7 results on '"J.L. Duke"'
Search Results
2. Purification and characterization of active and latent forms of recombinant plasminogen activator inhibitor 1 produced in Escherichia coli
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Kamerkar Sm, Seetharam R, Walton Hl, J.L. Duke, Huckins Nr, Corman Ji, Wilk Rr, Woodeshick Rw, Hayman Ac, and Anil M. Dwivedi
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Circular dichroism ,Molecular Sequence Data ,Gene Expression ,Serpin ,Guanidines ,Biochemistry ,Protein Structure, Secondary ,chemistry.chemical_compound ,Drug Stability ,Affinity chromatography ,Plasminogen Activator Inhibitor 1 ,Escherichia coli ,Trypsin ,Amino Acid Sequence ,Vitronectin ,Guanidine ,Chromatography, High Pressure Liquid ,Glycoproteins ,chemistry.chemical_classification ,Chromatography ,biology ,Circular Dichroism ,Molecular biology ,Peptide Fragments ,Recombinant Proteins ,Amino acid ,Spectrometry, Fluorescence ,chemistry ,Plasminogen activator inhibitor-1 ,biology.protein ,Plasminogen activator - Abstract
Plasminogen activator inhibitor 1 (PAI-1), the principal physiological inhibitor of tissue plasminogen activator (tPA), is a protein of 379 amino acids and belongs to the SERPIN family of serine protease inhibitors. We have previously described methods to express [Sisk et al. (1990) Gene 96, 305-309] and purify [Reilly et al. (1990) J. Biol. Chem. 265, 9570-9574] a highly active form of the protein in substantial amounts, from Escherichia coli. Further analyses of this material showed the presence of small but significant amounts of latent rPAI-1. The present paper describes for the first time purification of latent and active forms of rPAI-1 from a single preparation, as well as the functional and structural characteristics of the two forms. Latent rPAI-1, which has properties similar to the latent forms described by other groups, was separated from active rPAI-1 by high-resolution ion-exchange chromatography or by affinity chromatography using immobilized anhydrotrypsin. It had low intrinsic activity (< 5% of active rPAI-1) and was partially reactivated by guanidine hydrochloride treatment or by incubation with vitronectin. Conversion of the active rPAI-1 to the latent form was influenced by temperature and additives including sucrose, EDTA, and arginine. Active and latent rPAI-1 did not show any obvious differences in their primary structures and displayed remarkably similar secondary structures as determined by circular dichroism spectral analyses. However, they did exhibit differences in tryptophan fluorescence, suggesting tertiary structural differences between the two forms.
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- 1992
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3. Vitronectin effects on recombinant plasminogen activator inhibitor-1: structural and functional analysis
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Thomas M. Reilly, Anil M. Dwivedi, J.L. Duke, Shaker A. Mousa, Harry L. Walton, R.M. Knabb, D.L. Wade, and Mark S. Forsythe
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biology ,Binding protein ,Tryptophan ,Hematology ,Fluorescence spectroscopy ,law.invention ,chemistry.chemical_compound ,Biochemistry ,chemistry ,law ,Plasminogen activator inhibitor-1 ,Recombinant DNA ,biology.protein ,Vitronectin ,Plasminogen activator ,Incubation - Abstract
Functionally active recombinant plasminogen activator inhibitor-1 (rPAI-1) has been purified from bacterial cells in the absence of any discrete binding protein. In the present study, vitronectin, which is known to stabilise the natural form of PAI-1, was evaluated for its effects on the activity and structure of rPAI-1. As assessed by PAI-1 activity assays, purified human vitronectin was shown to stabilise rPAI-1, by doubling its half-life at 25° and 37°C, and to enhance the activity of rPAI-1 in concentration-dependent fashion. Vitronectin also restored partial activity to a latent form of rPAI-1 prepared by a 72h incubation at 37°C. Fluorescence spectroscopy studies revealed that rPAI-1 in association with vitronectin displayed a higher tryptophan emission signal than the sum of the emissions of the individual components. These results suggest that vitronectin effects on rPAI-1 activity may result from specific conformational effects induced upon binding of the two proteins.
