cardiomyocytes  (Worthington Biochemical)


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    Name:
    Neonatal Cardiomyocyte Isolation System
    Description:
    Kit for performing five separate tissue dissociations each containing up to twelve hearts Contains single use vials of purified collagenase and trypsin CMF HBSS Leibovitz L 15 media and Falcon cell strainers along with a detailed protocol The kit is use tested by Worthington to assure performance
    Catalog Number:
    lk003300
    Price:
    256
    Size:
    1 kt
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    see components
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    Structured Review

    Worthington Biochemical cardiomyocytes
    Phosphorylation of MnSOD by p38β kinase. ( A ) In-vitro kinase assay is performed with purified MnSOD in the indicated amount as a substrate in the presence of activated, GST-tagged purified p38β kinase in the presence or absence of the specific kinase inhibitor, SB203580. As a positive control, the kinase activity is also assayed on a well known substrate, ATF2. ( B ) In-vitro kinase assay is performed with endogenous p38β kinase immunoprecipitated from cultured rat <t>cardiomyocytes</t> and 1 µg of MnSOD or ATF2 as substrate in the presence or absence of the kinase inhibitor. A western blot of the isolated kinase is shown as a control.
    Kit for performing five separate tissue dissociations each containing up to twelve hearts Contains single use vials of purified collagenase and trypsin CMF HBSS Leibovitz L 15 media and Falcon cell strainers along with a detailed protocol The kit is use tested by Worthington to assure performance
    https://www.bioz.com/result/cardiomyocytes/product/Worthington Biochemical
    Average 99 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    cardiomyocytes - by Bioz Stars, 2021-03
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    Images

    1) Product Images from "Mitochondrial p38? and Manganese Superoxide Dismutase Interaction Mediated by Estrogen in Cardiomyocytes"

    Article Title: Mitochondrial p38? and Manganese Superoxide Dismutase Interaction Mediated by Estrogen in Cardiomyocytes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0085272

    Phosphorylation of MnSOD by p38β kinase. ( A ) In-vitro kinase assay is performed with purified MnSOD in the indicated amount as a substrate in the presence of activated, GST-tagged purified p38β kinase in the presence or absence of the specific kinase inhibitor, SB203580. As a positive control, the kinase activity is also assayed on a well known substrate, ATF2. ( B ) In-vitro kinase assay is performed with endogenous p38β kinase immunoprecipitated from cultured rat cardiomyocytes and 1 µg of MnSOD or ATF2 as substrate in the presence or absence of the kinase inhibitor. A western blot of the isolated kinase is shown as a control.
    Figure Legend Snippet: Phosphorylation of MnSOD by p38β kinase. ( A ) In-vitro kinase assay is performed with purified MnSOD in the indicated amount as a substrate in the presence of activated, GST-tagged purified p38β kinase in the presence or absence of the specific kinase inhibitor, SB203580. As a positive control, the kinase activity is also assayed on a well known substrate, ATF2. ( B ) In-vitro kinase assay is performed with endogenous p38β kinase immunoprecipitated from cultured rat cardiomyocytes and 1 µg of MnSOD or ATF2 as substrate in the presence or absence of the kinase inhibitor. A western blot of the isolated kinase is shown as a control.

    Techniques Used: In Vitro, Kinase Assay, Purification, Positive Control, Activity Assay, Immunoprecipitation, Cell Culture, Western Blot, Isolation

