70 results on '"Hart, SM"'
Search Results
2. A failure of M-Entropy to correctly detect burst suppression leading to sevoflurane overdosage.
- Author
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Hart SM, Buchannan CR, Sleigh JW, Hart, S M, Buchannan, C R, and Sleigh, J W
- Abstract
Electroencephalogram depth of anaesthesia monitors are increasingly being used, with the aim of reducing awareness during anaesthesia. Most literature concentrates on the ability of these monitors to predict when a patient is likely to be aware. This case report highlights the opposite problem, where the monitor (M-Entropy) indicated an awake state but the patient was in fact deeply anaesthetised. If the anaesthetist is unable to interpret the raw electroencephalogram and understand the limitations of the monitor being used, excessive doses of anaesthetic may be given with potentially serious consequences. [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
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3. Treatment of B-cell lymphomas with anti-idiotype antibodies alone and in combination with alpha interferon
- Author
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Brown, SL, Miller, RA, Horning, SJ, Czerwinski, D, Hart, SM, McElderry, R, Basham, T, Warnke, RA, Merigan, TC, and Levy, R
- Abstract
Idiotypes are distinct clonal markers for B-cell lymphomas. Previously we reported the use of anti-idiotype antibodies in the therapy of patients with B-cell malignancies. Because synergy was demonstrated with the addition of alpha interferon to anti-idiotype antibodies in a murine lymphoma model, we performed a clinical trial combining these two agents. Here we provide an update of the original trial of anti- idiotype antibodies alone and report the outcome of the new combination trial. In 16 treatment courses of anti-idiotype antibodies alone there were seven partial responses and one complete response. In 12 courses of combination anti-idiotype antibody and alpha interferon there were two complete responses and seven partial responses. Substantial tumor regressions occurred with minimal toxicity in both trials even in patients refractory to conventional chemotherapy. Tumor specimens obtained at the time of disease progression often contained a preponderance of idiotype-negative lymphoma cells, suggesting that anti- idiotype antibody treatment exerted a strong antitumor effect against antigen-positive cells. Anti-idiotype antibodies have reproducible objective antitumor activity in B-cell lymphoma. The addition of alpha interferon may improve the initial rate of response to this treatment. Strategies that deal effectively with idiotype-negative lymphoma cells should improve the extent and duration of these responses.
- Published
- 1989
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4. Biomarkers of Intestinal Permeability are Associated with Inflammation in Metabolically Healthy Obesity, but not Normal-Weight Obesity.
- Author
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Keirns BH, Medlin AR, Maki K, McClanahan K, Fruit SE, Sciarrillo CM, Hart SM, Joyce J, Lucas EA, and Emerson SR
- Abstract
Systemic inflammation is reported in normal-weight obesity (NWO) and metabolically healthy obesity (MHO), which may be linked to their increased cardiovascular disease (CVD) risk. Yet, drivers of this inflammation remain unclear. We characterized factors known to influence inflammatory status (i.e., intestinal permeability, adipose tissue, diet quality, microbiota) - and their relationships with measured inflammation - in NWO and MHO, healthy controls (CON) and metabolically unhealthy obesity (MUO; N=80; n=20/group). Serum indicators of intestinal permeability and inflammation were assessed using ELISA and/or multiplex. Total, visceral, and percent body fat were measured with dual-energy X-ray absorptiometry (DXA). Fecal microbiota composition was assessed via 16S rRNA sequencing (n=9-10/group). For C-reactive protein (CRP), MUO > NWO > CON ( p <0.0001). In MHO, CRP was intermediate - and similar to - both MUO and NWO. Lipopolysaccharide binding protein (LBP) and the ratio of LBP to soluble CD14 (sCD14) were higher in MHO and MUO versus CON/NWO ( p 's<0.0001). Across correlation and regression analyses, LBP consistently displayed the strongest relationships with CRP in the entire sample (r=0.78;β = 0.57; p 's<0.0001) and in MHO (r=0.74; p <0.01), but not NWO (r=0.37; p = 0.11). Shannon index was higher in CON compared to MUO ( p <0.05) and inversely correlated with CRP in the full sample (r=-0.37; p <0.05). These data are consistent with the notion that intestinal permeability is associated with low-grade inflammation in MHO, which could be implicated in this population's reported CVD risk.
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- 2024
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5. Peer support during in vivo exposure homework increases likelihood of prolonged exposure therapy completion.
- Author
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Hernandez-Tejada MA, Bruce MJ, Muzzy W, Birks A, Macedo E Cordeiro G, Hart SM, Hamski S, and Acierno R
- Abstract
Exposure-based treatments such as prolonged exposure therapy (PE) are effective for veterans with PTSD. However, dropout rates as high as 50% are common. The Department of Veterans Affairs employs peers to increase mental health treatment engagement, however peers are not routinely used to help patients complete PE homework assignments. The present study included 109 veterans who decided to drop out from exposure-based treatment after completing seven or fewer sessions and used a randomized controlled design to compare PE treatment completion rates in response to 2 forms of peer support: (1) standard weekly telephone-based peer support vs. (2) peer-assisted in vivo exposure, wherein peers accompanied veterans (virtually or in person) during a limited number of in vivo exposure assignments. There were no differences between instrumental vs general peer support conditions as randomized. However, post hoc analyses indicated that 87% of those who completed at least one peer-assisted in vivo exposure completed treatment, compared to 56% of those not completing any peer-assisted in vivo exposure. The dose effect of peer-assisted in vivo exposure increased to 93% with 2 or more peer-assisted exposures, and 97% with 3 or more peer-assisted exposures. The present study suggests that augmenting PE with instrumental peer support during in vivo exposure homework may reduce dropout if completed. Future research should test whether the impact of peer-assisted in vivo exposure is enhanced when offered at the beginning of treatment as opposed to waiting until the point of dropout.
- Published
- 2024
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6. Sculpting photoproducts with DNA origami.
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Gorman J, Hart SM, John T, Castellanos MA, Harris D, Parsons MF, Banal JL, Willard AP, Schlau-Cohen GS, and Bathe M
- Abstract
Natural light-harvesting systems spatially organize densely packed dyes in different configurations to either transport excitons or convert them into charge photoproducts, with high efficiency. In contrast, artificial photosystems like organic solar cells and light-emitting diodes lack this fine structural control, limiting their efficiency. Thus, biomimetic multi-dye systems are needed to organize dyes with the sub-nanometer spatial control required to sculpt resulting photoproducts. Here, we synthesize 11 distinct perylene diimide (PDI) dimers integrated into DNA origami nanostructures and identify dimer architectures that offer discrete control over exciton transport versus charge separation. The large structural-space and site-tunability of origami uniquely provides controlled PDI dimer packing to form distinct excimer photoproducts, which are sensitive to interdye configurations. In the future, this platform enables large-scale programmed assembly of dyes mimicking natural systems to sculpt distinct photophysical products needed for a broad range of optoelectronic devices, including solar energy converters and quantum information processors., Competing Interests: Declaration of interests The authors declare no competing interests.
- Published
- 2024
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7. Cardiorespiratory fitness and submaximal exercise dynamics in normal-weight obesity and metabolically healthy obesity.
- Author
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Hart SM, Keirns BH, Sciarrillo CM, Malin SK, Kurti SP, and Emerson SR
- Subjects
- Humans, Body Mass Index, Health Status, Obesity, Phenotype, Risk Factors, Cardiorespiratory Fitness, Metabolic Syndrome, Obesity, Metabolically Benign
- Abstract
Purpose: Cardiorespiratory fitness (CRF) is critical for cardiovascular health. Normal-weight obesity (NWO) and metabolically healthy obesity (MHO) may be at increased risk for cardiovascular disease, but a comparison of CRF and submaximal exercise dynamics against rigorously defined low- and high-risk groups is lacking., Methods: Four groups (N = 40; 10/group) based on body mass index (BMI), body fat %, and metabolic syndrome (MetS) risk factors were recruited: healthy controls (CON; BMI 18.5-24.9 kg/m
2 , body fat < 25% [M] or < 35% [F], 0-1 risk factors), NWO (BMI 18.5-24.9 kg/m2 , body fat ≥ 25% [M] or ≥ 35% [F]), MHO (BMI > 30 kg/m2 , body fat ≥ 25% [M] or ≥ 35% [F], 0-1 risk factors), or metabolically unhealthy obesity (MUO; BMI > 30 kg/m2 , body fat ≥ 25% [M] or ≥ 35% [F], 2 + risk factors). All participants completed a V ˙ O2peak test on a cycle ergometer., Results: V ˙ O2peak was similarly low in NWO (27.0 ± 4.8 mL/kg/min), MHO (25.4 ± 6.7 mL/kg/min) and MUO (24.6 ± 10.0 mL/kg/min) relative to CON (44.2 ± 11.0 mL/kg/min) when normalized to total body mass (p's < 0.01), and adjusting for fat mass or lean mass did not alter these results. This same differential V ˙ O2 pattern was apparent beginning at 25% of the exercise test (PGroup*Time < 0.01)., Conclusions: NWO and MHO had similar peak and submaximal CRF to MUO, despite some favorable health traits. Our work adds clarity to the notion that excess adiposity hinders CRF across BMI categories., Clinicaltrials: gov registration: NCT05008952., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)- Published
- 2024
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8. Postprandial triglycerides across the aging spectrum: A secondary analysis utilizing an abbreviated fat tolerance test.
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Keirns BH, Sciarrillo CM, Poindexter KL, Dixon MD, Medlin AR, Koemel NA, Hart SM, Geist CH, Jenkins NDM, Lucas EA, and Emerson SR
- Subjects
- Adult, Aged, Humans, Young Adult, Aging, Fasting, Postprandial Period physiology, Triglycerides, Middle Aged, Hypertriglyceridemia diagnosis, Hypertriglyceridemia epidemiology
- Abstract
Background & Aims: Elevated postprandial triglycerides are an independent cardiovascular disease risk factor and observed in older adults. However, differences in postprandial triglycerides across the spectrum of adulthood remain unclear., Methods and Results: We performed a secondary analysis of six studies where adults (aged 18-84 years; N = 155) completed an abbreviated fat tolerance test (9 kcal/kg; 70% fat). Differences in postprandial triglycerides were compared in those ≥50 and <50 years and by decade of life, adjusting for sex and BMI. Compared to those <50 years, participants ≥50 years had higher fasting, 4 h, and Δ triglycerides from baseline (p's < 0.05). When examining triglyceride parameters by decade, no differences were observed for fasting triglycerides, but 50 s, 60 s, and 70s-80 s displayed greater 4 h and Δ triglycerides versus 20 s (p's ≤ 0.001). The frequency of adverse postprandial triglyceride responses (i.e., ≥220 mg/dL) was higher in participants ≥50 versus <50 years (p < 0.01), and in 60 s compared to all other decades (p = 0.01)., Conclusion: Older age was generally associated with higher postprandial triglycerides, with no divergence across the spectrum of older adulthood. In our sample, postprandial triglyceride differences in older and younger adults were driven by those >50 years relative to young adults in their 20 s., Registration: N/A (secondary analysis)., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to disclose., (Copyright © 2023 The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)
- Published
- 2024
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9. Observation of parallel intersystem crossing and charge transfer-state dynamics in [Fe(bpy) 3 ] 2+ from ultrafast 2D electronic spectroscopy.
