Briefly, isolated arterioles were cannulated on glass micropipettes and secured in an organ chamber with circulating Kreb’s buffer. Following equilibration, endothelin-1 was added to assess viability and to preconstrict the vessels by 30-50% of their passive diameter. Internal diameters were measured at steady-state before and during intraluminal flow at pressure gradients of 5 to 100cmH2O. The following inhibitors were added to the bath 30 minutes prior to initiation of flow; nitric oxide synthase (NOS) inhibitor Nω-nitro-l -arginine (L-NAME; 10−4 mol/L), H2O2 scavenger polyethylene glycol-catalase (PEG-Catalase; 500U/mL), mitochondrial antioxidant Mito-TEMPO (10−5 mol/L), or electron transport chain (ETC) complex I inhibitor rotenone (10−6 mol/L ). C-2 Ceramide (10−5 mol/L ) or the neutral sphingomyelinase inhibitor GW4869 (4×10−6 mol/L) was incubated with arterioles from healthy patients and patients with CAD, respectively, for 16-20hrs prior to the experiment. Papaverine, (10−4 mol/L ) an endothelium-independent vasodilator, was added at the end of each experiment to determine the vessel’s maximal diameter, then the direction of maximal flow was reversed in the presence of maximal dilation, to confirm matched impedance between pipettes.
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Sphingomyelinase
Sphingomyelinase
Sphingomyelinase is an enzyme that catalyzes the hydrolysis of sphingomyelin, a major component of cell membranes, into ceramide and phosphocholine.
This process is crucial for cellular signaling and regulating various biological functions, including apoptosis, inflammation, and lipid metabolism.
Sphingomyelinase plays a key role in the pathogenesis of several diseases, such as Niemann-Pick disease, Alzheimer's disease, and cancer.
Researchers utilize sophisticated analytical techniques, including mass spectrometry and fluorescence-based assays, to study the structure, function, and regulation of this important enzyme.
Understandig the role of Sphingomyelinase in health and disease continues to be an active area of scientific enquiry, with potential implications for developing novel therapeutic strategies.
This process is crucial for cellular signaling and regulating various biological functions, including apoptosis, inflammation, and lipid metabolism.
Sphingomyelinase plays a key role in the pathogenesis of several diseases, such as Niemann-Pick disease, Alzheimer's disease, and cancer.
Researchers utilize sophisticated analytical techniques, including mass spectrometry and fluorescence-based assays, to study the structure, function, and regulation of this important enzyme.
Understandig the role of Sphingomyelinase in health and disease continues to be an active area of scientific enquiry, with potential implications for developing novel therapeutic strategies.
Most cited protocols related to «Sphingomyelinase»
Antioxidants
Arginine
Arterioles
Bath
Blood Vessel
Buffers
Catalase
catalase-polyethylene glycol
Ceramides
Dilatation
Electron Transport Complex I
Endothelin-1
Endothelium
Gas Scavengers
GW 4869
inhibitors
Mitochondria
Mitomycin
NG-Nitroarginine Methyl Ester
Nitric Oxide Synthase
Papaverine
Patients
Peroxide, Hydrogen
Polyethylene Glycols
Pressure
Rotenone
Sphingomyelinase
Vasodilator Agents
Development of a method to quantify person-to-person variability in LDL aggregation-susceptibility is available in Supplementary material online , Methods and Figures S1–S3. In the assay we developed, LDL isolated from human plasma was extensively dialysed against 20 mM MES buffer, pH 5.5, containing 150 mM NaCl, 2 mM MgCl2, 2 mM CaCl2, and 50 µM ZnCl2. The sample was diluted with the same buffer to give a final concentration of 0.2 mg of apoB-100/mL, then human recombinant secretory sphingomyelinase (hrSMase) was added to to give a final enzyme concentration of 75 µg/mL, and the mixture was incubated at +37°C. The size of LDL was determined immediately and the hourly up to 6 h. An aliquot was taken at the same time points for phosphorylcholine measurement. The degree of sphingomyelins (SM) hydrolysis was determined by measuring the phosphorylcholine content from the samples with Amplex Red reagent (Invitrogen).
