This study investigated the effects of short-term exposure to elevated D-glucose levels on L-arginine transport and nitric oxide (NO) synthesis in human umbilical vein endothelial cells (HUVECs). The authors found that exposing HUVECs to 25 mmol/L D-glucose for just 2 minutes increased L-arginine transport and NO production through phosphorylation of endothelial NO synthase (eNOS) via p42/44 MAP kinases and phosphatidylinositol 3-kinase pathways. These rapid effects of D-glucose on the L-arginine/NO signaling pathway could be important in conditions with fluctuations in blood glucose levels and may underlie vasodilation seen in
This study investigated how elevated glucose levels affect adenosine transport in human umbilical vein endothelial cells (HUVECs). The researchers found that incubating HUVECs in 25 mmol/L glucose or ATP reduced adenosine transport by inhibiting the human equilibrative nucleoside transporter 1 (hENT1). This inhibition was mediated by activation of P2Y2 purinoceptors and involved reduced hENT1 expression and activity. The effects of glucose and nucleotides were blocked by P2Y receptor antagonists, demonstrating that glucose stimulates P2Y2 receptors by increasing ATP release, providing a potential mechanism for how glucose impacts adenosine regulation and vascular function.
This document describes site-directed mutagenesis experiments performed on the global transcription factor cAMP receptor protein (CRP) in Escherichia coli. Specifically, lysine residue 36 of CRP was mutated to alanine using site-directed mutagenesis to investigate how protein acetylation may affect CRP function and gene regulation. The document provides background on CRP and its role in transcription, as well as an overview of protein acetylation and its effects. It then describes the methods used to mutate lysine 36 to alanine in CRP and plans to analyze the mutant protein.
This document discusses phospholipase C (PLC) and its role in neutrophil degranulation and T cell cytotoxicity. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate. PLC signaling is required for lytic granule polarization and effective T cell killing through T cell antigen receptor activation. Neutrophil degranulation involves increases in calcium and PLC production of phosphatidylinositol, which is essential for granule exocytosis. Different intracellular signaling pathways regulate the release of primary, secondary, and tertiary granules from neutroph
This document describes a study characterizing a novel gene cluster involved in the degradation of 4-chlorocatechol by Pseudomonas reinekei MT1. The researchers found that during growth on 5-chlorosalicylate, a novel (chloro)catechol 1,2-dioxygenase (C12OccaA) and a novel (chloro)muconate cycloisomerase (MCIccaB) were induced. MCIccaB was found to transform 3-chloromuconate into equal amounts of cis-dienelactone and protoanemonin, acting as a functional intermediate between known chloromuconate cycloisomerases and muconate cycloisomerases
This document presents a structure/function model for the enzyme 1-pyrroline-5’ carboxylate reductase (P5CR) based on similarities to the β-hydroxyacid dehydrogenase enzyme family. The model is supported by evidence that recombinant E. coli P5CR has similar secondary structure to a β-hydroxyacid dehydrogenase and contains conserved functional domains. Site-directed mutagenesis of conserved residues in the proposed substrate-binding domain reduced catalytic efficiency. Chemical modification studies also provided insights into active site residues. The model proposes γ-glutamate semialdehyde as a potential true substrate based on inhibition studies.
This proposal seeks funding to develop an assay to determine the efficiency of the E. coli γ-complex clamp loader loading the E. coli β-clamp onto DNA. The researcher will purify proteins and assemble the γ-complex. An oligonucleotide will be biotinylated and annealed to bind to streptavidin beads. The γ-complex will load β-clamp onto the DNA using varying ratios and times. Analyzing samples by SDS-PAGE will optimize conditions and verify loading. Developing this assay will allow future study of the β-clamp-clamp loader complex role in DNA replication. Challenges include optimizing protein ratios and times to achieve sufficient loading without unloading.
A reading report for <A Secreted Slit2 Fragment Regulates Adipose Tissue Ther...星云 王
A reading report for <A Secreted Slit2 Fragment Regulates Adipose Tissue Thermogenesis and Metabolic Function
>, only for private study use, please do not use it for profit or public.
Treatment of mucopolysaccharidosis type IIIB (MPS IIIB) with enzyme replacement therapy has been hindered by the inadequate intracellular uptake of recombinantly produced alpha-N-acetylglucosaminidase (NAGLU). The authors generated an improved form of NAGLU by fusing it to insulin-like growth factor II (IGF-II), which binds to the mannose-6-phosphate receptor. They expressed and purified the NAGLU-IGF-II fusion protein from Chinese hamster ovary cells. Testing in MPS IIIB fibroblasts showed the fusion protein was readily taken up via receptor-mediated endocytosis and reduced glycosaminoglycan storage, supporting its potential as a therapeutic candidate for M
This study investigated how elevated glucose levels affect adenosine transport in human umbilical vein endothelial cells (HUVECs). The researchers found that incubating HUVECs in 25 mmol/L glucose or ATP reduced adenosine transport by inhibiting the human equilibrative nucleoside transporter 1 (hENT1). This inhibition was mediated by activation of P2Y2 purinoceptors and involved reduced hENT1 expression and activity. The effects of glucose and nucleotides were blocked by P2Y receptor antagonists, demonstrating that glucose stimulates P2Y2 receptors by increasing ATP release, providing a potential mechanism for how glucose impacts adenosine regulation and vascular function.
This document describes site-directed mutagenesis experiments performed on the global transcription factor cAMP receptor protein (CRP) in Escherichia coli. Specifically, lysine residue 36 of CRP was mutated to alanine using site-directed mutagenesis to investigate how protein acetylation may affect CRP function and gene regulation. The document provides background on CRP and its role in transcription, as well as an overview of protein acetylation and its effects. It then describes the methods used to mutate lysine 36 to alanine in CRP and plans to analyze the mutant protein.
This document discusses phospholipase C (PLC) and its role in neutrophil degranulation and T cell cytotoxicity. PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) into two second messengers, diacylglycerol and inositol 1,4,5-trisphosphate. PLC signaling is required for lytic granule polarization and effective T cell killing through T cell antigen receptor activation. Neutrophil degranulation involves increases in calcium and PLC production of phosphatidylinositol, which is essential for granule exocytosis. Different intracellular signaling pathways regulate the release of primary, secondary, and tertiary granules from neutroph
This document describes a study characterizing a novel gene cluster involved in the degradation of 4-chlorocatechol by Pseudomonas reinekei MT1. The researchers found that during growth on 5-chlorosalicylate, a novel (chloro)catechol 1,2-dioxygenase (C12OccaA) and a novel (chloro)muconate cycloisomerase (MCIccaB) were induced. MCIccaB was found to transform 3-chloromuconate into equal amounts of cis-dienelactone and protoanemonin, acting as a functional intermediate between known chloromuconate cycloisomerases and muconate cycloisomerases
This document presents a structure/function model for the enzyme 1-pyrroline-5’ carboxylate reductase (P5CR) based on similarities to the β-hydroxyacid dehydrogenase enzyme family. The model is supported by evidence that recombinant E. coli P5CR has similar secondary structure to a β-hydroxyacid dehydrogenase and contains conserved functional domains. Site-directed mutagenesis of conserved residues in the proposed substrate-binding domain reduced catalytic efficiency. Chemical modification studies also provided insights into active site residues. The model proposes γ-glutamate semialdehyde as a potential true substrate based on inhibition studies.
This proposal seeks funding to develop an assay to determine the efficiency of the E. coli γ-complex clamp loader loading the E. coli β-clamp onto DNA. The researcher will purify proteins and assemble the γ-complex. An oligonucleotide will be biotinylated and annealed to bind to streptavidin beads. The γ-complex will load β-clamp onto the DNA using varying ratios and times. Analyzing samples by SDS-PAGE will optimize conditions and verify loading. Developing this assay will allow future study of the β-clamp-clamp loader complex role in DNA replication. Challenges include optimizing protein ratios and times to achieve sufficient loading without unloading.
A reading report for <A Secreted Slit2 Fragment Regulates Adipose Tissue Ther...星云 王
A reading report for <A Secreted Slit2 Fragment Regulates Adipose Tissue Thermogenesis and Metabolic Function
>, only for private study use, please do not use it for profit or public.
Treatment of mucopolysaccharidosis type IIIB (MPS IIIB) with enzyme replacement therapy has been hindered by the inadequate intracellular uptake of recombinantly produced alpha-N-acetylglucosaminidase (NAGLU). The authors generated an improved form of NAGLU by fusing it to insulin-like growth factor II (IGF-II), which binds to the mannose-6-phosphate receptor. They expressed and purified the NAGLU-IGF-II fusion protein from Chinese hamster ovary cells. Testing in MPS IIIB fibroblasts showed the fusion protein was readily taken up via receptor-mediated endocytosis and reduced glycosaminoglycan storage, supporting its potential as a therapeutic candidate for M
N-acetyl-D-glucosamine kinase (NAGK) interacts with dynein light-chain roadblock type 1 (DYNLRB1) at dendritic branch points in neurons. Immunocytochemistry and proximity ligation assays showed colocalization of NAGK and DYNLRB1 on microtubule fibers at dendritic branches. NAGK was also found to interact with Golgi outposts and DYNLRB1 at branch points, indicating a tripartite interaction between NAGK, dynein, and Golgi that regulates dendritic growth and branching. Introduction of a peptide derived from DYNLRB1 stunted dendrite development in cultured neurons.
This study examined the secretion and intracellular trafficking of apolipoprotein A-V (apoA-V) using transfected cell lines. The key findings were:
1) Only about 20% of newly synthesized apoA-V was secreted from cells within 3 hours, with about 65% undergoing degradation before secretion.
2) When cells were treated with oleic acid to stimulate triglyceride synthesis, apoA-V secretion decreased while cell-associated apoA-V increased and moved to cytosolic lipid droplets.
3) Expression of apoA-V in hepatoma cells inhibited triglyceride secretion by 50% and reduced the diameter of secreted VLDL particles, but did not impact apolip
This document summarizes an experiment to engineer ascorbate peroxidase (APX) activity into cytochrome c peroxidase (CCP) by introducing the APX ascorbate-binding site into CCP. Specifically, the researchers replaced the ascorbate-binding loop and a critical arginine residue in CCP with the corresponding residues from APX to create a mutant called CCP2APX. While wild-type CCP showed no APX activity, CCP2APX was able to catalyze the peroxidation of ascorbate, demonstrating that the engineered ascorbate-binding site could bind ascorbate. Crystal structures of CCP2APX confirmed that the engineered binding site
This document discusses Polycomb group (PcG) proteins, which are important repressor proteins that regulate gene expression during development. It describes two main PcG complexes, PRC1 and PRC2, and their mechanisms of action, including histone modifications and chromatin compaction. The document also examines a case study on the interaction between the PcG protein LHP1 and deubiquitinating enzymes UBP12 and UBP13 in Arabidopsis thaliana. The study found that UBP12 and UBP13 interact with and help recruit LHP1 to target genes, and are involved in histone deubiquitination, which impacts gene silencing by PcG proteins.
Rational design of transition state analogue inhibitor for PvdQ enzyme.
PvdQ enzyme plays a key role in biosynthesis of siderophore pyoverdine in Pseudomonas aeruginosa and shows substrate preference for acyl chains 12-14 carbons. A series of alkylboronic acids were designed and found to be reversible, competitive inhibitors with picomolar affinity. X-ray crystal structure showed 1-tridecylboronic acid forms covalent bond with active site serine, mimicking substrate transition state. This inhibitor blocked bacterial growth in iron-limited conditions, reproducing genetic pvdQ disruption phenotype.
The document summarizes genetic and mutational characterization of the relV gene of Vibrio cholerae, which encodes a small alarmone synthetase protein called RelV. Key findings include:
1) Site-directed mutagenesis identified five amino acid residues (K107, D129, R132, L150, E188) in the RelA-SpoT domain of RelV that are essential for its (p)ppGpp synthetase activity.
