Delayed cyclin A and B1 degradation in non-transformed mammalian cells

1995 ◽  
Vol 108 (7) ◽  
pp. 2599-2608 ◽  
Author(s):  
F. Girard ◽  
A. Fernandez ◽  
N. Lamb
Keyword(s):  
Mammalian Cells ◽  
Marine Invertebrates ◽  
Cyclin A ◽  
Cdc2 Kinase ◽  
Marked Contrast ◽  
Anaphase Onset ◽  
Significant Difference ◽  
Primary Fibroblasts ◽  
Transformed Cell Lines ◽  
Derivatives Of

Cyclins A and B are known to exhibit significant differences in their function, cellular distribution and timing of degradation at mitosis. On the basis of observations in marine invertebrates and Xenopus, it was proposed that cyclin destruction triggers cdc2 kinase inactivation and anaphase onset. However, this model has recently been questioned, both in Xenopus and in budding yeast. In this report, we present evidence for delayed degradation of both cyclins A and B1 in non-transformed mammalian cells. Indeed, by means of indirect immunofluorescence and confocal microscopy, we show that cyclins A and B1 are present up to anaphase in REF52, Hs68, human primary fibroblasts and NRK epithelial cells. In marked contrast, cyclin A is shown to be degraded within metaphase and cyclin B just at the transition to anaphase in HeLa and two transformed cell lines, derivatives of normal NRK and REF52. These results further support the notion that cyclin destruction might be not correlated with anaphase onset in normal cells and highlight a significant difference in the fate of mitotic cyclins between transformed and non-transformed cells.

Inactivation of cdc2 kinase during mitosis requires regulated and constitutive proteins in a cell-free system

1993 ◽  
Vol 104 (3) ◽  
pp. 873-881
Author(s):  
F.A. Suprynowicz
Keyword(s):  
Mammalian Cells ◽  
Protein Phosphatase 1 ◽  
Cdc2 Kinase ◽  
Free System ◽  
Culture Cells ◽  
Anaphase Onset ◽  
Tissue Culture Cells ◽  
A Cell ◽  
Protein Components

Inactivation of the cyclin-p34cdc2 protein kinase complex is a major requirement for anaphase onset and exit from mitosis. To facilitate identification of specific molecules that regulate this event in mammalian cells, I have developed a cell-free assay in which cdc2 kinase associated with a chromosomal fraction from metaphase tissue culture cells is inactivated by a cell-cycle-regulated cytosolic system. In vitro kinase inactivation requires ATP, Mg2+ and the dephosphorylation of one or more sites in the chromosomal fraction by protein phosphatase 1 and/or 2A. Cyclin B is destroyed during inactivation, while the level of p34cdc2 remains constant. Ammonium sulfate fractionation resolves the cytosolic inactivating system into at least two distinct protein components that are both required for inactivation and are differentially regulated during mitosis.

ets-2 Regulates cdc2 Kinase Activity in Mammalian Cells: Coordinated Expression of cdc2 and Cyclin A

1995 ◽  
Vol 217 (1) ◽  
pp. 8-14 ◽  
Author(s):  
Shau-Ching Wen ◽  
De-Hui Ku ◽  
Antonio De Luca ◽  
Pier Paolo Claudio ◽  
Antonio Giordano ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Kinase Activity ◽  
Cyclin A ◽  
Cdc2 Kinase ◽  
Coordinated Expression

scholarly journals In vitro fusion of endocytic vesicles is inhibited by cyclin A-cdc2 kinase.

1993 ◽  
Vol 4 (5) ◽  
pp. 541-553 ◽  
Author(s):  
P G Woodman ◽  
J P Adamczewski ◽  
T Hunt ◽  
G Warren
Keyword(s):  
Indirect Effect ◽  
Mammalian Cells ◽  
Cyclin A ◽  
Vesicle Fusion ◽  
Cyclin B ◽  
Endocytic Vesicle ◽  
Cdc2 Kinase ◽  
Histone Kinase ◽  
Endocytic Vesicles

Receptor-mediated endocytosis and recycling are inhibited in mitotic mammalian cells, and previous studies have shown that inhibition of endocytic vesicle fusion in vitro occurs via cyclin B-cdc2 kinase. To test for the ability of cyclin A-cdc2 kinase to inhibit endocytic vesicle fusion, we employed recombinant cyclin A proteins. Addition of cyclin A to interphase extracts activated a histone kinase and markedly reduced the efficiency of endocytic vesicle fusion. By a number of criteria, inhibition of fusion was shown to be due to the action of cyclin A, via the mitosis-specific cdc2 kinase, and not an indirect effect through cyclin B. Two-stage incubations were used to demonstrate that at least one target of cyclin A-cdc2 kinase is a cytosolic component of the fusion apparatus. Reconstitution experiments showed that this component was also modified in mitotic cytosols and was unaffected by N-ethyl maleimide treatment.

