Biomedical subjects

Leslie Wilson

Publications and source records attributed to Leslie Wilson.

At least 19 recordsLinked to original sources

PubMed2007

Caregiving time in sickle cell disease: psychological effects in maternal caregivers.

BACKGROUND: Providing home care for a child with a chronic illness can be stressful for the family. The purpose of this paper is to examine patterns of caregiving and the associated psychological impact on maternal caregivers of children with sickle cell disease (SCD). PROCEDURE: Fourteen maternal caregivers of children with SCD were interviewed as part of a larger study of maternal caregivers of children with chronic illness. Forty-four caregivers of children with HIV and 36 caregivers of healthy children were included as comparison groups. Interviews included questions regarding amount of time spent providing care for the child (technical care, non-technical care, health care management), hospitalization, emergency room visits, illness stigma, and mental health of the caregiver. RESULTS: Children with SCD had significantly lower functional status and significantly more hospitalizations in the previous 3 months than children with HIV. Caregivers of children with SCD were more likely to work full-time and had higher incomes than caregivers of children with HIV. The three caregiving groups did not differ significantly on amount of total care, although caregivers of children with SCD and caregivers of children with HIV both reported significantly more time spent in technical care than caregivers of healthy children. Despite lower functional status of the children in the SCD group, when group comparisons on caregiving time variables were adjusted for child's functional status, the differences between groups increased. This appeared to be due to the fact that caregivers in the HIV group spent more time in all caregiving categories except skin, crisis, and other care. In terms of caregiver mental health, caregivers of children with HIV and SCD had significantly higher depressive mood scores than caregivers of healthy children but the groups did not differ on caregiving burden. CONCLUSIONS: The perceived care burden of caregivers of children with SCD may be related to the unpredictable nature of the crisis care they provide. Additional attention is warranted to developing adequate resources for caregivers of children with SCD to mitigate the stress of unexpected crises.

Adaptation, Psychological↗

PubMed2006

Microtubule protofilament number is modulated in a stepwise fashion by the charge density of an enveloping layer.

Microtubules are able to adjust their protofilament (PF) number and, as a consequence, their dynamics and function, to the assembly conditions and presence of cofactors. However, the principle behind such variations is poorly understood. Using synchrotron x-ray scattering and transmission electron microscopy, we studied how charged membranes, which under certain conditions can envelop pre-assembled MTs, regulate the PF number of those MTs. We show that the mean PF number, , is modulated primarily by the charge density of the membranes. decreases in a stepwise fashion with increasing membrane charge density. does not depend on the membrane-protein stoichiometry or the solution ionic strength. We studied the effect of taxol and found that increases logarithmically with taxol/tubulin stoichiometry. We present a theoretical model, which by balancing the electrostatic and elastic interactions in the system accounts for the trends in our findings and reveals an effective MT bending stiffness of order 10-100 k(B)T/nm, associated with the observed changes in PF number.

Animals↗

PubMed2006

Antimitotic sulfonamides inhibit microtubule assembly dynamics and cancer cell proliferation.

Several sulfonamides have antitumor activities and are currently undergoing clinical evaluation for the treatment of cancer. In this study, we have elucidated the antiproliferative mechanism of action of five indole sulfonamides. The indole sulfonamides inhibited the polymerization of microtubule protein into microtubules in vitro. In addition, three representative derivatives, ER-68378 (2), ER-68384 (4) and ER-68394 (5), suppressed the dynamic instability behavior at the plus ends of individual steady-state microtubules in vitro. The analogues inhibited HeLa cell proliferation with half-maximal inhibitory concentrations in the range of 6-17 microM. The compounds blocked cell cycle progression at mitosis. At their lowest effective antimitotic concentrations, they depolymerized the spindle microtubules and disorganized the chromosomes but did not affect the microtubules in interphase cells. However, at relatively high concentrations, interphase microtubules were also depolymerized by these sulfonamides. Furthermore, all five compounds were found to induce apoptosis in the cells in association with the phosphorylation of bcl-2. The results suggest that the indole sulfonamides inhibit cell proliferation at mitosis by perturbing the assembly dynamics of spindle microtubules and that they can kill cancer cells by inducing apoptosis through the bcl-2-dependent pathway.

Aminophenols↗

PubMed2006

FTDP-17 mutations compromise the ability of tau to regulate microtubule dynamics in cells.