2) Progressive deletion analysis determined the functional N-terminal boundary of RelV to be amino acid 59 and the C-terminal boundary to be amino acid 248, indicating that flanking sequences of the RelA-SpoT
The document discusses research on using phospholipase A2 (PLA2) antibodies to protect cells from radiation damage. PLA2 is an enzyme that releases fatty acids from phospholipids in cell membranes. Exposure to ionizing radiation activates PLA2, leading to cell death. The research proposal is to test whether antibodies that block PLA2 can inhibit this activation and thereby reduce the toxic effects of radiation exposure and increase radiation survival. Blocking PLA2 with antibodies may provide a new radiation protection strategy and tool for diagnosing and monitoring acute radiation sickness.
This study examined the interaction between the Abl kinase domain and the cancer drug Gleevec (imatinib) using site-directed mutagenesis to introduce a mutation (S417Y) associated with drug resistance. Researchers used PCR, protein purification techniques, and kinase assays to compare the inhibitory effect of Gleevec on wild-type Abl and the S417Y mutant. Their results supported the hypothesis that the S417Y mutation decreases Gleevec's ability to inhibit Abl kinase activity, though protein impurities limited the strength of the conclusions. The findings help explain why some cancers become resistant to Gleevec treatment.
1) Ubiad1 is an antioxidant enzyme that regulates eNOS activity through cytosolic CoQ10 synthesis. Mutation of Ubiad1 in zebrafish bar mutants leads to increased oxidative stress and cardiovascular failure.
2) Experiments show that Ubiad1 produces CoQ10 in a biosynthetic pathway, likely in the Golgi apparatus. Exogenous CoQ10 rescues bar mutants, while statins that block CoQ10 synthesis cause oxidative stress phenotypes.
3) Localization studies indicate Ubiad1 is located in the Golgi apparatus, where it produces a non-mitochondrial pool of CoQ10 that regulates eNOS activity and nitric oxide signaling.
1. Plasmids containing fragments of the yeast prion protein Sup35 and GFP were mutated using site-directed mutagenesis to incorporate the unnatural amino acid p-benzoyl-L-phenylalanine (pBpa) at specific tyrosine sites.
2. The plasmids were transformed into E. coli cells containing the machinery for pBpa incorporation, and protein expression was induced to produce Sup35-GFP fusions containing pBpa.
3. SDS-PAGE and fluorescence microscopy confirmed the expression of full-length and truncated fusion proteins, and that GFP fluorescence was maintained after mutagenesis, suggesting pBpa incorporation did not greatly perturb protein structure.
This document appears to be a midterm exam for a university course on biochemistry. It contains multiple choice and true/false questions about topics like glycolysis, gluconeogenesis, the citric acid cycle, and related metabolic pathways. It also asks students to fill in missing information about the glycolysis pathway and answer short questions about enzymatic deficiencies and how the utilization of glucose-6-phosphate is regulated.
This document summarizes the expression of recombinant β-lactoglobulin (rBLG) in prokaryotic and eukaryotic cells. In Escherichia coli, rBLG was expressed with a pET26 vector and was found predominantly in a denatured form, even when expressed in the periplasm. In eukaryotic cells like COS-7 and mouse tibialis muscle, rBLG was expressed in its native conformation. The authors quantified rBLG expression using immunoassays that distinguish between native and denatured rBLG. They found higher expression levels and native folding of rBLG in eukaryotic systems compared to prokaryotic expression
NQO1 modulates redox status and H2O2 levels in pancreatic β-cells. The study aimed to determine the role of NQO1 in β-cell health and metabolism. It was hypothesized that 1) NQO1 reduces oxidative stress in β-cells by lowering quinone-dependent H2O2 production and 2) NQO1 modulates the NAD(P)H-to-NAD(P)+ ratio in β-cells. Results showed that overexpressing NQO1 decreased menadione-dependent H2O2 production in INS-1 cells and isolated rodent islets, while NQO1 knockout increased H2O2 production. N
Taxol and 10-deacetylbaccatinIII induce distinct changesNeesar Ahmed
The document summarizes a study comparing the effects of Taxol, 10-deacetylbaccatinIII (10-DAB), and BaccatinIII (BacIII) on caveolae dynamics in HeLa cells expressing GFP-Caveolin-1. The key findings are:
1) Taxol treatment caused a transient recruitment of Caveolin-1 to the cell surface followed by internalization, while 10-DAB increased the "kiss and run" dynamics of caveolae.
2) Sustained Caveolin-1 phosphorylation was observed with both Taxol and 10-DAB treatments, with Taxol also inducing phosphorylation of Raf-1, ERK1/2,
System response of metabolic networks in Chlamydomonas reinhardtii during Nit...Nishikant Wase
Nitrogen starvation induces a global stress response in microalga that results in the accumulation of triglycerides in lipid bodies. To identify components and mechanisms leading to lipid accumulation during nitrogen stress, we used GC-MS based metabolite profiling and iTRAQ based quantitative proteomics to examine Chlamydomonas reinhardtii cultured up to 144 hours without nitrogen. When nitrogen is limiting, starch and lipid accumulated rapidly, with lipid becoming the major storage compound by 144 hours. Our FAMEs data showed that the percentage of highly unsaturated fatty acids was reduced and the percentage of saturated and monounsaturated fatty acids were increased. Using information from the GC-MS based metabolite profiling; a Partial Least Squares Discriminant Analysis model was created to evaluate the role of different intracellular metabolites during lipid accumulation. We observed decreased abundance of key amino acids whereas some important metabolites including citric acid, trehalose, triethanolamine, nicotinamine, methnionine, citramalic acid and sorbitol were increased in abundance. Addition of citric acid (from 4 mM to 6 mM) to the growth media significantly improves the lipid yield in Chlamydomonas reinhardtii while growing in TAP media containing nitrogen. Examination of differentially expressed proteins revealed that 100 of 793 identified proteins were induced after 144 hours, while 77 proteins were reduced in abundance. Proteins involved in nitrogen assimilation, oxidative phosphorylation, the glycolytic pathway, TCA cycle, starch, and lipid metabolism were found to be higher in abundance than in non-stressed cultures. Another effect of nitrogen starvation was reduction of proteins of the photosynthetic apparatus (including PS-I and PS-II) and light harvesting complex proteins. We conclude that during nitrogen starvation, carbon availability is the most important factor controlling oil biosynthesis and that there is carbon partitioning between starch and oil synthesis.
Set7/9 is a lysine methyltransferase that interacts with the transcription factor Pdx1. This study found that:
1) Set7/9 methylates two lysine residues (Lys-123 and Lys-131) on Pdx1. Methylation only occurred with full-length Pdx1, indicating structural requirements.
2) Lys-131 methylation by Set7/9 augments Pdx1's transcriptional activity in luciferase reporter assays.
3) Mice with beta-cell specific deletion of Set7/9 (SetΔβ mice) showed glucose intolerance and impaired insulin secretion from islets, similar to Pdx1-deficient mice. Target genes
- The document describes research into identifying and characterizing silaffin-like proteins (SFLPs) in the diatom Thalassiosira pseudonana.
- The researcher aimed to study the intracellular localization of four SFLPs (SFLP-25, -26, -63, and -28) by creating fusions with GFP and expressing them in T. pseudonana.
- Methods included constructing transformation vectors with each SFLP fused to GFP, transforming T. pseudonana via microparticle bombardment, and screening transformants for fluorescence to determine SFLP localization.
This document describes the identification of a putative human mitochondrial thymidine monophosphate kinase (TMPK2). The researchers cloned and characterized a cDNA encoding TMPK2 that contains a mitochondrial targeting sequence. They showed that TMPK2 localizes to mitochondria and overexpression increases cellular dTTP levels and the conversion of radioactive thymidine and dTMP to dTDP and dTTP in mitochondria. TMPK2 expression was detected in various tissues and cell lines, and was upregulated during monocyte/macrophage differentiation, suggesting its role in mitochondrial DNA synthesis during cellular differentiation.
This document summarizes research on engineering glycosylation of the Campylobacter jejuni protein OmpH1 for surface display in Gram-negative bacteria. Key points:
- C. jejuni naturally glycosylates OmpH1 at asparagine residue 139 via the pgl locus glycosylation machinery.
- Researchers introduced artificial glycosylation sites in OmpH1 and showed it could be glycosylated when expressed in E. coli or Salmonella containing the C. jejuni glycosylation machinery. Multi-glycosylated OmpH1 variants were produced.
- Glycosylation efficiency was highest for sites introduced in exposed loop regions of OmpH1's structure
This document summarizes research on the role of calcium and motility in sperm cell viability and function. It discusses how calcium regulates flagellar movement and capacitation processes like the acrosome reaction. Maintaining low motility through modulating calcium channels may increase sperm cell viability by reducing reactive oxygen species production from flagellar activity. The document also reviews various ion channels involved in calcium signaling in sperm and how environmental factors like temperature can impact sperm cell physiology and preservation.
Este documento compara la motilidad y viabilidad de espermatozoides de mamíferos y peces. Analiza parámetros como la motilidad, acrosoma, calcio intracelular y viabilidad de espermatozoides bajo diferentes condiciones como refrigeración, activación y envejecimiento. Los resultados muestran que la regulación del calcio intracelular es fundamental para el movimiento y función de los espermatozoides en estas especies.
N-acetyl-D-glucosamine kinase (NAGK) interacts with dynein light-chain roadblock type 1 (DYNLRB1) at dendritic branch points in neurons. Immunocytochemistry and proximity ligation assays showed colocalization of NAGK and DYNLRB1 on microtubule fibers at dendritic branches. NAGK was also found to interact with Golgi outposts and DYNLRB1 at branch points, indicating a tripartite interaction between NAGK, dynein, and Golgi that regulates dendritic growth and branching. Introduction of a peptide derived from DYNLRB1 stunted dendrite development in cultured neurons.
This study examined the secretion and intracellular trafficking of apolipoprotein A-V (apoA-V) using transfected cell lines. The key findings were:
1) Only about 20% of newly synthesized apoA-V was secreted from cells within 3 hours, with about 65% undergoing degradation before secretion.
2) When cells were treated with oleic acid to stimulate triglyceride synthesis, apoA-V secretion decreased while cell-associated apoA-V increased and moved to cytosolic lipid droplets.
3) Expression of apoA-V in hepatoma cells inhibited triglyceride secretion by 50% and reduced the diameter of secreted VLDL particles, but did not impact apolip
This document summarizes an experiment to engineer ascorbate peroxidase (APX) activity into cytochrome c peroxidase (CCP) by introducing the APX ascorbate-binding site into CCP. Specifically, the researchers replaced the ascorbate-binding loop and a critical arginine residue in CCP with the corresponding residues from APX to create a mutant called CCP2APX. While wild-type CCP showed no APX activity, CCP2APX was able to catalyze the peroxidation of ascorbate, demonstrating that the engineered ascorbate-binding site could bind ascorbate. Crystal structures of CCP2APX confirmed that the engineered binding site
This document discusses Polycomb group (PcG) proteins, which are important repressor proteins that regulate gene expression during development. It describes two main PcG complexes, PRC1 and PRC2, and their mechanisms of action, including histone modifications and chromatin compaction. The document also examines a case study on the interaction between the PcG protein LHP1 and deubiquitinating enzymes UBP12 and UBP13 in Arabidopsis thaliana. The study found that UBP12 and UBP13 interact with and help recruit LHP1 to target genes, and are involved in histone deubiquitination, which impacts gene silencing by PcG proteins.
Rational design of transition state analogue inhibitor for PvdQ enzyme.
PvdQ enzyme plays a key role in biosynthesis of siderophore pyoverdine in Pseudomonas aeruginosa and shows substrate preference for acyl chains 12-14 carbons. A series of alkylboronic acids were designed and found to be reversible, competitive inhibitors with picomolar affinity. X-ray crystal structure showed 1-tridecylboronic acid forms covalent bond with active site serine, mimicking substrate transition state. This inhibitor blocked bacterial growth in iron-limited conditions, reproducing genetic pvdQ disruption phenotype.
The document summarizes genetic and mutational characterization of the relV gene of Vibrio cholerae, which encodes a small alarmone synthetase protein called RelV. Key findings include:
1) Site-directed mutagenesis identified five amino acid residues (K107, D129, R132, L150, E188) in the RelA-SpoT domain of RelV that are essential for its (p)ppGpp synthetase activity.