scholarly journals Involvement of CGRP receptor RAMP1 in cluster headache: A Swedish case-control study

2019 ◽  
Vol 2 ◽  
pp. 251581631987988 ◽  
Author(s):  
Julia M Michalska ◽  
Caroline Ran ◽  
Carmen Fourier ◽  
Anna Steinberg ◽  
Christina Sjöstrand ◽  
...  
Keyword(s):  
Cluster Headache ◽  
Mrna Expression ◽  
Quantitative Polymerase Chain Reaction ◽  
Active Phase ◽  
Nucleotide Polymorphisms ◽  
Cgrp Receptor ◽  
Receptor Component ◽  
Significant Difference ◽  
Potent Vasodilator ◽  
Primary Fibroblasts

Background: Increased levels of the potent vasodilator calcitonin gene-related peptide (CGRP) have been found in ipsilateral jugular vein blood during the active phase of cluster headache (CH) and this is hypothesized to cause distinctive vasodilation. The receptor activity-modifying protein 1 (RAMP1) is part of the CGRP receptor complex responsible for ligand binding and specificity and therefore constitutes a promising candidate gene for CH. The aim of this study was to investigate the possible genetic association of RAMP1 with CH in Sweden, with focus on two RAMP1 single nucleotide polymorphisms, rs3754701 and rs7590387, and quantify RAMP1 mRNA expression levels in biological tissue from CH patients and controls. Methods: rs3754701 and rs7590387 were genotyped by quantitative polymerase chain reaction (qPCR) in 542 CH patients and 585 control subjects. RAMP1 mRNA expression was determined by reverse transcription qPCR in tissue from 12 CH patients and 12 controls. Results: We identified a significant difference between the CH patient and control groups for rs3754701 ( p = 0.0088). In addition, RAMP1 mRNA expression was enhanced in primary fibroblasts from CH patients compared to controls ( p = 0.0073). Conclusion: The association between rs3754701 and CH and the enhanced RAMP1 mRNA expression in CH patients support the hypothesis that CGRP and its receptor component RAMP1 are involved in CH pathophysiology.

scholarly journals Mutational Analysis of the Cy Motif from p21 Reveals Sequence Degeneracy and Specificity for Different Cyclin-Dependent Kinases

2001 ◽  
Vol 21 (15) ◽  
pp. 4868-4874 ◽  
Author(s):  
James A. Wohlschlegel ◽  
Brian T. Dwyer ◽  
David Y. Takeda ◽  
Anindya Dutta
Keyword(s):  
Inhibitory Activity ◽  
Mammalian Cells ◽  
Mutational Analysis ◽  
Cyclin E ◽  
Cyclin A ◽  
Binding Motif ◽  
Binding Studies ◽  
Alanine Scanning Mutagenesis ◽  
Cyclin Dependent Kinases

ABSTRACT Inhibitors, activators, and substrates of cyclin-dependent kinases (cdks) utilize a cyclin-binding sequence, known as a Cy or RXL motif, to bind directly to the cyclin subunit. Alanine scanning mutagenesis of the Cy motif of the cdk inhibitor p21 revealed that the conserved arginine or leucine (constituting the conserved RXL sequence) was important for p21's ability to inhibit cyclin E-cdk2 activity. Further analysis of mutant Cy motifs showed, however, that RXL was neither necessary nor sufficient for a functional cyclin-binding motif. Replacement of either of these two residues with small hydrophobic residues such as valine preserved p21's inhibitory activity on cyclin E-cdk2, while mutations in either polar or charged residues dramatically impaired p21's inhibitory activity. Expressing p21N with non-RXL Cy sequences inhibited growth of mammalian cells, providing in vivo confirmation that RXL was not necessary for a functional Cy motif. We also show that the variant Cy motifs identified in this study can effectively target substrates to cyclin-cdk complexes for phosphorylation, providing additional evidence that these non-RXL motifs are functional. Finally, binding studies using p21 Cy mutants demonstrated that the Cy motif was essential for the association of p21 with cyclin E-cdk2 but not with cyclin A-cdk2. Taking advantage of this differential specificity toward cyclin E versus cyclin A, we demonstrate that cell growth inhibition was absolutely dependent on the ability of a p21 derivative to inhibit cyclin E-cdk2.