The neural microtubule-associated protein Tau binds directly to microtubules and regulates their dynamic behavior. In addition to being required for normal development, maintenance, and function of the nervous system, Tau is associated with several neurodegenerative diseases, including Alzheimer disease. One group of neurodegenerative dementias known as FTDP-17 (fronto-temporal dementia with Parkinsonism linked to chromosome 17) is directly linked genetically to mutations in the tau gene, demonstrating that Tau misfunction can cause neuronal cell death and dementia. These mutations result either in amino acid substitutions in Tau or in altered Tau mRNA splicing that skews the expression ratio of wild-type 3-repeat and 4-repeat Tau isoforms. Because wild-type Tau regulates microtubule dynamics, one possible mechanism underlying Tau-mediated neurodegeneration is aberrant regulation of microtubule behavior. In this study, we microinjected normal and mutated Tau protein into cultured cells expressing fluorescent tubulin and measured the effects on the dynamic instability of individual microtubules. We found that the FTDP-17 amino acid substitutions G272V (in both 3-repeat and 4-repeat Tau contexts), DeltaK280, and P301L all exhibited markedly reduced abilities to regulate dynamic instability relative to wild-type Tau. In contrast, the FTDP-17 R406W mutation (which maps in a regulatory region outside the microtubule binding domain of Tau) did not significantly alter the ability of 3-repeat or 4-repeat Tau to regulate microtubule dynamics. Overall, these data are consistent with a loss-of-function model in which both amino acid substitutions and altered mRNA splicing in Tau lead to neurodegeneration by diminishing the ability of Tau to properly regulate microtubule dynamics.

Alternative Splicing↗

PubMed2006

2-Methoxyestradiol suppresses microtubule dynamics and arrests mitosis without depolymerizing microtubules.

2-Methoxyestradiol (2ME2), a metabolite of estradiol-17beta, is a novel antimitotic and antiangiogenic drug candidate in phase I and II clinical trials for the treatment of a broad range of tumor types. 2ME2 binds to tubulin at or near the colchicine site and inhibits the polymerization of tubulin in vitro, suggesting that it may work by interfering with normal microtubule function. However, the role of microtubule depolymerization in its antitumor mechanism of action has been controversial. To determine the mechanism by which 2ME2 induces mitotic arrest, we analyzed its effects on microtubule polymerization in vitro and its effects on dynamic instability both in vitro and in living MCF7 cells. In vitro, 2ME2 (5-100 micromol/L) inhibited assembly of purified tubulin in a concentration-dependent manner, with maximal inhibition (60%) at 200 micromol/L 2ME2. However, with microtubule-associated protein-containing microtubules, significantly higher 2ME2 concentrations were required to depolymerize microtubules, and polymer mass was reduced by only 13% at 500 micromol/L 2ME2. In vitro, dynamic instability was inhibited at lower concentrations. Specifically, 4 micromol/L 2ME2 reduced the mean growth rate by 17% and dynamicity by 27%. In living interphase MCF7 cells at the IC50 for mitotic arrest (1.2 micromol/L), 2ME2 significantly suppressed the mean microtubule growth rate, duration and length, and the overall dynamicity, consistent with its effects in vitro, and without any observable depolymerization of microtubules. Taken together, the results suggest that the major mechanism of mitotic arrest at the lowest effective concentrations of 2ME2 is suppression of microtubule dynamics rather than microtubule depolymerization per se.

2-Methoxyestradiol↗

PubMed2005

Stathmin strongly increases the minus end catastrophe frequency and induces rapid treadmilling of bovine brain microtubules at steady state in vitro.

Stathmin is a ubiquitous microtubule destabilizing protein that is believed to play an important role linking cell signaling to the regulation of microtubule dynamics. Here we show that stathmin strongly destabilizes microtubule minus ends in vitro at steady state, conditions in which the soluble tubulin and microtubule levels remain constant. Stathmin increased the minus end catastrophe frequency approximately 13-fold at a stathmin:tubulin molar ratio of 1:5. Stathmin steady-state catastrophe-promoting activity was considerably stronger at the minus ends than at the plus ends. Consistent with its ability to destabilize minus ends, stathmin strongly increased the treadmilling rate of bovine brain microtubules. By immunofluorescence microscopy, we also found that stathmin binds to purified microtubules along their lengths in vitro. Co-sedimentation of purified microtubules polymerized in the presence of a 1:5 initial molar ratio of stathmin to tubulin yielded a binding stoichiometry of 1 mol of stathmin per approximately 14.7 mol of tubulin in the microtubules. The results firmly establish that stathmin can increase the steady-state catastrophe frequency by a direct action on microtubules, and furthermore, they indicate that an important regulatory action of stathmin in cells may be to destabilize microtubule minus ends.