- Author
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Lee A, Son M, Deegbey M, Woodhouse MD, Hart SM, Beissel HF, Cesana PT, Jakubikova E, McCusker JK, and Schlau-Cohen GS
- Abstract
Transition metal-based charge-transfer complexes represent a broad class of inorganic compounds with diverse photochemical applications. Charge-transfer complexes based on earth-abundant elements have been of increasing interest, particularly the canonical [Fe(bpy)
3 ]2+ . Photoexcitation into the singlet metal-ligand charge transfer (1 MLCT) state is followed by relaxation first to the ligand-field manifold and then to the ground state. While these dynamics have been well-studied, processes within the MLCT manifold that facilitate and/or compete with relaxation have been more elusive. We applied ultrafast two-dimensional electronic spectroscopy (2DES) to disentangle the dynamics immediately following MLCT excitation of this compound. First, dynamics ascribed to relaxation out of the initially formed1 MLCT state was found to correlate with the inertial response time of the solvent. Second, the additional dimension of the 2D spectra revealed a peak consistent with a ∼20 fs1 MLCT →3 MLCT intersystem crossing process. These two observations indicate that the complex simultaneously undergoes intersystem crossing and direct conversion to ligand-field state(s). Resolution of these parallel pathways in this prototypical earth-abundant complex highlights the ability of 2DES to deconvolve the otherwise obscured excited-state dynamics of charge-transfer complexes., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)- Published
- 2023
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10. Engineering Exciton Dynamics with Synthetic DNA Scaffolds.
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Hart SM, Gorman J, Bathe M, and Schlau-Cohen GS
- Subjects
- Energy Transfer, DNA chemistry, Photosynthesis, Fluorescent Dyes
- Abstract
Excitons are the molecular-scale currency of electronic energy. Control over excitons enables energy to be directed and harnessed for light harvesting, electronics, and sensing. Excitonic circuits achieve such control by arranging electronically active molecules to prescribe desired spatiotemporal dynamics. Photosynthetic solar energy conversion is a canonical example of the power of excitonic circuits, where chromophores are positioned in a protein scaffold to perform efficient light capture, energy transport, and charge separation. Synthetic systems that aim to emulate this functionality include self-assembled aggregates, molecular crystals, and chromophore-modified proteins. While the potential of this approach is clear, these systems lack the structural precision to control excitons or even test the limits of their power. In recent years, DNA origami has emerged as a designer material that exploits biological building blocks to construct nanoscale architectures. The structural precision afforded by DNA origami has enabled the pursuit of naturally inspired organizational principles in a highly precise and scalable manner. In this Account, we describe recent developments in DNA-based platforms that spatially organize chromophores to construct tunable excitonic systems. The high fidelity of DNA base pairing enables the formation of programmable nanoscale architectures, and sequence-specific placement allows for the precise positioning of chromophores within the DNA structure. The integration of a wide range of chromophores across the visible spectrum introduces spectral tunability. These excitonic DNA-chromophore assemblies not only serve as model systems for light harvesting, solar conversion, and sensing but also lay the groundwork for the integration of coupled chromophores into larger-scale nucleic acid architectures.We have used this approach to generate DNA-chromophore assemblies of strongly coupled delocalized excited states through both sequence-specific self-assembly and the covalent attachment of chromophores. These strategies have been leveraged to independently control excitonic coupling and system-bath interaction, which together control energy transfer. We then extended this framework to identify how scaffold configurations can steer the formation of symmetry-breaking charge transfer states, paving the way toward the design of dual light-harvesting and charge separation DNA machinery. In an orthogonal application, we used the programmability of DNA chromophore assemblies to change the optical emission properties of strongly coupled dimers, generating a series of fluorophore-modified constructs with separable emission properties for fluorescence assays. Upcoming advances in the chemical modification of nucleotides, design of large-scale DNA origami, and predictive computational methods will aid in constructing excitonic assemblies for optical and computing applications. Collectively, the development of DNA-chromophore assemblies as a platform for excitonic circuitry offers a pathway to identifying and applying design principles for light harvesting and molecular electronics.
- Published
- 2023
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11. Elucidating interprotein energy transfer dynamics within the antenna network from purple bacteria.
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Wang D, Fiebig OC, Harris D, Toporik H, Ji Y, Chuang C, Nairat M, Tong AL, Ogren JI, Hart SM, Cao J, Sturgis JN, Mazor Y, and Schlau-Cohen GS
- Subjects
- Photosynthesis, Spectrum Analysis, Energy Transfer, Proteobacteria metabolism, Light-Harvesting Protein Complexes metabolism
- Abstract
In photosynthesis, absorbed light energy transfers through a network of antenna proteins with near-unity quantum efficiency to reach the reaction center, which initiates the downstream biochemical reactions. While the energy transfer dynamics within individual antenna proteins have been extensively studied over the past decades, the dynamics between the proteins are poorly understood due to the heterogeneous organization of the network. Previously reported timescales averaged over such heterogeneity, obscuring individual interprotein energy transfer steps. Here, we isolated and interrogated interprotein energy transfer by embedding two variants of the primary antenna protein from purple bacteria, light-harvesting complex 2 (LH2), together into a near-native membrane disc, known as a nanodisc. We integrated ultrafast transient absorption spectroscopy, quantum dynamics simulations, and cryogenic electron microscopy to determine interprotein energy transfer timescales. By varying the diameter of the nanodiscs, we replicated a range of distances between the proteins. The closest distance possible between neighboring LH2, which is the most common in native membranes, is 25 Å and resulted in a timescale of 5.7 ps. Larger distances of 28 to 31 Å resulted in timescales of 10 to 14 ps. Corresponding simulations showed that the fast energy transfer steps between closely spaced LH2 increase transport distances by ∼15%. Overall, our results introduce a framework for well-controlled studies of interprotein energy transfer dynamics and suggest that protein pairs serve as the primary pathway for the efficient transport of solar energy.
- Published
- 2023
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12. Whole-body bone mineral density and markers of bone homeostasis in adults with normal-weight obesity.
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Keirns BH, Sciarrillo CM, Medlin AR, Hart SM, Cronic EM, and Emerson SR
- Abstract
Background: Normal-weight obesity (NWO) describes individuals with a normal body mass index (BMI), but high body fat percent. NWO are at-risk for cardiometabolic diseases, but little is known about their bone health., Methods: Adults (N = 24) were classified as NWO (n = 12; 5M/7F) or low body fat percent controls (Con; n = 12; 6M/6F). Body composition and whole-body bone mineral density (BMD) were assessed using DXA. A serum bioplex assay was performed to examine markers related to bone formation and resorption., Results: In addition to higher body fat percent and visceral fat, NWO had lower whole-body BMD relative to Con ( p 's < 0.05). Circulating leptin was higher in NWO than Con ( p < 0.05). Two biomarkers generally associated with lower bone mass - sclerostin and parathyroid hormone - were higher in NWO compared to Con ( p 's < 0.05)., Conclusion: In this preliminary study, adults with NWO displayed lower whole-body BMD alongside evidence of bone resorption. Impaired bone health may be another subclinical risk factor present in NWO., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2023 The Authors.)
- Published
- 2023
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13. Activating charge-transfer state formation in strongly-coupled dimers using DNA scaffolds.
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Hart SM, Banal JL, Castellanos MA, Markova L, Vyborna Y, Gorman J, Häner R, Willard AP, Bathe M, and Schlau-Cohen GS
- Abstract
Strongly-coupled multichromophoric assemblies orchestrate the absorption, transport, and conversion of photonic energy in natural and synthetic systems. Programming these functionalities involves the production of materials in which chromophore placement is precisely controlled. DNA nanomaterials have emerged as a programmable scaffold that introduces the control necessary to select desired excitonic properties. While the ability to control photophysical processes, such as energy transport, has been established, similar control over photochemical processes, such as interchromophore charge transfer, has not been demonstrated in DNA. In particular, charge transfer requires the presence of close-range interchromophoric interactions, which have a particularly steep distance dependence, but are required for eventual energy conversion. Here, we report a DNA-chromophore platform in which long-range excitonic couplings and short-range charge-transfer couplings can be tailored. Using combinatorial screening, we discovered chromophore geometries that enhance or suppress photochemistry. We combined spectroscopic and computational results to establish the presence of symmetry-breaking charge transfer in DNA-scaffolded squaraines, which had not been previously achieved in these chromophores. Our results demonstrate that the geometric control introduced through the DNA can access otherwise inaccessible processes and program the evolution of excitonic states of molecular chromophores, opening up opportunities for designer photoactive materials for light harvesting and computation., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)
- Published
- 2022
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14. Postprandial triglycerides, endothelial function, and inflammatory cytokines as potential candidates for early risk detection in normal-weight obesity.
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Keirns BH, Hart SM, Sciarrillo CM, Poindexter KL, Clarke SL, and Emerson SR
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- Humans, Triglycerides, Cytokines, Tumor Necrosis Factor-alpha, Obesity complications, Postprandial Period, Glucose, Body Mass Index, Metabolic Syndrome complications, Hypertriglyceridemia, Cardiovascular Diseases etiology
- Abstract
Problem: Normal-weight obesity (NWO) is associated with increased cardiovascular disease (CVD) risk. However, NWO's clinical presentation is often unremarkable based on common risk factors. We examined whether CVD risk factors not routinely measured clinically including postprandial triglycerides, flow-mediated dilation (FMD), and inflammatory cytokines would be abnormal in NWO, consistent with their future risk., Methods: Individuals were recruited into 3 groups (n = 10/ group): controls (Con), NWO, and metabolic syndrome (MetS). Con was defined as a normal body mass index (BMI), < 25% (M) or < 35% (F) body fat, and < 1 International Diabetes Federation (IDF) criteria. NWO were above this body fat cutoff while maintaining a normal BMI and MetS was defined per the IDF. Participants underwent an abbreviated fat tolerance test (i.e., difference in fasting and 4 h triglycerides following a high-fat meal [9 kcal/kg; 73% fat)] and fasting and postprandial lipid and glucose metrics, as well as FMD were measured. A T cell cytokine bioplex was also performed using fasting serum., Results: NWO and MetS had similar body fat% and both were higher than Con (p < 0.0001). Despite having similar fasting triglycerides to Con, NWO had 4-hour triglycerides 66% greater than Con, but 46% lower than MetS (p < 0.01). FMD decreased in all groups after the high-fat meal (p < 0.0001). MetS displayed lower fasting FMD than Con, and NWO was similar to both groups (p < 0.05). No group differences were observed with postprandial FMD and the majority of fasting cytokines assessed. However, MetS exhibited higher fasting TNF-α than Con (p < 0.05), and NWO was similar to both groups., Conclusions: Overall, NWO was associated with higher postprandial triglycerides than Con, but displayed little evidence of impaired vascular health or inflammation., Competing Interests: Declaration of interest The authors declare there are no conflicts of interest to disclose., (Copyright © 2022 Asia Oceania Association for the Study of Obesity. Published by Elsevier Ltd. All rights reserved.)
- Published
- 2022
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15. Tuning Optical Absorption and Emission Using Strongly Coupled Dimers in Programmable DNA Scaffolds.
- Author
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Hart SM, Wang X, Guo J, Bathe M, and Schlau-Cohen GS
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- Carbocyanines metabolism, Circular Dichroism, DNA metabolism, Dimerization, Fluorescent Dyes, Nucleic Acid Conformation, Carbocyanines chemistry, DNA chemistry
- Abstract
Molecular materials for light harvesting, computing, and fluorescence imaging require nanoscale integration of electronically active subunits. Variation in the optical absorption and emission properties of the subunits has primarily been achieved through modifications to the chemical structure, which is often synthetically challenging. Here, we introduce a facile method for varying optical absorption and emission properties by changing the geometry of a strongly coupled Cy3 dimer on a double-crossover (DX) DNA tile. Leveraging the versatility and programmability of DNA, we tune the length of the complementary strand so that it "pushes" or "pulls" the dimer, inducing dramatic changes in the photophysics including lifetime differences observable at the ensemble and single-molecule level. The separable lifetimes, along with environmental sensitivity also observed in the photophysics, suggest that the Cy3-DX tile constructs could serve as fluorescence probes for multiplexed imaging. More generally, these constructs establish a framework for easily controllable photophysics via geometric changes to coupled chromophores, which could be applied in light-harvesting devices and molecular electronics.