2) Progressive deletion analysis determined the functional N-terminal boundary of RelV to be amino acid 59 and the C-terminal boundary to be amino acid 248, indicating that flanking sequences of the RelA-SpoT
The document discusses research on using phospholipase A2 (PLA2) antibodies to protect cells from radiation damage. PLA2 is an enzyme that releases fatty acids from phospholipids in cell membranes. Exposure to ionizing radiation activates PLA2, leading to cell death. The research proposal is to test whether antibodies that block PLA2 can inhibit this activation and thereby reduce the toxic effects of radiation exposure and increase radiation survival. Blocking PLA2 with antibodies may provide a new radiation protection strategy and tool for diagnosing and monitoring acute radiation sickness.
This study examined the interaction between the Abl kinase domain and the cancer drug Gleevec (imatinib) using site-directed mutagenesis to introduce a mutation (S417Y) associated with drug resistance. Researchers used PCR, protein purification techniques, and kinase assays to compare the inhibitory effect of Gleevec on wild-type Abl and the S417Y mutant. Their results supported the hypothesis that the S417Y mutation decreases Gleevec's ability to inhibit Abl kinase activity, though protein impurities limited the strength of the conclusions. The findings help explain why some cancers become resistant to Gleevec treatment.
1) Ubiad1 is an antioxidant enzyme that regulates eNOS activity through cytosolic CoQ10 synthesis. Mutation of Ubiad1 in zebrafish bar mutants leads to increased oxidative stress and cardiovascular failure.
2) Experiments show that Ubiad1 produces CoQ10 in a biosynthetic pathway, likely in the Golgi apparatus. Exogenous CoQ10 rescues bar mutants, while statins that block CoQ10 synthesis cause oxidative stress phenotypes.
3) Localization studies indicate Ubiad1 is located in the Golgi apparatus, where it produces a non-mitochondrial pool of CoQ10 that regulates eNOS activity and nitric oxide signaling.
1. Plasmids containing fragments of the yeast prion protein Sup35 and GFP were mutated using site-directed mutagenesis to incorporate the unnatural amino acid p-benzoyl-L-phenylalanine (pBpa) at specific tyrosine sites.
2. The plasmids were transformed into E. coli cells containing the machinery for pBpa incorporation, and protein expression was induced to produce Sup35-GFP fusions containing pBpa.
3. SDS-PAGE and fluorescence microscopy confirmed the expression of full-length and truncated fusion proteins, and that GFP fluorescence was maintained after mutagenesis, suggesting pBpa incorporation did not greatly perturb protein structure.
This document appears to be a midterm exam for a university course on biochemistry. It contains multiple choice and true/false questions about topics like glycolysis, gluconeogenesis, the citric acid cycle, and related metabolic pathways. It also asks students to fill in missing information about the glycolysis pathway and answer short questions about enzymatic deficiencies and how the utilization of glucose-6-phosphate is regulated.
This document summarizes the expression of recombinant β-lactoglobulin (rBLG) in prokaryotic and eukaryotic cells. In Escherichia coli, rBLG was expressed with a pET26 vector and was found predominantly in a denatured form, even when expressed in the periplasm. In eukaryotic cells like COS-7 and mouse tibialis muscle, rBLG was expressed in its native conformation. The authors quantified rBLG expression using immunoassays that distinguish between native and denatured rBLG. They found higher expression levels and native folding of rBLG in eukaryotic systems compared to prokaryotic expression
NQO1 modulates redox status and H2O2 levels in pancreatic β-cells. The study aimed to determine the role of NQO1 in β-cell health and metabolism. It was hypothesized that 1) NQO1 reduces oxidative stress in β-cells by lowering quinone-dependent H2O2 production and 2) NQO1 modulates the NAD(P)H-to-NAD(P)+ ratio in β-cells. Results showed that overexpressing NQO1 decreased menadione-dependent H2O2 production in INS-1 cells and isolated rodent islets, while NQO1 knockout increased H2O2 production. N
Taxol and 10-deacetylbaccatinIII induce distinct changesNeesar Ahmed
The document summarizes a study comparing the effects of Taxol, 10-deacetylbaccatinIII (10-DAB), and BaccatinIII (BacIII) on caveolae dynamics in HeLa cells expressing GFP-Caveolin-1. The key findings are:
1) Taxol treatment caused a transient recruitment of Caveolin-1 to the cell surface followed by internalization, while 10-DAB increased the "kiss and run" dynamics of caveolae.
2) Sustained Caveolin-1 phosphorylation was observed with both Taxol and 10-DAB treatments, with Taxol also inducing phosphorylation of Raf-1, ERK1/2,
System response of metabolic networks in Chlamydomonas reinhardtii during Nit...Nishikant Wase
Nitrogen starvation induces a global stress response in microalga that results in the accumulation of triglycerides in lipid bodies. To identify components and mechanisms leading to lipid accumulation during nitrogen stress, we used GC-MS based metabolite profiling and iTRAQ based quantitative proteomics to examine Chlamydomonas reinhardtii cultured up to 144 hours without nitrogen. When nitrogen is limiting, starch and lipid accumulated rapidly, with lipid becoming the major storage compound by 144 hours. Our FAMEs data showed that the percentage of highly unsaturated fatty acids was reduced and the percentage of saturated and monounsaturated fatty acids were increased. Using information from the GC-MS based metabolite profiling; a Partial Least Squares Discriminant Analysis model was created to evaluate the role of different intracellular metabolites during lipid accumulation. We observed decreased abundance of key amino acids whereas some important metabolites including citric acid, trehalose, triethanolamine, nicotinamine, methnionine, citramalic acid and sorbitol were increased in abundance. Addition of citric acid (from 4 mM to 6 mM) to the growth media significantly improves the lipid yield in Chlamydomonas reinhardtii while growing in TAP media containing nitrogen. Examination of differentially expressed proteins revealed that 100 of 793 identified proteins were induced after 144 hours, while 77 proteins were reduced in abundance. Proteins involved in nitrogen assimilation, oxidative phosphorylation, the glycolytic pathway, TCA cycle, starch, and lipid metabolism were found to be higher in abundance than in non-stressed cultures. Another effect of nitrogen starvation was reduction of proteins of the photosynthetic apparatus (including PS-I and PS-II) and light harvesting complex proteins. We conclude that during nitrogen starvation, carbon availability is the most important factor controlling oil biosynthesis and that there is carbon partitioning between starch and oil synthesis.
Set7/9 is a lysine methyltransferase that interacts with the transcription factor Pdx1. This study found that:
1) Set7/9 methylates two lysine residues (Lys-123 and Lys-131) on Pdx1. Methylation only occurred with full-length Pdx1, indicating structural requirements.
2) Lys-131 methylation by Set7/9 augments Pdx1's transcriptional activity in luciferase reporter assays.
3) Mice with beta-cell specific deletion of Set7/9 (SetΔβ mice) showed glucose intolerance and impaired insulin secretion from islets, similar to Pdx1-deficient mice. Target genes
- The document describes research into identifying and characterizing silaffin-like proteins (SFLPs) in the diatom Thalassiosira pseudonana.
- The researcher aimed to study the intracellular localization of four SFLPs (SFLP-25, -26, -63, and -28) by creating fusions with GFP and expressing them in T. pseudonana.
- Methods included constructing transformation vectors with each SFLP fused to GFP, transforming T. pseudonana via microparticle bombardment, and screening transformants for fluorescence to determine SFLP localization.
This document describes the identification of a putative human mitochondrial thymidine monophosphate kinase (TMPK2). The researchers cloned and characterized a cDNA encoding TMPK2 that contains a mitochondrial targeting sequence. They showed that TMPK2 localizes to mitochondria and overexpression increases cellular dTTP levels and the conversion of radioactive thymidine and dTMP to dTDP and dTTP in mitochondria. TMPK2 expression was detected in various tissues and cell lines, and was upregulated during monocyte/macrophage differentiation, suggesting its role in mitochondrial DNA synthesis during cellular differentiation.
This document summarizes research on engineering glycosylation of the Campylobacter jejuni protein OmpH1 for surface display in Gram-negative bacteria. Key points:
- C. jejuni naturally glycosylates OmpH1 at asparagine residue 139 via the pgl locus glycosylation machinery.
- Researchers introduced artificial glycosylation sites in OmpH1 and showed it could be glycosylated when expressed in E. coli or Salmonella containing the C. jejuni glycosylation machinery. Multi-glycosylated OmpH1 variants were produced.
- Glycosylation efficiency was highest for sites introduced in exposed loop regions of OmpH1's structure
This document summarizes research on the role of calcium and motility in sperm cell viability and function. It discusses how calcium regulates flagellar movement and capacitation processes like the acrosome reaction. Maintaining low motility through modulating calcium channels may increase sperm cell viability by reducing reactive oxygen species production from flagellar activity. The document also reviews various ion channels involved in calcium signaling in sperm and how environmental factors like temperature can impact sperm cell physiology and preservation.
Este documento compara la motilidad y viabilidad de espermatozoides de mamíferos y peces. Analiza parámetros como la motilidad, acrosoma, calcio intracelular y viabilidad de espermatozoides bajo diferentes condiciones como refrigeración, activación y envejecimiento. Los resultados muestran que la regulación del calcio intracelular es fundamental para el movimiento y función de los espermatozoides en estas especies.
Algo nuevo, algo viejo y algo prestado en alzheimer 2014 jparodiJorge Parodi
Este documento presenta una revisión de diferentes enfoques para comprender la enfermedad de Alzheimer, incluyendo la hipótesis del "poro amiloide", estudios sobre el efecto del etanol en agregados de beta-amiloide, y experimentos en ovocitos de rana para analizar la actividad sináptica. También resume diferentes estudios sobre los mecanismos por los cuales los agregados de beta-amiloide podrían inducir disfunción sináptica y neurodegeneración.
Este documento compara la motilidad y viabilidad de espermatozoides de mamíferos y peces usando diferentes modelos. Analiza parámetros como la motilidad, acrosoma, ondas de calcio y viabilidad de espermatozoides bajo diferentes condiciones como refrigeración, activación y envejecimiento. También estudia el efecto de xenobióticos como el didymo en estos parámetros. Los resultados sugieren que la regulación del calcio intracelular es importante para el funcionamiento normal de los espermatozoides.
Este documento presenta una revisión de diferentes enfoques para comprender la enfermedad de Alzheimer, incluyendo la hipótesis del "poro amiloide", estudios sobre el efecto del etanol en agregados de beta-amiloide, y experimentos en ovocitos de rana para analizar la actividad sináptica. También resume diferentes estudios sobre los mecanismos por los cuales los agregados de beta-amiloide podrían inducir disfunción sináptica y neurodegeneración.
Glucose transport into cells is mediated by glucose transporter (GLUT) proteins. [1] There are five main GLUT transporters that are involved in glucose transport and each has a distinct tissue distribution and function. [2] GLUT transporters use a flip-flop mechanism to transport glucose across the cell membrane according to the concentration gradient. [3] Insulin regulates glucose transport by stimulating the translocation of GLUT4 and GLUT1 transporters from intracellular vesicles to the cell membrane, increasing the influx of glucose into cells.
Sodium glucose co transporter( SGLT2) Inhibitors Philip Vaidyan
This document discusses sodium-glucose co-transporter 2 (SGLT2) inhibitors, a new class of drugs for treating type 2 diabetes. SGLT2 inhibitors work by blocking glucose reabsorption in the kidney, increasing urinary glucose excretion in a glucose-dependent manner. Three SGLT2 inhibitors have been approved by the FDA - canagliflozin, dapagliflozin, and empagliflozin. SGLT2 inhibitors offer advantages over other anti-diabetic drugs in that they are non-insulin dependent and can be used throughout the course of diabetes. However, long-term safety studies are still needed to fully assess their risk-benefit profile.
