Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, reversibly inhibits chromatin condensation during in vitro maturation of porcine oocytes

Zygote ◽  
2001 ◽  
Vol 9 (4) ◽  
pp. 309-316 ◽  
Author(s):  
Carsten Krischek ◽  
Burkhard Meinecke
Keyword(s):  
Map Kinase ◽  
Protein Kinases ◽  
Chromatin Condensation ◽  
Specific Inhibitor ◽  
Histone H1 ◽  
Dependent Manner ◽  
Free Medium ◽  
Concentration Dependent Manner ◽  
Dependent Protein

In the present study the effects of roscovitine on the in vitro nuclear maturation of porcine oocytes were investigated. Roscovitine, a specific inhibitor of cyclin-dependent protein kinases, prevented chromatin condensation in a concentration-dependent manner. This inhibition was reversible and was accompanied by non-activation of p34cdc2/histone H1 kinase. It also decreased enzyme activity of MAP kinase, suggesting a correlation between histone H1 kinase activation and the onset of chromatin condensation. The addition of roscovitine (50 μM) to extracts of metaphase II oocytes revealed that the MAP kinase activity was not directly affected by roscovitine, which indicates a possible link between histone H1 and MAP kinase. Chromatin condensation occurred between 20 and 28 h of culture of cumulus-oocyte complexes (COCs) in inhibitor-free medium (germinal vesicle stage I, GV1: 74.6% and 13.7%, respectively). Nearly the same proportion of chromatin condensation was detected in COCs incubated initially in inhibitor-free medium for 20-28 h and subsequently in roscovitine-supplemented medium (50 μM) for a further 2-10 h (GV I: 76.2% and 18.8%, respectively). This observation indicates that roscovitine prevents chromatin condensation even after an initial inhibitor-free cultivation for 20 h. Extending this initial incubation period to ≥22 h led to an activation of histone H1 and MAP kinase and increasing proportions of oocytes exhibiting chromatin condensation in the presence of roscovitine. It is concluded that histone H1 kinase is involved in the induction of chromatin condensation during in vitro maturation of porcine oocytes.

Escherichia coli LPS induces heat shock protein 25 in intestinal epithelial cells through MAP kinase activation

2004 ◽  
Vol 286 (4) ◽  
pp. G645-G652 ◽  
Author(s):  
Keishi Kojima ◽  
Mark W. Musch ◽  
Mark J. Ropeleski ◽  
David L. Boone ◽  
Averil Ma ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Heat Shock ◽  
Map Kinase ◽  
Enteric Bacteria ◽  
Dependent Manner ◽  
Intestinal Epithelial ◽  
Filamentous Actin ◽  
Concentration Dependent Manner ◽  
Epithelial Integrity

Protection of colonic epithelial integrity and function is critical, because compromises in mucosal functions can lead to adverse and potentially life-threatening effects. The gut flora may contribute to this protection, in part, through the sustained induction of cytoprotective heat shock proteins (HSPs) in surface colonocytes. In this study, we investigated whether Escherichia coli LPS mediates bacteria-induced HSP by using cultured young adult mouse colon (YAMC) cells, an in vitro model of the colonic epithelium. E. coli LPS led to an epithelial cell-type specific induction of HSP25 in a time- and concentration-dependent manner, an effect that did not involve changes in HSP72. YAMC cells expressed the toll-like receptors (TLR)2 and TLR4 but not the costimulatory CD14 molecule. Whereas LPS stimulated both the p38 and ERK1/2 but not the stress-activated protein kinase/c-Jun NH2-terminal kinase, signaling pathways in the YAMC cells, all three were stimulated in RAW macrophage cells (in which no LPS-induced HSP25 expression was observed). The p38 inhibitor SB-203580 and the MAP kinase kinase-1 inhibitor PD-98059 inhibited HSP25 induction by LPS. LPS treatment also conferred protection against actin depolymerization induced by the oxidant monochloramine. The HSP25 dependence of the LPS protective effect was outlined in inhibitor studies and through adenovirus-mediated overexpression of HSP25. In conclusion, LPS may be an important mediator of enteric bacteria-induced expression of intestinal epithelial HSP25, an effect that may contribute to filamentous actin stabilization under physiological as well as pathophysiological conditions and thus protection of colonic epithelial integrity.

