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Leslie Wilson - One of the best experts on this subject based on the ideXlab platform.
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maytansinoid antibody conjugates induce mitotic arrest by suppressing microtubule Dynamic Instability
Molecular Cancer Therapeutics, 2010Co-Authors: Emin Oroudjev, Leslie Wilson, Manu Lopus, Charlene Audette, Carmela Provenzano, Hans K Erickson, Yelena Kovtun, Ravi V J Chari, Mary Ann JordanAbstract:Maytansine and its analogues (maytansinoids) are potent microtubule-targeted compounds that inhibit proliferation of cells at mitosis. Antibody-maytansinoid conjugates consisting of maytansinoids (DM1 and DM4) attached to tumor-specific antibodies have shown promising clinical results. To determine the mechanism by which the antibody-DM1 conjugates inhibit cell proliferation, we examined the effects of the cleavable anti-EpCAM-SPP-DM1 and uncleavable anti-EpCAM-SMCC-DM1 conjugates on MCF7 human breast tumor cells. We also examined the effects of the free maytansinoids, maytansine and S-methyl DM1 (a version of DM1 that is stable in cell culture medium), for comparison. Both the conjugates and free maytansinoids potently inhibited MCF7 cell proliferation at nanomolar and subnanomolar concentrations, respectively, by arresting the cells in mitotic prometaphase/metaphase. Arrest occurred in concert with the internalization and intracellular processing of both conjugates under conditions that induced abnormal spindle organization and suppressed microtubule Dynamic Instability. Microtubule depolymerization occurred only at significantly higher drug concentrations. The results indicate that free maytansinoids, antibody-maytansinoid conjugates, and their metabolites exert their potent antimitotic effects through a common mechanism involving suppression of microtubule Dynamic Instability.
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eribulin binds at microtubule ends to a single site on tubulin to suppress Dynamic Instability
Biochemistry, 2010Co-Authors: Jennifer A Smith, Leslie Wilson, Olga Azarenko, Bryan M Lewis, Bruce A Littlefield, Mary Ann JordanAbstract:Eribulin mesylate (E7389), a synthetic analogue of the marine natural product halichondrin B, is in phase III clinical trials for the treatment of cancer. Eribulin targets microtubules, suppressing Dynamic Instability at microtubule plus ends through an inhibition of microtubule growth with little or no effect on shortening [Jordan, M. A., et al. (2005) Mol. Cancer Ther. 4, 1086−1095]. Using [3H]eribulin, we found that eribulin binds soluble tubulin at a single site; however, this binding is complex with an overall Kd of 46 μM, but also showing a real or apparent very high affinity (Kd = 0.4 μM) for a subset of 25% of the tubulin. Eribulin also binds microtubules with a maximum stoichiometry of 14.7 ± 1.3 molecules per microtubule (Kd = 3.5 μM), strongly suggesting the presence of a relatively high-affinity binding site at microtubule ends. At 100 nM, the concentration that inhibits microtubule plus end growth by 50%, we found that one molecule of eribulin is bound per two microtubules, indicating that th...
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eribulin binds at microtubule ends to a single site on tubulin to suppress Dynamic Instability
Biochemistry, 2010Co-Authors: Jennifer A Smith, Leslie Wilson, Olga Azarenko, Bryan M Lewis, Bruce A Littlefield, Xiaojie Zhu, Mary Ann JordanAbstract:Eribulin mesylate (E7389), a synthetic analogue of the marine natural product halichondrin B, is in phase III clinical trials for the treatment of cancer. Eribulin targets microtubules, suppressing Dynamic Instability at microtubule plus ends through an inhibition of microtubule growth with little or no effect on shortening [Jordan, M. A., et al. (2005) Mol. Cancer Ther. 4, 1086−1095]. Using [3H]eribulin, we found that eribulin binds soluble tubulin at a single site; however, this binding is complex with an overall Kd of 46 μM, but also showing a real or apparent very high affinity (Kd = 0.4 μM) for a subset of 25% of the tubulin. Eribulin also binds microtubules with a maximum stoichiometry of 14.7 ± 1.3 molecules per microtubule (Kd = 3.5 μM), strongly suggesting the presence of a relatively high-affinity binding site at microtubule ends. At 100 nM, the concentration that inhibits microtubule plus end growth by 50%, we found that one molecule of eribulin is bound per two microtubules, indicating that th...