Mutations that reduce outer membrane permeability in Escherichia coli lead to increased tolerance of the bacterium to the antimicrobial lactoperoxidase enzyme system. The study identified two E. coli mutants with increased tolerance to lactoperoxidase due to mutations in genes involved in lipopolysaccharide synthesis. These mutants had reduced amounts of porins in their outer membrane, indicating decreased outer membrane permeability. Knockout mutants of specific porin genes also showed increased tolerance, suggesting the antimicrobial activity of lactoperoxidase relies on uptake of its oxidized substrates through porins.
Translational Control of Autism Spectrum Disorders in Eif4ebp2 knockout Mouse...Rey Christian Pacis
1) NKCC1, a chloride importer implicated in autism, is upregulated in Eif4ebp2 knockout mice compared to wildtype. This may cause excitatory-inhibitory imbalances linked to autism.
2) Phosphorylated forms of eIF4E, mTOR, and ERK are also upregulated in knockouts, and may be important in translational control of NKCC1. Total eIF4E is downregulated.
3) Total levels of mTOR, ERK, and Akt, as well as phosphorylated Akt, show trends of change but are not significantly different between knockout and wildtype mice.
NQO1, a cytosolic oxidoreductase, plays an important role in maintaining proper redox status and insulin secretion in pancreatic β-cells. The study found that in insulin-secreting cells and isolated rodent islets, overexpressing NQO1 decreased reactive oxygen species production from quinones and the NAD(P)H/NAD(P)+ ratio, while NQO1 knockout cells had increased reactive oxygen species formation. This suggests NQO1 protects β-cells from oxidative stress by facilitating the full, two-electron reduction of quinones.
1) The study investigated the effects of dopamine (DA), dihydroxyphenylacetaldehyde (DOPAL), and dihydroxyphenylacetic acid (DOPAC) on oligomerization of wild-type α-synuclein and mutated forms (A53T, A30P).
2) DOPAL was found to potently oligomerize α-synuclein, around 10 times more than DA. DOPAC had little effect.
3) DOPAL also oligomerized the mutated forms of α-synuclein and appeared to aggregate the A53T form to the point that it did not migrate in gels.
- A series of point mutations were made to NPC2 to neutralize charged residues on its surface. Kinetic studies of these mutants showed that certain residues are necessary for NPC2's cholesterol transport function.
- Turbidity assays suggested that NPC2 may bind to more than one membrane simultaneously. This led to the hypothesis that NPC2 transports cholesterol by forming inter-membrane bridges between the inner endo/lysosomal membranes and other membranes, facilitating cholesterol egress from the compartment.
- Methods like tryptophan fluorescence quenching and resonance energy transfer were used to quantify sterol transfer rates of NPC2 and its mutants between membranes. Preliminary results support the hypothesis that NPC2 can bind two membranes at once to
This study investigated the effects of antioxidants such as alpha-lipoic acid, vitamin C, and resveratrol on the overexpression of cytochrome P450 2E1 in streptozotocin-induced diabetic rat tissues. Real-time PCR and RT-PCR analysis showed that administration of vitamin C to diabetic rats had the most significant effect in reducing CYP2E1 overexpression, while administration of vitamin C together with alpha-lipoic acid caused the second most decrease. Administration of alpha-lipoic acid alone resulted in lower reduction of CYP2E1 expression compared to vitamin C and the combination of vitamins.
This document summarizes a study that developed a simplified method for purifying the Thermus aquaticus (Taq) DNA polymerase expressed in Escherichia coli. Key steps included:
1) Overproducing the Taq DNA polymerase in E. coli by cloning the Taq gene into an expression vector and inducing high-level expression.
2) Lysing the E. coli cells and heat-treating the lysate to inactivate endogenous nucleases.
3) Precipitating the Taq polymerase from the lysate using polyethyleneimine and eluting it from an ion exchange resin.
4) Obtaining a purified Taq polymerase preparation in a single-day procedure yielding 40-60 mg of
This document summarizes Richard Rodriguez's final lab report on analyzing the L34P mutation in the gap junction protein Cx31 using site-directed mutagenesis. Key findings include:
1) The L34P mutation is associated with Erythrokeratoderma vairabilis (EKV) skin disorder and causes the disease through a recessive inheritance pattern by preventing formation of normal gap junctions between keratinocytes.
2) Site-directed mutagenesis was used to introduce the L34P mutation into a Cx31 plasmid, which was then transformed into E. coli cells. Over 200 colonies grew on each transformation plate.
3) The mutated Cx31 plasmid will be used
Signal transducing machinery as targets for potential drugs.
Drugs:-
a). Diclofenac- for treating cholera toxin
b). Fasentin- for treating insulin signalling
- A single episomal plasmid containing Oct4, Sox2, KLF4, L-Myc driven by a hybrid CAG promoter can reprogram human fibroblasts into induced pluripotent stem cells (iPSCs).
- The inclusion of 5 small molecules that enhance reprogramming results in the appearance of proto-colonies by 7 days and mature colonies by 21 days under low oxygen conditions.
- The iPSCs express pluripotency markers, can differentiate into neurons and cardiomyocytes, and grow robustly in a new xeno-free media. Further work will examine residual vector presence and gene expression profiles.
Characterization of the human HCN1 channel and its inhibition by capsazepineShahnaz Yusaf
1. The human hyperpolarization-activated cyclic nucleotide-gated 1 (hHCN1) subunit was expressed in mammalian cell lines and its electrophysiological properties were characterized using patch-clamp recordings.
2. Activation of hHCN1 generated a slowly activating, non-inactivating inward current similar to native hyperpolarization-activated currents (Ih). Ih was blocked by known blockers Cs+, ZD 7288, and zatebradine.
3. The VR1 receptor antagonist capsazepine inhibited hHCN1-mediated currents in a concentration-dependent, reversible manner by shifting the activation curve and slowing current activation kinetics.
- The study characterized the kinetic properties of wild-type human Δ1-pyrroline-5-carboxylate reductase 2 (HsPYCR2) and its R251C mutant found in patients.
- Kinetic analysis showed the R251C mutant had slightly lower catalytic efficiency (4-5 fold) for NADH and NADPH substrates compared to wild-type, suggesting the mutation impacts proline biosynthesis.
- Additional studies on protein structure may help understand how the R251C mutation contributes to neurological disease phenotypes seen in patients.
1. Second messengers are small intracellular molecules that transmit signals within cells after extracellular signaling molecules (hormones or neurotransmitters) bind to cell surface receptors.
2. There are three main types of second messenger systems: cyclic AMP (cAMP), cyclic GMP (cGMP), and inositol trisphosphate (IP3)/diacylglycerol (DAG). These systems activate protein kinases or trigger the release of calcium ions to produce a physiological response.
3. Second messengers amplify and diversify extracellular signals, allowing for precise regulation of multiple cellular processes. Their roles are important for understanding cell signaling, disease mechanisms, and potential drug targets.
This document discusses secondary messengers, which are intracellular signaling molecules that transmit signals inside a cell in response to primary messengers binding to cell surface receptors. There are several types of secondary messengers, including hydrophilic messengers like cAMP and cGMP, hydrophobic messengers like diacylglycerol, and gaseous messengers like nitric oxide. The document describes key secondary messengers like cAMP, cGMP, IP3, calcium ions, nitric oxide, and diacylglycerol in detail and explains their roles in cellular signaling pathways. Secondary messengers work by amplifying and relaying signals from cell surface receptors to target proteins and genes inside the cell.
This summary provides the key points from the document in 3 sentences:
This study explored how sulforaphane affects calcium levels and cell viability in renal tubular cells. The results showed that sulforaphane increased calcium levels in the cells in a concentration-dependent manner by promoting calcium entry from outside the cell rather than calcium release from internal stores. Sulforaphane also decreased cell viability in a concentration-dependent manner, which was independent of changes in calcium levels and may have involved the induction of apoptosis.
The document discusses using the Burmese python as a model to study lipid homeostasis. After eating, pythons show a doubling in size of major organs and a 52-fold increase in triglycerides without signs of lipotoxicity. This suggests a more efficient mechanism of lipid homeostasis than humans. The nuclear receptor PPARα is identified as a master regulator of lipid homeostasis and is the gene of interest. Methods are described to isolate RNA from python liver, synthesize cDNA, validate and test primers for PPARα and other genes, and perform quantitative real-time PCR to analyze gene expression levels at different time points after feeding.
This study characterized the function of ALL2, a homolog of ALL1, in the fungal pathogen Cryptococcus neoformans. The key findings were:
1) Unlike deletion of ALL1, deletion of ALL2 attenuated virulence in a pulmonary infection model.
2) The all2Δ mutant shed a less viscous exopolysaccharide and was more sensitive to hydrogen peroxide but more resistant to macrophage killing than the wild type.
3) Transcriptome analysis supported distinct functions for ALL1 and ALL2, with ALL2 uniquely involved in maintaining intracellular pH under low-pH conditions.
This document describes a study investigating changes in acid sphingomyelinase (ASM) activity levels in Niemann-Pick disease (NPD) and the effects of cyclodextrin (CD) treatment. The study aims to determine if reduced ASM activity in human NPD cells is due to decreased levels of SMPD1 mRNA, which encodes ASM, and if CD treatment alters ASM activity in hamster cells by changing SMPD1 mRNA levels. Primers were designed and tested to quantify SMPD1 mRNA levels in CD-treated hamster cells and human wildtype and NPD cells using quantitative RT-PCR. Initial results showed the primers perform efficiently between 59.9-
This document summarizes a study investigating the epigenetic changes that occur during the transdifferentiation of pancreatic acinar cells (HPACs) to hepatocyte-like cells. The study found that DNA methylation in HPACs peaks at 24 hours after treatment with dexamethasone, and the cells display phenotypic changes associated with hepatocytes after 7 days. The study also examined the promoter sequence of the SGK1 gene, which is involved in the transdifferentiation process. The results suggest therapeutic potential for producing hepatocytes from pancreatic cells to treat liver disease in a more cost-effective manner than current alternatives.
This document discusses the role of NQO1, an NAD(P)H-dependent oxidoreductase, in pancreatic beta cells. The study found that NQO1 protects beta cells from oxidative stress by reducing quinone-dependent reactive oxygen species production. It also found that NQO1 lowers the NADH-to-NAD+ ratio in beta cells, indicating it modulates cellular redox status. Overexpression of NQO1 decreased hydrogen peroxide levels while knockout of NQO1 increased hydrogen peroxide levels in beta cells exposed to oxidative stress. Therefore, NQO1 enhances beta cell health and metabolism by protecting from oxidative damage and regulating redox cycling.
This document discusses the development of an ex vivo model to study the effects of Didymosphenia geminata, commonly known as rock snot, on freshwater fish cells and gametes. It first reviews D. geminata as an invasive nuisance species found in rivers internationally. It then describes preliminary work maintaining D. geminata in an artificial river system and observing effects on cell lines. The document proposes building upon this to explore chronic effects of contaminated water and extracts on cells. It also reviews maintaining D. geminata viability in the lab and the need for a complete river model to better understand its growth requirements and impacts on aquatic systems.
1) The study developed a low-cost screening system using freely available ImageJ software to analyze video recordings of the swimming behavior and kinetics of different life stages of the sea louse Caligus rogercresseyi exposed to potential drug compounds.
2) Results showed that taurine, glutamate, GABA, and iodine all impacted the motility of C. rogercresseyi planktonic forms in a time- and dose-dependent manner, with iodine having the strongest inhibiting effects.
3) The system provides a way to efficiently and inexpensively study the pharmacological effects of potential treatments on C. rogercresseyi before conducting more complex in vitro
Tipos de modelos de cultivos celulares iiJorge Parodi
Este documento describe los diferentes tipos de modelos de cultivos celulares, incluyendo cultivos primarios, subcultivos, líneas celulares, explantes, cultivos de órganos, cultivos organotípicos y cultivos tridimensionales. Explica que cada modelo mantiene algunas propiedades fisiológicas y morfológicas pero pierde propiedades estructurales con el tiempo, y que los modelos más avanzados como los cultivos organotípicos requieren múltiples tipos de células. También resume algunas aplicaciones generales
Dolores de la industria salmonicultura en chile vJorge Parodi
El documento discute varias enfermedades que afectan a la salmonicultura en Chile. La septicemia rickettsial salmonidea (SRS), causada por la bacteria Piscirickettsia salmonis, es el principal problema y se transmite horizontalmente. Otras enfermedades importantes son la enfermedad del riñón del salmón y la tenacibaculosis. Se ha observado una disminución de SRS del 30% en 2018, pero ha aumentado la caligidosis. Más investigación es necesaria sobre la patología y transmisión de la tenacibaculosis.