Animals↗

PubMed2005

Radial compression of microtubules and the mechanism of action of taxol and associated proteins.

Microtubules (MTs) are hollow cylindrical polymers composed of alphabeta-tubulin heterodimers that align head-to-tail in the MT wall, forming linear protofilaments that interact laterally. We introduce a probe of the interprotofilament interactions within MTs and show that this technique gives insight into the mechanisms by which MT-associated proteins (MAPs) and taxol stabilize MTs. In addition, we present further measurements of the mechanical properties of MT walls, MT-MT interactions, and the entry of polymers into the MT lumen. These results are obtained from a synchrotron small angle x-ray diffraction (SAXRD) study of MTs under osmotic stress. Above a critical osmotic pressure, P(cr), we observe rectangular bundles of MTs whose cross sections have buckled to a noncircular shape; further increases in pressure continue to distort MTs elastically. The P(cr) of approximately 600 Pa provides, for the first time, a measure of the bending modulus of the interprotofilament bond within an MT. The presence of neuronal MAPs greatly increases P(cr), whereas surprisingly, the cancer chemotherapeutic drug taxol, which suppresses MT dynamics and inhibits MT depolymerization, does not affect the interprotofilament interactions. This SAXRD-osmotic stress technique, which has enabled measurements of the mechanical properties of MTs, should find broad application for studying interactions between MTs and of MTs with MAPs and MT-associated drugs.

Animals↗

PubMed2005

Cationic liposome-microtubule complexes: pathways to the formation of two-state lipid-protein nanotubes with open or closed ends.

Intermolecular interactions between charged membranes and biological polyelectrolytes, tuned by physical parameters, which include the membrane charge density and bending rigidity, the membrane spontaneous curvature, the biopolymer curvature, and the overall charge of the complex, lead to distinct structures and morphologies. The self-assembly of cationic liposome-microtubule (MT) complexes was studied, using synchrotron x-ray scattering and electron microscopy. Vesicles were found to either adsorb onto MTs, forming a "beads on a rod" structure, or undergo a wetting transition and coating the MT. Tubulin oligomers then coat the external lipid layer, forming a tunable lipid-protein nanotube. The beads on a rod structure is a kinetically trapped state. The energy barrier between the states depends on the membrane bending rigidity and charge density. By controlling the cationic lipid/tubulin stoichiometry it is possible to switch between two states of nanotubes with either open ends or closed ends with lipid caps, a process that forms the basis for controlled chemical and drug encapsulation and release.

Cytoplasmic Vesicles↗

PubMed2005

Kinetic suppression of microtubule dynamic instability by griseofulvin: implications for its possible use in the treatment of cancer.

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 G(2)/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 IC(50) of 20 muM, 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 microM) were required to inhibit MT polymerization in vitro. However, much lower drug concentrations (1-20 microM) 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.

Animals↗

PubMed2005

BetaIII-tubulin induces paclitaxel resistance in association with reduced effects on microtubule dynamic instability.

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.

Animals↗

PubMed2005

Three- and four-repeat tau regulate the dynamic instability of two distinct microtubule subpopulations in qualitatively different manners. Implications for neurodegeneration.

The microtubule-associated protein tau is implicated in the pathogenesis of many neurodegenerative diseases, including fronto-temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), in which both RNA splicing and amino acid substitution mutations in tau cause dominantly inherited early onset dementia. RNA-splicing FTDP-17 mutations alter the wild-type approximately 50:50 3-repeat (3R) to 4-repeat (4R) tau isoform ratio, usually resulting in an excess of 4R tau. To examine further how splicing mutations might cause dysfunction by misregulation of microtubule dynamics, we used video microscopy to determine the in vitro behavior of individual microtubules stabilized by varying amounts of human 4R and 3R tau. At low tau:tubulin ratios (1:55 and 1:45), all 3R isoforms reduced microtubule growth rates relative to the no-tau control, whereas all 4R isoforms increased them; however, at a high tau:tubulin ratio (1:20), both 4R and 3R tau increased the growth rates. Further analysis revealed two distinct subpopulations of growing microtubules in the absence of tau. Increasing concentrations of both 4R and 3R tau resulted in an increase in the size of the faster growing subpopulation of microtubules; however, 4R tau caused a redistribution to the faster growing subpopulation at lower tau:tubulin ratios than 3R tau. This modulation of discrete growth rate subpopulations by tau suggests that tau causes a conformational shift in the microtubule resulting in altered dynamics. Quantitative and qualitative differences observed between 4R and 3R tau are consistent with a "microtubule misregulation" model in which abnormal tau isoform expression results in the inability to properly regulate microtubule dynamics, leading to neuronal death and dementia.