scholarly journals In Vitro Evidence for Effects of Magnesium Supplementation on Quinolone-treated Horse and Dog Chondrocytes

2001 ◽  
Vol 38 (2) ◽  
pp. 143-148 ◽  
Author(s):  
M. Egerbacher ◽  
B. Wolfesberger ◽  
C. Gabler
Keyword(s):  
Cell Proliferation ◽  
Magnesium Deficiency ◽  
Great Part ◽  
Control Group ◽  
Dependent Manner ◽  
Free Medium ◽  
Culture Dish ◽  
Magnesium Supplementation ◽  
Concentration Dependent Manner

Quinolones and magnesium deficiency cause similar lesions in joint cartilage of young animals. Chondrocytes cultivated in the presence of quinolones and in Mg-free medium show severe alterations in cytoskeleton and decreased ability to adhere to the culture dish. We investigated whether Mg2+ supplementation can prevent quinolone-mediated effects on chondrocytes in vitro. Chondrocytes cultivated in Dulbecco's modified Eagle's medium/HAM's F-12 medium were treated with ciprofloxacin (80 and 160 μg/ml) and enrofloxacin (100 and 150 μg/ml). Mg2+ was added at a concentration of 0.0612 mg/ml (MgCl) and 0.0488 mg/ml (MgSO4) or a triple dose. In addition, cells were cultivated in Mg-free medium and accordingly treated with Mg2+ supplementation. After 5 days in culture, the number of adherent cells per milliliter was determined. The number of chondrocytes in quinolone-treated groups decreased to 12-36% that of the control group within the culture period. With Mg2+ supplementation, the number of attached cells increased to 40-70% that of control cells. The threefold dose of Mg2+ led to better results than did the single dose. Cell proliferation tested by immunohistochemical staining with Ki67 (clone MIB5) decreased from 70% in control groups to 55%, 48%, and 30% in enrofloxacintreated groups in a concentration dependent manner (50, 100, and 150 μg/ml). Addition of Mg2+ did not increase the rate of cell proliferation. These results suggest that a great part of quinolone-induced damage is due to magnesium complex formation, as Mg2+ supplementation is able to reduce the effects in vitro. However, quinolone effects on cell proliferation seem to be an independent process that is not influenced by magnesium supplementation.

scholarly journals The anti-malarial drug atovaquone potentiates platinum-mediated cancer cell death by increasing oxidative stress

2020 ◽  
Vol 6 (1) ◽  
Author(s):  
James T. T. Coates ◽  
Gonzalo Rodriguez-Berriguete ◽  
Rathi Puliyadi ◽  
Thomas Ashton ◽  
Remko Prevo ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Cell Death ◽  
Cancer Cell ◽  
Tumor Hypoxia ◽  
Specific Inhibitor ◽  
Cytotoxic Agents ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Cancer Cell Death

Abstract Platinum chemotherapies are highly effective cytotoxic agents but often induce resistance when used as monotherapies. Combinatorial strategies limit this risk and provide effective treatment options for many cancers. Here, we repurpose atovaquone (ATQ), a well-tolerated & FDA-approved anti-malarial agent by demonstrating that it potentiates cancer cell death of a subset of platinums. We show that ATQ in combination with carboplatin or cisplatin induces striking and repeatable concentration- and time-dependent cell death sensitization in vitro across a variety of cancer cell lines. ATQ induces mitochondrial reactive oxygen species (mROS), depleting intracellular glutathione (GSH) pools in a concentration-dependent manner. The superoxide dismutase mimetic MnTBAP rescues ATQ-induced mROS production and pre-loading cells with the GSH prodrug N-acetyl cysteine (NAC) abrogates the sensitization. Together, these findings implicate ATQ-induced oxidative stress as key mediator of the sensitizing effect. At physiologically achievable concentrations, ATQ and carboplatin furthermore synergistically delay the growth of three-dimensional avascular spheroids. Clinically, ATQ is a safe and specific inhibitor of the electron transport chain (ETC) and is concurrently being repurposed as a candidate tumor hypoxia modifier. Together, these findings suggest that ATQ is deserving of further study as a candidate platinum sensitizing agent.

scholarly journals Uterotonic Effects of Aqueous and Methanolic Extracts of Lannea Acida in Wistar Rats: An in Vitro Study.