IDN5706 is a semi-synthetic derivative of hyperforin that has neuroprotective effects. The document reports on experiments examining the effects of IDN5706 on field excitatory postsynaptic potentials (fEPSPs) in mouse hippocampal slices. The results suggest that IDN5706 activates TRPC3/6/7 channels, improving synaptic response and protecting against reductions in fEPSPs caused by amyloid beta oligomers. IDN5706 also improved spatial memory in mice, and this effect was blocked by a TRPC channel blocker. Computational modeling further supported the idea that IDN5706 activates TRPC channels.
The document discusses reactive oxygen species (ROS) and their effects on boar sperm physiology. It notes that ROS can negatively impact sperm motility, viability, and fertilization potential by damaging lipids, proteins, DNA, and inducing premature capacitation. This is detrimental to sperm quality and lifespan. The document also examines the use of seminal fluid diluents for short or long-term sperm preservation. Diluents aim to maintain sperm concentration and viability during storage and transport. However, ROS levels may still increase and damage sperm over time. The inclusion of macromolecules that stabilize sperm membranes could help protect sperm from ROS effects and better maintain motility and function during preservation.
This document summarizes research on sperm cell motility, viability, and calcium regulation. It discusses how sperm motility is regulated by calcium levels, which are controlled by ion channels such as CATsper. Modulating calcium channels and reducing motility may increase sperm cell viability by decreasing reactive oxygen species production. The document also reviews the roles of factors like temperature, membrane potential, and ion currents in modulating calcium concentrations and sperm cell functions like capacitation and the acrosome reaction.
Este documento describe un estudio in vitro de Didymo realizado en el Laboratorio de Fisiología de la Reproducción de la Universidad Católica de Temuco. El estudio utilizó microscopía de campo claro, Nomarsky y confocal para observar Didymo en condiciones de control a 38°C y después de 30 minutos, 24 horas, congelación y almacenamiento seco durante 1 y 2 meses. El resumen discute la viabilidad de Didymo basada en la presencia de granulos llenos o vacíos y propone el uso de un protocolo
1) A study investigated the vasodilatory and toxic effects of a crude extract of Ruta graveolens (Ruta) on rat aortas and CRL1730 endothelial cells.
2) The Ruta extract generated vasodilation in rat aortas at subtoxic concentrations, partially dependent on the endothelium. It caused a loss of cell viability in CRL1730 cells at high concentrations but did not induce oxidative stress or DNA fragmentation.
3) The results suggest Ruta extract regulates vascular tone through a complex, partially endothelium-dependent mechanism and has vasodilatory activity at subtoxic levels without damaging cell membranes or viability.
The document summarizes research on the effects of Wnt ligands on synaptic plasticity. It discusses how Wnt-5a modulates potassium channel activity and miniature synaptic currents. Wnt-5a increases AMPA miniature currents but does not affect NMDA miniature currents. Wnt-5a enhances long-term potentiation induced by theta burst stimulation in the hippocampus in a manner dependent on calcium influx and nitric oxide signaling. Wnt-5a also increases neuronal viability and mitochondrial ATP production. The findings suggest Wnt ligands play complex roles in synaptic plasticity.
1) Xenopus laevis oocytes exposed to amyloid-β aggregate developed oscillatory electric activity (blips) recorded via two-microelectrode voltage clamp.
2) The blips increased in amplitude over time from 3.8 nA initially to 6.8 nA after 15 minutes of amyloid-β aggregate exposure, similar to the effects of channel-forming agents amphotericin B and gramicidin.
3) The amyloid-β aggregate-induced currents were dependent on extracellular calcium and disrupted calcium-dependent currents between oocytes and surrounding follicular cells. Electron microscopy also revealed dissociation of follicular cells from the oocytes.
The document discusses alternative treatments and therapies for Alzheimer's disease, including herbal medicines, supplements, and clinical trials. It summarizes several studies that tested supplements like ginkgo biloba, omega-3 fatty acids, and phosphatidylserine against placebos but found no reduction in cognitive decline. Another study showed that continued treatment with donepezil provided cognitive and functional benefits over 12 months for patients with moderate to severe Alzheimer's. The document acknowledges further research is still needed to understand alternative treatments for Alzheimer's disease.
El documento resume la investigación sobre los canales iónicos en los espermatozoides, destacando que el canal CatSper no es el único relevante y que existen otros canales iónicos como canales de potasio y calcio que también juegan un papel importante. El documento también discute cómo estos canales modulan el movimiento de calcio intracelular y la capacitación de los espermatozoides, y proporciona algunos ejemplos de estudios que han investigado el efecto de bloquear diferentes canales. Finalmente, reconoce que queda mucho por aprender sobre
Este documento resume las actividades del Laboratorio de Fisiología de la Reproducción dirigido por Jorge Parodi. El laboratorio realiza estudios de electrofisiología y morfofuncionales en espermatozoides, incluyendo la medición del calcio intracelular. También evalúa el efecto de diferentes condiciones ambientales y tratamientos en parámetros como el tamaño y perímetro de ovocitos de salmón.
Este documento resume investigaciones sobre los mecanismos subyacentes a la enfermedad de Alzheimer. Describe cómo las placas amiloides causan daño sináptico al modular canales iónicos y receptores, lo que conduce a la pérdida de masa cerebral y cambios cognitivos. También explora posibles terapias, incluido el bloqueo del poro amiloide y el uso de compuestos como el etanol para prevenir la agregación amiloide y proteger la función sináptica.
Tetrahydrohyperforin (IDN5706), a derivative of the active molecule hyperforin in St. John's Wort, was examined for its ability to prevent cognitive deficits and synaptic impairment in an Alzheimer's disease mouse model. Five-month old APPswe/PSEN1DE9 mice were treated with IDN5706 for 10 weeks. IDN5706 improved memory, prevented decreases in synaptic proteins and LTP, and reduced amyloid-beta plaque burden, tau hyperphosphorylation, and astrogliosis. In cell cultures, IDN5706 decreased amyloid precursor protein processing leading to amyloid-beta peptide generation. The results suggest IDN5706 may be a potential therapeutic for treating Alzheimer's
In tech perforated-patch_clamp_in_non_neuronal_cells_the_model_of_mammalian_s...Jorge Parodi
This document outlines the steps to perform perforated patch-clamp recordings on mammalian sperm cells. It describes how perforated patch-clamp allows for better preservation of the intracellular environment compared to whole-cell recordings. The key steps include selecting motile sperm cells using a swim-up method, incubating the cells in a hyposmotic solution, and applying nystatin or other drugs in the recording pipette to gradually form holes in the plasma membrane. With practice, tight seals of over 1 GΩ can be obtained in about 45% of attempts, allowing recordings of ion channel currents in the sperm cell membrane.
El documento lista varias obras de arte famosas y sus creadores, incluyendo estatuas como "El David" de Donatello y Miguel Ángel, pinturas como "La creación de Adán" de Miguel Ángel, "Noche estrellada" de Van Gogh y "El Pifano" de Monet, y retratos como "Oscar Wilde" de Toulouse-Lautrec, "Pasteur" de Albert Edelfelt, y "Retrato de Darwin" de John Collier. También menciona a autores importantes como Dante, Maquiavelo y Einstein.
Este documento discute el derecho a la educación en Chile según la constitución de 1980 y cómo la responsabilidad de la educación recae principalmente en los padres. También describe brevemente la historia de la educación en Chile desde la creación de la Universidad de Chile en 1842 y cómo la constitución de 1925 separó la iglesia del estado, haciendo al estado responsable de la educación. Finalmente, analiza las condiciones actuales que llevaron al movimiento estudiantil chileno, incluyendo el aumento del costo de la educación en relación con los su
1. This study examined the effects of insulin on adenosine transport in human umbilical artery smooth muscle cells from normal pregnancies and those with gestational diabetes.
2. Insulin increased adenosine transport in cells from normal pregnancies by increasing nitric oxide and cGMP levels. However, insulin decreased the already elevated adenosine transport in cells from diabetic pregnancies by activating adenylyl cyclase.
3. The results suggest that insulin differentially modulates adenosine transport in these cell types, and that adenosine signaling may be altered under conditions of sustained hyperglycemia in diabetes.
In the realm of cybersecurity, offensive security practices act as a critical shield. By simulating real-world attacks in a controlled environment, these techniques expose vulnerabilities before malicious actors can exploit them. This proactive approach allows manufacturers to identify and fix weaknesses, significantly enhancing system security.
This presentation delves into the development of a system designed to mimic Galileo's Open Service signal using software-defined radio (SDR) technology. We'll begin with a foundational overview of both Global Navigation Satellite Systems (GNSS) and the intricacies of digital signal processing.
The presentation culminates in a live demonstration. We'll showcase the manipulation of Galileo's Open Service pilot signal, simulating an attack on various software and hardware systems. This practical demonstration serves to highlight the potential consequences of unaddressed vulnerabilities, emphasizing the importance of offensive security practices in safeguarding critical infrastructure.
Best 20 SEO Techniques To Improve Website Visibility In SERPPixlogix Infotech
Boost your website's visibility with proven SEO techniques! Our latest blog dives into essential strategies to enhance your online presence, increase traffic, and rank higher on search engines. From keyword optimization to quality content creation, learn how to make your site stand out in the crowded digital landscape. Discover actionable tips and expert insights to elevate your SEO game.
HCL Notes und Domino Lizenzkostenreduzierung in der Welt von DLAUpanagenda
Webinar Recording: https://www.panagenda.com/webinars/hcl-notes-und-domino-lizenzkostenreduzierung-in-der-welt-von-dlau/
DLAU und die Lizenzen nach dem CCB- und CCX-Modell sind für viele in der HCL-Community seit letztem Jahr ein heißes Thema. Als Notes- oder Domino-Kunde haben Sie vielleicht mit unerwartet hohen Benutzerzahlen und Lizenzgebühren zu kämpfen. Sie fragen sich vielleicht, wie diese neue Art der Lizenzierung funktioniert und welchen Nutzen sie Ihnen bringt. Vor allem wollen Sie sicherlich Ihr Budget einhalten und Kosten sparen, wo immer möglich. Das verstehen wir und wir möchten Ihnen dabei helfen!
Wir erklären Ihnen, wie Sie häufige Konfigurationsprobleme lösen können, die dazu führen können, dass mehr Benutzer gezählt werden als nötig, und wie Sie überflüssige oder ungenutzte Konten identifizieren und entfernen können, um Geld zu sparen. Es gibt auch einige Ansätze, die zu unnötigen Ausgaben führen können, z. B. wenn ein Personendokument anstelle eines Mail-Ins für geteilte Mailboxen verwendet wird. Wir zeigen Ihnen solche Fälle und deren Lösungen. Und natürlich erklären wir Ihnen das neue Lizenzmodell.
Nehmen Sie an diesem Webinar teil, bei dem HCL-Ambassador Marc Thomas und Gastredner Franz Walder Ihnen diese neue Welt näherbringen. Es vermittelt Ihnen die Tools und das Know-how, um den Überblick zu bewahren. Sie werden in der Lage sein, Ihre Kosten durch eine optimierte Domino-Konfiguration zu reduzieren und auch in Zukunft gering zu halten.
Diese Themen werden behandelt
- Reduzierung der Lizenzkosten durch Auffinden und Beheben von Fehlkonfigurationen und überflüssigen Konten
- Wie funktionieren CCB- und CCX-Lizenzen wirklich?
- Verstehen des DLAU-Tools und wie man es am besten nutzt
- Tipps für häufige Problembereiche, wie z. B. Team-Postfächer, Funktions-/Testbenutzer usw.