Amplex Red
Apolipoprotein B-100
Biological Assay
Buffers
Enzymes
Homo sapiens
Hydrolysis
Magnesium Chloride
Phosphorylcholine
Plasma
secretion
Sodium Chloride
Sphingomyelinase
Sphingomyelins
Susceptibility, Disease
MVs were isolated from the supernatants of HIV-1-infected macrophages and LPS-activated microglia through differential centrifugations with or without neutral sphingomyelinases inhibitor GW4869 (Sigma) at different dosages, 2, 5 and 10 μM. Briefly, the supernatants were first centrifuged at 300 × g for 10 min to remove free cells, at 3000 × g for 20 min to remove cellular debris and then 10,000 × g for 30 min to remove free organelles. Lastly, MVs were collected by ultracentrifugation at 100,000 × g for 2 h at 4 °C. To prepare MVs for Western blot, the MVs pellets were lysed in M-PER mammalian protein extraction reagent (Thermo Scientific, Pittsburgh, PA). For negative staining, MVs were fixed in 2 % glutaraldehyde and 2 % paraformaldehyde. For glutaminase activity assay and neurotoxicity, the MVs were resuspended in 1 ml of glutamine-free neurobasal medium.
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Biological Assay
Cells
Centrifugation
Glutaminase
Glutamine
Glutaral
GW 4869
HIV-1
Macrophage
Mammals
Microglia
Neurotoxicity Syndromes
Organelles
paraform
Pellets, Drug
Proteins
Sphingomyelinase
Ultracentrifugation
Western Blotting
The library of pharmacologically active compounds (LOPAC, Sigma) and a subset of the Spectrum Collection (Food and Drug Administration, MicroSource Discovery Systems) were screened against human nSMase2. This screening was done in 384-well format at a single concentration (10 μM) in duplicate using the fluorescence-based activity assay. Positive control inhibitors known to block nSMase2 activity were used in all assays: 150 μM GW4869 (Sigma), 100 μM manumycin A (Enzo Lifescience) and 100 μM altenusin (Enzo Lifescience). A negative control, 100 μM zoledronic acid (Enzo Lifescience), known to inhibit acid but not neutral sphingomyelinase, was also included in all plates. Compounds that showed at least 50% inhibitory activity selective for nSMase2 were considered hits. The compound physicochemical properties and drug-like features were used to evaluate the hits and select those to be tested in the confirmatory direct activity assay using 14C-labeled substrate.
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Acids
altenusin
Biological Assay
Cardiac Arrest
cDNA Library
Fluorescence
GW 4869
Homo sapiens
inhibitors
manumycin
Pharmaceutical Preparations
Psychological Inhibition
Sphingomyelinase
Zoledronic Acid
Acids
Alkaline Phosphatase
Biological Assay
Buffers
choline oxidase
Kidney Cortex
Medical Devices
Molecular Probes
Peroxide, Hydrogen
SMPD1 protein, human
Sphingomyelinase
Sphingomyelins
Tissues
Most recents protocols related to «Sphingomyelinase»
Duplicate aliquots of 5 μg surface membrane extract proteins of untreated and ARA (50 μM, 30 min)-treated cells were evaluated for nSMase content using the Sphingomyelinase Assay kit of Abcam, ab287874 following the manufacturer’s instruction. Fluorescence was measured at Ex/Em of 540/590 nm, at 30, 60, and 120 min after adding reagents, and data were displayed after subtracting background values (Victor X4 Multi-Label Plate Reader).
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Biological Assay
Cells
Fluorescence
Membrane Proteins
Sphingomyelinase
During the isolation of exosomes, co-elution of proteins may occur and constitute a source of contamination. This can lead to a misinterpretation of the association of these proteins with exosomal fractions [57 (link)]. To avoid this bias, it is usual to inhibit exosome biogenesis, in order to check the level of contamination during fractionation. We used GW4869 to inhibit exosome production. GW4869 is a strong inhibitor of neutral sphingomyelinases that, by preventing the formation of intraluminal vesicles, is able to block exosome production. Cells were treated overnight with 10 µM GW4869. In parallel, control cells were treated with the same volume of DMSO. CCS were collected and exosome fractions were prepared using the Exoeasy Maxi kit®.
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Cardiac Arrest
Cells
Exosomes
GW 4869
isolation
Proteins
Radiotherapy Dose Fractionations
Sphingomyelinase
Staphylococcal Protein A
Sulfoxide, Dimethyl
To predict B-cell linear epitopes and perform in silico analyses, we used the entire sequence of Sph2 of L. interrogans serogroup Icterohaemorrhagiae and serovar Lai (Uniprot ID: P59116), obtained from the Uniprot database https://www.uniprot.org/ (accessed on 24 March 2020), as the sequence reference to this study. Moreover, to evaluate the similarity of Sph2 among pathogenic leptospires, we compared the used sequence with the described sequences of Sph2 from L. interrogans (serogroups: Australis, Bataviae, and Pyrogenes); L. alexanderi (serogroup: Manhao); L. alstonii (serogroup: Ranarum); L. borgpetersenii (serogroups: Pomona and Sejroe); L. noguchii (serogroups: Autumnalis and Panama); L. kirschneri (serogroup: Autumnalis); L. santarosai (serogroup: Javanica); and L. weilii (serogroups: Ranarum and Tarassovi). In addition, to investigate the conservation of reference Sph2 and other Leptospira spp. sphingomyelinases, this sequence was also aligned and compared with sequences of SphH (Uniprot ID: O34095), Sph1 (Uniprot ID: P59115), Sph3 (Uniprot ID: A0A0E2DC81), and Sph4 (Uniprot ID: A0A0E2DCF7) from L. interrogans serogroup Icterohaemorrhagiae and serovar Lai. Finally, the Sph2 reference sequence was also compared with sphingomyelinases from Listeria ivanovii (Uniprot ID: Q9RLV9), Bacillus cereus (Uniprot ID: P09599), Staphylococcus aureus (Uniprot ID: A0A7U4AUV1), and Pseudomonas sp. (Uniprot ID: Q93HR5). All studied sequences were accessed on 24 March 2020 and are summarized in Table 1 .
