjun Sun
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Lipid Membranes ◽  
Effect Of Temperature ◽  
In Vitro Assays ◽  
Vitro Model ◽  
First Time ◽  
Dose Dependent Increase ◽  
Dose Dependent

EsxA and EsxB are secreted as a heterodimer and have been shown to play critical roles in phagosome rupture and translocation of Mycobacterium tuberculosis into the cytosol. Recent in vitro studies have suggested that the EsxAB heterodimer is dissociated upon acidification, which might allow EsxA insertion into lipid membranes. While the membrane permeabilizing activity (MPA) of EsxA has been well characterized in liposomes composed of di-oleoyl-phosphatidylcholine (DOPC), the MPA of EsxAB heterodimer has not been detected through in vitro assays due to its negligible activity with DOPC liposomes. In this study, we established a new in vitro membrane assay to test the MPA activity of N-terminal acetylated EsxA (N-EsxA). We established that a dose-dependent increase in anionic charged lipids enhances the MPA of N-EsxA. The MPA of both N-EsxA and EsxAB were significantly increased with this new liposome system and made it possible to characterize the MPA of EsxAB in more physiologically-relevant conditions. We tested, for the first time, the effect of temperature on the MPA of N-EsxA and EsxAB in this new system. Interestingly, the MPA of N-EsxA was lower at 37 °C than at RT, and on the contrary, the MPA of EsxAB was higher at 37 °C than at RT. Surprisingly, after incubation at 37 °C, the MPA of N-EsxA continuously decreased over time, while MPA of EsxAB remained stable, suggesting EsxB plays a key role in stabilizing N-EsxA to preserve its MPA at 37 °C. In summary, this study established a new in vitro model system that characterizes the MPA of EsxAB and the role of EsxB at physiological-relevant conditions.

scholarly journals Evolutionarily ancient role of cholecystokinin-type neuropeptide signalling as an inhibitory regulator of feeding-related processes revealed in an echinoderm

2020 ◽  
Author(s):  
Ana B. Tinoco ◽  
Antón Barreiro-Iglesias ◽  
Luis Alfonso Yañez-Guerra ◽  
Jérôme Delroisse ◽  
Ya Zhang ◽  
...  
Keyword(s):  
Body Wall ◽  
Oral Feeding ◽  
Cardiac Stomach ◽  
First Time ◽  
Dose Dependent ◽  
Type Receptor ◽  
Tube Feet

AbstractCholecystokinin (CCK) / sulfakinin (SK)-type neuropeptides regulate feeding and digestion in chordates and protostomes (e.g. insects). Here we characterised CCK/SK-type signalling for the first time in a non-chordate deuterostome - the starfish Asterias rubens (phylum Echinodermata). In this species, two neuropeptides (ArCCK1, ArCCK2) derived from the precursor protein ArCCKP act as ligands for a CCK/SK-type receptor (ArCCKR) and are expressed in the nervous system, digestive system, tube feet and body wall. Furthermore, ArCCK1 and ArCCK2 cause dose-dependent contraction of cardiac stomach, tube foot and body wall apical muscle preparations in vitro and injection of these neuropeptides in vivo triggers cardiac stomach retraction and inhibition of the onset of feeding in A. rubens. Thus, an evolutionarily ancient role of CCK/SK-type neuropeptides as inhibitory regulators of feeding-related processes in the Bilateria has been conserved in the unusual and unique context of the extra-oral feeding behaviour and pentaradial body plan of an echinoderm.

scholarly journals Ancient role of sulfakinin/cholecystokinin-type signalling in inhibitory regulation of feeding processes revealed in an echinoderm

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Ana B Tinoco ◽  
Antón Barreiro-Iglesias ◽  
Luis Alfonso Yañez Guerra ◽  
Jérôme Delroisse ◽  
Ya Zhang ◽  
...  
Keyword(s):  
Oral Feeding ◽  
Cardiac Stomach ◽  
First Time ◽  
Dose Dependent ◽  
Type Receptor ◽  
Tube Feet ◽  
Inhibitory Regulation