- Published
- 2022
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16. Comparison of a Standardized High-Fat Meal versus a High-Fat Meal Scaled to Body Mass for Measuring Postprandial Triglycerides: A Randomized Crossover Study.
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Keirns BH, Sciarrillo CM, Hart SM, and Emerson SR
- Abstract
Post-meal triglycerides are an independent cardiovascular disease (CVD) risk factor, but the ideal high-fat meal formulation has yet to be standardized and is one challenge prohibiting widespread clinical adoption of postprandial triglyceride assessment. Two general approaches often used are giving individuals a high-fat meal scaled to body weight or a standardized high-fat meal containing a set fat bolus. A recent expert panel statement has endorsed the latter, specifying 75 g of fat as an appropriate fat dosage. Despite this recommendation, no study to date has tested whether there is a difference in postprandial triglycerides or if risk classification is affected based on these different approaches. We recruited 16 generally healthy individuals with roughly equal distribution among body mass index (BMI)class ( n = 5-6/per BMI category) and sex ( n = 2-3 M/F) within each BMI class. Each participant underwent two abbreviated fat tolerance tests separated by ~1 week: one with a scaled to body weight high-fat meal (9 kcal/kg; 70% fat) and a standardized meal containing 75 g of fat (70% fat). Fasting, 4 h, and absolute change in triglycerides across the entire sample and within each BMI category were similar regardless of high-fat meal. Only one participant with obesity had discordant postprandial responses between the fat tolerance tests (i.e., different CVD risk classification). These findings suggest that, within a certain range of fat intake, generally healthy individuals will have a similar postprandial triglyceride response. Considering the greater convenience of utilizing standardized high-fat meals, our data suggest that a standardized high-fat meal may be acceptable for large-scale studies and clinical implementation.
- Published
- 2022
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17. Identification of Nonradiative Decay Pathways in Cy3.
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Hart SM, Banal JL, Bathe M, and Schlau-Cohen GS
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- Carbocyanines radiation effects, Fluorescent Dyes radiation effects, Light, Spectrum Analysis methods, Stereoisomerism, Vibration, Carbocyanines chemistry, Fluorescent Dyes chemistry
- Abstract
Photoexcited fluorescent markers are extensively used in spectroscopy, imaging, and analysis of biological systems. The performance of fluorescent markers depends on high levels of emission, which are limited by competing nonradiative decay pathways. Small-molecule fluorescent dyes have been increasingly used as markers due to their high and stable emission. Despite their prevalence, the nonradiative decay pathways of these dyes have not been determined. Here, we investigate these pathways for a widely used indocarbocyanine dye, Cy3, using transient grating spectroscopy. We identify a nonradiative decay pathway via a previously unknown dark state formed within ∼1 ps of photoexcitation. Our experiments, in combination with electronic structure calculations, suggest that the generation of the dark state is mediated by picosecond vibrational mode coupling, likely via a conical intersection. We further identify the vibrational modes, and thus structural elements, responsible for the formation and dynamics of the dark state, providing insight into suppressing nonradiative decay pathways in fluorescent markers such as Cy3.
- Published
- 2020
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18. Hydration Control of Gel-Adhesion and Muco-Adhesion.
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McGhee EO, Hart SM, Urueña JM, and Sawyer WG
- Abstract
Protective mucin gel layers established by epithelial cell surfaces in biology have water contents above 90% and provide a low-shear stress nonadhesive interfacial boundary on epithelial surfaces throughout the body. Adhesion between gels and mucin layers, muco-adhesion, is an important aspect of drug delivery, biocompatibility, and the prevention of damage during insertion, use, and removal of medical devices in contact with moist epithelial surfaces. This manuscript develops a simple mathematical model to suggest that gel-adhesion and muco-adhesion are controlled by dehydration. For a fully swollen gel, the osmotic pressure is balanced by the elastic stress in the polymer gel, and differences in the elastic modulus are used to calculate dehydration stresses. A model based on Winkler contact mechanics gives a closed form expression for the force of adhesion that is dependent on the contact radius and gel thickness, inversely proportional to the mucin layer stiffness, and proportional to the square of the differences in elastic modulus. Submerged contact experiments conducted on Gemini gel interfaces of polyacrylamide aqueous gels showed increasing adhesion with increasing dehydration of the probe. Additionally, experiments conducted against mucinated epithelial cell monolayers found mucin transfer onto the most dehydrated gels and no transfer on swollen gels. The model and experiments reveal that high water content fully swollen gels are not intrinsically muco-adhesive, which is consistent with previous tribological experience showing increased lubricity with increasing water content and mesh size.
- Published
- 2019
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19. Effect of nanotube coupling on exciton transport in polymer-free monochiral semiconducting carbon nanotube networks.
- Author
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Arias DH, Sulas-Kern DB, Hart SM, Kang HS, Hao J, Ihly R, Johnson JC, Blackburn JL, and Ferguson AJ
- Abstract
Semiconducting single-walled carbon nanotubes (s-SWCNTs) are attractive light-harvesting components for solar photoconversion schemes and architectures, and selective polymer extraction has emerged as a powerful route to obtain highly pure s-SWCNT samples for electronic applications. Here we demonstrate a novel method for producing electronically coupled thin films of near-monochiral s-SWCNTs without wrapping polymer. Detailed steady-state and transient optical studies on such samples provide new insights into the role of the wrapping polymer on controlling intra-bundle nanotube-nanotube interactions and exciton energy transfer within and between bundles. Complete removal of polymer from the networks results in rapid exciton trapping within nanotube bundles, limiting long-range exciton transport. The results suggest that intertube electronic coupling and associated exciton delocalization across multiple tubes can limit diffusive exciton transport. The complex relationship observed here between exciton delocalization, trapping, and long-range transport, helps to inform the design, preparation, and implementation of carbon nanotube networks as active elements for optical and electronic applications.
- Published
- 2019
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20. Emulsion droplet crystallinity attenuates early in vitro digestive lipolysis and beta-carotene bioaccessibility.
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Hart SM, Lin XL, Thilakarathna SH, and Wright AJ
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- Dietary Fats, Digestion, Duodenum drug effects, Duodenum metabolism, Emulsions pharmacokinetics, Humans, Lipolysis drug effects, Particle Size, Polysorbates chemistry, Triglycerides chemistry, beta Carotene chemistry, Emulsions chemistry, Lipid Droplets chemistry, beta Carotene pharmacokinetics
- Abstract
The impacts of lipid crystallinity on in vitro digestive lipolysis and bioaccessibility of encapsulated (0.1 wt%) beta-carotene (BC) were investigated for a 15 wt% cocoa butter emulsion prepared as crystalline (i.e. solid emulsions, SE & SE-BC) or undercooled (liquid emulsions, LE & LE-BC) droplets at 25 °C. Particle size distributions (D
4,3 ∼0.7 μm), morphology (spherical), polymorphism (beta-V), thermal behavior (peak melting ∼30 °C), zeta potential (∼-44 mV) and BC degradation under accelerated lighting conditions were similarly extensive. Following exposure to simulated gastric conditions, duodenal hydrolysis and BC bioaccessibility were lower for SE-BC up to 2 h (P < 0.05). Ultimately, samples with both solid and liquid droplets were hydrolyzed extensively and BC bioaccessibility did not differ (P > 0.05). Therefore, for compositionally equivalent emulsions, lipid droplet solid state delayed digestive lipolysis and bioactive solubilization. These results help to clarify the role of lipid physical state on dietary lipid digestion and bioactive release., (Copyright © 2018 Elsevier Ltd. All rights reserved.)- Published
- 2018
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21. Femtosecond stimulated Raman evidence for charge-transfer character in pentacene singlet fission.
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Hart SM, Silva WR, and Frontiera RR
- Abstract
Singlet fission is a spin-allowed process in which an excited singlet state evolves into two triplet states. We use femtosecond stimulated Raman spectroscopy, an ultrafast vibrational technique, to follow the molecular structural evolution during singlet fission in order to determine the mechanism of this process. In crystalline pentacene, we observe the formation of an intermediate characterized by pairs of excited state peaks that are red- and blue-shifted relative to the ground state features. We hypothesize that these features arise from the formation of cationic and anionic species due to partial transfer of electron density from one pentacene molecule to a neighboring molecule. These observations provide experimental evidence for the role of states with significant charge-transfer character which facilitate the singlet fission process in pentacene. Our work both provides new insight into the singlet fission mechanism in pentacene and demonstrates the utility of structurally-sensitive time-resolved spectroscopic techniques in monitoring ultrafast processes.
- Published
- 2017
- Full Text
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22. Discovery of 6,7-dihydro-5H-pyrrolo[2,3-a]pyrimidines as orally available G protein-coupled receptor 119 agonists.
- Author
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Katamreddy SR, Carpenter AJ, Ammala CE, Boros EE, Brashear RL, Briscoe CP, Bullard SR, Caldwell RD, Conlee CR, Croom DK, Hart SM, Heyer DO, Johnson PR, Kashatus JA, Minick DJ, Peckham GE, Ross SA, Roller SG, Samano VA, Sauls HR, Tadepalli SM, Thompson JB, Xu Y, and Way JM
- Subjects
- Administration, Oral, Animals, Cell Line, Colon metabolism, Glucagon-Like Peptide 1 metabolism, Glucose metabolism, Glucose Tolerance Test, Humans, Hypoglycemic Agents pharmacokinetics, Hypoglycemic Agents pharmacology, Incretins metabolism, Intestinal Mucosa metabolism, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Piperidines pharmacokinetics, Piperidines pharmacology, Pyrimidines pharmacokinetics, Pyrimidines pharmacology, Pyrroles pharmacokinetics, Pyrroles pharmacology, Rats, Rats, Sprague-Dawley, Rats, Wistar, Stereoisomerism, Structure-Activity Relationship, Hypoglycemic Agents chemical synthesis, Piperidines chemical synthesis, Pyrimidines chemical synthesis, Pyrroles chemical synthesis, Receptors, G-Protein-Coupled agonists
- Abstract
GPR119 is a 7-transmembrane receptor that is expressed in the enteroendocrine cells in the intestine and in the islets of Langerhans in the pancreas. Indolines and 6,7-dihydro-5H-pyrrolo[2,3-a]pyrimidines were discovered as G protein-coupled receptor 119 (GPR119) agonists, and lead optimization efforts led to the identification of 1-methylethyl 4-({7-[2-fluoro-4-(methylsulfonyl)phenyl]-6,7-dihydro-5H-pyrrolo[2,3-d]pyrimidin-4-yl}oxy)-1-piperidinecarboxylate (GSK1104252A) (3), a potent and selective GPR119 agonist. Compound 3 showed excellent pharmacokinetic properties and sufficient selectivity with in vivo studies supporting a role for GPR119 in glucose homeostasis in the rodent. Thus, 3 appeared to modulate the enteroinsular axis, improve glycemic control, and strengthen previous suggestions that GPR119 agonists may have utility in the treatment of type 2 diabetes.
- Published
- 2012
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23. Identification of potent and selective glucosylceramide synthase inhibitors from a library of N-alkylated iminosugars.
- Author
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Ghisaidoobe A, Bikker P, de Bruijn AC, Godschalk FD, Rogaar E, Guijt MC, Hagens P, Halma JM, Van't Hart SM, Luitjens SB, van Rixel VH, Wijzenbroek M, Zweegers T, Donker-Koopman WE, Strijland A, Boot R, van der Marel G, Overkleeft HS, Aerts JM, and van den Berg RJ
- Abstract
Glucosylceramide synthase (GCS) is an important target for clinical drug development for the treatment of lysosomal storage disorders and a promising target for combating type 2 diabetes. Iminosugars are useful leads for the development of GCS inhibitors; however, the effective iminosugar type GCS inhibitors reported have some unwanted cross-reactivity toward other glyco-processing enzymes. In particular, iminosugar type GCS inhibitors often also inhibit to some extent human acid glucosylceramidase (GBA1) and the nonlysosomal glucosylceramidase (GBA2), the two enzymes known to process glucosylceramide. Of these, GBA1 itself is a potential drug target for the treatment of the lysosomal storage disorder, Gaucher disease, and selective GBA1 inhibitors are sought after as potential chemical chaperones. The physiological importance of GBA2 in glucosylceramide processing in relation to disease states is less clear, and here, selective inhibitors can be of use as chemical knockout entities. In this communication, we report our identification of a highly potent and selective N-alkylated l-ido-configured iminosugar. In particular, the selectivity of 27 for GCS over GBA1 is striking.
