This document summarizes research on nicotinic acetylcholine receptors (nAChRs) in mouse brain. The researchers developed assays to measure nAChR binding and function, including assays for dopamine, GABA, and ion flux. Comparing results across assays and between wildtype and nAChR subunit knockout mice revealed diversity in nAChR subtypes. At least six nAChR binding sites were identified with different pharmacological properties. Assays of function suggested different nAChR subtypes mediate dopamine vs. GABA release and ion flux. Deletion of the beta2 subunit eliminated most nAChR binding and function, confirming its role in major brain nAChR subtypes.
This study examined the sensitivity of six neuronal nicotinic acetylcholine receptor (nAChR) combinations to various nicotinic agonists when expressed in Xenopus oocytes. Each receptor combination displayed a distinct sensitivity profile. For example, the α2β2 combination was 10 times more sensitive to nicotine than acetylcholine, while α3β2 was 17 times less sensitive to nicotine. Both the α and β subunits contributed to the pharmacological properties of neuronal nAChRs.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.
The document summarizes a study that analyzed the expression of the At1g17950 gene in diploid and tetraploid Arabidopsis thaliana plants under salt stress conditions. The study found that the At1g17950 gene, which encodes a MYB transcription factor involved in abscisic acid production and response to salt stress, was expressed more highly in diploid plants than in tetraploid plants. This suggests that tetraploid A. thaliana have greater resistance to salt stress due to lower expression of the At1g17950 gene, while diploid plants are more sensitive to salt stress due to higher expression of this gene.
The document summarizes a study that investigated the effects of low molecular weight components from the venom of the Chilean Black Widow spider (Latrodectus mactans) on synaptic activity. Whole-cell patch clamp techniques were used to study the effects of purified venom on rat hippocampal neuronal cultures. The venom increased neuronal membrane resistance, prolonged action potentials, and increased spontaneous synaptic activity in a concentration- and time-dependent manner. This demonstrates that the venom has pharmacological effects through low molecular weight fragments, despite L. mactans venom not containing alpha-latrotoxin like the European Black Widow spider venom.
JACS-CD38 localization using a fluorescent probeJonathan Shrimp
This document describes the development of a fluorescent small molecule probe called SR101−F-araNMN that can label the enzyme CD38 in live cells in a mechanism-based manner. The probe was used to investigate the cellular localization of CD38 in leukemia (HL-60 and K562) and lymphoma (Raji) cell lines. The results showed that CD38 is predominantly localized to the plasma membrane in Raji and RA-treated HL-60 cells, with very little intracellular CD38. Additionally, no CD38 expression was detected in K562 cells. This suggests that the major function of CD38 is to hydrolyze extracellular rather than intracellular NAD.
This document summarizes research investigating the biosynthesis and processing of succinate dehydrogenase (SDH) subunits in cultured mammalian cells. The key points are:
1. Antisera were produced against purified bovine heart SDH and its large and small subunits, which detected precursor and mature forms of the subunits in rat, pig, and bovine cell lines.
2. In pig kidney cells, newly synthesized precursors of the large and small SDH subunits were detected that were 1000-2000 and 4000-5000 Da larger than the mature forms, respectively.
3. Pulse-chase experiments showed the precursor forms were fully processed to the mature subunits within 45 minutes when uncouplers of
Expression of Genetically Engineered Chitinase Gene of Pyrococcus furiosusIJERDJOURNAL
ABSTRACT: Wild-type Pyrococcus furiosus is most likely unable to grow on chitin in the natural biotope due to a nucleotide insertion which separates the chitinase gene into two ORFs, whereas a genetically engineered strain with the deleted nucleotide is able to grow on chitin. In the latest studies, the recombinant enzyme activity against the crystal chitins was examined. But there are still some conflictions. In our study, to shed a light on whether the construct composed of a catalytic domain and a chitin binding domain show any activity against crystalline chitin, the construct was created in the pET 28b (+) expression vector and expressed in Escherichia coli. The chitinase with an approximately 55 kDa molecular weight was determined. The activity of the enzyme was measured spectrophotometrically. Despite the presence of enzyme activity against the colloidal chitin, no significant activity against the crystal chitin has been measured.