- Praxisbeispiele und Best Practices zum sofortigen Umsetzen
For the full video of this presentation, please visit: https://www.edge-ai-vision.com/2024/06/temporal-event-neural-networks-a-more-efficient-alternative-to-the-transformer-a-presentation-from-brainchip/
Chris Jones, Director of Product Management at BrainChip , presents the “Temporal Event Neural Networks: A More Efficient Alternative to the Transformer” tutorial at the May 2024 Embedded Vision Summit.
The expansion of AI services necessitates enhanced computational capabilities on edge devices. Temporal Event Neural Networks (TENNs), developed by BrainChip, represent a novel and highly efficient state-space network. TENNs demonstrate exceptional proficiency in handling multi-dimensional streaming data, facilitating advancements in object detection, action recognition, speech enhancement and language model/sequence generation. Through the utilization of polynomial-based continuous convolutions, TENNs streamline models, expedite training processes and significantly diminish memory requirements, achieving notable reductions of up to 50x in parameters and 5,000x in energy consumption compared to prevailing methodologies like transformers.
Integration with BrainChip’s Akida neuromorphic hardware IP further enhances TENNs’ capabilities, enabling the realization of highly capable, portable and passively cooled edge devices. This presentation delves into the technical innovations underlying TENNs, presents real-world benchmarks, and elucidates how this cutting-edge approach is positioned to revolutionize edge AI across diverse applications.
Programming Foundation Models with DSPy - Meetup SlidesZilliz
Prompting language models is hard, while programming language models is easy. In this talk, I will discuss the state-of-the-art framework DSPy for programming foundation models with its powerful optimizers and runtime constraint system.
Fueling AI with Great Data with Airbyte WebinarZilliz
This talk will focus on how to collect data from a variety of sources, leveraging this data for RAG and other GenAI use cases, and finally charting your course to productionalization.
Trusted Execution Environment for Decentralized Process MiningLucaBarbaro3
Presentation of the paper "Trusted Execution Environment for Decentralized Process Mining" given during the CAiSE 2024 Conference in Cyprus on June 7, 2024.
A Comprehensive Guide to DeFi Development Services in 2024Intelisync
DeFi represents a paradigm shift in the financial industry. Instead of relying on traditional, centralized institutions like banks, DeFi leverages blockchain technology to create a decentralized network of financial services. This means that financial transactions can occur directly between parties, without intermediaries, using smart contracts on platforms like Ethereum.
In 2024, we are witnessing an explosion of new DeFi projects and protocols, each pushing the boundaries of what’s possible in finance.
In summary, DeFi in 2024 is not just a trend; it’s a revolution that democratizes finance, enhances security and transparency, and fosters continuous innovation. As we proceed through this presentation, we'll explore the various components and services of DeFi in detail, shedding light on how they are transforming the financial landscape.
At Intelisync, we specialize in providing comprehensive DeFi development services tailored to meet the unique needs of our clients. From smart contract development to dApp creation and security audits, we ensure that your DeFi project is built with innovation, security, and scalability in mind. Trust Intelisync to guide you through the intricate landscape of decentralized finance and unlock the full potential of blockchain technology.
Ready to take your DeFi project to the next level? Partner with Intelisync for expert DeFi development services today!
Introduction of Cybersecurity with OSS at Code Europe 2024Hiroshi SHIBATA
I develop the Ruby programming language, RubyGems, and Bundler, which are package managers for Ruby. Today, I will introduce how to enhance the security of your application using open-source software (OSS) examples from Ruby and RubyGems.
The first topic is CVE (Common Vulnerabilities and Exposures). I have published CVEs many times. But what exactly is a CVE? I'll provide a basic understanding of CVEs and explain how to detect and handle vulnerabilities in OSS.
Next, let's discuss package managers. Package managers play a critical role in the OSS ecosystem. I'll explain how to manage library dependencies in your application.
I'll share insights into how the Ruby and RubyGems core team works to keep our ecosystem safe. By the end of this talk, you'll have a better understanding of how to safeguard your code.
Your One-Stop Shop for Python Success: Top 10 US Python Development Providersakankshawande
Simplify your search for a reliable Python development partner! This list presents the top 10 trusted US providers offering comprehensive Python development services, ensuring your project's success from conception to completion.
GraphRAG for Life Science to increase LLM accuracyTomaz Bratanic
GraphRAG for life science domain, where you retriever information from biomedical knowledge graphs using LLMs to increase the accuracy and performance of generated answers
Taking AI to the Next Level in Manufacturing.pdfssuserfac0301
Read Taking AI to the Next Level in Manufacturing to gain insights on AI adoption in the manufacturing industry, such as:
1. How quickly AI is being implemented in manufacturing.
2. Which barriers stand in the way of AI adoption.
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2. Flores et al D-Glucose Acutely Activates L-Arginine/NO Pathway 65
L-Arginine Transport Intracellular Ca2
L-Arginine transport (1 Ci/mL, 37°C, 1 minute) was determined in Cells on glass coverslips were loaded (30 minutes, 23°C) with the
cells preincubated (15 seconds to 5 minutes) with Krebs solution acetoxymethyl derivative of fluo 3 (5 mol/L). Coverslips were
(mmol/L: NaCl 131, KCl 5.6, NaHCO3 25, NaH2PO4 1, HEPES 20, transferred to an experimental bath with Krebs solution containing 5
CaCl2 2.5, and MgCl2 1 [pH 7.4, 37°C]) containing 5 or 25 mmol/L or 25 mmol/L D-glucose, and Ca2 was imaged using a Zeiss LSM
D-glucose, 25 mmol/L L-glucose, or 5 mmol/L D-glucose plus 410 confocal microscope.4
20 mmol/L D-mannitol (osmotic controls).2– 4 L-Arginine transport
was also determined in Krebs solution in which NaCl was replaced Western Blots
by equimolar concentrations of choline chloride2– 4 or in cells After pretreatment with 10 mol/L PD-98059 (30 minutes), 100
incubated (30 minutes) with KCl (5.5 to 131 mmol/L), with NaCl mol/L SNAP (2 to 5 minutes), or 30 nmol/L wortmannin (30
decreased equivalently, or with 131 mmol/L KCl for 2, 4, 10, 20, or minutes), the cells were incubated with 5 or 25 mmol/L D-glucose (2
30 minutes. In trans-stimulation experiments, cells were preincu- minutes). Cell protein extracts were probed with a primary poly-
bated (2 hours) with primary culture medium containing 10 mmol/L clonal mouse antiphosphorylated (1:1000) or nonphosphorylated
L-lysine. Cell-associated radioactivity and data analyses were per-
(1:1500) p44/p42mapk, rabbit anti-eNOS (1:2500) or anti-phosphory-
formed as described.2– 4 lated Ser1177– eNOS (1:2500) antibodies, and horseradish peroxidase–
conjugated goat secondary antibodies as described.3,5 Primary poly-
L-Arginine transport was assayed in cells preincubated (30 min-
clonal mouse anti-actin (1:2000) served as the internal control.
utes) with 100 mol/L NG-nitro-L-arginine methyl ester (L-NAME,
Proteins were detected by enhanced chemiluminescence and quan-
eNOS inhibitor),2,3 10 mol/L PD-98059 (MAP kinase kinase 1/2
tified by densitometry (Ultroscan XL enhanced laser densitometer,
[MEK1/2] inhibitor), 19 100 mol/L S-nitroso-N-acetyl- L , D - LKB Instruments).3,5
penicillamine (SNAP, NO donor), 1 mol/L KT-5823 (protein
kinase G [PKG] inhibitor),20 100 nmol/L calphostin C (PKC inhib-
Semiquantitative PCR
itor),21 10 mol/L glibenclamide (ATP-sensitive K [K ATP] channel
Extracted mRNA (Dynal) was reversed-transcribed into cDNA using
blocker),22 1 mol/L levcromakalim (K ATP activator),23 or 30
oligo(dT18) plus random hexamers (10-mer) and M-MLV reverse
nmol/L wortmannin (PI3-k inhibitor).24 transcriptase (Promega) for 1 hour at 37°C.3 Polymerase chain
reactions (PCRs) were performed in 20- L samples (2 L of 10
Membrane Potential PCR buffer, 0.8 L of 50 mmol/L Mg2 , 0.4 L dNTPs, 13.6 L
[3H]Tetraphenylphosphonium ([3H]TPP ) influx (46 nmol/L, 0.5 RNase-free H2O, 0.2 L Taq DNA polymerase, and 0.5 mol/L
Ci/mL, 15 to 120 seconds), a membrane potential–sensitive sequence-specific oligonucleotide primers for human CAT-1, CAT-
probe,2,3,25 was determined in Krebs solution containing 5 or 2A, or CAT-2B). Samples were incubated (4 minutes, 95°C),
25 mmol/L D-glucose, 100 mol/L L-arginine, and 5.5 or followed by 35 cycles of 30 seconds at 95°C, 30 seconds at 57°C, 30
131 mmol/L KCl.2,3 Resting membrane potential (Em, whole-cell seconds at 72°C, and a final extension for 7 minutes at 72°C. -Actin
patch clamp) was recorded using an EPC-7 amplifier (List Medical) expression was used as a reference value. Reverse transcription
as described.2 Em was measured for at least 1 minute in the presence (RT)-PCR products were sequenced in both directions by Taq
of 5 or 25 mmol/L D-glucose. Only recordings with 0.1-mV dideoxy terminator cycle sequencing (automated DNA sequencer
variations were considered. Em was also determined in cells prein- 373A, Applied Biosystems).3
cubated for 2 hours with 10 mmol/L L-lysine. Oligonucleotide primers were as follows: hCAT-1 (sense)
5 -CCAGTACTTCCGACGAGTTAGA-3 , hCAT-1 (antisense)
5 -CATCCACACAGCAAACCGGACC-3 , hCAT-2A (sense) 5 -
PKC Activity TATCCCGATTTTTTTGCTGTGTGC-3 , hCAT-2A (antisense)
PKC activity (32P incorporation from [ -32P]ATP into a synthetic 5 -TGCAGTCAACGTGGCAGCAACT-3 , hCAT-2B (sense) 5 -
PKC substrate peptide analogue5) was determined in cells exposed to TCCCAATGCCTCGTGTAATCTA-3 , hCAT-2B (antisense)
5 or 25 mmol/L D-glucose (2 minutes) after pretreatment with 100 5 -GCATGCTGAAGCCCTGTCTCTGC-3 , -actin (sense) 5 -
nmol/L phorbol 12-myristate 13-acetate (5 minutes, PKC activator), AACCGCGAGAAGATGACCCAGATCATCTTT-3 , and -actin
100 nmol/L 4 -phorbol 12,13-didecanoate (5 minutes, less active (antisense) 5 -AGCAGCCGTGGCCATCTCTTGCTCGAAGTC-3 .
phorbol 12-myristate 13-acetate analogue), 100 nmol/L calphostin C Expected size products were as follows: hCAT-1, 450 bp; hCAT-2A,
(30 minutes), 10 mol/L PD-98059 (30 minutes), 100 mol/L 690 bp; hCAT-2B, 360 bp; and -actin, 350 bp.