The Polo-like kinase Plx1 is a component of the MPF amplification loop at the G2/M-phase transition of the cell cycle in Xenopus eggs

1998 ◽  
Vol 111 (12) ◽  
pp. 1751-1757 ◽  
Author(s):  
A. Abrieu ◽  
T. Brassac ◽  
S. Galas ◽  
D. Fisher ◽  
J.C. Labbe ◽  
...  
Keyword(s):  
Phase Transition ◽  
Cell Cycle ◽  
Cyclin A ◽  
Cyclin B ◽  
Cdc2 Kinase ◽  
C Terminus ◽  
M Phase ◽  
Egg Extracts ◽  
Amplification Loop

We have investigated whether Plx1, a kinase recently shown to phosphorylate cdc25c in vitro, is required for activation of cdc25c at the G2/M-phase transition of the cell cycle in Xenopus. Using immunodepletion or the mere addition of an antibody against the C terminus of Plx1, which suppressed its activation (not its activity) at G2/M, we show that Plx1 activity is required for activation of cyclin B-cdc2 kinase in both interphase egg extracts receiving recombinant cyclin B, and cycling extracts that spontaneously oscillate between interphase and mitosis. Furthermore, a positive feedback loop allows cyclin B-cdc2 kinase to activate Plx1 at the G2/M-phase transition. In contrast, activation of cyclin A-cdc2 kinase does not require Plx1 activity, and cyclin A-cdc2 kinase fails to activate Plx1 and its consequence, cdc25c activation in cycling extracts.

scholarly journals Distinct Peculiarities of In Planta Synthesis of Isoprenoid and Aromatic Cytokinins

Biomolecules ◽  
2020 ◽  
Vol 10 (1) ◽  
pp. 86
Author(s):  
Vladimir Oslovsky ◽  
Ekaterina Savelieva ◽  
Mikhail Drenichev ◽  
Georgy Romanov ◽  
Sergey Mikhailov
Keyword(s):  
Arabidopsis Thaliana ◽  
Plant Cells ◽  
Cytokinin Activity ◽  
Hormonal Activity ◽  
In Planta ◽  
Significant Difference ◽  
Aromatic Cytokinins ◽  
Derivatives Of ◽  
Ribose Moiety

The biosynthesis of aromatic cytokinins in planta, unlike isoprenoid cytokinins, is still unknown. To compare the final steps of biosynthesis pathways of aromatic and isoprenoid cytokinins, we synthesized a series of nucleoside derivatives of natural cytokinins starting from acyl-protected ribofuranosyl-, 2′-deoxyribofuranosyl- and 5′-deoxyribofuranosyladenine derivatives using stereoselective alkylation with further deblocking. Their cytokinin activity was determined in two bioassays based on model plants Arabidopsis thaliana and Amaranthus caudatus. Unlike active cytokinins-bases, cytokinin nucleosides lack the hormonal activity until the ribose moiety is removed. According to our experiments, ribo-, 2′-deoxyribo- and 5′-deoxyribo-derivatives of isoprenoid cytokinin N6-isopentenyladenine turned in planta into active cytokinins with clear hormonal activity. As for aromatic cytokinins, both 2′-deoxyribo- and 5′-deoxyribo-derivatives did not exhibit analogous activity in Arabidopsis. The 5′-deoxyribo-derivatives cannot be phosphorylated enzymatically in vivo; therefore, they cannot be “activated” by the direct LOG-mediated cleavage, largely occurring with cytokinin ribonucleotides in plant cells. The contrasting effects exerted by deoxyribonucleosides of isoprenoid (true hormonal activity) and aromatic (almost no activity) cytokinins indicates a significant difference in the biosynthesis of these compounds.

scholarly journals Nonclassical Biological Activities of Quinolone Derivatives

2011 ◽  
Vol 15 (1) ◽  
pp. 52 ◽  
Author(s):  
Mohsen Daneshtalab ◽  
Abeer Ahmed
Keyword(s):  
Mammalian Cells ◽  
Antibacterial Agents ◽  
Biological Activities ◽  
Antiviral Agent ◽  
Bioactive Molecules ◽  
Structural Modifications ◽  
Further Development ◽  
Derivatives Of ◽  
Ns5b Polymerase

Quinolones are considered as a big family of multi-faceted drugs; their chemical synthesis is flexible and can be easily adapted to prepare new congeners with rationally devised structures. This is shown by the description of many thousands of derivatives in the literature. Scientists could accurately describe their QSAR, which is essential for effective drug design. This also gave them the chance to discover new and unprecedented activities, which makes quinolones an endless source of hope and enables further development of new clinically useful drugs. Quinolones are among the most common frameworks present in the bioactive molecules that have dominated the market for more than four decades. Since 1962, 4(1H)-quinolone-3-carboxylic acid derivatives are widely used as antibacterial agents. Quinolones have a broad and potent spectrum of activity and are also used as second-line drugs to treat tuberculosis (TB). Recently, quinolones have been reported to display “nonclassical” biological activities, such as antitumor, anti-HIV-1 integrase, anti- HCV-NS3 helicase and -NS5B-polymerase activities. The present review focuses on the structural modifications responsible for the transformation of an antibacterial into an anticancer agent and/or an antiviral agent. Indeed, quinolones’ antimicrobial action is distinguishable among antibacterial agents, because they target different type II topoisomerase enzymes. Many derivatives of this family show high activity against bacterial topoisomerases and eukaryotic topoisomerases, and are also toxic to cultured mammalian cells and in vivo tumor models. Moreover, quinolones have shown antiviral activity against HIV and HCV viruses. In this context the quinolones family of drugs seem to link three different biological activities (antibacterial, anticancer, and the antiviral profiles) and the review will also provide an insight into the different mechanisms responsible for these activities among different species. This article is open to POST-PUBLICATION REVIEW. Registered readers (see “For Readers”) may comment by clicking on ABSTRACT on the issue’s contents page.