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Bacillus cereus
Congenital Spherocytosis
Epitopes, B-Lymphocyte
Leptospira
Listeria ivanovii
Pathogenicity
Pseudomonas
Spherocytosis, Type 3
Spherocytosis, Type 4
Sphingomyelinase
Staphylococcus aureus
Sequence alignment and identification of conserved patterns among sphingomyelinases from L. interrogans serovar Lai [Sph2 (Uniprot ID: P59116), Sph1 (Uniprot ID: P59115), Sph3 (Uniprot ID: A0A0E2DC81), Sph4 (Uniprot ID: A0A0E2DCF7), and SphH (Uniprot ID: O34095)] and other bacteria including Listeria ivanovii (SmcLUniprot ID: Q9RLV9), Bacillus cereus (BC SMase, Uniprot ID: P09599), Staphylococcus aureus (Uniprot ID: A0A7U4AUV1), and Pseudomonas spp. strain TK4 (Uniprot ID: Q93HR5) were conducted by MAFFT [40 (link)], using the software MegAlign pro. In the same way, to verify the conservation degree of Sph2 among pathogenic Leptospira spp., the reference Sph2 was aligned and compared with all other 17 Sph2 described in Table 1 . To investigate the conservation degree of predicted epitopes among pathogenic leptospires, each predicted sequence was aligned and compared with all Sph2 sequences described in Table 1 . Values of identity (%) represent the percentage of equal amino acids aligned. Moreover, we compared the conservation of amino acids present in the catalytic sites, and metal-binding sites were compared among Sph2 from pathogenic leptospires and other sphingomyelinases, based on the study of Narayanavari S.A. and collaborators [18 (link)].
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Amino Acids
Bacillus cereus
Bacteria
Binding Sites
Congenital Spherocytosis
Epitopes
Leptospira
Listeria ivanovii
Metals
Pathogenicity
Pseudomonas
Sequence Alignment
Spherocytosis, Type 3
Spherocytosis, Type 4
Sphingomyelinase
Staphylococcus aureus
Strains
In total, 1 × 105 SH-SY5Y cells were seeded into the lower chambers. For the cell treatment, approximately 5 × 105 NSCs were seeded into the upper chambers of 6-well cell culture inserts. Exosomes were added to the culture medium at 2 μg of exosomes per 1 × 105 recipient cells. GW4869 is a neutral sphingomyelinase inhibitor, and is the most widely used pharmacological agent for blocking exosome generation (Reiner et al. 2017 (link)). GW4869 inhibits the inward budding of multivesicular bodies and release of mature exosomes; thus, GW4869 was used in the present study to inhibit exosome secretion from NSCs. Twenty micromolar of GW4869 (#D1692, Sigma) was used to treat NSCs.
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Cardiac Arrest
Cell Culture Techniques
Cells
Exosomes
GW 4869
Multivesicular Body
PER1 protein, human
secretion
Sphingomyelinase
Top products related to «Sphingomyelinase»
Sourced in United States, Italy, Japan
The Amplex Red Sphingomyelinase assay kit is a fluorometric assay used to measure sphingomyelinase activity. The kit utilizes the Amplex Red reagent, which reacts with the hydrogen peroxide produced by the sphingomyelinase-catalyzed hydrolysis of sphingomyelin, to generate a red-fluorescent oxidation product.
Sourced in United States, Germany, China, Macao, United Kingdom
GW4869 is a laboratory reagent manufactured by Merck Group. It is a small molecule inhibitor that functions as a neutral sphingomyelinase 2 (nSMase2) inhibitor. The core function of GW4869 is to inhibit the activity of nSMase2, an enzyme involved in the metabolism of sphingolipids.
Sourced in United States
GW4869 is a potent and selective inhibitor of neutral sphingomyelinase 2 (nSMase2). It is a commonly used tool compound for the study of sphingolipid metabolism and signaling pathways involving nSMase2.