- Published
- 2010
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24. The JAK3-selective inhibitor PF-956980 reverses the resistance to cytotoxic agents induced by interleukin-4 treatment of chronic lymphocytic leukemia cells: potential for reversal of cytoprotection by the microenvironment.
- Author
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Steele AJ, Prentice AG, Cwynarski K, Hoffbrand AV, Hart SM, Lowdell MW, Samuel ER, and Wickremasinghe RG
- Subjects
- Apoptosis drug effects, Bone Marrow Cells drug effects, Bone Marrow Cells metabolism, Calcium-Calmodulin-Dependent Protein Kinases antagonists & inhibitors, Cytochromes c metabolism, Extracellular Signal-Regulated MAP Kinases metabolism, Flavonoids pharmacology, Gene Expression Regulation, Leukemic drug effects, Humans, Interleukin-4 pharmacology, Janus Kinase 3 metabolism, Myeloid Cell Leukemia Sequence 1 Protein, Phosphorylation drug effects, Proto-Oncogene Proteins c-bcl-2 genetics, STAT6 Transcription Factor metabolism, Stromal Cells drug effects, Stromal Cells metabolism, T-Lymphocytes drug effects, T-Lymphocytes metabolism, Tumor Cells, Cultured, Tumor Suppressor Protein p53 metabolism, bcl-X Protein genetics, Drug Resistance, Neoplasm drug effects, Interleukin-4 therapeutic use, Janus Kinase 3 antagonists & inhibitors, Leukemia, Lymphocytic, Chronic, B-Cell drug therapy, Pyrimidines pharmacology, Pyrroles pharmacology
- Abstract
Extensive evidence suggests that the malignant cells of chronic lymphocytic leukemia (CLL) patients are in close contact with activated T lymphocytes, which secrete a range of cytoprotective cytokines including interleukin-4 (IL-4). IL-4 induced the rapid phosphorylation and activation of the signal transducer and activator of transcription 6 transcription factor in CLL cells in vitro. Longer incubation with IL-4 resulted in up-regulation of the antiapoptotic proteins, Mcl-1 and Bcl-X(L). All of these events were blocked by the JAK3-selective inhibitor, PF-956980. A dye reduction cytotoxicity assay showed that IL-4 induced resistance to the cytotoxic drugs fludarabine and chlorambucil and to the novel p53-elevating agent nutlin 3. IL-4-induced drug resistance was reversed by PF-956980. These conclusions were confirmed by independent assays for apoptosis induction (annexin V binding, cleavage of poly[ADP-ribose] polymerase, and morphologic analysis). Coculture with bone marrow stromal cells in the presence of supernatants derived from activated T-lymphocyte cultures also protected CLL cells from apoptosis induction by chlorambucil. Protection by these combined signals was reversed by PF-956980. The data here provide a preclinical rationale for the possible therapeutic use of PF-956980 in conjunction with conventional cytotoxic drugs to achieve more extensive killing of CLL cells by overcoming antiapoptotic signaling by the microenvironment.
- Published
- 2010
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25. 2-Phenylacetylenesulfonamide (PAS) induces p53-independent apoptotic killing of B-chronic lymphocytic leukemia (CLL) cells.
- Author
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Steele AJ, Prentice AG, Hoffbrand AV, Yogashangary BC, Hart SM, Lowdell MW, Samuel ER, North JM, Nacheva EP, Chanalaris A, Kottaridis P, Cwynarski K, and Wickremasinghe RG
- Subjects
- Aged, Aged, 80 and over, Annexin A5 metabolism, Caspase 3 metabolism, Cytochromes c metabolism, Cytosol metabolism, Cytosol pathology, Dose-Response Relationship, Drug, Drug Resistance drug effects, Drug Screening Assays, Antitumor, Female, Hematopoietic Stem Cells metabolism, Hematopoietic Stem Cells pathology, Humans, Leukemia, Lymphocytic, Chronic, B-Cell metabolism, Leukemia, Lymphocytic, Chronic, B-Cell pathology, Male, Middle Aged, Mitochondria metabolism, Mitochondria pathology, Poly(ADP-ribose) Polymerases metabolism, Protein Transport drug effects, T-Lymphocytes metabolism, T-Lymphocytes pathology, Time Factors, Tumor Cells, Cultured, Up-Regulation drug effects, bcl-2-Associated X Protein metabolism, Antineoplastic Agents pharmacokinetics, Apoptosis drug effects, Gene Expression Regulation, Leukemic drug effects, Leukemia, Lymphocytic, Chronic, B-Cell drug therapy, Proto-Oncogene Proteins c-bcl-2 biosynthesis, Sulfonamides pharmacology, Tumor Suppressor Protein p53 metabolism
- Abstract
We studied the actions of 2-phenylacetylenesulfonamide (PAS) on B-chronic lymphocytic leukemia (CLL) cells. PAS (5-20 microM) initiated apoptosis within 24 hours, with maximal death at 48 hours asassessed by morphology, cleavage of poly(ADP-ribose) polymerase (PARP), caspase 3 activation, and annexin V staining. PAS treatment induced Bax proapoptotic conformational change, Bax movement from the cytosol to the mitochondria, and cytochrome c release, indicating that PAS induced apoptosis via the mitochondrial pathway. PAS induced approximately 3-fold up-regulation of proapoptotic Noxa protein and mRNA levels. In addition, Noxa was found unexpectedly to be bound to Bcl-2 in PAS-treated cells. PAS treatment of CLL cells failed to up-regulate p53, suggesting that PAS induced apoptosis independently of p53. Furthermore, PAS induced apoptosis in CLL isolates with p53 gene deletion in more than 97% of cells. Normal B lymphocytes were as sensitive to PAS-induced Noxa up-regulation and apoptosis as were CLL cells. However, both T lymphocytes and bone marrow hematopoietic progenitor cells were relatively resistant to PAS. Our data suggest that PAS may represent a novel class of drug that induces apoptosis in CLL cells independently of p53 status by a mechanism involving Noxa up-regulation.
- Published
- 2009
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26. Optimization of a digoxigenin-based immunoassay system for gene detection in Arabidopsis thaliana.
- Author
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Hart SM and Basu C
- Subjects
- DNA Probes, DNA, Plant, Genome, Plant, Immunoassay methods, In Situ Hybridization methods, Peptide Elongation Factor 1 genetics, Plasmids genetics, Sensitivity and Specificity, Arabidopsis genetics, Digoxigenin, Genes, Plant
- Abstract
Digoxigenin is derived from a plant steroid hormone digoxin found in the plants Digitalis sp. Digoxigenin has been used successfully in labeling nucleic acids. In this experiment we optimized minimum probe requirement for a nonradioactive digoxigenin-based gene detection system in the model plant Arabidopsis thaliana. We showed that 1 microL of labeled probe was sufficient to hybridize onto 1-10 microg of target plasmid DNA. We also examined the sensitivity of labeled probe and showed that 2 microL of labeled probe was not able to hybridize with 1 microg of target DNA, although 2 microL of labeled probe was able to detect target DNA ranging from 2 to 10 microg. To test the efficacy of our optimization protocol, we used 1 microL of labeled plasmid DNA pU16893 harboring an Arabidopsis housekeeping gene elongation factor-1 and showed that the elongation factor-1 gene could be detected in Arabidopsis genome under various environmental conditions. This paper describes a nonradioactive in situ hybridization technique to detect nucleic acids in plants.
- Published
- 2009
27. p53-mediated apoptosis of CLL cells: evidence for a transcription-independent mechanism.
- Author
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Steele AJ, Prentice AG, Hoffbrand AV, Yogashangary BC, Hart SM, Nacheva EP, Howard-Reeves JD, Duke VM, Kottaridis PD, Cwynarski K, Vassilev LT, and Wickremasinghe RG
- Subjects
- Adult, Aged, Aged, 80 and over, Apoptosis drug effects, Apoptosis genetics, Apoptosis Regulatory Proteins metabolism, Benzothiazoles pharmacology, Chlorambucil pharmacology, Female, Humans, In Vitro Techniques, Leukemia, Lymphocytic, Chronic, B-Cell genetics, Male, Middle Aged, Mitochondria drug effects, Mitochondria metabolism, Proto-Oncogene Proteins metabolism, Proto-Oncogene Proteins c-bcl-2 metabolism, Toluene analogs & derivatives, Toluene pharmacology, Transcription, Genetic drug effects, Tumor Suppressor Protein p53 antagonists & inhibitors, Vidarabine analogs & derivatives, Vidarabine pharmacology, Apoptosis physiology, Leukemia, Lymphocytic, Chronic, B-Cell metabolism, Leukemia, Lymphocytic, Chronic, B-Cell pathology, Tumor Suppressor Protein p53 metabolism
- Abstract
The p53 protein plays a key role in securing the apoptotic response of chronic lymphocytic leukemia (CLL) cells to genotoxic agents. Transcriptional induction of proapoptotic proteins including Puma are thought to mediate p53-dependent apoptosis. In contrast, recent studies have identified a novel nontranscriptional mechanism, involving direct binding of p53 to antiapoptotic proteins including Bcl-2 at the mitochondrial surface. Here we show that the major fraction of p53 induced in CLL cells by chlorambucil, fludarabine, or nutlin 3a was stably associated with mitochondria, where it binds to Bcl-2. The Puma protein, which was constitutively expressed in a p53-independent manner, was modestly up-regulated following p53 induction. Pifithrin alpha, an inhibitor of p53-mediated transcription, blocked the up-regulation of Puma and also of p21(CIP1). Surprisingly, pifithrin alpha dramatically augmented apoptosis induction by p53-elevating agents and also accelerated the proapoptotic conformation change of the Bax protein. These data suggest that direct interaction of p53 with mitochondrial antiapoptotic proteins including Bcl-2 is the major route for apoptosis induction in CLL cells and that p53's transcriptional targets include proteins that impede this nontranscriptional pathway. Therefore, strategies that block up-regulation of p53-mediated transcription may be of value in enhancing apoptosis induction of CLL cells by p53-elevating drugs.
- Published
- 2008
- Full Text
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28. Bcr-Abl signaling through the PI-3/S6 kinase pathway inhibits nuclear translocation of the transcription factor Bach2, which represses the antiapoptotic factor heme oxygenase-1.
- Author
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Yoshida C, Yoshida F, Sears DE, Hart SM, Ikebe D, Muto A, Basu S, Igarashi K, and Melo JV
- Subjects
- Apoptosis, Basic-Leucine Zipper Transcription Factors physiology, Cell Line, Humans, Leukemia, Myelogenous, Chronic, BCR-ABL Positive, Phosphorylation, Protein Processing, Post-Translational physiology, Repressor Proteins, Signal Transduction, Active Transport, Cell Nucleus, Basic-Leucine Zipper Transcription Factors metabolism, Fusion Proteins, bcr-abl physiology, Heme Oxygenase-1 antagonists & inhibitors, Phosphatidylinositol 3-Kinases metabolism, Ribosomal Protein S6 Kinases metabolism
- Abstract
The malignant phenotype of chronic myeloid leukemia (CML) is due to the abnormal tyrosine kinase activity of the Bcr-Abl oncoprotein. We have previously reported that expression of the Bach2 transcription factor, which induces apoptosis in response to oxidative stress, is greatly reduced in CML cells. Because these cells are resistant to apoptosis, we tested whether Bach2 could also be regulated through posttranslational mechanisms that promote inhibition of the apoptotic response to mutagenic stimuli in CML. We found that Bach2 is phosphorylated on S521 via the phosphatidylinositol-3/S6 kinase pathway, and substitution of this site to alanine leads to nuclear accumulation of the protein, indicating that this phosphorylation is important for its subcellular localization. Ectopic expression of the S521 mutant imparts greater impairment to CML cell growth than the wild-type factor. Furthermore, we showed that Bach2 transcriptionally represses heme oxygenase-1, an antiapoptotic factor up-regulated in CML. Because CML cells are known to produce high levels of intracellular reactive oxygen species, overexpression of heme oxygenase-1 resulting from inhibition of Bach2 activity may contribute to their genomic instability and leukemic phenotype.
