Pub Date : 2001-05-01DOI: 10.1177/153331750101600308
H A Shanks-McElroy, J Strobino
Caregiving for persons with Alzheimer's disease (AD) has been shown to pose a challenge to the health of the spousal caregiver. Because most of the caregiving literature focuses on the female caregiver, there is some question about the generalizability of such literature to the male caregiver. This report focuses on male caregivers of spouses with AD and represents a subsample from a larger descriptive study that examined the relationship between risk factors and the health status of spousal caregivers. Twenty-nine male caregivers affiliated with Alzheimer's organizations in Pennsylvania and Ontario, Canada, returned mail surveys. On average, physical health symptoms increased by one-third when comparing pre- and post-caregiving data. Caregivers also were experiencing moderate to severe depression and burden. Male caregivers generally rated their physical health as fair to excellent and exhibited fewer than expected physical health symptoms. Caregiver health was related to perceptions of stress surrounding the provision of activities of daily living (ADL) assistance, the frequency of behavioral problems, perceptions of stress associated with the AD spouse's dysfunctional behaviors, and satisfaction with leisure opportunities. The identification of the role that caregiver perceptions of stressfulness associated with caregiving and the need for leisure satisfaction offer important implications for community-based education and respite services to maintain health status for spousal caregivers.
{"title":"Male caregivers of spouses with Alzheimer's disease: risk factors and health status.","authors":"H A Shanks-McElroy, J Strobino","doi":"10.1177/153331750101600308","DOIUrl":"10.1177/153331750101600308","url":null,"abstract":"<p><p>Caregiving for persons with Alzheimer's disease (AD) has been shown to pose a challenge to the health of the spousal caregiver. Because most of the caregiving literature focuses on the female caregiver, there is some question about the generalizability of such literature to the male caregiver. This report focuses on male caregivers of spouses with AD and represents a subsample from a larger descriptive study that examined the relationship between risk factors and the health status of spousal caregivers. Twenty-nine male caregivers affiliated with Alzheimer's organizations in Pennsylvania and Ontario, Canada, returned mail surveys. On average, physical health symptoms increased by one-third when comparing pre- and post-caregiving data. Caregivers also were experiencing moderate to severe depression and burden. Male caregivers generally rated their physical health as fair to excellent and exhibited fewer than expected physical health symptoms. Caregiver health was related to perceptions of stress surrounding the provision of activities of daily living (ADL) assistance, the frequency of behavioral problems, perceptions of stress associated with the AD spouse's dysfunctional behaviors, and satisfaction with leisure opportunities. The identification of the role that caregiver perceptions of stressfulness associated with caregiving and the need for leisure satisfaction offer important implications for community-based education and respite services to maintain health status for spousal caregivers.</p>","PeriodicalId":15087,"journal":{"name":"Journal of Biomolecular Screening","volume":"12 1","pages":"167-75"},"PeriodicalIF":0.0,"publicationDate":"2001-05-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10833828/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"87384563","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1089/108705701750160282
K. Subbaramaiah, P. Bulic, Y. Lin, A. Dannenberg, D. Pasco
Cyclooxygenase-2 (COX-2) is a recognized target for cancer prevention and possibly treatment. To identify novel inhibitors of COX-2, we developed a high throughput reporter gene assay that utilizes a region of the human COX-2 promoter to drive luciferase expression. A total of 968 extracts from 266 plants were screened. Extracts from 12 plants (4.5%), including Arnebia euchroma, a medicinal plant used in the Far East to treat inflammation, inhibited the stimulation of COX-2 promoter activity. The gene promoter assay then was used to identify shikonin, a compound with known anti-inflammatory and chemopreventive properties, as an active compound in A. euchroma. To complement the gene promoter studies, we determined the effects of a mixture of shikonins on phorbol 12-myristate 13-acetate (PMA)-mediated induction of COX-2 in transformed human mammary epithelial cells. Shikonins inhibited PMA-mediated induction of COX-2 mRNA, protein, and prostaglandin E(2) synthesis. In transient transfections, PMA caused a severalfold increase in COX-2 promoter activity, an effect that was suppressed by shikonins. Shikonins also inhibited PMA-mediated stimulation of extracellular signal-regulated kinase1/2 mitogen-activated protein kinases and activator protein-1 activity. Collectively, these results demonstrate the successful development and use of a high throughput reporter gene assay for the identification of a novel inhibitor of COX-2 expression.