    Physical association between p38β and MnSOD. ( A ) Endogenous p38β is immunoprecipitated (IP: p38β) from cardiomyocytes after H/R with or without E2 at 10 nM, and the p38β-containing immunocomplex blotted for MnSOD. A western blot of p38β protein is also shown as a loading control. Nonspecific IgG is used as a specificity control in place of antibody specific to p38β in immunoprecipitation (IP: NS IgG). ( B ) Endogenous MnSOD is immunoprecipitated (IP: MnSOD) from treated cardiomyocytes as indicated, then the MnSOD-containing immunocomplex is blotted for p38β. Nonspecific IgG is used as a specificity control in place of antibody specific to MnSOD in immunoprecipitation (IP: NS IgG).
    Figure Legend Snippet: Physical association between p38β and MnSOD. ( A ) Endogenous p38β is immunoprecipitated (IP: p38β) from cardiomyocytes after H/R with or without E2 at 10 nM, and the p38β-containing immunocomplex blotted for MnSOD. A western blot of p38β protein is also shown as a loading control. Nonspecific IgG is used as a specificity control in place of antibody specific to p38β in immunoprecipitation (IP: NS IgG). ( B ) Endogenous MnSOD is immunoprecipitated (IP: MnSOD) from treated cardiomyocytes as indicated, then the MnSOD-containing immunocomplex is blotted for p38β. Nonspecific IgG is used as a specificity control in place of antibody specific to MnSOD in immunoprecipitation (IP: NS IgG).

    Techniques Used: Immunoprecipitation, Western Blot

    Identification of mitochondrial p38β. ( A ) Upper , Immnoblots of total p38β and phosphorylated p38β (p-p38β) from mitochondria (Mito) and cytosol (Cyto) of rat cardiomyocytes are shown. Final [E2] = 10 nM. Immunoblots of CoxIV and actin are shown also as loading controls for mitochondrial and cytosolic fractions, respectively. Lower Left , CoxIV activity. Mitochondria were isolated and assayed for Cox IV activity and citrate synthase activity. CoxIV activity was then normalized to that of citrate synthase, and is presented with quantitative analysis. Lower Right , NAO fluorescence. Cells were stained with NAO, a marker of mitochondrial biogenesis, and analyzed by flow cytometry. A representative graph from triplicate experiments is presented ( B ) Mitochondrial p-p38β. Upper , the ratio of dual phosphorylated p38β (p-p38β) over total p38β in mitochondria after indicated treatments is expressed in graph. Lower , the percentage of mitochondrial p38β localized to mitochondria is shown in graph as the total mitochondrial p38β over total cellular p38β pool. ( C ) Immunocytochemistry of p38β in cardiomyocytes. Primary antibody specific to p38β is used to stain for intracellular p38β ( left ), and the cells are co-stained with MitoTracker, a fluorescent probe for mitochondria ( middle ). The two images are then merged to demonstrate co-localization of p38β with mitochondria ( right ). The white scale bar represents 25 µm. ( D ) Mitochondrial membrane potential by JC-1 fluorescence. Upper , Flow cytometry analysis of JC-1 stained cells after the indicated treatments shows mitochondrial membrane potential changes reflected by the ratio of the red (FL2 district) and the green (FL1) fluorescent signals. Lower , Red/green JC-1 fluorescence ratio is represented in graph with quantitative analysis. * p
    Figure Legend Snippet: Identification of mitochondrial p38β. ( A ) Upper , Immnoblots of total p38β and phosphorylated p38β (p-p38β) from mitochondria (Mito) and cytosol (Cyto) of rat cardiomyocytes are shown. Final [E2] = 10 nM. Immunoblots of CoxIV and actin are shown also as loading controls for mitochondrial and cytosolic fractions, respectively. Lower Left , CoxIV activity. Mitochondria were isolated and assayed for Cox IV activity and citrate synthase activity. CoxIV activity was then normalized to that of citrate synthase, and is presented with quantitative analysis. Lower Right , NAO fluorescence. Cells were stained with NAO, a marker of mitochondrial biogenesis, and analyzed by flow cytometry. A representative graph from triplicate experiments is presented ( B ) Mitochondrial p-p38β. Upper , the ratio of dual phosphorylated p38β (p-p38β) over total p38β in mitochondria after indicated treatments is expressed in graph. Lower , the percentage of mitochondrial p38β localized to mitochondria is shown in graph as the total mitochondrial p38β over total cellular p38β pool. ( C ) Immunocytochemistry of p38β in cardiomyocytes. Primary antibody specific to p38β is used to stain for intracellular p38β ( left ), and the cells are co-stained with MitoTracker, a fluorescent probe for mitochondria ( middle ). The two images are then merged to demonstrate co-localization of p38β with mitochondria ( right ). The white scale bar represents 25 µm. ( D ) Mitochondrial membrane potential by JC-1 fluorescence. Upper , Flow cytometry analysis of JC-1 stained cells after the indicated treatments shows mitochondrial membrane potential changes reflected by the ratio of the red (FL2 district) and the green (FL1) fluorescent signals. Lower , Red/green JC-1 fluorescence ratio is represented in graph with quantitative analysis. * p