- Published
- 2007
- Full Text
- View/download PDF
29. Generational diversity: impact on recruitment and retention of registered nurses.
- Author
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Hart SM
- Subjects
- Adult, Aged, Humans, Middle Aged, Nursing Staff supply & distribution, United States, Intergenerational Relations, Nursing Staff organization & administration, Personnel Selection
- Published
- 2006
- Full Text
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30. T:G mismatch-specific thymine-DNA glycosylase (TDG) as a coregulator of transcription interacts with SRC1 family members through a novel tyrosine repeat motif.
- Author
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Lucey MJ, Chen D, Lopez-Garcia J, Hart SM, Phoenix F, Al-Jehani R, Alao JP, White R, Kindle KB, Losson R, Chambon P, Parker MG, Schär P, Heery DM, Buluwela L, and Ali S
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Animals, COS Cells, Chlorocebus aethiops, Histone Acetyltransferases, Humans, Molecular Sequence Data, Nuclear Receptor Coactivator 1, Repetitive Sequences, Amino Acid, Trans-Activators chemistry, Transcription Factors chemistry, Tyrosine analysis, Thymine DNA Glycosylase chemistry, Thymine DNA Glycosylase metabolism, Trans-Activators metabolism, Transcription Factors metabolism
- Abstract
Gene activation involves protein complexes with diverse enzymatic activities, some of which are involved in chromatin modification. We have shown previously that the base excision repair enzyme thymine DNA glycosylase (TDG) acts as a potent coactivator for estrogen receptor-alpha. To further understand how TDG acts in this context, we studied its interaction with known coactivators of nuclear receptors. We find that TDG interacts in vitro and in vivo with the p160 coactivator SRC1, with the interaction being mediated by a previously undescribed motif encoding four equally spaced tyrosine residues in TDG, each tyrosine being separated by three amino acids. This is found to interact with two motifs in SRC1 also containing tyrosine residues separated by three amino acids. Site-directed mutagenesis shows that the tyrosines encoded in these motifs are critical for the interaction. The related p160 protein TIF2 does not interact with TDG and has the altered sequence, F-X-X-X-Y, at the equivalent positions relative to SRC1. Substitution of the phenylalanines to tyrosines is sufficient to bring about interaction of TIF2 with TDG. These findings highlight a new protein-protein interaction motif based on Y-X-X-X-Y and provide new insight into the interaction of diverse proteins in coactivator complexes.
- Published
- 2005
- Full Text
- View/download PDF
31. Effects of moxifloxacin on QT interval in conscious dogs.
- Author
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Mittelstadt SW and Hart SM
- Subjects
- Animals, Anti-Arrhythmia Agents administration & dosage, Anti-Arrhythmia Agents pharmacokinetics, Aza Compounds administration & dosage, Aza Compounds pharmacokinetics, Blood Pressure drug effects, Body Temperature, Cardiac Output drug effects, Dose-Response Relationship, Drug, Electrocardiography, Fluoroquinolones, Infusions, Intravenous veterinary, Long QT Syndrome chemically induced, Long QT Syndrome physiopathology, Male, Moxifloxacin, Quinolines administration & dosage, Quinolines pharmacokinetics, Reference Values, Anti-Arrhythmia Agents pharmacology, Aza Compounds pharmacology, Dogs physiology, Heart Conduction System drug effects, Quinolines pharmacology
- Abstract
Moxifloxacin has been shown to induce QT prolongation in both clinical and preclinical models. However, the ability to observe this effect at clinically relevant concentration in normal conscious dogs has not been reported. The purpose of this study was to investigate the effects of moxifloxacin on the QT interval in conscious, healthy dogs. Four male mongrel dogs were chronically instrumented for the measurement of arterial blood pressure, left ventricular blood pressure, cardiac output, electrocardiograms (ECGs), and body temperature. Animals were administered a 1-h i.v. infusion of moxifloxacin once per day via a catheter in the cephalic vein. Each dog received all doses (0, 1, 10, 25 and 50 mg/kg) in an escalating fashion. Moxifloxacin caused a statistically significant increase in arterial blood pressure at 50 mg/kg. A dose-response effect on QT and QTc prolongation was observed. A statistically significant prolongation in the QT interval was observed at 10, 25 and 50 mg/kg and a prolongation of QTc was observed at 25 and 50 mg/kg. These effects occurred at clinically relevant plasma concentrations. This study demonstrate that a study design with four dogs was sensitive enough to measure moxifloxacin-induced QT prolongation at clinically relevant plasma concentrations.
- Published
- 2005
- Full Text
- View/download PDF
32. Inhibiting estrogen responses in breast cancer cells using a fusion protein encoding estrogen receptor-alpha and the transcriptional repressor PLZF.
- Author
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Buluwela L, Pike J, Mazhar D, Kamalati T, Hart SM, Al-Jehani R, Yahaya H, Patel N, Sarwar N, Heathcote DA, Schwickerath O, Phoenix F, Hill R, Aboagye E, Shousha S, Waxman J, Lemoine NR, Zelent A, Coombes RC, and Ali S
- Subjects
- Animals, Cell Line, Tumor, DNA-Binding Proteins metabolism, Estrogen Receptor alpha metabolism, Female, Gene Expression Regulation, Humans, Kruppel-Like Transcription Factors, Luciferases genetics, Mice, Mice, Nude, Promyelocytic Leukemia Zinc Finger Protein, Transcription Factors metabolism, Transfection methods, beta-Galactosidase genetics, Breast Neoplasms metabolism, Breast Neoplasms therapy, DNA-Binding Proteins genetics, Estrogen Receptor alpha genetics, Estrogens metabolism, Genetic Therapy methods, Recombinant Fusion Proteins therapeutic use, Transcription Factors genetics
- Abstract
Estrogen receptor alpha (ERalpha) is a ligand-inducible transcription factor that acts to regulate gene expression by binding to palindromic DNA sequence, known as the estrogen response element, in promoters of estrogen-regulated genes. In breast cancer ERalpha plays a central role, where estrogen-regulated gene expression leads to tumor initiation, growth and survival. As an approach to silencing estrogen-regulated genes, we have studied the activities of a fusion protein between ERalpha and the promyelocytic leukemia zinc-finger (PLZF) protein, a transcriptional repressor that acts through chromatin remodeling. To do this, we have developed lines from the estrogen-responsive MCF-7 breast cancer cell line in which the expression of the fusion protein PLZF-ERalpha is conditionally regulated by tetracycline and shows that these feature long-term silencing of the expression of several well-characterized estrogen-regulated genes, namely pS2, cathepsin-D and the progesterone receptor. However, the estrogen-regulated growth of these cells is not inhibited unless PLZF-ERalpha expression is induced, an observation that we have confirmed both in vitro and in vivo. Taken together, these results show that PLZF-ERalpha is a potent repressor of estrogen-regulated gene expression and could be useful in distinguishing estrogen-regulated genes required for the growth of breast cancer cells.
- Published
- 2005
- Full Text
- View/download PDF
33. ICI182,780 induces p21Waf1 gene transcription through releasing histone deacetylase 1 and estrogen receptor alpha from Sp1 sites to induce cell cycle arrest in MCF-7 breast cancer cell line.
- Author
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Varshochi R, Halim F, Sunters A, Alao JP, Madureira PA, Hart SM, Ali S, Vigushin DM, Coombes RC, and Lam EW
- Subjects
- Binding Sites, Cell Line, Tumor, Cyclin-Dependent Kinase Inhibitor p21, Enzyme Inhibitors pharmacology, Fulvestrant, G1 Phase drug effects, Gene Silencing, Histone Deacetylase Inhibitors, Humans, Hydroxamic Acids pharmacology, Promoter Regions, Genetic physiology, RNA, Small Interfering, Sp1 Transcription Factor metabolism, Transcriptional Activation drug effects, Up-Regulation drug effects, Up-Regulation genetics, Antineoplastic Agents, Hormonal pharmacology, Breast Neoplasms, Cell Cycle Proteins genetics, Estradiol analogs & derivatives, Estradiol pharmacology, Estrogen Receptor alpha metabolism, Histone Deacetylases metabolism
- Abstract
We used the estrogen-responsive MCF-7 breast cancer cell line as a relevant model to study the anti-proliferative effects of ICI182,780 and identified the negative cell cycle regulator p21Waf1 as a specific target of ICI182,780. Furthermore, silencing of the p21Waf1 expression by small interfering RNA overcame the G0/G1 cell cycle arrest induced by ICI182,780, suggesting that the induction of p21Waf1 expression has a direct role in mediating the ICI182,780-induced G0/G1 arrest. We further demonstrated that the induction of p21Waf1 by ICI182,780 is mediated at transcriptional and gene promoter levels through the proximal Sp1 sites located near the transcription start site. Co-immunoprecipitation, DNA "pull-down," and chromatin immunoprecipitation experiments together showed that in cycling cells, estrogen receptor alpha and histone deacetylase 1 (HDAC1) are recruited to the proximal Sp1 sites of the promoter to repress p21Waf1 expression. In the presence of ICI182,780, estrogen receptor alpha and HDACs are dissociated from Sp1, resulting in increased histone acetylation and de-repression of the p21Waf1 promoter and induction of p21Waf1 expression. The fact that p21Waf1 expression is normally repressed by HDAC activity in cycling cells is further demonstrated by the finding that p21Waf1 transcription can be induced by the silencing of HDACs with small interfering RNA or treatment with HDAC inhibitors.
- Published
- 2005
- Full Text
- View/download PDF
34. Miniaturization of the structure elucidation of novel natural products--two trace antibacterial acylated caprylic alcohol glycosides from Arctostaphylos pumila.
- Author
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Hu JF, Yoo HD, Williams CT, Garo E, Cremin PA, Zeng L, Vervoort HC, Lee CM, Hart SM, Goering MG, O'Neil-Johnson M, and Eldridge GR
- Subjects
- Anti-Bacterial Agents administration & dosage, Anti-Bacterial Agents chemistry, Anti-Bacterial Agents therapeutic use, Glycosides administration & dosage, Glycosides chemistry, Glycosides pharmacology, Glycosides therapeutic use, Humans, Methicillin Resistance, Microbial Sensitivity Tests, Plant Extracts administration & dosage, Plant Extracts chemistry, Plant Extracts therapeutic use, Anti-Bacterial Agents pharmacology, Arctostaphylos, Phytotherapy, Plant Extracts pharmacology, Staphylococcus aureus drug effects
- Abstract
High-throughput isolation, purification and analysis methods applied to natural products libraries from plants gave rise to the discovery of two novel acylated caprylic alcohol glycosides (1, 2) produced by Arctostaphylos pumila. The NMR spectra were acquired using the CapNMR probe and performed on mass-limited samples, which enabled us to elucidate the structures of 2,6-diacetyl-3,4-diisobutyl-1- O-octylglucopyranoside (1, 200 microg) and 2,6-diacetyl-3,4-dimethylbutyl-1- O-octylglucopyranosid (2, 70 microg). Compounds 1 and 2 exhibited antibacterial activity against Gram-positive methicillin-resistant Staphylococcus aureus with an MIC of 128 microg/mL and 64 microg/mL, respectively.