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regulation of microtubule Dynamic Instability in vitro by differentially phosphorylated stathmin
Journal of Biological Chemistry, 2009Co-Authors: Tapas Manna, Srinivas Honnappa, Douglas Thrower, Michel O. Steinmetz, Leslie WilsonAbstract:Stathmin is an important regulator of microtubule polymerization and Dynamics. When unphosphorylated it destabilizes microtubules in two ways, by reducing the microtubule polymer mass through sequestration of soluble tubulin into an assembly-incompetent T2S complex (two α:β tubulin dimers per molecule of stathmin), and by increasing the switching frequency (catastrophe frequency) from growth to shortening at plus and minus ends by binding directly to the microtubules. Phosphorylation of stathmin on one or more of its four serine residues (Ser16, Ser25, Ser38, and Ser63) reduces its microtubule-destabilizing activity. However, the effects of phosphorylation of the individual serine residues of stathmin on microtubule Dynamic Instability have not been investigated systematically. Here we analyzed the effects of stathmin singly phosphorylated at Ser16 or Ser63, and doubly phosphorylated at Ser25 and Ser38, on its ability to modulate microtubule Dynamic Instability at steady-state in vitro. Phosphorylation at either Ser16 or Ser63 strongly reduced or abolished the ability of stathmin to bind to and sequester soluble tubulin and its ability to act as a catastrophe factor by directly binding to the microtubules. In contrast, double phosphorylation of Ser25 and Ser38 did not affect the binding of stathmin to tubulin or microtubules or its catastrophe-promoting activity. Our results indicate that the effects of stathmin on Dynamic Instability are strongly but differently attenuated by phosphorylation at Ser16 and Ser63 and support the hypothesis that selective targeting by Ser16-specific or Ser63-specific kinases provides complimentary mechanisms for regulating microtubule function.
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suppression of microtubule Dynamic Instability by the tip protein eb1 and its modulation by the cap gly domain of p150glued
Biochemistry, 2008Co-Authors: Tapas Manna, Srinivas Honnappa, Michel O. Steinmetz, Leslie WilsonAbstract:The EB1+TIP protein family and its binding partners track growing plus ends of microtubules in cells and are thought to regulate their Dynamics. Here we determined the effects of EB1 and the N-terminal CAP-Gly domain (p150n) of one of its major binding partners, p150Glued, both separately and together, on the Dynamic Instability parameters at plus ends of purified steady-state microtubules. With EB1 alone, the shortening rate, the extent of shortening, and the catastrophe frequency were suppressed in the absence of significant effects on the growth rate or rescue frequency. The effects of EB1 on Dynamics were significantly different when p150n was added together with EB1. The rate and extent of shortening and the catastrophe frequency were suppressed 3−4 times more strongly than with EB1 alone. In addition, the EB1−p150n complex increased the rescue frequency and the mean length the microtubules grew, parameters that were not significantly affected by EB1 alone. Similarly, deletion of EB1's C-terminal tai...
Mary Ann Jordan - One of the best experts on this subject based on the ideXlab platform.
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maytansinoid antibody conjugates induce mitotic arrest by suppressing microtubule Dynamic Instability
Molecular Cancer Therapeutics, 2010Co-Authors: Emin Oroudjev, Leslie Wilson, Manu Lopus, Charlene Audette, Carmela Provenzano, Hans K Erickson, Yelena Kovtun, Ravi V J Chari, Mary Ann JordanAbstract:Maytansine and its analogues (maytansinoids) are potent microtubule-targeted compounds that inhibit proliferation of cells at mitosis. Antibody-maytansinoid conjugates consisting of maytansinoids (DM1 and DM4) attached to tumor-specific antibodies have shown promising clinical results. To determine the mechanism by which the antibody-DM1 conjugates inhibit cell proliferation, we examined the effects of the cleavable anti-EpCAM-SPP-DM1 and uncleavable anti-EpCAM-SMCC-DM1 conjugates on MCF7 human breast tumor cells. We also examined the effects of the free maytansinoids, maytansine and S-methyl DM1 (a version of DM1 that is stable in cell culture medium), for comparison. Both the conjugates and free maytansinoids potently inhibited MCF7 cell proliferation at nanomolar and subnanomolar concentrations, respectively, by arresting the cells in mitotic prometaphase/metaphase. Arrest occurred in concert with the internalization and intracellular processing of both conjugates under conditions that induced abnormal spindle organization and suppressed microtubule Dynamic Instability. Microtubule depolymerization occurred only at significantly higher drug concentrations. The results indicate that free maytansinoids, antibody-maytansinoid conjugates, and their metabolites exert their potent antimitotic effects through a common mechanism involving suppression of microtubule Dynamic Instability.