- 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.
This study examined the sensitivity of six neuronal nicotinic acetylcholine receptor (nAChR) combinations to various nicotinic agonists when expressed in Xenopus oocytes. Each receptor combination displayed a distinct sensitivity profile. For example, the α2β2 combination was 10 times more sensitive to nicotine than acetylcholine, while α3β2 was 17 times less sensitive to nicotine. Both the α and β subunits contributed to the pharmacological properties of neuronal nAChRs.
The IOSR Journal of Pharmacy (IOSRPHR) is an open access online & offline peer reviewed international journal, which publishes innovative research papers, reviews, mini-reviews, short communications and notes dealing with Pharmaceutical Sciences( Pharmaceutical Technology, Pharmaceutics, Biopharmaceutics, Pharmacokinetics, Pharmaceutical/Medicinal Chemistry, Computational Chemistry and Molecular Drug Design, Pharmacognosy & Phytochemistry, Pharmacology, Pharmaceutical Analysis, Pharmacy Practice, Clinical and Hospital Pharmacy, Cell Biology, Genomics and Proteomics, Pharmacogenomics, Bioinformatics and Biotechnology of Pharmaceutical Interest........more details on Aim & Scope).
All manuscripts are subject to rapid peer review. Those of high quality (not previously published and not under consideration for publication in another journal) will be published without delay.
The document summarizes a study that analyzed the expression of the At1g17950 gene in diploid and tetraploid Arabidopsis thaliana plants under salt stress conditions. The study found that the At1g17950 gene, which encodes a MYB transcription factor involved in abscisic acid production and response to salt stress, was expressed more highly in diploid plants than in tetraploid plants. This suggests that tetraploid A. thaliana have greater resistance to salt stress due to lower expression of the At1g17950 gene, while diploid plants are more sensitive to salt stress due to higher expression of this gene.
The document summarizes a study that investigated the effects of low molecular weight components from the venom of the Chilean Black Widow spider (Latrodectus mactans) on synaptic activity. Whole-cell patch clamp techniques were used to study the effects of purified venom on rat hippocampal neuronal cultures. The venom increased neuronal membrane resistance, prolonged action potentials, and increased spontaneous synaptic activity in a concentration- and time-dependent manner. This demonstrates that the venom has pharmacological effects through low molecular weight fragments, despite L. mactans venom not containing alpha-latrotoxin like the European Black Widow spider venom.
JACS-CD38 localization using a fluorescent probeJonathan Shrimp
This document describes the development of a fluorescent small molecule probe called SR101−F-araNMN that can label the enzyme CD38 in live cells in a mechanism-based manner. The probe was used to investigate the cellular localization of CD38 in leukemia (HL-60 and K562) and lymphoma (Raji) cell lines. The results showed that CD38 is predominantly localized to the plasma membrane in Raji and RA-treated HL-60 cells, with very little intracellular CD38. Additionally, no CD38 expression was detected in K562 cells. This suggests that the major function of CD38 is to hydrolyze extracellular rather than intracellular NAD.
This document summarizes research investigating the biosynthesis and processing of succinate dehydrogenase (SDH) subunits in cultured mammalian cells. The key points are:
1. Antisera were produced against purified bovine heart SDH and its large and small subunits, which detected precursor and mature forms of the subunits in rat, pig, and bovine cell lines.
2. In pig kidney cells, newly synthesized precursors of the large and small SDH subunits were detected that were 1000-2000 and 4000-5000 Da larger than the mature forms, respectively.
3. Pulse-chase experiments showed the precursor forms were fully processed to the mature subunits within 45 minutes when uncouplers of
Expression of Genetically Engineered Chitinase Gene of Pyrococcus furiosusIJERDJOURNAL
ABSTRACT: Wild-type Pyrococcus furiosus is most likely unable to grow on chitin in the natural biotope due to a nucleotide insertion which separates the chitinase gene into two ORFs, whereas a genetically engineered strain with the deleted nucleotide is able to grow on chitin. In the latest studies, the recombinant enzyme activity against the crystal chitins was examined. But there are still some conflictions. In our study, to shed a light on whether the construct composed of a catalytic domain and a chitin binding domain show any activity against crystalline chitin, the construct was created in the pET 28b (+) expression vector and expressed in Escherichia coli. The chitinase with an approximately 55 kDa molecular weight was determined. The activity of the enzyme was measured spectrophotometrically. Despite the presence of enzyme activity against the colloidal chitin, no significant activity against the crystal chitin has been measured.