- Published
- 2005
- Full Text
- View/download PDF
35. T:G mismatch-specific thymine-DNA glycosylase potentiates transcription of estrogen-regulated genes through direct interaction with estrogen receptor alpha.
- Author
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Chen D, Lucey MJ, Phoenix F, Lopez-Garcia J, Hart SM, Losson R, Buluwela L, Coombes RC, Chambon P, Schär P, and Ali S
- Subjects
- Amino Acid Motifs, Amino Acid Sequence, Animals, Base Pair Mismatch, COS Cells, Cell Nucleus metabolism, Chloramphenicol O-Acetyltransferase metabolism, Chromatin metabolism, DNA metabolism, DNA Glycosylases, DNA Repair, Estrogen Receptor alpha, Genetic Vectors, Glutathione Transferase metabolism, Humans, Immunoblotting, Ligands, Models, Genetic, Molecular Sequence Data, Mutagenesis, Site-Directed, Precipitin Tests, Protein Binding, Protein Biosynthesis, RNA metabolism, RNA Splicing, Receptors, Estrogen metabolism, Recombinant Fusion Proteins metabolism, Sequence Homology, Amino Acid, Thymidine chemistry, Transcription, Genetic, Transcriptional Activation, Transfection, Two-Hybrid System Techniques, beta-Galactosidase metabolism, Estrogens metabolism, N-Glycosyl Hydrolases chemistry, Receptors, Estrogen chemistry
- Abstract
Nuclear receptors (NR) classically regulate gene expression by stimulating transcription upon binding to their cognate ligands. It is now well established that NR-mediated transcriptional activation requires the recruitment of coregulator complexes, which facilitate recruitment of the basal transcription machinery through direct interactions with the basal transcription machinery and/or through chromatin remodeling. However, a number of recently described NR coactivators have been implicated in cross-talk with other nuclear processes including RNA splicing and DNA repair. T:G mismatch-specific thymine DNA glycosylase (TDG) is required for base excision repair of deaminated methylcytosine. Here we show that TDG is a coactivator for estrogen receptor alpha (ERalpha). We demonstrate that TDG interacts with ERalpha in vitro and in vivo and suggest a separate role for TDG to its established role in DNA repair. We show that this involves helix 12 of ERalpha. The region of interaction in TDG is mapped to a putative alpha-helical motif containing a motif distinct from but similar to the LXXLL motif that mediates interaction with NR. Together with recent reports linking TFIIH in regulating NR function, our findings provide new data to further support an important link between DNA repair proteins and nuclear receptor function.
- Published
- 2003
- Full Text
- View/download PDF
36. Core binding factor genes and human leukemia.
- Author
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Hart SM and Foroni L
- Subjects
- Animals, Core Binding Factor Alpha 2 Subunit, Core Binding Factor beta Subunit, DNA-Binding Proteins physiology, Hematopoiesis, Humans, Leukemia etiology, Mutation, Transcription Factor AP-2, Transcription Factors physiology, DNA-Binding Proteins genetics, Leukemia genetics, Proto-Oncogene Proteins, Transcription Factors genetics
- Abstract
Background: The core binding factor (CBF) transcription complex, consisting of the interacting proteins RUNX1 and CBFb, is essential for normal hematopoiesis. Recent studies have shown that mutations and gene rearrangements involving this complex are frequently implicated in leukemogenesis. Understanding the molecular events leading to the disruption of CBF has provided important insights into our understanding of the normal regulatory pathways that control hematopoiesis and has begun to reveal how alterations in these pathways induce leukemia., Information Sources: Both authors are involved in the identification and characterization of chromosomal abnormalities associated with hematologic malignancy. This has led to contributions to multicenter clinical and laboratory investigations as well as publications in peer-reviewed journals. All of the references cited in this review are published in journals covered by Medline. State of the Art. The core binding factor (CBF) is a heterodimeric transcription factor composed of the RUNX1 and CBFb subunits. RUNX1 is the DNA binding element of the complex and its affinity is greatly increased in the presence of CBFb. Knock-out studies in mice have demonstrated that both RUNX1 and CBFb are necessary for definitive hematopoiesis. Furthermore, reciprocal chromosomal translocations involving both partners have been directly implicated in leukemogenesis. Evidence is now emerging that at least some of the resulting fusion proteins, namely ETV6-RUNX1, RUNX1-MTG8 and CBFb-MYH11 dominantly inhibit the function of native CBF by recruiting transcriptional co-repressor complexes. However, knock-in studies have shown that whilst expression of these fusion genes may disrupt normal hematopoiesis, this, by itself, is not sufficient for the subsequent development of leukemia. Mutations of RUNX1 have been identified in familial platelet disorder (FDP), in which there is a congenital predisposition to the development of AML and heterozygous point mutations have been identified in the RUNX1 gene in some leukemias. Moreover, a small number of cases have been reported in which amplification of RUNX1 has been detected in childhood ALL suggesting mechanisms other than loss of function, such as gene dosage may also play a role., Conclusions: Understanding the role CBF plays in normal hematopoiesis and hematologic malignancies has provided critical reagents for the accurate identification of the broad group of leukemias harboring alterations of CBF. The application of these molecular approaches has already shown an impact on the clinical management of these patients and as more information becomes available, the ability to tailor therapy to improve each patient's chance of a cure becomes feasible.
- Published
- 2002
37. High-throughput method for the production and analysis of large natural product libraries for drug discovery.
- Author
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Eldridge GR, Vervoort HC, Lee CM, Cremin PA, Williams CT, Hart SM, Goering MG, O'Neil-Johnson M, and Zeng L
- Subjects
- Chromatography, High Pressure Liquid, Humans, Paclitaxel analogs & derivatives, Plant Extracts chemistry, Spectrometry, Mass, Electrospray Ionization, Taxus chemistry, Tumor Cells, Cultured, Biological Factors chemistry, Combinatorial Chemistry Techniques, Drug Screening Assays, Antitumor methods
- Abstract
High-throughput methods were applied to the production, analysis, and characterization of libraries of natural products in order to accelerate the drug discovery process for high-throughput screening in the pharmaceutical and biotechnology industries. Library production integrates automated flash chromatography, solid-phase extraction, filtration, and high-throughput parallel four-channel preparative high-performance liquid chromatography to obtain the libraries in 96- or 384-well plates. Libraries consist of purified fractions with approximately one to five compounds per well. Libraries are analyzed prior to biological screening by a high-throughput parallel eight-channel liquid chromatography-evaporative light scattering detection-mass spectrometry system to determine the molecular weight, number, and quantity of compounds in a fraction. After biological screening, active fractions are rapidly purified at the microgram level and individual compounds are rescreened for confirmation of activity. Structures of active compounds are elucidated by NMR spectroscopy and mass spectrometry. Utilization of a novel microcoil probe allows NMR data to be gathered on 50 microg. As a demonstration, a library was made from the stem bark of Taxus brevifolia. Biological screening in the National Cancer Institute's in vitro panel of three cancer cell lines demonstrates that the process enables the discovery of active anticancer compounds not detected in the flash fractions from which the library originates.
- Published
- 2002
- Full Text
- View/download PDF
38. Actions of the selective protein kinase C inhibitor PKC412 on B-chronic lymphocytic leukemia cells in vitro.
- Author
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Ganeshaguru K, Wickremasinghe RG, Jones DT, Gordon M, Hart SM, Virchis AE, Prentice HG, Hoffbrand AV, Man A, Champain K, Csermak K, and Mehta AB
- Subjects
- 2-Chloroadenosine pharmacology, ATP Binding Cassette Transporter, Subfamily B, Member 1 metabolism, Aged, Aged, 80 and over, Apoptosis drug effects, B-Lymphocytes drug effects, B-Lymphocytes enzymology, Calcium Channel Blockers pharmacology, Chlorambucil pharmacology, Deoxyadenosines pharmacology, Drug Resistance, Multiple, Drug Resistance, Neoplasm, Drug Screening Assays, Antitumor, Female, Humans, In Situ Nick-End Labeling, Male, Middle Aged, Palatine Tonsil cytology, Tumor Cells, Cultured drug effects, Tumor Cells, Cultured enzymology, Verapamil pharmacology, Vidarabine analogs & derivatives, Vidarabine pharmacology, 2-Chloroadenosine analogs & derivatives, Antineoplastic Agents pharmacology, Enzyme Inhibitors pharmacology, Leukemia, Lymphocytic, Chronic, B-Cell pathology, Neoplasm Proteins antagonists & inhibitors, Protein Kinase C antagonists & inhibitors, Staurosporine analogs & derivatives, Staurosporine pharmacology
- Abstract
Background and Objectives: The staurosporine derivative PKC412 (CGP41251) is a more selective inhibitor of the conventional isoforms of protein kinase C (PKC) than is the parent compound. In addition to its growth inhibitory properties, PKC412 reverses the efflux function of the multidrug resistance (MDR)-1 gene product, P-glycoprotein (P-gp)., Design and Methods: The in vitro actions of PKC412 were investigated in peripheral blood lymphocytes (PBL) from 4 normal volunteers, B-cell isolates from 3 normal tonsils and 31 patients with B-cell chronic lymphocytic leukemia (B-CLL). Following incubation with PKC412 for 2 days, the viability of B-CLL cells was decreased relative to that of controls (63+/-23% at 1 micromole/L; 52+/-30% at 10 micromole/L; n=20). Normal PBL were significantly more resistant to the drug (91+/-5% viable cells at 1 micromole/L; 73+/-18% at 10 micromole/L; n=4). Thirteen of the B-CLL patients were treated with oral PKC412 in a phase II trial., Results: PKC activity in malignant cells from these patients showed a reduction post-treatment of 25-96% of their respective pre-treatment levels. Morphologic analysis, as well as in situ assay for DNA strand breaks (TUNEL assay) showed that B-CLL cells were killed by an apoptotic mechanism. In B-CLL cells the mean IC50, for PKC412, as measured by the reduction of 3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), was 2.1 micromol/L in 16 samples in which the IC50 were below the maximum concentration of PKC412 used for the assay. In tonsillar B-cells, the mean IC50 was 11 micromol/L whereas PBL cells were resistant. Four of eight and 1/3 B-CLL samples that were resistant to chlorambucil and fludarabine, respectively, were sensitive to PKC412. In 15/31 B-CLL samples a dose-dependent reversal of P-gp-mediated drug efflux by PKC412 was observed. A statistically significant correlation (p<0.001) was observed between P-gp protein expression as measured by FACScan analysis and the reversal of efflux activity by either PKC412 or verapamil. PKC412 increased the sensitivity of B-CLL cells to 2'-chlorodeoxyadenosine and chlorambucil., Interpretation and Conclusions: This study establishes the in vitro cytotoxic and multidrug resistance (MDR) modulatory properties of PKC412 towards malignant cells from B-CLL patients. The direct antitumor activity combined with the potential for P-gp modulation make PKC412 an attractive drug for the treatment of malignancies expressing the MDR phenotype, or in combination with conventional drugs.
- Published
- 2002
39. Modulation of nuclear receptor dependent transcription.
- Author
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Hart SM
- Subjects
- Humans, Ligands, Receptors, Cytoplasmic and Nuclear chemistry, Structure-Activity Relationship, Transcription, Genetic, Eukaryotic Cells physiology, Gene Expression Regulation physiology, Receptors, Cytoplasmic and Nuclear physiology, Transcription Factors physiology
- Abstract
Nuclear receptors comprise a family of transcription factors that regulate gene expression in a ligand dependent manner. They can activate or repress target genes by binding directly to DNA response elements as homo- or hetero-dimers or by binding to other classes of DNA-bound transcription factors. These activities have been linked to the formation of complexes with molecules that appear to serve as coactivators or corepressors, causing local modification of chromatin structure in order to regulate expression of their target genes. Several members of nuclear receptor family are directly associated with human malignancies including breast cancer, prostate cancer and leukaemia. The pathogenesis of each of these diseases is underpinned by the activities of a member of the superfamily; estrogen receptor-alpha (ER alpha) in breast cancer, androgen receptor (AR) in prostate cancer, and retinoic acid receptor alpha (RAR alpha) in acute promyelocytic leukaemia.