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eribulin binds at microtubule ends to a single site on tubulin to suppress Dynamic Instability
Biochemistry, 2010Co-Authors: Jennifer A Smith, Leslie Wilson, Olga Azarenko, Bryan M Lewis, Bruce A Littlefield, Mary Ann JordanAbstract:Eribulin mesylate (E7389), a synthetic analogue of the marine natural product halichondrin B, is in phase III clinical trials for the treatment of cancer. Eribulin targets microtubules, suppressing Dynamic Instability at microtubule plus ends through an inhibition of microtubule growth with little or no effect on shortening [Jordan, M. A., et al. (2005) Mol. Cancer Ther. 4, 1086−1095]. Using [3H]eribulin, we found that eribulin binds soluble tubulin at a single site; however, this binding is complex with an overall Kd of 46 μM, but also showing a real or apparent very high affinity (Kd = 0.4 μM) for a subset of 25% of the tubulin. Eribulin also binds microtubules with a maximum stoichiometry of 14.7 ± 1.3 molecules per microtubule (Kd = 3.5 μM), strongly suggesting the presence of a relatively high-affinity binding site at microtubule ends. At 100 nM, the concentration that inhibits microtubule plus end growth by 50%, we found that one molecule of eribulin is bound per two microtubules, indicating that th...
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eribulin binds at microtubule ends to a single site on tubulin to suppress Dynamic Instability
Biochemistry, 2010Co-Authors: Jennifer A Smith, Leslie Wilson, Olga Azarenko, Bryan M Lewis, Bruce A Littlefield, Xiaojie Zhu, Mary Ann JordanAbstract:Eribulin mesylate (E7389), a synthetic analogue of the marine natural product halichondrin B, is in phase III clinical trials for the treatment of cancer. Eribulin targets microtubules, suppressing Dynamic Instability at microtubule plus ends through an inhibition of microtubule growth with little or no effect on shortening [Jordan, M. A., et al. (2005) Mol. Cancer Ther. 4, 1086−1095]. Using [3H]eribulin, we found that eribulin binds soluble tubulin at a single site; however, this binding is complex with an overall Kd of 46 μM, but also showing a real or apparent very high affinity (Kd = 0.4 μM) for a subset of 25% of the tubulin. Eribulin also binds microtubules with a maximum stoichiometry of 14.7 ± 1.3 molecules per microtubule (Kd = 3.5 μM), strongly suggesting the presence of a relatively high-affinity binding site at microtubule ends. At 100 nM, the concentration that inhibits microtubule plus end growth by 50%, we found that one molecule of eribulin is bound per two microtubules, indicating that th...
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βiii tubulin induces paclitaxel resistance in association with reduced effects on microtubule Dynamic Instability
Journal of Biological Chemistry, 2005Co-Authors: Kathy Kamath, Leslie Wilson, Fernando Cabral, Mary Ann JordanAbstract:The development of resistance to paclitaxel in tumors is one of the most significant obstacles to successful therapy. Overexpression of the betaIII-tubulin isotype has been associated with paclitaxel resistance in a number of cancer cell lines and in tumors, but the mechanism of resistance has remained unclear. Paclitaxel inhibits cancer cell proliferation by binding to the beta-subunit of tubulin in microtubules and suppressing microtubule Dynamic Instability, leading to mitotic arrest and cell death. We hypothesized that betaIII-tubulin overexpression induces resistance to paclitaxel either by constitutively enhancing microtubule Dynamic Instability in resistant cells or by rendering the microtubules less sensitive to the suppression of Dynamics by paclitaxel. Using Chinese hamster ovary cells that inducibly overexpress either betaI- or betaIII-tubulin, we analyzed microtubule Dynamic Instability during interphase by microinjection of rhodamine-labeled tubulin and time-lapse fluorescence microscopy. In the absence of paclitaxel, there were no differences in any aspect of Dynamic Instability between the two beta-tubulin-overexpressing cell types. However, in the presence of 150 nm paclitaxel, Dynamic Instability was suppressed to a significantly lesser extent (suppressed only 12%) in cells overexpressing betaIII-tubulin than in cells overexpressing similar levels of betaI-tubulin (suppressed 47%). The results suggest that overexpression of betaIII-tubulin induces paclitaxel resistance by reducing the ability of paclitaxel to suppress microtubule Dynamics. The results also suggest that endogenous regulators of microtubule Dynamics may differentially interact with individual tubulin isotypes, supporting the idea that differential expression of tubulin isotypes has functional consequences in cells.