2020 ◽  
Author(s):  
esther simo ngadjui ◽  
Jibril Yves Kouam ◽  
Georges Romeo Fozin Bonsou ◽  
Aimé Césaire Tetsatsi Momo ◽  
Patrick Brice Defo Deeh ◽  
...  
Keyword(s):  
Wistar Rats ◽  
Methanolic Extract ◽  
Female Rats ◽  
Organ Bath ◽  
Dependent Manner ◽  
Free Medium ◽  
Concentration Dependent Manner ◽  
Methanolic Extracts ◽  
Oxytocin Receptors

Abstract Background: Lannea acida (Anacardiaceae), commonly called Kikié in the Noun division (West-Cameroon), is a tree whose bark is used locally to solve difficult childbirth. This study aimed to evaluate the in vitro uterotonic effects of aqueous and methanolic extracts of L. acida in female Wistar rats. Uterine strips isolated from female rats pretreated (48h) with oestradiol (5μg) were mounted in a single-organ bath containing a well aerated and thermostated De Jalon solution (37°C). The effects of L. acida extracts were recorded in a non-cumulative manner after application. The effect of the methanolic extract (the most active extract) was monitored in the presence of atosiban (a competitive antagonist of oxytocin receptors), atropine (a specific type 3 muscarinic receptor antagonist), nifedipine (an L-type calcium channel antagonist) and 2-Aminoethoxydiphenyl borate (2-ADB, a specific antagonist of inositol 1,4,5-triphosphate receptors type 1), and in calcium-free medium containing EGTA. Results: L. acidainduced uterine contraction in a concentration-dependent manner with the methanolic extract (1.506 ± 0.032 gf) being the most effective. Administration of atosiban (2 μmol/l), atropine (1 μmol/l), nifedipine (5 μmol), 2-APB (100 μmol), and calcium free medium containing EGTA (2 mmol) reduced the contractile effect of L. acida. Complete inhibition was observed with nifedipine, 2-APB, and calcium free medium containing EGTA.Conclusions: These results suggest that L. acida possesses an uterotonic effect mediated through oxytocin receptors with mobilization of extracellular calcium.

scholarly journals Synapsins as major neuronal Ca2+/S100A1-interacting proteins

1999 ◽  
Vol 344 (2) ◽  
pp. 577-583 ◽  
Author(s):  
Jörg HEIERHORST ◽  
Ken I. MITCHELHILL ◽  
Richard J. MANN ◽  
Tony TIGANIS ◽  
Andrew J. CZERNIK ◽  
...  
Keyword(s):  
Protein Kinases ◽  
Double Labelling ◽  
Dependent Manner ◽  
Interacting Proteins ◽  
Confocal Immunofluorescence Microscopy ◽  
Confocal Immunofluorescence ◽  
Brain Extracts ◽  
Dependent Protein

The mammalian S100A1 protein can activate the invertebrate myosin-associated giant protein kinase twitchin in a Ca2+-dependent manner by more than 1000-fold in vitro; however, no mammalian S100-dependent protein kinases are known. In an attempt to identify novel mammalian Ca2+/S100A1-regulated protein kinases, brain extracts were subjected to combined Ca2+-dependent affinity chromatography with S100A1 and an ATP analogue. This resulted in the purification to near-homogeneity of the four major synapsin isoforms Ia, Ib, IIa and IIb. All four synapsins were specifically affinity-labelled with the ATP analogue 5′-p-fluorosulphonylbenzoyladenosine. S100A1 bound to immobilized synapsin IIa in BIAcore experiments in a Ca2+-dependent and Zn2+-enhanced manner with submicromolar affinity; this interaction could be competed for with synthetic peptides of the proposed S100A1-binding sites of synapsin. Double-labelling confocal immunofluorescence microscopy demonstrated that synapsins and S100A1 are both present in the soma and neurites of PC12 cells, indicating their potential to interact in neurons in vivo.

scholarly journals Induction of a cytoplasmic activator of DNA synthesis in lymphocytes is mediated through a membrane-associated protein kinase.