scholarly journals Inhibition of Chromosomal Separation Provides Insights into Cleavage Furrow Stimulation in Cultured Epithelial Cells

1998 ◽  
Vol 9 (8) ◽  
pp. 2173-2184 ◽  
Author(s):  
Sally P. Wheatley ◽  
Christopher B. O’Connell ◽  
Yu-li Wang
Keyword(s):  
Epithelial Cells ◽  
Mammalian Cells ◽  
Topoisomerase Ii ◽  
Rat Kidney ◽  
Myosin Ii ◽  
Daughter Cells ◽  
Irregular Shapes ◽  
Anaphase Onset ◽  
Cultured Epithelial Cells ◽  
Normal Rat

While astral microtubules are believed to be primarily responsible for the stimulation of cytokinesis in Echinodermembryos, it has been suggested that a signal emanating from the chromosomal region and mediated by the interzonal microtubules stimulates cytokinesis in cultured mammalian cells. To test this hypothesis, we examined cytokinesis in normal rat kidney cells treated with an inhibitor of topoisomerase II, (+)-1,2-bis(3,5-dioxopiperaz-inyl-1-yl)propane, which prevents the separation of sister chromatids and the formation of a spindle interzone. The majority of treated cells showed various degrees of abnormality in cytokinesis. Furrows frequently deviated from the equatorial plane, twisting daughter cells into irregular shapes. Some cells developed furrows in regions outside the equator or far away from the spindle. In addition, F-actin and myosin II accumulated at the lateral ingressing margins but did not form a continuous band along the equator as in control cells. Imaging of microinjected 5- (and 6-) carboxymtetramethylrhodamine-tubulin revealed that a unique set of microtubules projected out from the chromosomal vicinity upon anaphase onset. These microtubules emanated toward the lateral cortex, where they delineated sites of microtubule bundle formation, cortical ingression, and F-actin and myosin II accumulation. As centrosome integrity and astral microtubules appeared unperturbed by (+)-1,2-bis(3,5-dioxopiperaz-inyl-1-yl)propane treatment, the present observations cannot be easily explained by the conventional model involving astral microtubules. We suggest that in cultured epithelial cells the organization of the chromosomes dictates the organization of midzone microtubules, which in turn determines and maintains the cleavage activity.

Optical and geometric isomers of some fatty acids with vicinal hydroxy groups

1967 ◽  
Vol 45 (11) ◽  
pp. 1259-1265 ◽  
Author(s):  
D. F. Ewing ◽  
C. Y. Hopkins
Keyword(s):  
Chemical Shift ◽  
Seed Oil ◽  
Optically Active ◽  
Geometric Isomers ◽  
Nuclear Magnetic Resonance Spectra ◽  
Significant Difference ◽  
Isopropylidene Derivatives ◽  
Hydroxy Groups ◽  
Derivatives Of ◽  
Long Chain

Racemic threo-9,10-dihydroxypalmitic acid was resolved by means of the brucine salts into the optically active forms. Levorotatory threo-11,12-dihydroxyeicosanoic acid was obtained from the corresponding racemate by crystallization of the ephedrine salts.The conformation of the geometric isomers of certain dihydroxy long-chain acids and their derivatives was studied by means of nuclear magnetic resonance spectra. There was a small but significant difference in the chemical shift of the CH protons in the ( ± )-threo acid as compared with those in the ( ± )-erythro acid. This was observed in 9,10-dihydroxypalmitic, 9,10-dihydroxystearic, and 11,12-dihydroxyeicosanoic acids. A similar but larger difference in the chemical shift (0.49 p.p.m.) was observed for the CH protons in the O-isopropylidene derivatives of the same acids. These differences are discussed and correlated with the stereochemistry of the dihydroxy acids and the corresponding 1,3-dioxolanes.cis-( + )-12,13-Epoxyoleic acid was isolated from the seed oil of Vernonia colorata. ( − )-threo-12,13-Dihydroxyoleic acid was prepared from the oil of V. cinerea.

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