Jennifer A Smith - One of the best experts on this subject based on the ideXlab platform.
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eribulin binds at microtubule ends to a single site on tubulin to suppress Dynamic Instability
Biochemistry, 2010Co-Authors: Jennifer A Smith, Leslie Wilson, Olga Azarenko, Bryan M Lewis, Bruce A Littlefield, Mary Ann JordanAbstract:Eribulin mesylate (E7389), a synthetic analogue of the marine natural product halichondrin B, is in phase III clinical trials for the treatment of cancer. Eribulin targets microtubules, suppressing Dynamic Instability at microtubule plus ends through an inhibition of microtubule growth with little or no effect on shortening [Jordan, M. A., et al. (2005) Mol. Cancer Ther. 4, 1086−1095]. Using [3H]eribulin, we found that eribulin binds soluble tubulin at a single site; however, this binding is complex with an overall Kd of 46 μM, but also showing a real or apparent very high affinity (Kd = 0.4 μM) for a subset of 25% of the tubulin. Eribulin also binds microtubules with a maximum stoichiometry of 14.7 ± 1.3 molecules per microtubule (Kd = 3.5 μM), strongly suggesting the presence of a relatively high-affinity binding site at microtubule ends. At 100 nM, the concentration that inhibits microtubule plus end growth by 50%, we found that one molecule of eribulin is bound per two microtubules, indicating that th...
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eribulin binds at microtubule ends to a single site on tubulin to suppress Dynamic Instability
Biochemistry, 2010Co-Authors: Jennifer A Smith, Leslie Wilson, Olga Azarenko, Bryan M Lewis, Bruce A Littlefield, Xiaojie Zhu, Mary Ann JordanAbstract:Eribulin mesylate (E7389), a synthetic analogue of the marine natural product halichondrin B, is in phase III clinical trials for the treatment of cancer. Eribulin targets microtubules, suppressing Dynamic Instability at microtubule plus ends through an inhibition of microtubule growth with little or no effect on shortening [Jordan, M. A., et al. (2005) Mol. Cancer Ther. 4, 1086−1095]. Using [3H]eribulin, we found that eribulin binds soluble tubulin at a single site; however, this binding is complex with an overall Kd of 46 μM, but also showing a real or apparent very high affinity (Kd = 0.4 μM) for a subset of 25% of the tubulin. Eribulin also binds microtubules with a maximum stoichiometry of 14.7 ± 1.3 molecules per microtubule (Kd = 3.5 μM), strongly suggesting the presence of a relatively high-affinity binding site at microtubule ends. At 100 nM, the concentration that inhibits microtubule plus end growth by 50%, we found that one molecule of eribulin is bound per two microtubules, indicating that th...
Dulal Panda - One of the best experts on this subject based on the ideXlab platform.