    Techniques Used: Western Blot, Activity Assay, Isolation, Fluorescence, Staining, Marker, Flow Cytometry, Cytometry, Immunocytochemistry

    Mitochondrial p38β activity and viability. ( A ) p38β is immunoprecipitated from mitochondria isolated from cultured rat cardiomyocytes and subjected to kinase assays using 32 P-labeled ATP and ATF2 as its substrate after indicated treatments. Thus radiolabeled ATF2 is shown with quantitative analysis of the band intensity. Representative western blots of ATF2 protein and immunoprecipitated mitochondrial p38β are shown at the bottom as a control. [E2] = 10 nM. * p
    Figure Legend Snippet: Mitochondrial p38β activity and viability. ( A ) p38β is immunoprecipitated from mitochondria isolated from cultured rat cardiomyocytes and subjected to kinase assays using 32 P-labeled ATP and ATF2 as its substrate after indicated treatments. Thus radiolabeled ATF2 is shown with quantitative analysis of the band intensity. Representative western blots of ATF2 protein and immunoprecipitated mitochondrial p38β are shown at the bottom as a control. [E2] = 10 nM. * p

    Techniques Used: Activity Assay, Immunoprecipitation, Isolation, Cell Culture, Labeling, Western Blot

    MnSOD activity and anion superoxide. ( A ) After indicated treatments, mitochondria isolated from cardiomyocytes are assayed for MnSOD activity. The final dismutase activity is expressed relative to that under normoxia (control), with quantitative analysis. * p
    Figure Legend Snippet: MnSOD activity and anion superoxide. ( A ) After indicated treatments, mitochondria isolated from cardiomyocytes are assayed for MnSOD activity. The final dismutase activity is expressed relative to that under normoxia (control), with quantitative analysis. * p

    Techniques Used: Activity Assay, Isolation

    Related Articles

    Isolation:

    Article Title: Construction of Cardiac Tissue Rings Using a Magnetic Tissue Fabrication Technique
    Article Snippet: .. Primary Culture of Neonatal Rat Cardiomyocytes Primary neonatal rat cardiomyocytes were isolated using a neonatal cardiomyocyte isolation kit (Worthington Biochemical, Lakewood, NJ, USA) according to the published procedure [ ]. .. Briefly, ventricles from 2–4 day-old Sprague-Dawley rats (Japan SLC, Inc., Hamamatsu, Japan) were incubated for 16–20 h at 4 °C in Hank’s balanced salt solution containing trypsin (50–100 μg/mL), followed by digestion with collagenase (75 U/mL) for 40 min at 37 °C.

    Article Title: Mitoregulin: A lncRNA-Encoded Microprotein that Supports Mitochondrial Supercomplexes and Respiratory Efficiency
    Article Snippet: .. Neonatal rat cardiomyocytes (NRCMs) were isolated using the Worthington Neonatal Cardiomyocyte Isolation System (Worthington Biochemical Corporation, Cat. No ). ..