SNAP (5 minutes), 100 mol/L L-NAME (30 minutes), or 30
nmol/L wortmannin (30 minutes). SOD Activity and -Tocopherol Experiments
Cells were homogenized in buffer containing 50 mmol/L Tris-(hy-
cGMP Determination droxymethyl)-aminomethane, 100 mmol/L potassium chloride,
Cells preincubated (30 minutes) in Krebs solution (37°C) containing 0.02% Triton X-100, 100 mmol/L sodium pyrophosphate, and
L -arginine (100 mol/L) and 3-isobutyl-1-methylxanthine 100 mmol/L sodium fluoride (pH 7.4), which was supplemented with
(0.5 mmol/L, phosphodiesterase inhibitor),2,3 in the absence or trypsin inhibitors (4 mg/mL aprotinin, 1 mg/mL benzamidine, 5
presence of L-NAME (100 mol/L), were exposed to 5 or g/mL leupeptin, and 200 mol/L sodium orthovanadate). Aliquots
25 mmol/L D-glucose for the last 2 minutes of the 30-minute (1 mg protein/mL) were incubated (25°C, 2 minutes) with potassium
phosphate buffer (50 mmol/L, pH 10.2) containing adrenochrome
incubation period with 3-isobutyl-1-methylxanthine. cGMP was
(200 mol/L) and epinephrine (10 mol/L), and absorbance was
determined in HCl-cell extracts by radioimmunoassay.2,3
measured at 480 nm. Superoxide dismutase (SOD) activity was
calculated from the inhibition curve for epinephrine auto-oxidation
L-Citrulline Assay versus protein concentration. Basal absorbance (100% activity) was
Cells were incubated with L-[3H]arginine (100 mol/L, 4 Ci/mL, the reaction in the absence of cell extracts.26 Cells were also
30 minutes, 37°C) in the absence or presence of L-NAME (100 preincubated (30 minutes) with -tocopherol (500 g/mL, 1%
mol/L). Cells were exposed to 5 or 25 mmol/L D-glucose for 30 ethanol).27
minutes or for the last 2, 4, 10, or 20 minutes of this incubation
period. Some experiments were performed in cells exposed only to Materials
L-[3H]arginine (4 Ci/mL) and 5 or 25 mmol/L D-glucose. Formic Sera, agarose, and buffers were from GIBCO Life Technologies.
acid– digested cells (200 L) were passed through a sodium ion form Collagenase type II (Clostridium histolyticum) was from Boehring-
of the cation ion-exchange resin Dowex 50W (50X8-200), and er-Mannheim, and Bradford protein reagent was from Bio-Rad
L-[3H]citrulline concentration was determined in the H2O eluate.3 Laboratories. L-NAME and SNAP were from Calbiochem. Ethidium
3. 66 Circulation Research January 10/24, 2003
bromide, Dowex (50WX8-400), and all other reagents were from
Sigma Chemical Co. L-[2,3-3H]Arginine (36.1 Ci/mmol), D-[1-
14
C]mannitol (49.3 mCi/mmol), [ -32P]ATP, and [3H]TPP (37
Ci/mmol) were from NEN. 3 ,5 -cGMP-TME was from ICN. Anti-
bodies were from Cell Signaling, New England Biolabs.
Statistical Analysis
Values are mean SEM, where n indicates the number of different
cell cultures (4 to 8 replicates per experiment). Statistical analyses
were carried out on raw data using the Peritz F multiple-means
comparison test.28 A Student t test was applied for unpaired data, and
a value of P 0.05 was considered statistically significant.
Results
L-Arginine Transport
Elevated D-glucose (2 minutes), but not L-glucose or
D-mannitol, stimulated L-arginine transport (half-maximal
effect [K1/2] 13 2 mmol/L D-glucose) (Figure 1A). Basal
transport rates increased significantly after 30 seconds of
exposure to elevated D-glucose (K1/2 25 5 seconds), with
maximal rates achieved within 1 minute and sustained over 5
minutes (Figure 1B). Subsequent experiments were per-
formed using 25 mmol/L D-glucose for 2 minutes. D-Glu-
cose–stimulated L-arginine transport decreased to basal val-
ues within 5 minutes after reexposure of cells to 5 mmol/L
D-glucose (Figure 1B). RT-PCR analysis detected only
hCAT-1 and hCAT-2B mRNA in HUVECs (Figure 1C).
Elevated D-glucose had no effect on the nonsaturable
component (KD) of overall L-arginine transport but increased
Vmax, with no change in apparent Km (Figure 2A, Table 1).
Cell incubation with L-lysine (10 mmol/L, 2 hours) increased
6.5-fold the L-arginine transport in 5 mmol/L D-glucose
(Figure 2B). However, L-arginine transport was increased
2.9-fold in cells exposed to 25 mmol/L D-glucose (last 2
minutes of the 2-hour incubation with L-lysine) (Figure 2B).
D-Glucose stimulation and trans-stimulation by L-lysine of
L-arginine transport was unaltered in Na -free Krebs solution
(not shown).
TPP Influx and Membrane Potential
Elevated D-glucose increased TPP influx (1.8-fold) and
caused membrane hyperpolarization (Table 2). TPP influx
and L-arginine transport were inhibited by KCl-induced
membrane depolarization. Glibenclamide (K ATP channel
blocker) blocked D-glucose–increased L-arginine transport
Figure 1. Effect of D-glucose on L-arginine transport and CAT
and changes in TPP influx and Em (Table 2). Levcromakalim expression. A, L-Arginine transport (100 mol/L, 1 minute, 37°C)
(K ATP channel activator) hyperpolarized the plasma mem- in HUVECs incubated (2 minutes) with increasing concentrations
brane and increased L-arginine transport and TPP influx only of D-glucose ( ), L-glucose ( ), or 5 mmol/L D-glucose plus 5 to
in 5 mmol/L D-glucose; effects were blocked by gliben- 20 mmol/L D-mannitol (▫). *P 0.05 and **P 0.04 vs 5 or
7.5 mmol/L D-glucose and corresponding values in L-glucose
clamide (Table 2). The effects of D-glucose were also blocked and D-glucose D-mannitol. B, Time course of effect of
by wortmannin (not shown). Preloading cells with L-lysine D-glucose on L-arginine transport (as in panel A) in cells incu-
did not alter Em ( 66.1 0.3 mV, P 0.05; n 29 cells) bated for 0 to 5 minutes in 5 mmol/L D-glucose ( ), 25 mmol/L
D-glucose ( ), or 5 mmol/L D-glucose 20 mmol/L D-mannitol (▫).
compared with control cells ( 67.5 0.5 mV, P 0.05; n 45 High D-glucose– containing Krebs solution was replaced (arrows)
cells). L-Arginine transport was inhibited by KCl with half- by 5 mmol/L D-glucose, and cells were incubated for 10 min-
maximal inhibition at 12 2 and 17 4 mmol/L KCl for 5 and utes. *P 0.04 vs all other values. Values are mean SEM
(n 13). C, RT-PCR for mRNA from cells in 5 mmol/L D-glucose.
25 mmol/L D-glucose, respectively. The time needed to mRNA was reversed-transcribed into cDNA, and PCR was per-
induce half-maximal inhibition of transport with 131 mmol/L formed for human CAT-1 (lane 2, 449 bp), CAT-2A (lane 3, 690
KCl was similar in cells in 5 mmol/L D-glucose (6.5 0.6 bp), or CAT-2B (lane 4, 357 bp). Lane 5 is -actin (350 bp), and
minutes) compared with 25 mmol/L D-glucose (7.2 0.6 lane 1 is DNA ladder (100 to 2000 bp). Data are representative
of 14 cell cultures.
minutes).
4. Flores et al D-Glucose Acutely Activates L-Arginine/NO Pathway 67
lation at Ser1177, cGMP, and L-[3H]citrulline formation. Sim-
ilar results were found in cells exposed to 25 mmol/L
D-glucose for the last 4, 10, or 20 minutes of the 30-minute
incubation period with L-[3H]arginine (not shown). Intracel-
lular Ca2 in cells incubated with 25 mmol/L D-glucose for 2
minutes (42 7 nmol/L) was not statistically different
(P 0.05, n 125 cells) from values in cells incubated with
5 mmol/L D-glucose (35 5 nmol/L).
L-NAME also inhibited the effect of D-glucose on
L-arginine transport Vmax (Table 1), TPP influx, and Em
(Table 2); however, L-NAME did not alter TPP influx or Em
in 5 mmol/L D-glucose. Other experiments show that SNAP
(NO donor) induces TPP influx, L-arginine transport, and
membrane hyperpolarization only in 5 mmol/L D-glucose
(Table 2) and that dibutyryl cGMP (dbcGMP) increased
L-arginine transport (Figure 4A) and TPP influx (Figure 4B).
The effects of D-glucose and dbcGMP were blocked by
KT-5823, a PKG inhibitor (Table 2).
PKC and MAP Kinase Involvement
PKC activity was unaltered at up to 5 minutes of incubation
with high D-glucose (Figure 5A) or after the addition of
SNAP (not shown). Furthermore, calphostin C (PKC inhibi-
tor) had no effect on D-glucose–increased L-arginine transport
(Figure 5B), TPP influx, or NO synthesis (not shown).
However, longer incubation with elevated D-glucose ( 10
Figure 2. Effect of D-glucose on kinetic parameters and trans- minutes) increased membrane PKC activity (not shown),
stimulation of L-arginine transport. A, Saturable L-arginine trans- confirming our previous observations in HUVECs.5 In con-
port (1 minute, 37°C) in HUVECs incubated (2 minutes) in trast, high D-glucose induced p42/p44mapk phosphorylation
5 mmol/L D-glucose ( ) or 25 mmol/L D-glucose ( ). B,
L-Arginine transport (100 mol/L, 1 minute, 37°C) in cells prein-
(Figure 6A), an effect blocked by PD-98059 and mimicked
cubated (2 hours) in medium 199 in the absence (control) or by brief exposure (2 minutes) to SNAP (Figure 6B). Inter-
presence of 10 mmol/L L-lysine. Transport assays were per- estingly, the effect of D-glucose on p42/44mapk phosphoryla-
formed in cells exposed to 5 mmol/L D-glucose (open bars) or tion was blocked by wortmannin (Figure 6C). PD-98059 also
25 mmol/L D-glucose (filled bars) for the last 2 minutes of the
2-hour incubation period with L-lysine. Values are mean SEM blocked the stimulatory effects of elevated D-glucose and
(n 16). *P 0.04 vs all other values. SNAP on TPP influx, L-arginine transport, and Em (Table 2).
NO Involvement SOD Activity and -Tocopherol Effect
Elevated D-glucose increased eNOS phosphorylation at SOD activity in cells in 5 mmol/L D-glucose (5.5 0.6 U/mL)
Ser1177 (Figure 3A), L-[3H]citrulline (Figure 3B), and cGMP was not significantly altered (P 0.05, n 5) by 25 mmol/L
accumulation (Figure 3C). L-NAME inhibited the effect of D-glucose (6 1 U/mL). Extracellular SOD or -tocopherol
3
D-glucose on cGMP and L-[ H]citrulline formation but did not did not block (P 0.05, n 4 to 8) the effect of D-glucose on
alter eNOS-Ser1177 phosphorylation (not shown). However, L-arginine transport (5.6 0.3 and 5.8 0.6 pmol/ g protein
wortmannin inhibited D-glucose–induced eNOS phosphory- per minute, respectively), L-citrulline formation (3.1 0.2 and
TABLE 1. D-Glucose Effect on L-Arginine Transport in HUVECs
Vmax /Km, pmol/ g KD, pmol/ g
Vmax, pmol/ g Protein per Minute Protein per Minute
Km, mol/L Protein per Minute per mol/L per mol/L
5 mmol/L D-Glucose 105 9 4.4 0.1 0.044 0.011 0.002 0.0007
PD-98059 123 12 4.2 0.2 0.034 0.012 0.003 0.0012
L-NAME 97 16 3.8 0.3 0.039 0.016 0.003 0.0009
25 mmol/L D-Glucose 111 16 6.1 0.7* 0.055 0.012* 0.003 0.0011
PD-98059 133 22 3.7 0.3† 0.028 0.009† 0.004 0.0012
L-NAME 125 22 3.6 0.5† 0.029 0.007† 0.003 0.0008
L-Arginine transport (100 mol/L, 37°C) in cells preincubated (30 minutes) with 5 mmol/L D-glucose, in absence
or presence of 100 mol/L PD-98059 or 100 mol/L L-NAME, and exposed (2 minutes) to Krebs containing 5 or
25 mmol/L D-glucose. Values are mean SEM, n 6.
*P 0.05, †P 0.05 vs 5 and 25 mmol/L D-glucose, respectively.