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curcumin suppresses the Dynamic Instability of microtubules activates the mitotic checkpoint and induces apoptosis in mcf 7 cells
FEBS Journal, 2010Co-Authors: Mithu Banerjee, Parminder Singh, Dulal PandaAbstract:In this study, curcumin, a potential anticancer agent, was found to dampen the Dynamic Instability of individual microtubules in living MCF-7 cells. It strongly reduced the rate and extent of shortening states, and modestly reduced the rate and extent of growing states. In addition, curcumin decreased the fraction of time microtubules spent in the growing state and strongly increased the time microtubules spent in the pause state. Brief treatment with curcumin depolymerized mitotic microtubules, perturbed microtubule-kinetochore attachment and disturbed the mitotic spindle structure. Curcumin also perturbed the localization of the kinesin protein Eg5 and induced monopolar spindle formation. Further, curcumin increased the accumulation of Mad2 and BubR1 at the kinetochores, indicating that it activated the mitotic checkpoint. In addition, curcumin treatment increased the metaphase/anaphase ratio, indicating that it can delay mitotic progression from the metaphase to anaphase. We provide evidence suggesting that the affected cells underwent apoptosis via the p53-dependent apoptotic pathway. The results support the idea that kinetic stabilization of microtubule Dynamics assists in the nuclear translocation of p53. Curcumin exerted additive effects when combined with vinblastine, a microtubule depolymerizing drug, whereas the combination of curcumin with paclitaxel, a microtubule-stabilizing drug, produced an antagonistic effect on the inhibition of MCF-7 cell proliferation. The results together suggested that curcumin inhibited MCF-7 cell proliferation by inhibiting the assembly Dynamics of microtubules.
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kinetic suppression of microtubule Dynamic Instability by griseofulvin implications for its possible use in the treatment of cancer
Proceedings of the National Academy of Sciences of the United States of America, 2005Co-Authors: Dulal Panda, Krishnan Rathinasamy, Manas Kumar Santra, Leslie WilsonAbstract:The antifungal drug griseofulvin inhibits mitosis strongly in fungal cells and weakly in mammalian cells by affecting mitotic spindle microtubule (MT) function. Griseofulvin also blocks cell-cycle progression at G2/M and induces apoptosis in human tumor cell lines. Despite extensive study, the mechanism by which the drug inhibits mitosis in human cells remains unclear. Here, we analyzed the ability of griseofulvin to inhibit cell proliferation and mitosis and to affect MT polymerization and organization in HeLa cells together with its ability to affect MT polymerization and Dynamic Instability in vitro. Griseofulvin inhibited cell-cycle progression at prometaphase/anaphase of mitosis in parallel with its ability to inhibit cell proliferation. At its mitotic IC50 of 20 μM, spindles in blocked cells displayed nearly normal quantities of MTs and MT organization similar to spindles blocked by more powerful MT-targeted drugs. Similar to previously published data, we found that very high concentrations of griseofulvin (>100 μM) were required to inhibit MT polymerization in vitro. However, much lower drug concentrations (1–20 μM) strongly suppressed the Dynamic Instability behavior of the MTs. We suggest that the primary mechanism by which griseofulvin inhibits mitosis in human cells is by suppressing spindle MT Dynamics in a manner qualitatively similar to that of much more powerful antimitotic drugs, including the vinca alkaloids and the taxanes. In view of griseofulvin's lack of significant toxicity in humans, we further suggest that it could be useful as an adjuvant in combination with more powerful drugs for the treatment of cancer.
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suppression of microtubule Dynamic Instability and treadmilling by deuterium oxide
Biochemistry, 2000Co-Authors: Dulal Panda, Leslie Wilson, Gopal Chakrabarti, Jon Hudson, Karli Pigg, Herbert P Miller, Richard H HimesAbstract:Deuterium oxide (D2O) is known to promote the assembly of tubulin into microtubules in vitro, to increase the volume of mitotic spindles and the number and length of spindle microtubules, and to inhibit mitosis. Reasoning that its actions on cellular microtubules could be due to modulation of microtubule Dynamics, we examined the effects of replacing H2O with D2O on microtubule Dynamic Instability, treadmilling, and steady-state GTPase activity. We found that replacing 50% or more of the H2O with D2O promoted microtubule polymerization and stabilized microtubules against dilution-induced disassembly. Using steady-state axoneme-seeded microtubules composed of pure tubulin and video microscopy, we found that 84% D2O decreased the catastrophe frequency by 89%, the shortening rate by 80%, the growing rate by 50%, and the Dynamicity by 93%. Sixty percent D2O decreased the treadmilling rate of microtubules composed of tubulin and microtubule-associated proteins by 42%, and 89% D2O decreased the steady-state GTP...
Airong Liu - One of the best experts on this subject based on the ideXlab platform.