Sulfakinin (SK)/cholecystokinin (CCK)-type neuropeptides regulate feeding and digestion in protostomes (e.g. insects) and chordates. Here, we characterised SK/CCK-type signalling for the first time in a non-chordate deuterostome – the starfish Asterias rubens (phylum Echinodermata). In this species, two neuropeptides (ArSK/CCK1, ArSK/CCK2) derived from the precursor protein ArSK/CCKP act as ligands for an SK/CCK-type receptor (ArSK/CCKR) and these peptides/proteins are expressed in the nervous system, digestive system, tube feet, and body wall. Furthermore, ArSK/CCK1 and ArSK/CCK2 cause dose-dependent contraction of cardiac stomach, tube foot, and apical muscle preparations in vitro, and injection of these neuropeptides in vivo triggers cardiac stomach retraction and inhibition of the onset of feeding in A. rubens. Thus, an evolutionarily ancient role of SK/CCK-type neuropeptides as inhibitory regulators of feeding-related processes in the Bilateria has been conserved in the unusual and unique context of the extra-oral feeding behaviour and pentaradial body plan of an echinoderm.

scholarly journals Loxosceles gaucho spider venom and its sphingomyelinase fraction trigger the main functions of human and rabbit platelets

2011 ◽  
Vol 30 (10) ◽  
pp. 1567-1574 ◽  
Author(s):  
Flávio L Tavares ◽  
María E Peichoto ◽  
Danieli de Morais Rangel ◽  
Kátia C Barbaro ◽  
Maria Cristina Cirillo ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Spider Venom ◽  
Acid Generation ◽  
Rabbit Platelets ◽  
In Vitro Effects ◽  
Induced Aggregation ◽  
Dose Dependent Increase ◽  
Dose Dependent

Loxosceles venoms can promote severe local and systemic damages. We have previously reported that Loxosceles gaucho spider venom causes a severe early thrombocytopenia in rabbits. Herein, we investigated the in vitro effects of this venom and its sphingomyelinase fraction on the main functions of platelets. Whole venom and its fraction induced aggregation of both human and rabbit platelets. Aggregation was dependent of plasma component(s) but independent of venom-induced lysophosphatidic acid generation. There was no increase in the levels of lactate dehydrogenase during platelet aggregation, ruling out the possibility of platelet lysis. The increased expression of ligand-induced binding site 1 (LIBS1) induced by L. gaucho venom and its sphingomyelinase fraction, as well as of P-selectin by the whole venom, evidenced the activation state of both human and rabbit platelets. Adhesion assays showed an irregular response when platelets were exposed to the whole venom, whereas the sphingomyelinase fraction induced a dose-dependent increase in the platelet adhesion to collagen. These findings evidence that L. gaucho venom and its sphingomyelinase fraction trigger adhesion, activation, and aggregation of both human and rabbit platelets. Thus, this work justifies the use of rabbits to investigate Loxosceles venom-induced platelet disturbances, and it also supports research on the role of platelets in the pathogenesis of loxoscelism.

scholarly journals Sls1p Stimulates Sec63p-Mediated Activation of Kar2p in a Conformation-Dependent Manner in the Yeast Endoplasmic Reticulum

2000 ◽  
Vol 20 (18) ◽  
pp. 6923-6934 ◽  
Author(s):  
Mehdi Kabani ◽  
Jean-Marie Beckerich ◽  
Claude Gaillardin
Keyword(s):  
Endoplasmic Reticulum ◽  
Protein Translocation ◽  
Synthetic Lethality ◽  
In Vitro Assays ◽  
Endoplasmic Reticulum Membrane ◽  
Dependent Manner ◽  
Dose Dependent

ABSTRACT We previously characterized the SLS1 gene in the yeastYarrowia lipolytica and showed that it interacts physically with YlKar2p to promote translocation across the endoplasmic-reticulum membrane (A. Boisramé, M. Kabani, J. M. Beckerich, E. Hartmann, and C. Gaillardin, J. Biol. Chem. 273:30903–30908, 1998). A Y. lipolytica Kar2p mutant was isolated that restored interaction with an Sls1p mutant, suggesting that the interaction with Sls1p could be nucleotide and/or conformation dependent. This result was used as a working hypothesis for more accurate investigations in Saccharomyces cerevisiae. We show by two-hybrid an in vitro assays that the S. cerevisiae homologue of Sls1p interacts with ScKar2p. Using dominant lethal mutants of ScKar2p, we were able to show that ScSls1p preferentially interacts with the ADP-bound conformation of the molecular chaperone. Synthetic lethality was observed between ΔScsls1 and translocation-deficientkar2 or sec63-1 mutants, providing in vivo evidence for a role of ScSls1p in protein translocation. Synthetic lethality was also observed with ER-associated degradation and folding-deficient kar2 mutants, strongly suggesting that Sls1p functions are not restricted to the translocation process. We show that Sls1p stimulates in a dose-dependent manner the binding ofScKar2p on the lumenal J domain of Sec63p fused to glutathione S-transferase. Moreover, Sls1p is shown to promote the Sec63p-mediated activation of Kar2p's ATPase activity. Our data strongly suggest that Sls1p could be the first GrpE-like protein described in the endoplasmic reticulum.