    Article Title: Single Cell Transcriptomics Reconstructs Fate Conversion from Fibroblast to Cardiomyocyte
    Article Snippet: For bulk RNA-seq experiments, neonatal CFs were similarly isolated except that MACS-isolated Thy1+ cells were directly lysed in TRIzol (Life Technology) without culturing. .. Neonatal CMs were isolated using the neonatal cardiomyocytes isolation system (Worthington Biochemical Corporation) except that all enzymes were used at a ¼ of the recommended concentration to increase cell viability. .. After a 1.5 hour pre-plating on uncoated surface to remove attached nonmyocytes, the unattached CMs were collected in TRIzol ( > 80% viability by Trypan blue staining).

    Article Title: ADAP1 limits neonatal cardiomyocyte hypertrophy by reducing integrin cell surface expression
    Article Snippet: Hexon-expressing cells immunostained using anti-hexon antibody (ab8249; Abcam) were quantified 36 h post-infection. .. RNVC were extracted from the hearts of Sprague Dawley rat pups (strain code 001; Charles River) 1–3 days after birth using the Neonatal Cardiomyocyte Isolation System (Worthington Biochemical Corporation). .. The isolated ventricles were incubated in calcium/magnesium-free Hank’s Balanced Salt Solution containing trypsin (50 µg/mL) in vented-cap tubes with slow agitation at 4 °C for 18 h. The trypsin was then inhibited by adding Soybean Trypsin Inhibitor (200 µg/mL), and the ventricles were further digested with collagenase (100 units/mL) at 37 °C for 30 min.

    Article Title: Pro-survival function of MEF2 in cardiomyocytes is enhanced by β-blockers
    Article Snippet: .. Cell culture Primary neonatal rat cardiomyocytes were prepared from 1- to 3-day old Sprague Dawley rats using the Neonatal Cardiomyocyte Isolation System (Worthington Biochemical Corp, Lakewood, NJ, USA). .. Briefly, whole hearts were dissociated with trypsin (Promega, Madison, WI, USA) and collagenase (Worthington Biochemical Corp).

    Article Title: Docosahexaenoic acid inhibits protein kinase C translocation/activation and cardiac hypertrophy in rat cardiomyocytes
    Article Snippet: .. Isolation of cardiomyocytes Neonatal cardiomyocytes were obtained using an isolation system from Worthington Biochemical Corporation. ..

    Article Title: Muscle ring finger protein-1 inhibits PKC? activation and prevents cardiomyocyte hypertrophy
    Article Snippet: Cell culture COS 7 cells were cultured in DME and transiently transfected using FuGENE (Roche) as described previously ( ). .. NRVM were isolated using the neonatal cardiomyocyte isolation kit (Worthington) and were plated on laminin. ..

    Article Title: Pivotal role of cardiac lineage protein-1 (CLP-1) in transcriptional elongation factor P-TEFb complex formation in cardiac hypertrophy
    Article Snippet: Confocal imaging was carried out using a BioRad Radiance microscope with LaserSharp 2000 software. .. Cardiac cells of 2–4-day-old WKY rats were isolated using a neonatal cardiomyocyte isolation system (Worthington Biochemical Corp, Lakewood, New Jersey, USA). .. After pre-plating to reduce contaminating non-muscle cells, cardiomyocytes were plated on Bioflex collagen 6-well plates (Flexcell, McKeesport, Pennsylvania, USA) and cultured in DMEM/F-12 (1:1) media supplemented with 10% fetal bovine serum for approximately 1.5 days prior to switching to serum-free medium.

    Concentration Assay:

    Article Title: Single Cell Transcriptomics Reconstructs Fate Conversion from Fibroblast to Cardiomyocyte
    Article Snippet: For bulk RNA-seq experiments, neonatal CFs were similarly isolated except that MACS-isolated Thy1+ cells were directly lysed in TRIzol (Life Technology) without culturing. .. Neonatal CMs were isolated using the neonatal cardiomyocytes isolation system (Worthington Biochemical Corporation) except that all enzymes were used at a ¼ of the recommended concentration to increase cell viability. .. After a 1.5 hour pre-plating on uncoated surface to remove attached nonmyocytes, the unattached CMs were collected in TRIzol ( > 80% viability by Trypan blue staining).