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experimental and analytical investigation on the in plane Dynamic Instability of arches owing to parametric resonance
Journal of Vibration and Control, 2018Co-Authors: Airong Liu, Zhicheng YangAbstract:When an arch is subjected to a periodic load, it may lose in-plane stability Dynamically owing to parametric resonance. Previous investigations have been concentrated on in-plane Dynamic buckling of pin-ended shallow arches. However, in engineering practice, fixed arches with different rise-to-span ratios are often encountered. Little research on in-plane Dynamic Instability of deep fixed arches has been reported in the literature. This paper is concerned with experimental and analytical investigations for in-plane Dynamic Instability of fixed circular arches with rise-to-span ratios 1/8–1/2 under a central periodic load owing to parametric resonance. Experiments are carried out to determine the in-plane frequency and damping ratio of arches, to investigate critical regions of frequencies and amplitudes of the periodic load for in-plane Dynamic Instability of arches, and to explore effects of the rise-to-span ratio and additional weights on Dynamic Instability. The analytical method for determining the re...
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assessment of lateral Dynamic Instability of columns under an arbitrary periodic axial load owing to parametric resonance
Journal of Sound and Vibration, 2017Co-Authors: Youqin Huang, Airong Liu, Wei GaoAbstract:Abstract The paper presents a method for assessing the Dynamic stability of simply supported columns with damping under arbitrary periodic axial loading owing to parametric resonance. The equation for determining critical excitation frequencies causing lateral parametric Instability of columns is derived using the Bolotin′s method. A method of matrix transformation is developed to solve the ill-condition problem in the even regions of Dynamic Instability of the column caused by inclusion of damping. The accuracy of the proposed method is carefully verified by comparing the results with those in the literature. New findings of this investigation are: (1) the critical excitation parameter for inducing Dynamic Instability under arbitrary periodic loading increases with increasing damping ratio; (2) in difference from columns under a simple harmonic load, the width of Instability regions of columns under an arbitrary periodic load does not necessarily decrease as the number of region increases, so the Instability regions can cover most of the parametric plane, and thus the Dynamic Instability is more likely to occur; (3) under an arbitrary periodic axial load, the first region of Instability of columns may not be the critical one, and the number of harmonic waves adopted in the computation is important for the location of the most critical region of Instability and the distribution of Instability regions in the parametric plane; and (4) the dimension of determinant in the equation of boundary frequencies rather than the number of harmonic waves is responsible for computation efficiency, and the sufficient large dimension of determinant should be retained to achieve the computational accuracy.
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analytical and experimental studies on out of plane Dynamic Instability of shallow circular arch based on parametric resonance
Nonlinear Dynamics, 2017Co-Authors: Airong Liu, Youqin HuangAbstract:Little research about the out-of-plane Dynamic stability of arches under in-plane loading has been reported in the literature hitherto. This paper presents analytical and experimental investigations of the out-of-plane Dynamic Instability of elastic shallow circular arches under an in-plane central concentrated periodic load owing to parametric resonance. Differential equations of out-of-plane motion of shallow arches are established using the Hamilton principle by accounting for the effects of geometric nonlinearity, additional concentrated weights and damping. The analytical solutions of the critical excitation frequencies of the concentrated periodic load for out-of-plane Dynamic Instability of arches are obtained. The corresponding experimental investigations are also carried out to verify the analytical solutions. Agreements between the analytical and experimental results are very good. In addition, the effects of the central concentrated weight and the in-plane excitation amplitude on out-of-plane Dynamic Instability of arches are investigated. It is found that as the weight increases, the bandwidth of the critical in-plane excitation frequencies for out-of-plane Dynamic Instability of the arch decreases. It is also found that the bandwidth of critical frequencies increases with an increase in the excitation amplitude. Furthermore, the nonlinear inertial force is derived, which is essential in determining the out-of-plane parametric resonance. It is shown that the curve of the excitation frequency versus amplitude of out-of-plane vibration bends toward the low-frequency region and that the “traction” out-of-plane Instability may occur owing to “amplitude” perturbation. To authors’ knowledge, the analytical solutions and experimental investigations for out-of-plane Dynamic Instability of arches owing to parametric resonance presented in the paper are first time reported in the literature. The new findings in the paper can provide an in-depth understanding of out-of-plane Dynamic Instability behavior of arches under a periodic load.