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- 1992
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4. Purification and characterization of recombinant plasminogen activator inhibitor-1 from Escherichia coli
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Gary L. Davis, D Kingsley, Susan K. Pierce, William Perry Sisk, R Seetharam, H L Walton, Thomas M. Reilly, and J.L. Duke
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Urokinase ,Gel electrophoresis ,Chromatography ,Chemistry ,Ion chromatography ,Cell Biology ,Biochemistry ,Tissue plasminogen activator ,High-performance liquid chromatography ,chemistry.chemical_compound ,Plasminogen activator inhibitor-1 ,medicine ,Sodium dodecyl sulfate ,Molecular Biology ,Plasminogen activator ,medicine.drug - Abstract
A recombinant form of plasminogen activator inhibitor-1 (rPAI-1) has been purified from lysates of pCE1200, a bacterial expression vector containing the full length PAI-1 gene, by utilizing sequential anion exchange and cation exchange chromatography on Q-Sepharose and S-Sepharose columns. Approximately 140 mg of rPAI-1, estimated at 98% purity on the basis of analytical high performance liquid chromatography, could be obtained from 200 g wet weight of cells. The purified protein exhibited a single Coomassie Blue-stainable band at the region of Mr = 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an NH2-terminal amino acid sequence consistent with the expected translation product of the pCE1200 PAI-1 insert. The rPAI-1 rapidly inhibited single- and two-chain tissue plasminogen activators, as well as urokinase, with apparent second order rate constants in the range of 2-5 x 10(7) M-1 s-1. A specific activity measurement of 250,000 units/mg was calculated for the rPAI-1 based on its ability to inhibit the enzymatic activity of a single-chain tissue plasminogen activator. Stability studies showed that the activity of the rPAI-1 was very stable when stored at temperatures of 25 degrees C or lower, but decayed within hours when stored at 37 degrees C. Sodium dodecyl sulfate treatment, which partially activates the latent form of natural PAI-1, inactivated rPAI-1. These results show that the purified rPAI-1 produced from pCE1200 displays many of the properties associated with the biologically active form of natural PAI-1.
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- 1990
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5. Computer automated electrooculography
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J.L. Duke, John R. Bourne, and P.R. Hudak
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genetic structures ,Computer science ,Medicine (miscellaneous) ,Adaptation (eye) ,Dark Adaptation ,Minicomputer ,law.invention ,law ,Oscillometry ,medicine ,Electroretinography ,Humans ,Computer vision ,Diagnosis, Computer-Assisted ,Simulation ,Autoanalysis ,medicine.diagnostic_test ,business.industry ,Adaptation, Ocular ,Computers ,Track (disk drive) ,Electrooculography ,Horizontal plane ,Automation ,Visual field ,Evaluation Studies as Topic ,Artificial intelligence ,Visual angle ,business - Abstract
Clinical electrooculography requires a patient to track two points in the visual field separated by at least 30° visual angle in a horizontal plane while recording the concomitant change in potential from electrodes placed at the external canthi of the two eyes. This test is relatively lengthy, requiring long periods of light and dark adaptation. A minicomputer program has been written to automate the test and to analyze and reduce the data. Time required to administer the test, as well as time needed to analyze the data, has been significantly reduced by computer automation.
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- 1972
6. Cognitive Effects of Body Temperature During Hypothermic Circulatory Arrest Trial (GOT ICE): A Randomized Clinical Trial Comparing Outcomes After Aortic Arch Surgery.