- Published
- 2002
- Full Text
- View/download PDF
40. Myonecrosis caused by Edwardsiella tarda: a case report and case series of extraintestinal E. tarda infections.
- Author
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Slaven EM, Lopez FA, Hart SM, and Sanders CV
- Subjects
- Adolescent, Adult, Female, Humans, Male, Middle Aged, Necrosis, Edwardsiella tarda isolation & purification, Enterobacteriaceae Infections microbiology, Muscles microbiology, Muscles pathology
- Abstract
Edwardsiella tarda is an unusual human pathogen. It is primarily associated with gastrointestinal disease, although recent reports of extraintestinal disease are broadening the current understanding of the clinical spectrum of E. tarda. A series of 11 cases of extraintestinal E. tarda infection is presented, including the first reported case of myonecrosis in an immunocompetent patient. Wound infections were the most common manifestation, and 3 of 5 patients with infected wounds had been exposed to a marine environment. One patient had bacteremia, and the remaining 5 patients developed abscesses that required surgical drainage. Four patients had E. tarda isolated in pure culture, including the patient with myonecrosis. Although it is often difficult to ascertain the contribution of E. tarda to infection when it is isolated as part of a mixed culture, this case series suggests that E. tarda is singularly capable of causing limb- and life-threatening infections.
- Published
- 2001
- Full Text
- View/download PDF
41. Influence of beta-blockers on mortality in chronic heart failure.
- Author
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Hart SM
- Subjects
- Bisoprolol therapeutic use, Carbazoles therapeutic use, Carvedilol, Chronic Disease, Heart Failure epidemiology, Heart Failure mortality, Humans, Metoprolol therapeutic use, Morbidity, Propanolamines therapeutic use, Randomized Controlled Trials as Topic, Treatment Outcome, Adrenergic beta-Antagonists therapeutic use, Heart Failure drug therapy
- Abstract
Objective: To briefly discuss the pathophysiology of heart failure and the rationale for the use of beta-blockers in the treatment of chronic heart failure. Key morbidity reduction trials are briefly mentioned, and recent mortality reduction trials are reviewed. General recommendations regarding the use of beta-blockers in heart failure are also included to guide clinical practice., Study Selection/data Extraction: Randomized, placebo-controlled clinical trials and meta-analyses evaluating mortality reduction with beta-blockers in the treatment of heart failure were identified using a MEDLINE search from January 1993 to March 2000. Abstracts and presented results from recent scientific meetings were also considered., Data Synthesis: Beta-blockers have been shown to decrease hospitalization for worsening heart failure, decrease the need for heart transplant, improve New York Heart Association (NYHA) functional class, and increase left-ventricular ejection fraction. A mortality benefit has recently been demonstrated for patients with chronic heart failure. Carvedilol, bisoprolol, and controlled-release/extended-release metoprolol decreased the risk of dying by 65%, 34%, and 34%, respectively, in patients with primarily NYHA functional class II or III and systolic dysfunction. The benefit of these agents in patients with class IV heart failure or determining whether one agent has an advantage over another is being investigated in ongoing clinical trials., Conclusions: Several beta-blockers have demonstrated mortality reduction in the treatment of patients with NYHA functional class II or III heart failure and systolic dysfunction. Beta-blockers should be considered in these patients when they are clinically stable and taking the current standard therapy of a diuretic plus an angiotensin-converting enzyme inhibitor or other vasodilator agent.
- Published
- 2000
- Full Text
- View/download PDF
42. Analysis of ETV6/AML1 abnormalities in acute lymphoblastic leukaemia: incidence, alternative spliced forms and minimal residual disease value.
- Author
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Codrington R, O'Connor HE, Jalali GR, Carrara P, Papaioannou M, Hart SM, Hoffbrand AV, Potter M, Prentice HG, Harrison CJ, and Foroni L
- Subjects
- Adolescent, Alternative Splicing, Amino Acid Sequence, Base Sequence, Child, Child, Preschool, Core Binding Factor Alpha 2 Subunit, Female, Humans, Incidence, Infant, Male, Molecular Sequence Data, Neoplasm, Residual genetics, Precursor B-Cell Lymphoblastic Leukemia-Lymphoma genetics, Proto-Oncogene Proteins c-ets, Reverse Transcriptase Polymerase Chain Reaction, ETS Translocation Variant 6 Protein, DNA-Binding Proteins genetics, Oncogene Proteins, Fusion genetics, Precursor Cell Lymphoblastic Leukemia-Lymphoma genetics, Proto-Oncogene Proteins, Repressor Proteins, Transcription Factors genetics
- Abstract
The t(12;21)(p13;q22) translocation, resulting in the fusion of the ETV6 and AML1 genes, occurs in 20-25% of paediatric B-lineage acute lymphoblastic leukaemias (ALL). The identification of the fusion product has important prognostic and therapeutic implications as the translocation has been associated with a favourable clinical outcome. The aim of this study was threefold: (i) to assess the frequency and clinical association of the fusion gene in patients with and without a cytogenetically detectable chromosome 12 and/or 21 abnormality or failed cytogenetic results, (ii) to characterize alternative forms of ETV6/AML1 transcripts, and (iii) to use ETV6/AML1 as a molecular marker for the investigation of minimal residual disease (MRD). ETV6/AML1 fusion was detected in 22 (39%) of 56 cases studied by reverse transcriptase polymerase chain reaction (RT-PCR). ETV6/AML1 fusion was found in nine out of 16 (56%) cases with a cytogenetically visible chromosome 12 abnormality, but also in nine out of 29 patients (31%) without a chromosome 12 abnormality or patients with failed cytogenetics (four out of 11 patients, 36%), making this the most common cytogenetic abnormality in childhood ALL. Alternatively spliced ETV6/AML1 forms were investigated in 14 of the positive patients. Exon 5 of ETV6 was fused in frame to all AML1 exons, except exon 4. Fusion to exon 6 of AML1 resulted in one amino acid change. The presence of ETV6/AML1 was associated with a lower white blood cell count (Student's t-test; P = 0.009) and common (c)ALL phenotype (chi(2) test; P > 0.001), but no better disease-free survival. Our study shows that (i) RT-PCR is the most effective approach for the detection of t(12;21) in childhood ALL, (ii) the association of ETV6/AML1 and chromosome 12 and/or 21, seen in 56% of our cases, further confirms existing data, (iii) overall survival of patients with t(12;21) was not better than other cytogenetics groups, and (d) MRD analysis using ETV6/AML1 fusion is specific, but not sensitive enough to avoid false negative results.
- Published
- 2000
- Full Text
- View/download PDF
43. Biochemical markers of bone turnover.
- Author
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Hart SM and Eastell R
- Subjects
- Alkaline Phosphatase blood, Animals, Biomarkers, Bone and Bones physiology, Circadian Rhythm, Humans, Kidney Failure, Chronic metabolism, Osteitis Deformans metabolism, Osteocalcin analysis, Seasons, Bone and Bones metabolism, Osteoporosis metabolism
- Abstract
Biochemical markers of bone turnover that are specific to bone allow study of the subtle changes in bone turnover associated with osteoporosis. They have been evaluated in Paget's disease of the bone and chronic renal failure. This review focuses on the use of biochemical markers of bone turnover in osteoporosis. The data in this review period are numerous and varied due to the growing interest in the use of biochemical markers of bone turnover in clinical practice. The data provide support for the use of the newer bone turnover markers for monitoring treatment of osteoporosis, if care is taken to minimize sources of variability.
- Published
- 1999
- Full Text
- View/download PDF
44. Economic evaluation of three methods of treating urogenital chlamydial infections in the emergency department.
- Author
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Petitta A, Hart SM, and Bailey EM
- Subjects
- Chlamydia Infections economics, Computer Simulation, Data Collection, Emergency Service, Hospital, Humans, Patient Compliance, Azithromycin administration & dosage, Chlamydia Infections drug therapy, Chlamydia trachomatis, Doxycycline administration & dosage, Female Urogenital Diseases drug therapy, Male Urogenital Diseases
- Abstract
We attempted to determine the economic impact of three alternatives for the treatment of chlamydial infections in the emergency department: a written prescription for 7 days of doxycycline therapy (D-RX); a prepacked 7-day supply of doxycycline (D-ED); or a single 1-g dose of azithromycin (AZI). Data inputs for the model were obtained from both patient experience and literature sources. Primary health outcomes of the model were number of infection relapses. Economic outcomes were costs for initial treatment, treatment of relapses, and treatment of complications of relapse. For every 1000 patients, D-ED and AZI resulted in 21.6 (-10 to -41) and 36.2 (-25 to -63) fewer relapses than D-RX, respectively; AZI resulted in 14.6 (-35 to -4) fewer relapses than D-ED. Total costs were decreased for D-ED and AZI versus D-RX by $18,879 (-$39,000 to -$8000) and $24,039 (-$59,000 to -$10,000), respectively, and AZI resulted in a total cost decrease of $5160 (-$35,000 to +$6000) versus D-ED. Both D-ER and AZI decreased infection relapses and overall health care costs compared with D-RX. Also, AZI resulted in additional decreases in relapses versus D-ED, although the incremental impact on cost was inconclusive.
- Published
- 1999
- Full Text
- View/download PDF
45. Herbimycin A accelerates the induction of apoptosis following etoposide treatment or gamma-irradiation of bcr/abl-positive leukaemia cells.
- Author
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Riordan FA, Bravery CA, Mengubas K, Ray N, Borthwick NJ, Akbar AN, Hart SM, Hoffbrand AV, Mehta AB, and Wickremasinghe RG
- Subjects
- Antibiotics, Antineoplastic pharmacology, Antineoplastic Agents, Phytogenic pharmacology, Apoptosis genetics, Benzoquinones, Fusion Proteins, bcr-abl drug effects, Fusion Proteins, bcr-abl radiation effects, HL-60 Cells, Humans, Lactams, Macrocyclic, Leukemia, Erythroblastic, Acute genetics, Leukemia, Myelogenous, Chronic, BCR-ABL Positive drug therapy, Leukemia, Myelogenous, Chronic, BCR-ABL Positive pathology, Proto-Oncogene Proteins c-bcl-2 biosynthesis, Proto-Oncogene Proteins c-bcl-2 drug effects, Rifabutin analogs & derivatives, Tumor Cells, Cultured, bcl-X Protein, Apoptosis drug effects, Apoptosis radiation effects, Etoposide pharmacology, Fusion Proteins, bcr-abl analysis, Gamma Rays, Leukemia, Erythroblastic, Acute drug therapy, Leukemia, Erythroblastic, Acute pathology, Quinones pharmacology
- Abstract
Philadelphia chromosome (Ph)-positive leukaemia cells express the chimeric bcr/abl oncoprotein, whose deregulated protein tyrosine kinase (PTK) activity antagonizes the induction of apoptosis by DNA damaging agents. Treatment of Ph-positive K562, TOM 1 and KCL-22 cells with etoposide for 2d induced cytosolic vacuolation, but not nuclear condensation or DNA fragmentation. The bcr/abl kinase-selective inhibitor herbimycin A increased the induction of nuclear apoptosis by etoposide or gamma-radiation. The concentration of herbimycin required to synergize with etoposide was similar to that required to decrease the level of tyrosine phosphorylated proteins or of the protein tyrosine kinase activity of anti-abl immune complexes in K562 cells. The ability of herbimycin A to sensitize K562, TOM 1 or KCL-22 cells to apoptosis induction correlated with its ability to decrease the cellular content of phosphotyrosyl proteins in these Philadelphia-positive lines. Enhancement of nuclear apoptosis by herbimycin was not attributable to downregulation of the bcl-2 or bcl-XL anti-apoptotic proteins. In contrast, herbimycin protected Philadelphia-negative HL60 cells from apoptosis induction by etoposide and did not affect killing of NC37 and CEM cells. The data suggest that the induction of apoptosis is blocked in cells expressing the bcr/abl oncoprotein and that herbimycin A increases induction of programmed cell death following DNA damage. Selective PTK inhibitors may therefore be of value in securing the genetic death of Ph-positive leukaemia cells.