{"title":"Development and use of a gene promoter-based screen to identify novel inhibitors of cyclooxygenase-2 transcription.","authors":"K. Subbaramaiah, P. Bulic, Y. Lin, A. Dannenberg, D. Pasco","doi":"10.1089/108705701750160282","DOIUrl":"https://doi.org/10.1089/108705701750160282","url":null,"abstract":"Cyclooxygenase-2 (COX-2) is a recognized target for cancer prevention and possibly treatment. To identify novel inhibitors of COX-2, we developed a high throughput reporter gene assay that utilizes a region of the human COX-2 promoter to drive luciferase expression. A total of 968 extracts from 266 plants were screened. Extracts from 12 plants (4.5%), including Arnebia euchroma, a medicinal plant used in the Far East to treat inflammation, inhibited the stimulation of COX-2 promoter activity. The gene promoter assay then was used to identify shikonin, a compound with known anti-inflammatory and chemopreventive properties, as an active compound in A. euchroma. To complement the gene promoter studies, we determined the effects of a mixture of shikonins on phorbol 12-myristate 13-acetate (PMA)-mediated induction of COX-2 in transformed human mammary epithelial cells. Shikonins inhibited PMA-mediated induction of COX-2 mRNA, protein, and prostaglandin E(2) synthesis. In transient transfections, PMA caused a severalfold increase in COX-2 promoter activity, an effect that was suppressed by shikonins. Shikonins also inhibited PMA-mediated stimulation of extracellular signal-regulated kinase1/2 mitogen-activated protein kinases and activator protein-1 activity. Collectively, these results demonstrate the successful development and use of a high throughput reporter gene assay for the identification of a novel inhibitor of COX-2 expression.","PeriodicalId":15087,"journal":{"name":"Journal of Biomolecular Screening","volume":"6 2 1","pages":"101-10"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/108705701750160282","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60620946","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 2001-01-01DOI: 10.1089/108705701753364922
M. Pantoliano, E. Petrella, Joseph D. Kwasnoski, V. S. Lobanov, J. Myslik, Edward Graf, T. Carver, E. Asel, B. Springer, Pamela Lane, F. Salemme
More general and universally applicable drug discovery assay technologies are needed in order to keep pace with the recent advances in combinatorial chemistry and genomics-based target generation. Ligand-induced conformational stabilization of proteins is a well-understood phenomenon in which substrates, inhibitors, cofactors, and even other proteins provide enhanced stability to proteins on binding. This phenomenon is based on the energetic coupling of the ligand-binding and protein-melting reactions. In an attempt to harness these biophysical properties for drug discovery, fully automated instrumentation was designed and implemented to perform miniaturized fluorescence-based thermal shift assays in a microplate format for the high throughput screening of compound libraries. Validation of this process and instrumentation was achieved by investigating ligand binding to more than 100 protein targets. The general applicability of the thermal shift screening strategy was found to be an important advantage because it circumvents the need to design and retool new assays with each new therapeutic target. Moreover, the miniaturized thermal shift assay methodology does not require any prior knowledge of a therapeutic target's function, making it ideally suited for the quantitative high throughput drug screening and evaluation of targets derived from genomics.