The Sphingomyelinase Fluorometric Assay kit is a laboratory tool designed to measure the activity of the enzyme sphingomyelinase. Sphingomyelinase is involved in the breakdown of the lipid sphingomyelin. The kit utilizes a fluorometric method to quantify the enzymatic activity.
Sourced in United States, Germany, Japan, United Kingdom
Methyl-β-cyclodextrin is a cyclic oligosaccharide compound commonly used as a laboratory reagent. It is a derivative of the natural compound β-cyclodextrin, with methyl groups attached to the hydroxyl groups. Methyl-β-cyclodextrin has the ability to form inclusion complexes with various organic molecules, which can be utilized in various applications involving solubilization, stabilization, and delivery of compounds in research and development settings.
Sourced in Germany, United Kingdom, France
The FLUOstar Optima is a fluorimeter manufactured by BMG Labtech. It is designed to measure fluorescence intensity in samples. The instrument provides a core function of fluorescence detection and quantification.
Sourced in United Kingdom
The Sphingomyelinase Assay kit is a laboratory tool designed to measure the activity of the enzyme sphingomyelinase. Sphingomyelinase plays a role in the breakdown of sphingomyelin, a component of cell membranes. The assay kit provides a quantitative method to assess sphingomyelinase activity in biological samples.
Sourced in United Kingdom
Sphingomyelinase is a laboratory product that catalyzes the hydrolysis of sphingomyelin, a type of phospholipid, into ceramide and phosphocholine. It is an important enzyme in the metabolism of sphingolipids, which are essential components of cell membranes.
Sourced in United States
The SMase is a laboratory instrument used for the analysis and study of sphingomyelinase (SMase) enzymes. SMase enzymes play a crucial role in lipid metabolism and signaling pathways. The SMase instrument provides researchers with the tools to measure and characterize the activity and properties of these enzymes in a controlled laboratory setting.
Sourced in United States
Sphingomyelinase from Bacillus cereus is an enzyme that catalyzes the hydrolysis of sphingomyelin, a component of cell membranes. This enzyme is isolated from the bacterium Bacillus cereus.
More about "Sphingomyelinase"
Sphingomyelinase (SMase) is a versatile enzyme that plays a crucial role in cellular signaling and lipid metabolism.
It catalyzes the hydrolysis of sphingomyelin, a major component of cell membranes, into ceramide and phosphocholine.
This process is integral for regulating various biological functions, including apoptosis, inflammation, and lipid homeostasis.
Researchers utilize advanced analytical techniques, such as mass spectrometry and fluorescence-based assays (e.g., Amplex Red Sphingomyelinase assay kit, Sphingomyelinase Fluorometric Assay kit), to study the structure, function, and regulation of this enzyme.
Inhibitors like GW4869 and Methyl-β-cyclodextrin have also been employed to investigate the role of SMase in cellular processes.
The dysregulation of Sphingomyelinase has been implicated in the pathogenesis of several diseases, including Niemann-Pick disease, Alzheimer's disease, and cancer.
Understanding the intricacies of SMase function and its impact on health and disease continues to be an active area of scientific enquiry, with potential implications for developing novel therapeutic strategies.
Researchers can leverage AI-driven platforms like PubCompare.ai to streamline their Sphingomyelinase research workflow.
These tools can help locate relevant protocols from literature, preprints, and patents, as well as identify the best protocols and products through intelligent comparisons, empowering scientists to optimize their research processes and advance the field of Sphingomyelinase study.
It catalyzes the hydrolysis of sphingomyelin, a major component of cell membranes, into ceramide and phosphocholine.
This process is integral for regulating various biological functions, including apoptosis, inflammation, and lipid homeostasis.
Researchers utilize advanced analytical techniques, such as mass spectrometry and fluorescence-based assays (e.g., Amplex Red Sphingomyelinase assay kit, Sphingomyelinase Fluorometric Assay kit), to study the structure, function, and regulation of this enzyme.
Inhibitors like GW4869 and Methyl-β-cyclodextrin have also been employed to investigate the role of SMase in cellular processes.
The dysregulation of Sphingomyelinase has been implicated in the pathogenesis of several diseases, including Niemann-Pick disease, Alzheimer's disease, and cancer.
Understanding the intricacies of SMase function and its impact on health and disease continues to be an active area of scientific enquiry, with potential implications for developing novel therapeutic strategies.
Researchers can leverage AI-driven platforms like PubCompare.ai to streamline their Sphingomyelinase research workflow.
These tools can help locate relevant protocols from literature, preprints, and patents, as well as identify the best protocols and products through intelligent comparisons, empowering scientists to optimize their research processes and advance the field of Sphingomyelinase study.