    Cell Culture:

    Article Title: Pro-survival function of MEF2 in cardiomyocytes is enhanced by β-blockers
    Article Snippet: .. Cell culture Primary neonatal rat cardiomyocytes were prepared from 1- to 3-day old Sprague Dawley rats using the Neonatal Cardiomyocyte Isolation System (Worthington Biochemical Corp, Lakewood, NJ, USA). .. Briefly, whole hearts were dissociated with trypsin (Promega, Madison, WI, USA) and collagenase (Worthington Biochemical Corp).

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    Worthington Biochemical dnase i activity
    Testing the inhibition of deoxyribonuclease I (DNase I) by previously known in vitro inhibitors. To generate dose–response curves, the change in product/min (pmole/min) by <t>DNase</t> I (1.72 nM) was monitored throughout a series of concentrations for
    Dnase I Activity, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    94
    Worthington Biochemical α amylase inhibitory activity
    <t>α‐Amylase</t> inhibitory activities of butternut squash residues (seed and skin extract). Values are means of duplicate determinations
    α Amylase Inhibitory Activity, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    93
    Worthington Biochemical fret activity assays nuc enzyme
    Activity of <t>Nuc/Nuc2</t> chimeric proteins. Chimeras consisting of combinations of Nuc and Nuc2 N- and C-terminal protein domains were constructed. Each chimera was constructed to include a His 6 -tag on the C-terminal protein domain. A . Schematic showing the Nuc/Nuc2 chimeric proteins constructed, with Nuc domains colored in teal and Nuc2 domains colored in purple (see Fig. 1A for additional description of domains and processing sites). Chimeras constructed include, from the top down: Nuc control (pMK12), Nuc2 control (pMK13), Nuc signal sequence fused to the Nuc2 active domain (pMK8), Nuc signal sequence and NucB leader fused to the Nuc2 active domain (pMK9), Nuc2 membrane anchor fused to NucA (pMK10) and Nuc2 membrane anchor fused to NucB (pMK11). Each construct was expressed in a S. aureus nuc mutant (strain AH1680) for 15 hr, and culture supernatant was saved and membranes purified. Anti-His immunoblots were performed to detect the presence of C-terminal His 6 -tag on chimeric proteins in culture supernatants ( B ) or membrane ( C ) fractions. <t>FRET</t> activity assays were performed to detect nuclease activity in culture supernatants ( D ) and membrane ( E) fractions. The nuc mutant strain was included as a negative control (called “no plasmid”).
    Fret Activity Assays Nuc Enzyme, supplied by Worthington Biochemical, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/fret activity assays nuc enzyme/product/Worthington Biochemical
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    Testing the inhibition of deoxyribonuclease I (DNase I) by previously known in vitro inhibitors. To generate dose–response curves, the change in product/min (pmole/min) by DNase I (1.72 nM) was monitored throughout a series of concentrations for

    Journal: Journal of biomolecular screening

    Article Title: Novel High-Throughput Deoxyribonuclease 1 Assay

    doi: 10.1177/1087057114555828

    Figure Lengend Snippet: Testing the inhibition of deoxyribonuclease I (DNase I) by previously known in vitro inhibitors. To generate dose–response curves, the change in product/min (pmole/min) by DNase I (1.72 nM) was monitored throughout a series of concentrations for

    Article Snippet: The percentage of DNase I activity was calculated using Equation 1: DNase\u00a0I\u00a0activity (%) =\u00a0 (mean\u00a0velocity\u00a0of\u00a0a\u00a0compound/mean\u00a0velocity\u00a0of\u00a0DMSO)\u00a0\u00d7\u00a0100 (1) In similar assays, recombinant murine EndoG (produced in-house) was used at a concentration of 0.14 μM in 0.1 mM MgCl2 , 10 mM Tris-HCl, pH 7.4; and DNase II (Worthington, Lakewood, NJ) (3.32 nM) was tested in 100 mM sodium citrate buffer, pH 5.0.