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Hughes GC, Chen EP, Browndyke JN, Szeto WY, DiMaio JM, Brinkman WT, Gaca JG, Blumenthal JA, Karhausen JA, Bisanar T, James ML, Yanez D, Li YJ, and Mathew JP
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- Humans, Retrospective Studies, Prospective Studies, Quality of Life, Single-Blind Method, Body Temperature, Circulatory Arrest, Deep Hypothermia Induced adverse effects, Perfusion adverse effects, Perfusion methods, Cognition, Cerebrovascular Circulation, Treatment Outcome, Aorta, Thoracic diagnostic imaging, Aorta, Thoracic surgery, Hypothermia
- Abstract
Background: Deep hypothermia has been the standard for hypothermic circulatory arrest (HCA) during aortic arch surgery. However, centers worldwide have shifted toward lesser hypothermia with antegrade cerebral perfusion. This has been supported by retrospective data, but there has yet to be a multicenter, prospective randomized study comparing deep versus moderate hypothermia during HCA., Methods: This was a randomized single-blind trial (GOT ICE [Cognitive Effects of Body Temperature During Hypothermic Circulatory Arrest]) of patients undergoing arch surgery with HCA plus antegrade cerebral perfusion at 4 US referral aortic centers (August 2016-December 2021). Patients were randomized to 1 of 3 hypothermia groups: DP, deep (≤20.0 °C); LM, low-moderate (20.1-24.0 °C); and HM, high-moderate (24.1-28.0 °C). The primary outcome was composite global cognitive change score between baseline and 4 weeks postoperatively. Analysis followed the intention-to-treat principle to evaluate if: (1) LM noninferior to DP on global cognitive change score; (2) DP superior to HM. The secondary outcomes were domain-specific cognitive change scores, neuroimaging findings, quality of life, and adverse events., Results: A total of 308 patients consented; 282 met inclusion and were randomized. A total of 273 completed surgery, and 251 completed the 4-week follow-up (DP, 85 [34%]; LM, 80 [34%]; HM, 86 [34%]). Mean global cognitive change score from baseline to 4 weeks in the LM group was noninferior to the DP group; likewise, no significant difference was observed between DP and HM. Noninferiority of LM versus DP, and lack of difference between DP and HM, remained for domain-specific cognitive change scores, except structured verbal memory, with noninferiority of LM versus DP not established and structured verbal memory better preserved in DP versus HM ( P = 0.036). There were no significant differences in structural or functional magnetic resonance imaging brain imaging between groups postoperatively. Regardless of temperature, patients who underwent HCA demonstrated significant reductions in cerebral gray matter volume, cortical thickness, and regional brain functional connectivity. Thirty-day in-hospital mortality, major morbidity, and quality of life were not different between groups., Conclusions: This randomized multicenter study evaluating arch surgery HCA temperature strategies found low-moderate hypothermia noninferior to traditional deep hypothermia on global cognitive change 4 weeks after surgery, although in secondary analysis, structured verbal memory was better preserved in the deep group. The verbal memory differences in the low- and high-moderate groups and structural and functional connectivity reductions from baseline merit further investigation and suggest opportunities to further optimize brain perfusion during HCA., Registration: URL: https://www.clinicaltrials.gov; Unique identifier: NCT02834065., Competing Interests: Disclosures None.
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- 2024
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7. Impact of US hospital center and interhospital transfer on spinal cord injury management: An analysis of the National Trauma Data Bank.
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Williamson T, Hodges S, Yang LZ, Lee HJ, Gabr M, Ugiliweneza B, Boakye M, Shaffrey CI, Goodwin CR, Karikari IO, Lad S, and Abd-El-Barr M
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- Adult, Aged, Databases, Factual statistics & numerical data, Female, Humans, Male, Middle Aged, Patient Admission statistics & numerical data, Retrospective Studies, Time-to-Treatment statistics & numerical data, Treatment Outcome, United States, Conservative Treatment statistics & numerical data, Neurosurgical Procedures statistics & numerical data, Patient Transfer statistics & numerical data, Spinal Cord Injuries therapy, Trauma Centers statistics & numerical data
- Abstract
Background: Traumatic spinal cord injury (SCI) is a serious public health problem. Outcomes are determined by severity of immediate injury, mitigation of secondary downstream effects, and rehabilitation. This study aimed to understand how the center type a patient presents to and whether they are transferred influence management and outcome., Methods: The National Trauma Data Bank was used to identify patients with SCI. The primary objective was to determine association between center type, transfer, and surgical intervention. A secondary objective was to determine association between center type, transfer, and surgical timing. Multivariable logistic regression models were fit on surgical intervention and timing of the surgery as binary variables, adjusting for relevant clinical and demographic variables., Results: There were 11,744 incidents of SCI identified. A total of 2,883 patients were transferred to a Level I center and 4,766 presented directly to a level I center. Level I center refers to level I trauma center. Those who were admitted directly to level I centers had a higher odd of receiving a surgery (odds ratio, 1.703; 95% confidence interval, 1.47-1.97; p < 0.001), but there was no significant difference in terms of timing of surgery. Patients transferred into a level I center were also more likely to undergo surgery than those at a level II/III/IV center, although this was not significant (odds ratio, 1.213; 95% confidence interval, 0.099-1.48; p = 0.059)., Conclusion: Patients with traumatic SCI admitted to level I trauma centers were more likely to have surgery, particularly if they were directly admitted to a level I center. This study provides insights into a large US sample and sheds light on opportunities for improving pre hospital care pathways for patients with traumatic SCI, to provide the timely and appropriate care and achieve the best possible outcomes., Level of Evidence: Care management, Level IV., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)
- Published
- 2021
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