Energetic dependence of NPY-induced LH secretion in a teleost fish (Dicentrarchus labrax)

1999 ◽  
Vol 277 (6) ◽  
pp. R1627-R1634 ◽  
Author(s):  
José Miguel Cerdá-Reverter ◽  
Lisa Ann Sorbera ◽  
Manuel Carrillo ◽  
Silvia Zanuy
Keyword(s):  
Glucose Metabolism ◽  
Luteinizing Hormone ◽  
Dicentrarchus Labrax ◽  
Sea Bass ◽  
Pituitary Cells ◽  
Lh Secretion ◽  
Dose Dependent Increase ◽  
Dose Dependent

The purpose of this work was to examine the role of energetic status in neuropeptide Y (NPY)-induced luteinizing hormone (LH) secretion and glucose metabolism in fish. Fasted juvenile sea bass ( Dicentrarchus labrax) were injected intraperitoneally with pig (p) NPY or pNPY + glucose, whereas fed animals were injected with pNPY alone and plasma glucose, insulin, and LH levels were examined. pNPY alone or in combination with glucose was found to induce a dose-dependent increase in LH secretion in fasted animals. Similar LH responses to pNPY were observed in vitro in dispersed pituitary cells isolated from fed and fasted animals incubated in L-15 and restricted media. Injection of pNPY + glucose in fasted animals resulted in depletion of glucose. Insulin plasma levels decreased in fasted animals coinjected with pNPY + glucose but remained stable when NPY was administrated alone to fed and fasted animals. Results suggest that 1) NPY-induced LH secretion in fish is dependent on energetic status and 2) NPY is capable of modifying glucose metabolism.

scholarly journals Inhibition of CYP21A2 Enzyme Activity Caused by Novel Missense Mutations Identified in Brazilian and Scandinavian Patients

2008 ◽  
Vol 93 (6) ◽  
pp. 2416-2420 ◽  
Author(s):  
F. C. Soardi ◽  
M. Barbaro ◽  
I. F. Lau ◽  
S. H. V. Lemos-Marini ◽  
M. T. M. Baptista ◽  
...  
Keyword(s):  
Binding Capacity ◽  
In Vitro Assays ◽  
Missense Mutations ◽  
Hydroxylase Deficiency ◽  
Rare Mutations ◽  
17 Hydroxyprogesterone ◽  
21 Hydroxylase Deficiency ◽  
First Time

Abstract Background: Most patients with 21-hydroxylase deficiency carry CYP21A1P-derived mutations, but an increasing number of novel and rare mutations have been reported in disease-causing alleles. Objective: Functional effects of three novel (p.G56R, p.L107R, p.L142P) and one recurrent (p.R408C) CYP21A2 mutations were investigated. The degree of enzyme impairment caused by p.H62L alone or combined to p.P453S was also analyzed. Design: The study included 10 Brazilian and two Scandinavian patients. To determine the deleterious role of each mutant protein, in vitro assays were performed in transiently transfected COS-1 cells. For a correct genotype-phenotype correlation, the enzymatic activities were evaluated toward the two natural substrates, 17-hydroxyprogesterone and progesterone. Results: Low levels of residual activities obtained for p.G56R, p.L107R, p.L142P, and p.R408C mutants classified them as classical congenital adrenal hyperplasia mutations, whereas the p.H62L showed an activity within the range of nonclassical mutations. Apparent kinetic constants for p.H62L confirmed the nonclassical classification as the substrate binding capacity was within the same magnitude for mutant and normal enzymes. A synergistic effect was observed for the allele bearing the p.H62L+p.P453S combination because it caused a significant reduction in the enzymatic activity. Conclusions: We describe the functional analysis of five rare missense mutations identified in Brazilian and Scandinavian patients. The p.G56R, p.L107R, and p.L142P are reported for the first time. Most probably these novel mutations are closer to null than the p.I172N, but for the p.G56R, that might not be the case, and the p.H62L is definitely a nonclassical mutation.