Alternative Splicing↗

PubMed2005

Inability of tau to properly regulate neuronal microtubule dynamics: a loss-of-function mechanism by which tau might mediate neuronal cell death.

Interest in the microtubule-associated protein tau stems from its critical roles in neural development and maintenance, as well as its role in Alzheimer's, FTDP-17 and related neurodegenerative diseases. Under normal circumstances, tau performs its functions by binding to microtubules and powerfully regulating their stability and growing and shortening dynamics. On the other hand, genetic analyses have established a clear cause-and-effect relationship between tau dysfunction/mis-regulation and neuronal cell death and dementia in FTDP-17, but the molecular basis of tau's destructive action(s) remains poorly understood. One attractive model suggests that the intracellular accumulation of abnormal tau aggregates causes cell death, i.e., a gain-of-toxic function model. Here, we describe the evidence and arguments for an alternative loss-of-function model in which tau-mediated neuronal cell death is caused by the inability of affected cells to properly regulate their microtubule dynamic due to mis-regulation by tau. In support of this model, our recent data demonstrate that missense FTDP-17 mutations that alter amino acid residues near tau's microtubule binding region strikingly modify the ability of tau to modulate microtubule dynamics. Additional recent data from our labs support the notion that the same dysfunction occurs in the FTDP-17 regulatory mutations that alter tau RNA splicing patterns. Our model posits that the dynamics of microtubules in neuronal cells must be tightly regulated to enable them to carry out their diverse functions, and that microtubules that are either over-stabilized or under-stabilized, that is, outside an acceptable window of dynamic activity, lead to neurodegeneration. An especially attractive aspect of this model is that it readily accommodates both the structural and regulatory classes of FTDP-17 mutations.

Cell Death↗

PubMed2005

New workers' compensation legislation: expected pharmaceutical cost savings.

BACKGROUND: California Workers' Compensation (WC) system costs are under review. With recently approved California State Assembly Bill (AB) 749 and Senate Bill (SB) 228, an assessment of proposed pharmaceutical cost savings is needed. METHODS: A large workers' compensation database provided by the California Workers' Compensation Institute (CWCI) and Medi-Cal pharmacy costs obtained from the State Drug Utilization Project are utilized to compare frequency, costs and savings to Workers' Compensation in 2002 with the new pharmacy legislation. RESULTS: Compared to the former California Workers' Compensation fee schedule, the newly implemented 100% Medi-Cal fee schedule will result in savings of 29.5% with a potential total pharmacy cost savings of $125 million. Further statistical analysis demonstrated that a large variability in savings across drugs could not be controlled with this drug pricing system. CONCLUSIONS: Despite the large savings in pharmaceuticals, inconsistencies between the two pharmaceutical payment systems could lead to negative incentives and uncertainty for long-term savings. Proposed alternative pricing systems could be considered. However, pain management implemented along with other cost containment strategies could more effectively reduce overall drug spending in the workers' compensation system.

California↗

PubMed2005

The primary antimitotic mechanism of action of the synthetic halichondrin E7389 is suppression of microtubule growth.

E7389, which is in phase I and II clinical trials, is a synthetic macrocyclic ketone analogue of the marine sponge natural product halichondrin B. Whereas its mechanism of action has not been fully elucidated, its main target seems to be tubulin and/or the microtubules responsible for the construction and proper function of the mitotic spindle. Like most microtubule-targeted antitumor drugs, it inhibits tumor cell proliferation in association with G(2)-M arrest. It binds to tubulin and inhibits microtubule polymerization. We examined the mechanism of action of E7389 with purified microtubules and in living cells and found that, unlike antimitotic drugs including vinblastine and paclitaxel that suppress both the shortening and growth phases of microtubule dynamic instability, E7389 seems to work by an end-poisoning mechanism that results predominantly in inhibition of microtubule growth, but not shortening, in association with sequestration of tubulin into aggregates. In living MCF7 cells at the concentration that half-maximally blocked cell proliferation and mitosis (1 nmol/L), E7389 did not affect the shortening events of microtubule dynamic instability nor the catastrophe or rescue frequencies, but it significantly suppressed the rate and extent of microtubule growth. Vinblastine, but not E7389, inhibited the dilution-induced microtubule disassembly rate. The results suggest that, at its lowest effective concentrations, E7389 may suppress mitosis by directly binding to microtubule ends as unliganded E7389 or by competition of E7389-induced tubulin aggregates with unliganded soluble tubulin for addition to growing microtubule ends. The result is formation of abnormal mitotic spindles that cannot pass the metaphase/anaphase checkpoint.