- 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.
This study compared the effects of three Toll-like receptor (TLR) agonists - P3CSK4 (TLR2 agonist), Lipid A (TLR4 agonist), and Poly I:C (TLR3 agonist) - on human monocytes and dendritic cells. It found that all three TLR agonists enhanced the expansion of IFN-γ producing CD4+ T cells when primed with dendritic cells, but only P3CSK4 and Lipid A enhanced T cell proliferation when primed with monocytes. Distinct molecular signatures induced in monocytes and dendritic cells by each TLR agonist correlated with their differential effects on T cell responses. TNF-α and CXCL
This study aimed to identify the site of ubiquitination on Activation-Induced Cytidine Deaminase (AID) by the ubiquitin ligase RING Finger Protein 126 (RNF126). Using site-directed mutagenesis, the researchers found that mutating all lysine residues (K0 mutant) prevented ubiquitination, suggesting ubiquitination occurs on a lysine. Analysis of single lysine mutants revealed the K22 mutant still showed ubiquitination, indicating K22 is the main site. However, RNF126 can ubiquitinate AID at other residues as well. Together, this suggests RNF126 favors K22 as the primary ubiquitination site on AID, though it can modify AID at additional
The document discusses cannabinoids and their effects on the brain. It describes how the main psychoactive compound in cannabis, tetrahydrocannabinol (THC), acts through the CB1 cannabinoid receptor. It notes that GABAergic neurons highly express CB1 receptors. Activation of these receptors inhibits the release of neurotransmitters. The document also discusses endogenous cannabinoids like anandamide and their role as retrograde synaptic signals. Finally, it reviews some central effects of cannabinoids like impaired memory and their medical applications.
This study tested the hypothesis that a base-pairing interaction between nucleotide A79 in the Hepatitis Delta Virus (HDV) ribozyme and nucleotide U(-1) in its substrate is necessary for catalytic activity. Mutant ribozymes and substrates with variations at these positions were created and their kinetic activity analyzed. While mutation of A79 significantly reduced activity, further experiments found the hypothesis was incorrect. Additional nucleotides like A78 may interact with the substrate and warrant further investigation.
The document summarizes the central dogma of biology and the discovery of DNA as the genetic material. It describes key experiments that showed DNA replicates in a semiconservative manner, with each parental strand serving as a template for a new complementary daughter strand. The process of DNA replication requires several enzymes including DNA polymerase, helicase, ligase and primase to unwind, copy and join new DNA strands.
This document describes a technique for bioassaying histamine in the presence of prostaglandins using an Amberlite XAD-2 column. The column removes prostaglandins, rabbit aorta contracting substance, and possibly slow reacting substance of anaphylaxis, but allows histamine to pass through. Two banks of bioassay organs (rabbit aorta strip, rat stomach strip, guinea pig ileum) are used, with one bank above the column and one below. This allows accurate measurement of histamine levels in samples also containing prostaglandins and other substances that interfere with histamine bioassay. The method was tested using effluent from shocked, perfused guinea pig lungs and showed improved accuracy
DNA damage repair Neil3 gene Knockout in MOLT-4iosrjce
RNAi is superannuated cellular mechanism that protect organism against viruses that replicate
through double- stranded RNA. RNAi can be used to diminish gene expression from plasmid expressing and
inserted sequence repeat. A stable harpin would be expressed after the vector was integrated into the genome.
In this paper a shiRNA expressing vector for Neil3 was designed and developed which is capable of replication
in MOLT-4. This shiRNA vector had the ability to arose the RNAi pathway, and reduce the gene expression of
Neil3. This was assessed by using pSilence 4.1CMV as a vector, and Gapdh as positive control.