{"title":"High-density miniaturized thermal shift assays as a general strategy for drug discovery.","authors":"M. Pantoliano, E. Petrella, Joseph D. Kwasnoski, V. S. Lobanov, J. Myslik, Edward Graf, T. Carver, E. Asel, B. Springer, Pamela Lane, F. Salemme","doi":"10.1089/108705701753364922","DOIUrl":"https://doi.org/10.1089/108705701753364922","url":null,"abstract":"More general and universally applicable drug discovery assay technologies are needed in order to keep pace with the recent advances in combinatorial chemistry and genomics-based target generation. Ligand-induced conformational stabilization of proteins is a well-understood phenomenon in which substrates, inhibitors, cofactors, and even other proteins provide enhanced stability to proteins on binding. This phenomenon is based on the energetic coupling of the ligand-binding and protein-melting reactions. In an attempt to harness these biophysical properties for drug discovery, fully automated instrumentation was designed and implemented to perform miniaturized fluorescence-based thermal shift assays in a microplate format for the high throughput screening of compound libraries. Validation of this process and instrumentation was achieved by investigating ligand binding to more than 100 protein targets. The general applicability of the thermal shift screening strategy was found to be an important advantage because it circumvents the need to design and retool new assays with each new therapeutic target. Moreover, the miniaturized thermal shift assay methodology does not require any prior knowledge of a therapeutic target's function, making it ideally suited for the quantitative high throughput drug screening and evaluation of targets derived from genomics.","PeriodicalId":15087,"journal":{"name":"Journal of Biomolecular Screening","volume":"6 6 1","pages":"429-40"},"PeriodicalIF":0.0,"publicationDate":"2001-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1089/108705701753364922","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60620661","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
A high throughput screening method for the analysis of 5-hydroxytryptamine(2A) (5-HT(2A)) receptor binding parameters has been developed, using 96-well filter plates of the Millipore MultiScreen system in combination with a MicroBeta PLUS microplate scintillation counter. MAFB filter plates (GF/B filter over a Durapore membrane) were used because of the lower nonspecific binding of the radioligand to GF/B filter material than to GF/C filters. Comparing different scintillation cocktails, highest counting efficiency and shortest equilibration time were detected with Betaplatescint, after drying the plates at 50 degrees C for 2 h. Measuring the plates without the plastic underdrain increased the counting efficiency by about 39% as compared with counting the plate with the underdrain intact. Presoaking the wells with 0.5% polyethyleneimine for 2 h reduced the nonspecific binding to the filter material by about 50%. A linear relationship of protein concentration and radioligand binding was established up to a protein concentration of 165 microg of protein/well. In the assays, 70 microg of protein/well was generally used, which has turned out to be favorable with respect to the number of counts obtained. When a higher concentration of protein was used, the period of time needed to aspirate the plate was too long because of obstruction of the filter material. Receptor-radioligand equilibration was reached after about 20 min at concentrations less than 0.05 nM [(3)H]ketanserin-HCl; at higher concentrations it was reached after about 10 min. Saturation analysis of [(3)H]ketanserin-HCl resulted in a mean B(max) of 393 fmol/mg protein and a K(D) of 2.0 nM using rat frontal cortex as a receptor source. Competition experiments with known 5-HT(2A) receptor ligands-DOB-HCl (K(i) = 59 nM), DOET-HCl (K(i) = 137 nM), DOM-HCl (K(i) = 533 nM), DMT (K(i) = 1,985 nM), and TMA-HCl (K(i) = 22,340 nM)-were in accordance with literature values.