1990 ◽  
Vol 1 (13) ◽  
pp. 1015-1025 ◽  
Author(s):  
M V Autieri ◽  
K L Fresa ◽  
F D Coffman ◽  
M E Katz ◽  
S Cohen
Keyword(s):  
Protein Kinase ◽  
Dna Synthesis ◽  
Protein Kinases ◽  
Phorbol Esters ◽  
Adenosine Monophosphate ◽  
Peripheral Blood Lymphocytes ◽  
Lymphoid Cells ◽  
Dependent Manner ◽  
Dependent Protein

We have shown previously that cytoplasmic extracts from actively dividing lymphoid cells are capable of inducing DNA synthesis in isolated nuclei. One of the factors involved in this activity, ADR, appears to be a greater than 90 kDa heat-labile protease. Cytoplasmic extracts prepared from nonproliferating lymphocytes express little to no ADR activity. However, ADR activity can be generated in these extracts by brief exposure to a membrane-enriched fraction of spontaneously proliferating, leukemic human T lymphoblastoid (MOLT-4) cells. This suggests that ADR activity is present in the resting cytoplasm in an inactive or precursor form. This in vitro generation of ADR activity can be inhibited in a dose-dependent manner by the isoquinolinesulfonamide derivative, H-7 (1-(5-isoquinoline-sulfonyl)-2-methylpiperazine dihydrochloride), an inhibitor of both cyclic adenosine monophosphate (cAMP)-dependent protein kinases and protein kinase C (PKC). However, more specific inhibitors of cAMP-dependent protein kinases, including N-[( 2-methylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride (H8) and N-(2-gua-nidinoethyl)-5-isoquinolinesulfonamide (HA-1004), had little to no effect on the in vitro generation of ADR activity. Furthermore, membranes from MOLT-4 cells depleted of PKC by long-term exposure (24 h) to phorbol esters and calcium ionophores were unable to induce ADR activity in resting peripheral blood lymphocytes extracts. The results of these studies suggest 1) ADR activity is present in resting cell cytoplasm in an inactive or precursor form; and 2) ADR activity can be induced in this resting cytoplasm through a mechanism involving a membrane-associated protein kinase, possibly PKC. The ability of alkaline phosphatase to deplete the activity of preformed ADR suggests the possibility that ADR itself is phosphoprotein.

Chickpea (Cicer arietinum L.) Lectin Exhibit Inhibition of ACE-I, α-amylase and α-glucosidase Activity

2019 ◽  
Vol 26 (7) ◽  
pp. 494-501 ◽  
Author(s):  
Sameer Suresh Bhagyawant ◽  
Dakshita Tanaji Narvekar ◽  
Neha Gupta ◽  
Amita Bhadkaria ◽  
Ajay Kumar Gautam ◽  
...  
Keyword(s):  
Biological Effects ◽  
Exchange Chromatography ◽  
Health Concern ◽  
Carbohydrate Binding ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Ic50 Values ◽  
Chickpea Lectin

Background: Diabetes and hypertension are the major health concern and alleged to be of epidemic proportions. This has made it a numero uno subject at various levels of investigation. Glucosidase inhibitor provides the reasonable option in treatment of Diabetes Mellitus (DM) as it specifically targets post prandial hyperglycemia. The Angiotensin Converting Enzyme (ACE) plays an important role in hypertension. Therefore, inhibition of ACE in treatment of elevated blood pressure attracts special interest of the scientific community. Chickpea is a food legume and seeds contain carbohydrate binding protein- a lectin. Some of the biological properties of this lectin hitherto been elucidated. Methods: Purified by ion exchange chromatography, chickpea lectin was tested for its in vitro antioxidant, ACE-I inhibitory and anti-diabetic characteristic. Results: Lectin shows a characteristic improvement over the synthetic drugs like acarbose (oral anti-diabetic drug) and captopril (standard antihypertensive drug) when, their IC50 values are compared. Lectin significantly inhibited α-glucosidase and α-amylase in a concentration dependent manner with IC50 values of 85.41 ± 1.21 ҝg/ml and 65.05 ± 1.2 µg/ml compared to acarbose having IC50 70.20 ± 0.47 value of µg/ml and 50.52 ± 1.01 µg/ml respectively. β-Carotene bleaching assay showed antioxidant activity of lectin (72.3%) to be as active as Butylated Hydroxylanisole (BHA). In addition, lectin demonstrated inhibition against ACE-I with IC50 value of 57.43 ± 1.20 µg/ml compared to captopril. Conclusion: Lectin demonstrated its antioxidant character, ACE-I inhibition and significantly inhibitory for α-glucosidase and α-amylase seems to qualify as an anti-hyperglycemic therapeutic molecule. The biological effects of chickpea lectin display potential for reducing the parameters of medically debilitating conditions. These characteristics however needs to be established under in vivo systems too viz. animals through to humans.