- Published
- 1998
- Full Text
- View/download PDF
46. Expression of the human major vault protein LRP in acute myeloid leukemia.
- Author
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Hart SM, Ganeshaguru K, Scheper RJ, Prentice HG, Hoffbrand AV, and Mehta AB
- Subjects
- ATP Binding Cassette Transporter, Subfamily B, Member 1 genetics, Acute Disease, Adult, Antineoplastic Agents pharmacology, Child, Gene Expression Regulation, Neoplastic drug effects, Humans, Leukocytes, Mononuclear metabolism, Multidrug Resistance-Associated Proteins, RNA, Messenger genetics, RNA, Neoplasm genetics, Tumor Cells, Cultured, ATP-Binding Cassette Transporters genetics, Drug Resistance, Multiple, Leukemia, Myeloid genetics, Neoplasm Proteins genetics, Vault Ribonucleoprotein Particles genetics
- Abstract
Overexpression of a 110-kD protein (lung resistance-related protein [LRP]) may predict a poor response to chemotherapy in patients with acute myeloid leukemia (AML) and ovarian carcinoma. The LRP gene has recently been mapped to chromosome 16, close to the multidrug resistance-associated protein (MRP) gene. Seventy-seven samples from 67 patients with AML were examined for expression of LRP, MRP, and multidrug resistance (MDR1) mRNA using a semiquantitative reverse transcription polymerase chain reaction (RT-PCR) assay. Results were compared with 29 normal samples (11 normal peripheral blood and 18 normal bone marrow). Thirty-three patients with untreated AML were evaluable for response to chemotherapy. Levels of LRP, but not of MRP or MDR1 mRNA, were significantly higher in eight patients who failed to achieve complete remission (CR) compared with 25 patients who achieved CR (p = 0.033). A positive correlation was demonstrated between LRP and MRP (R = 0.368, p = 0.001) and between MRP and MDR1 mRNA levels (R = 0.301, p = 0.01) in the 77 clinical samples analyzed. In AML samples, a significant difference in MDR1 mRNA levels was found between presentation (47 samples) and relapse (30 samples) (p = 0.031). No significant difference was seen in LRP mRNA levels between these two groups or in eight patients studied sequentially at both presentation and relapse. Thirteen samples (10 at presentation, 3 at relapse) were analyzed for LRP protein expression by flow cytometry. Eight (5 at presentation, 3 at relapse) displayed greater than 10% positive cells (range 15-86%). These data suggest that LRP gene overexpression may constitute a novel mechanism of multidrug resistance.
- Published
- 1997
47. Clinical and clinicopathological assessment of serial phlebotomy in the Sprague Dawley rat.
- Author
-
Scipioni RL, Diters RW, Myers WR, and Hart SM
- Subjects
- Animals, Body Weight, Cross-Over Studies, Erythrocyte Count veterinary, Erythrocyte Indices veterinary, Hematocrit veterinary, Hemoglobins metabolism, Leukocyte Count veterinary, Lymphocyte Count veterinary, Male, Neutrophils cytology, Organ Size, Phlebotomy adverse effects, Platelet Count veterinary, Rats, Reticulocyte Count veterinary, Survival Analysis, Phlebotomy veterinary, Rats, Sprague-Dawley blood
- Abstract
Two studies, designed to mimic a single-dose, cross-over pharmacokinetic protocol, were conducted to gain a better understanding of the rat's response to multiple, frequent blood sampling. Parameters evaluated included body weight, clinical signs of disease, hematologic and serum biochemical analytes, organ weights, and histopathologic features. Study groups consisted of either 6 or 8 male, viral antibody-free, Sprague Dawley rats. These included controls and blood-collection groups that represented withdrawal of 10, 15, 20, 30, and 40% of estimated total blood volume. Volume of blood collected per time point was based on the total volume to be withdrawn divided by the 13 samples that were collected over 24 h. This regimen was repeated 2 weeks later. Samples were taken for clinical pathologic evaluation on the days subsequent to blood collection for both studies as follows: 0, 1, 2, and 3 days; 7, 8, or 9 days; and either 13 or 14 days. In Study 1, samples were also taken on either days 15 or 16, and on 17 or 18 after the second blood collection. Approximately 2 weeks after the second blood collection regimen, animals were euthanized. Animals in one study were necropsied, and selected tissues were taken for histologic examination. Analysis of variance, based on changes from baseline, was used to assess group differences. Results indicate that the rate of body-weight gain for the bled groups was not significantly different from that of the controls. Group differences in multiple hematologic parameters were significant. Changes were typical of acute blood-loss anemia, with positive or negative trends relating to the volume of blood removed. In addition, these changes were characterized by recovery to control values within approximately 14 days. Few statistically significant group differences were detected in serum biochemical values, and those detected were not biologically relevant. Although organ weights of bled groups were similar to those of controls, minimal to mild splenic hematopoiesis was present in all bled groups, compared with controls. These data indicate that removal of up to 40% of a rat's total blood volume over a 24-h period, and repeated 2 weeks later, caused no gross ill effects.
- Published
- 1997
48. Role of double-stranded RNA-activated protein kinase in human hematological malignancies.
- Author
-
Basu S, Panayiotidis P, Hart SM, He LZ, Man A, Hoffbrand AV, and Ganeshaguru K
- Subjects
- Gene Expression Regulation, Enzymologic, Humans, Leukemia genetics, Leukocytes, Mononuclear enzymology, Phosphorylation, RNA, Double-Stranded metabolism, RNA, Messenger genetics, eIF-2 Kinase, Leukemia enzymology, Protein Serine-Threonine Kinases physiology
- Abstract
The double-stranded RNA (dsRNA)-activated protein kinase (PKR) is one of many genes induced by IFN. The PKR sequentially undergoes autophosphorylation and activation on binding to dsRNA. Previous studies have shown that PKR may be an important factor in the regulation of viral and cellular protein synthesis. Recent studies suggest that PKR may function as a tumor suppressor gene. The role of PKR in various human leukemic cells was therefore investigated. PKR mRNA levels by reverse transcription-PCR, protein expression by Western blot and FACScan analysis, and activity by phosphorylation status were studied. The expression of a known inhibitor of PKR, p58, was also investigated at mRNA and protein levels. A total of 24 samples from normal mononuclear cells (MNCs), 26 samples of acute lymphoblastic leukemia, 26 samples of acute myelogenous leukemia, 32 samples of chronic lymphocytic leukemia, and 5 samples of hairy cell leukemia was investigated. Mean mRNA levels were increased in acute lymphoblastic leukemia and acute myelogenous leukemia and decreased in chronic lymphocytic leukemia compared to normal MNCs. The mRNA levels in hairy cell leukemia were similar to those of normal MNCs. PKR protein was detectable in normal MNCs and leukemic cell extracts, and on FACScan analysis, more than 70% of cells stained positive for PKR. PKR activity was detectable in all samples investigated and was enhanced 4-23-fold in the presence of the synthetic dsRNA, poly(I) x poly(C). Protein expression of a known PKR inhibitor, p58, was barely detectable in normal MNCs and leukemic cells, with high expression in the HeLa cell line. These findings provide no evidence to support the hypothesis that PKR acts as a tumor suppressor in human leukemic cells.
- Published
- 1997
49. A practical look at the clinical usefulness of the beta-lactam/beta-lactamase inhibitor combinations.
- Author
-
Hart SM and Bailey EM
- Subjects
- Cross Infection drug therapy, Drug Therapy, Combination, Humans, beta-Lactams, Anti-Bacterial Agents therapeutic use, Enzyme Inhibitors therapeutic use, beta-Lactamase Inhibitors
- Abstract
Objective: To aid clinicians in developing an approach to the use of intravenous beta-lactam/beta-lactamase inhibitors on a patient-specific basis. To achieve this, the pharmacology, in vitro activity, and clinical use of the intravenous beta-lactam/beta-lactamase inhibitor combinations in the treatment of selected infections seen in hospitalized patients are discussed., Data Identification: An English-language literature search using MEDLINE (1987-1995); Index Medicus (1987-1995); program and abstracts of the 32nd (1992), 33rd (1993), 34th (1994), and 35th (1995) Interscience Conference on Antimicrobial Agents and Chemotherapy; bibliographic reviews of review articles; and package inserts., Study Selection: In vitro and in vivo studies on the pharmacokinetics, microbiology, pharmacology, and clinical effectiveness of ampicillin/sulbactam, ticarcillin/clavulanate, and piperacillin/tazobactam were evaluated., Data Synthesis: Many properties of the beta-lactam/beta-lactamase inhibitor combinations are similar. Differences in dosing, susceptibilities, and clinical applications are important considerations for clinicians. Potential roles for these agents in the clinical setting include pneumonia, intraabdominal infections, and soft tissue infections. A short discussion on susceptibility data interpretation is also presented., Conclusions: There are important differences among the available beta-lactam/beta-lactamase inhibitor combinations, such as spectra of activity, which need to be considered in choosing an agent for a patient-specific case. These products can be useful alternatives to conventional two- to three-drug regimens in mixed infections such as foot infections in patients with diabetes and hospital-acquired intraabdominal infections.
- Published
- 1996
- Full Text
- View/download PDF
50. Expression of the multidrug resistance-associated protein (MRP) in acute leukaemia.
- Author
-
Hart SM, Ganeshaguru K, Hoffbrand AV, Prentice HG, and Mehta AB
- Subjects
- ATP Binding Cassette Transporter, Subfamily B, Member 1 genetics, ATP-Binding Cassette Transporters metabolism, Acute Disease, Gene Expression, Humans, Leukemia drug therapy, Leukemia metabolism, Leukemia, Myeloid, Acute genetics, Leukemia, Myeloid, Acute metabolism, Multidrug Resistance-Associated Proteins, Polymerase Chain Reaction, Precursor Cell Lymphoblastic Leukemia-Lymphoma genetics, Precursor Cell Lymphoblastic Leukemia-Lymphoma metabolism, Prognosis, RNA, Messenger metabolism, Recurrence, Remission Induction, Transcription, Genetic, ATP-Binding Cassette Transporters genetics, Drug Resistance, Multiple genetics, Leukemia genetics
- Abstract
A semi-quantitative reverse transcription polymerase chain reaction (RT-PCR) was used to investigate and compare transcription levels of the human multidrug resistance gene (MDR1) and the recently described multidrug resistance-associated protein (MRP) in 105 samples from patients with acute leukaemia at presentation and relapse. MRP gene expression was significantly greater in samples from patients with acute lymphoblastic leukaemia (ALL) compared with samples from normal peripheral mononuclear cells (PBMC) and patients with de novo acute myeloid leukaemia (AML). MRP gene expression was found to be higher in patients with relapsed de novo AML compared to those at presentation but prior therapy did not affect MRP gene expression in ALL. MDR1 gene expression was significantly lower in ALL patients compared to normal PBMC and AML samples. Samples from patients with secondary AML had higher levels of MDR1 expression than those of de novo AML. No changes of MDR1 expression were observed in AML or ALL at relapse. No correlation was observed between MDR1 and MRP gene expression in this group of patients. Our results suggest that MRP expression may be of prognostic importance in AML but the significance of the increased levels we have detected remain unclear.
- Published
- 1994
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