{"title":"Development of a 5-hydroxytryptamine(2A) receptor binding assay for high throughput screening using 96-well microfilter plates.","authors":"A. Harms, D. Gündisch, C. Müller, K. Kovar","doi":"10.1089/108705700416146","DOIUrl":"https://doi.org/10.1089/108705700416146","url":null,"abstract":"A high throughput screening method for the analysis of 5-hydroxytryptamine(2A) (5-HT(2A)) receptor binding parameters has been developed, using 96-well filter plates of the Millipore MultiScreen system in combination with a MicroBeta PLUS microplate scintillation counter. MAFB filter plates (GF/B filter over a Durapore membrane) were used because of the lower nonspecific binding of the radioligand to GF/B filter material than to GF/C filters. Comparing different scintillation cocktails, highest counting efficiency and shortest equilibration time were detected with Betaplatescint, after drying the plates at 50 degrees C for 2 h. Measuring the plates without the plastic underdrain increased the counting efficiency by about 39% as compared with counting the plate with the underdrain intact. Presoaking the wells with 0.5% polyethyleneimine for 2 h reduced the nonspecific binding to the filter material by about 50%. A linear relationship of protein concentration and radioligand binding was established up to a protein concentration of 165 microg of protein/well. In the assays, 70 microg of protein/well was generally used, which has turned out to be favorable with respect to the number of counts obtained. When a higher concentration of protein was used, the period of time needed to aspirate the plate was too long because of obstruction of the filter material. Receptor-radioligand equilibration was reached after about 20 min at concentrations less than 0.05 nM [(3)H]ketanserin-HCl; at higher concentrations it was reached after about 10 min. Saturation analysis of [(3)H]ketanserin-HCl resulted in a mean B(max) of 393 fmol/mg protein and a K(D) of 2.0 nM using rat frontal cortex as a receptor source. Competition experiments with known 5-HT(2A) receptor ligands-DOB-HCl (K(i) = 59 nM), DOET-HCl (K(i) = 137 nM), DOM-HCl (K(i) = 533 nM), DMT (K(i) = 1,985 nM), and TMA-HCl (K(i) = 22,340 nM)-were in accordance with literature values.","PeriodicalId":15087,"journal":{"name":"Journal of Biomolecular Screening","volume":"5 4 1","pages":"269-78"},"PeriodicalIF":0.0,"publicationDate":"2000-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60621026","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 1998-03-01DOI: 10.1177/108705719800300206
J. Watson, J. Selkirk, A. Brown
With the exponential rate at which proposed drugs are being produced for disease therapy, it is now essential to automate assays used to screen these compounds and increase throughput. This has been rapidly adopted for simple radioligand binding assays but is less amenable for certain functional screens. [35S]GTPγS binding represents a convenient method for screening ligands that bind to G protein-coupled receptors and, ultimately, stimulate G-protein activation. In this study we have investigated the use of 96-well FlashPlates™ (NEN DuPont, Stevenage, England) to measure [35S]GTPγS binding to human 5-HT1B receptors expressed in Chinese hamster ovary cells. The cells were added to the individual wells of the FlashPlate and incubated with [35S]GTPγS in the presence or absence of test drug and bound radioactivity measured in a 96-well spectrometer. 5-HT produced a stimulation of basal [35S]GTPγS binding, which was robust within and between experiments, with pEC50 = 8.1. The 5-HT1B partial agonist GR127935 (2′n-methyl-4′-5-methyl-1,2,4 oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-amide) caused a partial stimulation (pEC5o = 8.3, intrinsic activity = 0.7), and the selective 5-HTIB receptor antagonist SB-224289 (2,3,6,7-tetrahydro-1′-methyl-5-{2′-methyl-4′-[(5-methyl-1,2,4-oxadiazole-3-yl)biphenyl-4-yl]carbonyl}furo[2,3-flindole-3-spiro-4′-piperidine oxalate) displayed inverse agonism with pEC50 = 7.6. These results are similar to those obtained using the conventional filtration method and indicate that FlashPlate technology can provide a rapid method for measuring [35S]GTPγS binding.