5. 68 Circulation Research January 10/24, 2003
TABLE 2. D-Glucose Effect on L-Arginine Transport, [3H]TPP Influx, and Em in HUVECs
L-Arginine Transport, pmol/ g [3H]TPP Influx, pmol/mg
Protein per Minute Protein per Minute Em, mV
5 mmol/L D-Glucose
Control 1.8 0.3 2.1 0.3 67.2 0.5
KCl 0.2 0.1* 0.1 0.04* 8.1 0.5*
Glibenclamide 1.3 0.3 1.5 0.3 63.9 0.6
Levcromakalim 4.6 0.3* 5.6 0.3* 74.2 0.3*
Levcromakalim glibenclamide 1.6 0.2† 1.4 0.6† 64.5 0.2†
PD-98059 2.1 0.1 1.7 0.2 64.2 0.5
L-NAME 1.6 0.1 1.5 0.3 65.1 0.3
SNAP 5.1 0.3* 3.9 0.4* 76.2 1.0*
SNAP PD-98059 2.1 0.3† 1.9 0.5† 65.3 1.0†
KT-5823 1.9 0.5 1.9 0.3 66.8 0.9
25 mmol/L D-Glucose
Control 4.0 0.5* 3.7 0.3* 77.1 0.2*
KCl 0.3 0.2*‡ 0.3 0.03*‡ 6.2 0.5*‡
Glibenclamide 2.3 0.4‡ 2.4 0.4‡ 62.7 0.5‡
Levcromakalim 4.3 0.4* 4.7 0.2* 75.1 0.3*
Levcromakalim glibenclamide 2.2 0.3†‡ 1.8 0.3†‡ 61.9 0.3†‡
PD-98059 0.9 0.3‡ 1.7 0.1‡ 69.9 1.0‡
L-NAME 1.4 0.1‡ 1.8 0.2‡ 65.7 0.7‡
SNAP 4.1 0.4* 3.2 0.2* 78.2 1.0*
SNAP PD-98059 1.1 0.5†‡ 1.9 0.2†‡ 65.7 1.0†‡
KT-5823 1.9 0.5‡ 1.9 0.3‡ 65.3 0.5‡
L-Arginine transport (100 mol/L), 3H TPP influx (46 nmol/L), and Em (whole-cell patch clamp) in cells
preincubated (30 minutes) with 5 mmol/L D-glucose and 5.5 mmol/L (control) or 131 mmol/L KCl. Cells in 5.5 mmol/L
KCl were preincubated with 10 mol/L glibenclamide (5 minutes), 1 mol/L levcromakalim (5 minutes), 10 mol/L
PD-98059 (30 minutes), 100 mol/L L-NAME (30 minutes), 10 mol/L KT-5823 (30 minutes), or 100 mol/L SNAP
(2 minutes) and exposed (2 minutes) to 5 or 25 mmol/L D-glucose. Values are mean SEM, n 17.
*P 0.05 vs control in 5 mmol/L D-glucose; †P 0.05 vs corresponding values in SNAP or levcromakalim; ‡P 0.05
vs control in 25 mmol/L D-glucose.
2.8 0.4 pmol/ g protein per 30 minutes, respectively), TPP confirmed here, is Na independent and inhibited by mem-
influx (4.4 0.2 and 4.1 0.3 pmol/mg protein per minute, brane depolarization.2,3,32 Because of their similar kinetic
respectively), or changes in Em ( 76 0.3 and 78 0.3 mV, properties, CAT-1 and CAT-2B are hard to distinguish at the
respectively). functional level.31 Our results show that both high-affinity
hCAT-1 (Km 100 to 200 mol/L) and hCAT-2B (Km 200
Discussion to 400 mol/L), but not the low-affinity hCAT-2A trans-
The present study establishes that D-glucose induces a rapid porter (Km 2 to 5 mmol/L), are present in HUVECs,
concentration-dependent stimulation of L-arginine transport confirming previous reports.3,33 CAT-1 is more sensitive than
in HUVECs. This effect requires NO synthesis associated CAT-2B to trans-stimulation by cationic amino acids.3,31,34
with increased phosphorylation of eNOS at Ser1177 and acti- When we preloaded HUVECs with L-lysine, L-arginine trans-
vation of p42/p44mapk and PI3-k, and it is independent of PKC port was increased by 7-fold in 5 mmol/L D-glucose.
and intracellular Ca2 changes. These findings provide the However, the L-lysine trans-stimulatory effect was less ef-
first evidence that short-term hyperglycemia activates the fective ( 3-fold) in cells exposed for 2 minutes to 25 mmol/L
L -arginine/NO signaling pathway in human fetal D-glucose. Because L-arginine transport is trans-stimulated
endothelium. by 9.8-fold or 1.8-fold in Xenopus oocytes injected with
L-Arginine transport is mediated by systems y /CATs,2,3,12 hCAT-1 or hCAT-2B mRNA, respectively, 34 trans-
y L,29,30 and b0, 12 in HUVECs, with the first likely predom- stimulation in HUVECs in 5 mmol/L D-glucose may be
inating at the physiological concentration of extracellular preferentially mediated by hCAT-1. The reduced trans-
L-arginine. The cDNAs for four potential human y trans- stimulation of transport in high D-glucose may result from a
porters (hCAT-1, hCAT-2B, hCAT-2A, and hCAT-4) have state of maximal activity of L-arginine transporters already
been sequenced.31 L-Arginine transport in HUVECs occurs induced by L-lysine; therefore, high D-glucose could not
with relatively high affinity (Km 80 to 100 mol/L) and, as further increase L-arginine transport. In addition, the possi-
6. Flores et al D-Glucose Acutely Activates L-Arginine/NO Pathway 69
Figure 4. cGMP involvement in the effect of D-glucose on
L-arginine transport and [3H]TPP influx. HUVECs preincubated
(30 minutes) with 5 mmol/L D-glucose in the absence (control) or
presence of KT-5823 or dbcGMP were exposed (2 minutes) to
5 mmol/L D-glucose (open bars) or 25 mmol/L D-glucose (filled
bars) in the presence of KT-5823 and/or dbcGMP, and
L-arginine transport (100 mol/L, 1 minute, 37°C) (A) and
[3H]TPP influx (46 nmol/L, 1 minute, 37°C) (B) were determined.
Values are mean SEM (n 12). *P 0.05 vs all other values.
Vmax for L-arginine transport without altering the apparent Km.
As noted above, L-arginine transport is sensitive to changes in
extracellular K and Em. Because high D-glucose induced
membrane hyperpolarization, stimulation of L-arginine trans-
port could result from changes in Em. The stimulatory effect
Figure 3. Effect of D-glucose on eNOS activity. A, Immunoblot of D-glucose on TPP influx and L-arginine transport was
for total eNOS and phosphorylated eNOS at Ser1177 (eNOS P- blocked by glibenclamide, a K ATP channel blocker. In addi-
Ser1177) in HUVECs preincubated (30 minutes) with 5 mmol/L
D-glucose in the absence or presence of wortmannin and then tion, levcromakalim (K ATP activator)23 mimics D-glucose–
exposed (2 minutes) to 5 or 25 mmol/L D-glucose (see Materials induced changes in Em, L-arginine transport, and TPP influx.
and Methods). Top panel shows densitometry ratios. Data are K ATP channels are expressed in the endothelium35 and are
representative of 6 cell cultures. B, L-[3H]Citrulline formation
from L-[3H]arginine (4 Ci/mL, 37°C) in cells preincubated (30 activated by D-glucose36; thus, the effects of D-glucose may
minutes) with Krebs solution in the absence (control) or pres- involve changes in the activity of glibenclamide-sensitive
ence of L-NAME and exposed for the last 2 minutes to Krebs K ATP channels.
solution containing 5 mmol/L D-glucose (open bars) or
D-Glucose (24 hours) also increases eNOS expression4 and
25 mmol/L D-glucose (filled bars). C, Intracellular cGMP under
same experimental conditions as in panel B. Values are activity2 in HUVECs. In the present study, eNOS activity was
mean SEM (n 17). *P 0.05 vs all other values. increased in HUVECs exposed for 1 to 5 minutes to high
D-glucose, an effect associated with increased phosphoryla-
bility that L-lysine–stimulated L-arginine transport was due to tion of eNOS at Ser1177, a residue known to be associated with
membrane hyperpolarization is unlikely because Em was eNOS activation.37 The rapid eNOS stimulatory effect of
unaltered in L-lysine–preloaded cells. D-glucose was not further increased by longer incubations
Long-term incubation (24 hours) of HUVECs with high periods with D-glucose, which could be due to a maximal and
D-glucose increases Vmax for L-arginine transport.4 The pres- sustained activation of eNOS acutely induced by elevated
ent study shows that acute (2-minute) D-glucose increases D-glucose. Because D-glucose did not alter basal intracellular
7. 70 Circulation Research January 10/24, 2003
Figure 5. Lack of effect of calphostin C in the effect of
D-glucose on L-arginine transport. A, PKC activity in cytosolic
(open symbols) and membrane (filled symbols) fractions from
HUVECs preincubated (30 minutes) with Krebs solution without
( , ) or with 100 nmol/L calphostin C (▫, ). Cells were incu-
bated (30 seconds) in 25 mmol/L D-glucose in the absence or
presence of calphostin C. B, Time-course effect of 25 mmol/L
D-glucose on L-arginine transport (100 mol/L, 1 minute, 37°C)
(as in panel A) in Krebs solution without ( ) or with 100 nmol/L
calphostin C ( ). Values are mean SEM (n 18). *P 0.05 vs all
other values.
Ca2 , it is likely that rapid eNOS activation by D-glucose is
Ca2 independent, supporting recent observations of Ca2 - Figure 6. NO and PI3-k involvement in effect of D-glucose on
independent eNOS activation in HUVECs.37,38 D-Glucose– p42/44mapk phosphorylation. A, Immunoblot for phosphorylated
p44mapk (p44 P) and p42mapk (p42 P) and nonphosphorylated
induced phosphorylation of eNOS at Ser1177, L-arginine trans- p44mapk (p44) or p42mapk (p42) in HUVECs preincubated (30 min-
port, and TPP influx were blocked by wortmannin, utes) with 5 mmol/L D-glucose in the absence or presence of
suggesting that the PI3-k pathway could be involved in the PD-98059 and then exposed (2 minutes) to 5 or 25 mmol/L
D-glucose. B, Effect of SNAP on p42/p42mapk in HUVECs in
effects of D-glucose. L-Arginine transport could be determi-
5 mmol/L D-glucose. C, Effect of wortmannin on p42/p42mapk in
nant for eNOS activity11; however, the possibility that the HUVECs (as in panel A). Data are representative of similar
D-glucose–induced increase of L-citrulline production was results in 17 cell cultures.
due to elevated L-arginine transport seems unlikely, inasmuch
as D-glucose–induced NO synthesis was unaltered in the synthase.2 NO causes membrane hyperpolarization in the
absence of extracellular L-arginine. endothelium,3,36 and NO (from SNAP) has been shown to
L-NAME blocked D-glucose–increased L-arginine trans- cause comparable increases in TPP influx and L-arginine
port and NO synthesis in HUVECs. This inhibitor does not transport and to cause membrane hyperpolarization to those
alter basal L-arginine transport in the endothelium2– 4,39; thus, caused by D-glucose, although SNAP treatment did not
NO most likely mediates changes in L-arginine transport, as further enhance the effects of high D-glucose, in HUVECs.
suggested in bovine aortic endothelium.40 This result is These findings support the hypothesis that NO acutely mod-
similar to that found in HUVECs from patients with gesta- ulates L-arginine transport by a mechanism that involves
tional diabetes; L-arginine transport in these cells is increased membrane hyperpolarization.
concomitantly with membrane hyperpolarization and NO NO-altered K channel activity may occur by both indirect
synthesis, and this increase is inhibited by blocking NO mechanisms via cGMP and direct NO action on channels.36
8. Flores et al D-Glucose Acutely Activates L-Arginine/NO Pathway 71
Our results show that the D-glucose increases in L-arginine fellowships. We thank the midwives of the Hospital Regional-
transport and TPP influx were mimicked by dbcGMP and Concepción (Chile) labor ward for the supply of umbilical cords and
Isabel Jara for secretarial assistance.
blocked by the PKG inhibitor KT-5823. In addition, D-
glucose–induced membrane hyperpolarization was also
blocked by KT-5823. Thus, modulation of ion channel
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