    Techniques: Inhibition, In Vitro

    Specificity of the high-throughput screening (HTS) assay to deoxyribonuclease I (DNase I). A Michaelis–Menten curve for each of 3 endonucleases (1.72 nM each) was generated for the assay to afford kinetic analysis. k cat / K m values of DNase I and

    Journal: Journal of biomolecular screening

    Article Title: Novel High-Throughput Deoxyribonuclease 1 Assay

    doi: 10.1177/1087057114555828

    Figure Lengend Snippet: Specificity of the high-throughput screening (HTS) assay to deoxyribonuclease I (DNase I). A Michaelis–Menten curve for each of 3 endonucleases (1.72 nM each) was generated for the assay to afford kinetic analysis. k cat / K m values of DNase I and

    Article Snippet: The percentage of DNase I activity was calculated using Equation 1: DNase\u00a0I\u00a0activity (%) =\u00a0 (mean\u00a0velocity\u00a0of\u00a0a\u00a0compound/mean\u00a0velocity\u00a0of\u00a0DMSO)\u00a0\u00d7\u00a0100 (1) In similar assays, recombinant murine EndoG (produced in-house) was used at a concentration of 0.14 μM in 0.1 mM MgCl2 , 10 mM Tris-HCl, pH 7.4; and DNase II (Worthington, Lakewood, NJ) (3.32 nM) was tested in 100 mM sodium citrate buffer, pH 5.0.

    Techniques: High Throughput Screening Assay, HTS Assay, Generated

    Identification of deoxyribonuclease I (DNase I) inhibitor candidates by screening of the chemical library of 1040 compounds. The library was screened at ( A ) 10 μM and ( B ) 1 μM concentrations, affording 45 and 5 hits, respectively. The

    Journal: Journal of biomolecular screening

    Article Title: Novel High-Throughput Deoxyribonuclease 1 Assay

    doi: 10.1177/1087057114555828

    Figure Lengend Snippet: Identification of deoxyribonuclease I (DNase I) inhibitor candidates by screening of the chemical library of 1040 compounds. The library was screened at ( A ) 10 μM and ( B ) 1 μM concentrations, affording 45 and 5 hits, respectively. The

    Article Snippet: The percentage of DNase I activity was calculated using Equation 1: DNase\u00a0I\u00a0activity (%) =\u00a0 (mean\u00a0velocity\u00a0of\u00a0a\u00a0compound/mean\u00a0velocity\u00a0of\u00a0DMSO)\u00a0\u00d7\u00a0100 (1) In similar assays, recombinant murine EndoG (produced in-house) was used at a concentration of 0.14 μM in 0.1 mM MgCl2 , 10 mM Tris-HCl, pH 7.4; and DNase II (Worthington, Lakewood, NJ) (3.32 nM) was tested in 100 mM sodium citrate buffer, pH 5.0.

    Techniques:

    Elaboration of the high-throughput screening (HTS) deoxyribonuclease I (DNase I) assay conditions. ( A ) A schematic diagram for the substrate, Cy5.5-end-labeled oligonucleotide, and principle of the assay. ( B ) The effects of the concentrations of calcium,

    Journal: Journal of biomolecular screening

    Article Title: Novel High-Throughput Deoxyribonuclease 1 Assay

    doi: 10.1177/1087057114555828

    Figure Lengend Snippet: Elaboration of the high-throughput screening (HTS) deoxyribonuclease I (DNase I) assay conditions. ( A ) A schematic diagram for the substrate, Cy5.5-end-labeled oligonucleotide, and principle of the assay. ( B ) The effects of the concentrations of calcium,

    Article Snippet: The percentage of DNase I activity was calculated using Equation 1: DNase\u00a0I\u00a0activity (%) =\u00a0 (mean\u00a0velocity\u00a0of\u00a0a\u00a0compound/mean\u00a0velocity\u00a0of\u00a0DMSO)\u00a0\u00d7\u00a0100 (1) In similar assays, recombinant murine EndoG (produced in-house) was used at a concentration of 0.14 μM in 0.1 mM MgCl2 , 10 mM Tris-HCl, pH 7.4; and DNase II (Worthington, Lakewood, NJ) (3.32 nM) was tested in 100 mM sodium citrate buffer, pH 5.0.