{"title":"Development of Flashplate™ Technology to Measure [35S]GTPyS Binding to Chinese Hamster Ovary Cell Membranes Expressing the Cloned Human 5-HTIB Receptor","authors":"J. Watson, J. Selkirk, A. Brown","doi":"10.1177/108705719800300206","DOIUrl":"https://doi.org/10.1177/108705719800300206","url":null,"abstract":"With the exponential rate at which proposed drugs are being produced for disease therapy, it is now essential to automate assays used to screen these compounds and increase throughput. This has been rapidly adopted for simple radioligand binding assays but is less amenable for certain functional screens. [35S]GTPγS binding represents a convenient method for screening ligands that bind to G protein-coupled receptors and, ultimately, stimulate G-protein activation. In this study we have investigated the use of 96-well FlashPlates™ (NEN DuPont, Stevenage, England) to measure [35S]GTPγS binding to human 5-HT1B receptors expressed in Chinese hamster ovary cells. The cells were added to the individual wells of the FlashPlate and incubated with [35S]GTPγS in the presence or absence of test drug and bound radioactivity measured in a 96-well spectrometer. 5-HT produced a stimulation of basal [35S]GTPγS binding, which was robust within and between experiments, with pEC50 = 8.1. The 5-HT1B partial agonist GR127935 (2′n-methyl-4′-5-methyl-1,2,4 oxadiazol-3-yl)-biphenyl-4-carboxylic acid [4-methoxy-3-(4-methyl-piperazin-1-yl)-phenyl]-amide) caused a partial stimulation (pEC5o = 8.3, intrinsic activity = 0.7), and the selective 5-HTIB receptor antagonist SB-224289 (2,3,6,7-tetrahydro-1′-methyl-5-{2′-methyl-4′-[(5-methyl-1,2,4-oxadiazole-3-yl)biphenyl-4-yl]carbonyl}furo[2,3-flindole-3-spiro-4′-piperidine oxalate) displayed inverse agonism with pEC50 = 7.6. These results are similar to those obtained using the conventional filtration method and indicate that FlashPlate technology can provide a rapid method for measuring [35S]GTPγS binding.","PeriodicalId":15087,"journal":{"name":"Journal of Biomolecular Screening","volume":"3 1","pages":"101 - 105"},"PeriodicalIF":0.0,"publicationDate":"1998-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1177/108705719800300206","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"65314785","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pub Date : 1998-03-01DOI: 10.1177/108705719800300207
Eleonora M. Hogan, A. Lipnik, Sarah Anstead, R. Kennedy
A program has been written to transfer a set of discontinuous samples in 96-well storage plates to archival plates using an 80-well format. One thousand samples per day can be processed in this manner. The software can handle different sample volumes, random sample locations, and varying concentrations. This program was implemented on a Beckman (Fullerton, CA) Biomek(r) 2000 Laboratory Automation Workstation* using Beckman's BioScript software, BioWorks 1.4A, Tool Command language, and Microsoft (Redmond, WA) Visual Basic™. This system provides an effective means of selecting and transferring discrete samples.
{"title":"A Solution for Discontinuous Sample Splitting and Dilution with the Biomek(r) 2000","authors":"Eleonora M. Hogan, A. Lipnik, Sarah Anstead, R. Kennedy","doi":"10.1177/108705719800300207","DOIUrl":"https://doi.org/10.1177/108705719800300207","url":null,"abstract":"A program has been written to transfer a set of discontinuous samples in 96-well storage plates to archival plates using an 80-well format. One thousand samples per day can be processed in this manner. The software can handle different sample volumes, random sample locations, and varying concentrations. This program was implemented on a Beckman (Fullerton, CA) Biomek(r) 2000 Laboratory Automation Workstation* using Beckman's BioScript software, BioWorks 1.4A, Tool Command language, and Microsoft (Redmond, WA) Visual Basic™. This system provides an effective means of selecting and transferring discrete samples.","PeriodicalId":15087,"journal":{"name":"Journal of Biomolecular Screening","volume":"3 1","pages":"107 - 138"},"PeriodicalIF":0.0,"publicationDate":"1998-03-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1177/108705719800300207","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"65314792","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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