Improved Method for Obtaining of Arctigenin from Arctium Lappa L. and its Antiproliferative Effect on Human Hepatocarcinoma HepG2 Cells

2020 ◽  
Vol 16 (3) ◽  
pp. 358-362
Author(s):  
Renan S. Teixeira ◽  
Paulo H.D. Carvalho ◽  
Jair A.K. Aguiar ◽  
Valquíria P. Medeiros ◽  
Ademar A. Da Silva Filho ◽  
...  
Keyword(s):  
Antitumor Activity ◽  
Crude Extract ◽  
Hepg2 Cells ◽  
Liver Carcinoma ◽  
Adhesion Assay ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Arctium Lappa ◽  
Nih 3T3

Background: Arctigenin is a lignan found in Arctium lappa L. (Asteraceae) that displays anti-inflammatory activities. Previous studies showed that the crude extract of A. Lappa has antitumor activity in human liver carcinoma, lung and stomach cancer cells. The aim of this study was to obtain arctigenin from A. lappa L., as well as to evaluate its antiproliferative effects in cells of liver carcinoma (HepG2) and fibroblasts (NIH/3T3). Methods: Arctigenin was obtained from the hydrolysis of arctiin, which was isolated from the crude extract of A. lappa. The effects of arctigenin and arctiin on HepG2 cell viability and cell adhesion were analyzed by MTT method. Adhesion assay was also carried out to evaluate the antitumor activity. Results: Our results showed that the analytical process to obtain arctigenin was fast and easy. In vitro experiments showed that arctigenin (107-269 μM) decreased HepG2 cells viability and did not cause cytotoxicity on NIH/3T3 cells. Arctigenin (27-269 μM) demonstrated anti-adhesion in HepG2 cells in a concentration-dependent manner, when compared with control. Conclusion: These results suggest a promising pharmacological activity for arctigenin as an antiproliferative compound.

scholarly journals Human Neuronal Cell Lines as An In Vitro Toxicological Tool for the Evaluation of Novel Psychoactive Substances

2021 ◽  
Vol 22 (13) ◽  
pp. 6785
Author(s):  
Valeria Sogos ◽  
Paola Caria ◽  
Clara Porcedda ◽  
Rafaela Mostallino ◽  
Franca Piras ◽  
...  
Keyword(s):  
Cell Death ◽  
Neuronal Cell ◽  
In Vitro Study ◽  
Dependent Manner ◽  
Novel Psychoactive Substances ◽  
Psychoactive Substances ◽  
Concentration Dependent Manner ◽  
Mechanisms Of Toxicity ◽  
Neuronal Cell Lines

Novel psychoactive substances (NPS) are synthetic substances belonging to diverse groups, designed to mimic the effects of scheduled drugs, resulting in altered toxicity and potency. Up to now, information available on the pharmacology and toxicology of these new substances is very limited, posing a considerable challenge for prevention and treatment. The present in vitro study investigated the possible mechanisms of toxicity of two emerging NPS (i) 4′-methyl-alpha-pyrrolidinoexanophenone (3,4-MDPHP), a synthetic cathinone, and (ii) 2-chloro-4,5-methylenedioxymethamphetamine (2-Cl-4,5-MDMA), a phenethylamine. In addition, to apply our model to the class of synthetic opioids, we evaluated the toxicity of fentanyl, as a reference compound for this group of frequently abused substances. To this aim, the in vitro toxic effects of these three compounds were evaluated in dopaminergic-differentiated SH-SY5Y cells. Following 24 h of exposure, all compounds induced a loss of viability, and oxidative stress in a concentration-dependent manner. 2-Cl-4,5-MDMA activates apoptotic processes, while 3,4-MDPHP elicits cell death by necrosis. Fentanyl triggers cell death through both mechanisms. Increased expression levels of pro-apoptotic Bax and caspase 3 activity were observed following 2-Cl-4,5-MDMA and fentanyl, but not 3,4-MDPHP exposure, confirming the different modes of cell death.

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