    Techniques: High Throughput Screening Assay, Labeling

    α‐Amylase inhibitory activities of butternut squash residues (seed and skin extract). Values are means of duplicate determinations

    Journal: Food Science & Nutrition

    Article Title: Evaluation of bioactivity of butternut squash (Cucurbita moschata D.) seeds and skin. Evaluation of bioactivity of butternut squash (Cucurbita moschata D.) seeds and skin

    doi: 10.1002/fsn3.1602

    Figure Lengend Snippet: α‐Amylase inhibitory activities of butternut squash residues (seed and skin extract). Values are means of duplicate determinations

    Article Snippet: The α‐amylase inhibitory activity was expressed as percentage inhibition (Worthington, Weddell, & Neilson, ).

    Techniques:

    Glucose metabolism in Cu− cerebellum. Cerebellar concentrations of glucose ( n = 5) and glucose-6-phosphate (G6P) ( n = 5) were found to be higher in Cu− rats without a corresponding increase in serum glucose ( n = 5) (Panel A). Levels of cerebellar glucose transporters Glut1 and Glut3 were not altered in Cu− rats (−) compared to Cu+ controls (+) (Panel B; n = 4). Values are means ± SEM; * P

    Journal: Experimental biology and medicine (Maywood, N.J.)

    Article Title: Fructose-2,6-Bisphosphate Is Lower in Copper Deficient Rat Cerebellum Despite Higher Content of Phosphorylated AMP-Activated Protein Kinase

    doi: 10.3181/0804-RM-132

    Figure Lengend Snippet: Glucose metabolism in Cu− cerebellum. Cerebellar concentrations of glucose ( n = 5) and glucose-6-phosphate (G6P) ( n = 5) were found to be higher in Cu− rats without a corresponding increase in serum glucose ( n = 5) (Panel A). Levels of cerebellar glucose transporters Glut1 and Glut3 were not altered in Cu− rats (−) compared to Cu+ controls (+) (Panel B; n = 4). Values are means ± SEM; * P

    Article Snippet: Cerebellar glucose 6-phosphate dehydrogenase was assayed in 50 mM Tris pH 7.8 containing 3 mM MgCl2 (Worthington Biochemical Corp. Manual).

    Techniques:

    Activity of Nuc/Nuc2 chimeric proteins. Chimeras consisting of combinations of Nuc and Nuc2 N- and C-terminal protein domains were constructed. Each chimera was constructed to include a His 6 -tag on the C-terminal protein domain. A . Schematic showing the Nuc/Nuc2 chimeric proteins constructed, with Nuc domains colored in teal and Nuc2 domains colored in purple (see Fig. 1A for additional description of domains and processing sites). Chimeras constructed include, from the top down: Nuc control (pMK12), Nuc2 control (pMK13), Nuc signal sequence fused to the Nuc2 active domain (pMK8), Nuc signal sequence and NucB leader fused to the Nuc2 active domain (pMK9), Nuc2 membrane anchor fused to NucA (pMK10) and Nuc2 membrane anchor fused to NucB (pMK11). Each construct was expressed in a S. aureus nuc mutant (strain AH1680) for 15 hr, and culture supernatant was saved and membranes purified. Anti-His immunoblots were performed to detect the presence of C-terminal His 6 -tag on chimeric proteins in culture supernatants ( B ) or membrane ( C ) fractions. FRET activity assays were performed to detect nuclease activity in culture supernatants ( D ) and membrane ( E) fractions. The nuc mutant strain was included as a negative control (called “no plasmid”).