Role of calcium and protein kinase C in ANP secretion by cultured rat cardiocytes

1988 ◽  
Vol 255 (3) ◽  
pp. H405-H409 ◽  
Author(s):  
H. Matsubara ◽  
Y. Hirata ◽  
H. Yoshimi ◽  
S. Takata ◽  
Y. Takagi ◽  
...  
Keyword(s):  
Protein Kinase C ◽  
Protein Kinase ◽  
Direct Action ◽  
Atrial Myocytes ◽  
Kinase C ◽  
Muscarinic Cholinergic ◽  
Dose Dependent Increase ◽  
Dose Dependent

The secretory mechanism of rat atrial natriuretic peptide (rANP) was studied in vitro with the use of primary culture of atrial myocytes from neonatal rats. Norepinephrine, phenylephrine, and carbamylcholine stimulated immunoreactive (IR) rANP secretion, whereas neither angiotensin II, arginine vasopressin, nor isoproterenol affected its secretion. The stimulatory effects of carbamylcholine and phenylephrine were blocked by atropine and prazosin, respectively. 12-O-tetradecanoylphorbol-beta-acetate (TPA), protein kinase C activator, induced a dose-dependent increase in IR rANP secretion, and TPA combined with Ca2+ ionophore ionomycin produced a synergistic effect. Ca2+-channel agonist BAY K 8644 also stimulated IR rANP secretion, the effect of which was blocked by Ca2+-channel antagonist nifedipine. These data suggest that alpha 1-adrenergic and muscarinic cholinergic agonists have direct action on rat cardiocytes to stimulate ANP secretion that involves receptor-mediated mobilization of intracellular Ca2+ and activation of protein kinase C.

scholarly journals An Alternative In Vitro Method for Examining Nanoparticle-Induced Cytotoxicity

2019 ◽  
Vol 38 (5) ◽  
pp. 385-394 ◽  
Author(s):  
Qiang Gu ◽  
Elvis Cuevas ◽  
Syed F. Ali ◽  
Merle G. Paule ◽  
Victor Krauthamer ◽  
...  
Keyword(s):  
Culture Media ◽  
Protective Role ◽  
In Vitro Assays ◽  
Ag Nps ◽  
Validation Data ◽  
Vitro Assay ◽  
Brain Microvessel ◽  
First Time

Conventional in vitro assays are often used as initial screens to identify potential toxic effects of nanoparticles (NPs). However, many NPs have shown interference with conventional in vitro assays, resulting in either false-positive or -negative outcomes. Here, we report an alternative method for the in vitro assessment of NP-induced cytotoxicity utilizing Fluoro-Jade C (FJ-C). To provide proof of concept and initial validation data, Ag-NPs and Au-NPs were tested in 3 different cell cultures including rat brain microvessel endothelial cells, mouse neural stem cells, and the human SH-SY5Y cell line. Conventional 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide (XTT) and lactate dehydrogenase (LDH) assays were run in parallel with the new method and served as references. The results demonstrate for the first time that FJ-C labeling can be a useful tool for assessing NP-induced cytotoxicity in vitro. Using these approaches, it was also demonstrated that removal of Ag-NPs—while keeping the Ag-ions that were released from the Ag-NPs in culture media—abolished the measured cytotoxicity, indicating that Ag-NPs rather than Ag-ions in solution contributed to the observed cytotoxic effects. Further, co-treatment of Ag-NPs with N-acetyl cysteine (NAC) prevented the observed cytotoxicity, suggesting a protective role of NAC in Ag-NP-induced cytotoxicity. Thus, this alternative in vitro assay is well suited for identify potential cytotoxicity associated with exposure to NPs.

Chondroitin Sulfate in Palatal Wound Healing

2004 ◽  
Vol 83 (11) ◽  
pp. 880-885 ◽  
Author(s):  
X.H. Zou ◽  
W.C. Foong ◽  
T. Cao ◽  
B.H. Bay ◽  
H.W. Ouyang ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Wound Healing ◽  
Cell Adhesion ◽  
Chondroitin Sulfate ◽  
Healing Process ◽  
Wound Healing Process ◽  
Dose Dependent Increase ◽  
Dose Dependent

Chondroitin sulfate is up-regulated in granulation tissue during wound healing. To investigate the role of chondroitin sulfate in the wound-healing process after surgical repair of cleft palate, we isolated and cultured rabbit palatal fibroblasts. Treatment with chondroitin-6-sulfate resulted in a dose-dependent increase in cell adhesion and cell proliferation, whereas the reverse effects were seen after chondroitinase degradation of chondroitin sulfate. The biological actions of chondroitin sulfate appeared to be dependent on the presence and position of sulfate groups. Inhibition of glycosaminoglycan sulfation by chlorate treatment led to reduced cell adhesion and cell proliferation and a slower rate of wound closure in vitro. Furthermore, exposure to chondroitin-4-sulfate resulted in a dose-dependent reduction in cell adhesion. Together, these results show that chondroitin sulfate is involved in palatal wound healing.

Sign in / Sign up

Export Citation Format

Share Document