Animals↗

PubMed2004

Higher-order assembly of microtubules by counterions: from hexagonal bundles to living necklaces.

Cellular factors tightly regulate the architecture of bundles of filamentous cytoskeletal proteins, giving rise to assemblies with distinct morphologies and physical properties, and a similar control of the supramolecular organization of nanotubes and nanorods in synthetic materials is highly desirable. However, it is unknown what principles determine how macromolecular interactions lead to assemblies with defined morphologies. In particular, electrostatic interactions between highly charged polyelectrolytes, which are ubiquitous in biological and synthetic self-assembled structures, are poorly understood. We have used a model system consisting of microtubules (MTs) and multivalent cations to examine how microscopic interactions can give rise to distinct bundle phases in biological polyelectrolytes. The structure of these supramolecular assemblies was elucidated on length scales from subnanometer to micrometer with synchrotron x-ray diffraction, transmission electron microscopy, and differential interference contrast microscopy. Tightly packed hexagonal bundles with controllable diameters were observed for large trivalent, tetravalent, and pentavalent counterions. Unexpectedly, in the presence of small divalent cations, we have discovered a living necklace bundle phase, comprised of 2D dynamic assemblies of MTs with linear, branched, and loop topologies. This new bundle phase is an experimental example of nematic membranes. The morphologically distinct MT assemblies give insight into general features of bundle formation and may be used as templates for miniaturized materials with applications in nanotechnology and biotechnology.

Animals↗

PubMed2004

Synchrotron X-ray diffraction study of microtubules buckling and bundling under osmotic stress: a probe of interprotofilament interactions.

Microtubules are hollow cylinders composed of tubulin heterodimers that stack into linear protofilaments that interact laterally to form the microtubule wall. Synchrotron x-ray diffraction of microtubules under increasing osmotic stress shows they transition to rectangular bundles with noncircular buckled cross sections, followed by hexagonally packed bundles. This new technique probes the strength of interprotofilamen bonds, yielding insight into the mechanism by which associated proteins and the chemotherapy drug taxol stabilize microtubules.

Guanosine Triphosphate↗

PubMed2004

Inhibition of DNA decatenation, but not DNA damage, arrests cells at metaphase.

DNA damage by double-strand breaks induces arrest during interphase in mammalian cells. It is not clear whether DNA damage can arrest cells in mitosis. We show here that three human cell lines, HeLa, U2OS, and HCT116, do not delay in mitosis in response to double-strand breaks induced during mitosis by gamma irradiation or by adriamycin. Durable arrest at metaphase occurs, however, with ICRF-193, a topoisomerase II inhibitor that does not damage DNA. Arrest with ICRF-193 is not accompanied by recruitment of Mad2 or Bub1 to kinetochores, nor by phosphorylation of the histone H2AX, indicating arrest by ICRF-193 is not due to activation of the spindle assembly checkpoint, nor is it a response to DNA damage. VP-16, another decatenation inhibitor, induces metaphase arrest only at concentrations well above those that induce DNA damage. We conclude that decatenation failure, but not DNA damage, creates metaphase arrest in mammalian cells.

Cell Line, Tumor↗

PubMed2004

MCAK, a Kin I kinesin, increases the catastrophe frequency of steady-state HeLa cell microtubules in an ATP-dependent manner in vitro.

Mitotic-centromere-associated kinesin (MCAK) is a member of the KIN I (internal motor domain) subfamily of kinesin related proteins. MCAK and its homologues destabilize microtubules both in cells and in vitro. Here, we analyzed the effects of MCAK in the presence and absence of ATP on the dynamic instability behavior of steady state microtubules assembled from purified HeLa cell tubulin. In the presence of ATP, substoichiometric levels of full length MCAK and a segment (A182) consisting of the motor and neck domains strongly increased the catastrophe frequency of the microtubules. These data demonstrate that MCAK is a microtubule-catastrophe promoting factor in vitro, and support the hypothesis that MCAK may serve as a catastrophe-promoting factor in cells.

Adenosine Triphosphate↗