    Journal: PLoS ONE

    Article Title: Staphylococcus aureus Nuc2 Is a Functional, Surface-Attached Extracellular Nuclease

    doi: 10.1371/journal.pone.0095574

    Figure Lengend Snippet: Activity of Nuc/Nuc2 chimeric proteins. Chimeras consisting of combinations of Nuc and Nuc2 N- and C-terminal protein domains were constructed. Each chimera was constructed to include a His 6 -tag on the C-terminal protein domain. A . Schematic showing the Nuc/Nuc2 chimeric proteins constructed, with Nuc domains colored in teal and Nuc2 domains colored in purple (see Fig. 1A for additional description of domains and processing sites). Chimeras constructed include, from the top down: Nuc control (pMK12), Nuc2 control (pMK13), Nuc signal sequence fused to the Nuc2 active domain (pMK8), Nuc signal sequence and NucB leader fused to the Nuc2 active domain (pMK9), Nuc2 membrane anchor fused to NucA (pMK10) and Nuc2 membrane anchor fused to NucB (pMK11). Each construct was expressed in a S. aureus nuc mutant (strain AH1680) for 15 hr, and culture supernatant was saved and membranes purified. Anti-His immunoblots were performed to detect the presence of C-terminal His 6 -tag on chimeric proteins in culture supernatants ( B ) or membrane ( C ) fractions. FRET activity assays were performed to detect nuclease activity in culture supernatants ( D ) and membrane ( E) fractions. The nuc mutant strain was included as a negative control (called “no plasmid”).

    Article Snippet: FRET activity assays Nuc enzyme was purchased from Worthington Biochemical (Lakewood, NJ).

    Techniques: Activity Assay, Construct, Sequencing, Mutagenesis, Purification, Western Blot, Negative Control, Plasmid Preparation

    Nuc2 is expressed at low levels during growth. A . Monitoring changes in fluorescence of a P nuc2 -sGFP transcriptional reporter over time (open circles) in comparison to growth (black squares). Dotted line indicates level of background fluorescence of growth media. B . Changes in surface Nuc2 activity (open circles) present in a S. aureus USA300 LAC nuc mutant over time as measured by whole-cell FRET assay.

    Journal: PLoS ONE

    Article Title: Staphylococcus aureus Nuc2 Is a Functional, Surface-Attached Extracellular Nuclease

    doi: 10.1371/journal.pone.0095574

    Figure Lengend Snippet: Nuc2 is expressed at low levels during growth. A . Monitoring changes in fluorescence of a P nuc2 -sGFP transcriptional reporter over time (open circles) in comparison to growth (black squares). Dotted line indicates level of background fluorescence of growth media. B . Changes in surface Nuc2 activity (open circles) present in a S. aureus USA300 LAC nuc mutant over time as measured by whole-cell FRET assay.

    Article Snippet: FRET activity assays Nuc enzyme was purchased from Worthington Biochemical (Lakewood, NJ).

    Techniques: Fluorescence, Activity Assay, Mutagenesis

    Nuc2 activity can be measured at the cell surface. A . Whole-cell FRET activity assay measuring Nuc2 surface function with UAMS-1 WT, nuc2 , nuc and nuc nuc2 double mutants. B . Cell surface nuclease activity in USA300 strain LAC WT, nuc2 , nuc and nuc nuc2 double mutants. Statistical significance (*p

    Journal: PLoS ONE

    Article Title: Staphylococcus aureus Nuc2 Is a Functional, Surface-Attached Extracellular Nuclease

    doi: 10.1371/journal.pone.0095574

    Figure Lengend Snippet: Nuc2 activity can be measured at the cell surface. A . Whole-cell FRET activity assay measuring Nuc2 surface function with UAMS-1 WT, nuc2 , nuc and nuc nuc2 double mutants. B . Cell surface nuclease activity in USA300 strain LAC WT, nuc2 , nuc and nuc nuc2 double mutants. Statistical significance (*p

    Article Snippet: FRET activity assays Nuc enzyme was purchased from Worthington Biochemical (Lakewood, NJ).

    Techniques: Activity Assay