This study compared the cytotoxicity and induction of genes encoding metabolic enzymes in cells exposed to different alkylated polycyclic aromatic hydrocarbons (PAHs). An SRB assay showed little cell death between exposures of 1uM naphthalene, 1uM dimethyl naphthalene, 1uM BAP, and the vehicle control, except for 5uM BAP. RT-PCR analysis found that 1uM BAP induced expression of CYP1A1 and CYP1B1 genes, but 1uM naphthalene and dimethyl naphthalene did not induce gene expression. Comparing responses to the parent naphthalene and dimethylated naphthalene,
This study aims to clone and analyze GAPDH genes from various plant species to compare amino acid sequences related to catalytic function. Previous work successfully cloned GAPDH from Oxalis corniculata and Plectranthus amboinicus but not Myrtaceae psidium. The current study will continue work on the cloned samples through midipreps, restriction enzyme digestion and sequencing. Sequence data will undergo bioinformatics analysis to compare conserved amino acids of the GAPDH protein between plant species. Results could provide new genetic information published in GenBank and further the understanding of energy production pathways in plants.
The document discusses DNA barcoding of Impatiens balsamina using chloroplast and nuclear markers. Genomic DNA was successfully extracted from I. balsamina leaves. The psbA-trnH marker was found to be the best barcode, amplifying a 337 bp fragment that showed 100% sequence identity to I. balsamina voucher sequences in GenBank. RbcL and ITS2 were also successfully amplified and sequenced, while matK amplification was unsuccessful. The sequences were verified through bioinformatics analysis, demonstrating DNA barcoding can authenticate I. balsamina.
Introduction to Genetic Material, Physical and Chemical properties of the same and various types of coiling mechanisms as well as information about chromosomal and extra-chromosomal DNA.
This document is a thesis presented by Archie Lovatt for the degree of Doctor of Philosophy at the University of Leicester in 1994. The thesis describes molecular analysis of a P-N-acetyl-hexosaminidase gene from the oral pathogen Porphyromonas gingivalis W83. Lovatt cloned the nahA gene encoding P-N-acetyl-hexosaminidase from P. gingivalis and characterized the gene and its protein product. Sequence analysis revealed that nahA is 2331 base pairs long and encodes a 111 amino acid protein with homology to hexosaminidases from other sources. The nahA gene appears to be conserved in P. gingivalis strains
Identification of perfect housekeeping genes for gene expression studies in p...ICRISAT
Gene expression analysis using quantitative real-time PCR (qRTPCR) is a very sensitive technique which completely depends on stable performance of reference genes used in the study.
3 July 2014
1. DREB genes play an important role in improving crop tolerance to stresses like drought, salt, and cold. DREB transcription factors regulate stress-responsive genes allowing plants to adapt.
2. Case studies showed overexpression of DREB genes enhanced stress tolerance in crops like rice and sugarcane. Co-transformation with DREB and other stress genes improved tolerance more than single DREB genes.
3. DREB genes respond differently to various stresses. DREB1 genes respond mainly to cold while DREB2 genes respond to dehydration and heat. Proper expression analysis of DREB genes is important for abiotic stress tolerance in transgenic crops.
This study examined the genetic attributes associated with the enhanced sensitivity of an HIV-1 clade C envelope protein (HVTR-PG80v2.eJ7) to autologous broadly neutralizing plasma antibodies from an elite neutralizer. The researchers found that mutations in the V3/C3 region of the envelope protein were associated with its increased sensitivity to neutralization by autologous plasma antibodies. Depletion experiments showed that these mutations altered the envelope conformation to better expose epitopes targeted by both neutralizing and non-neutralizing antibodies in the plasma. Therefore, distinct vulnerabilities associated with antibody evasion could be linked to mutations in the V3/C3 region of the HIV envelope.
Transcriptional profiling of Halobacterium sp. NRC-1 showed changes in gene expression in response to changes in salinity and temperature. Growth under high salt stress resulted in modulation of genes for ion transporters like potassium and phosphate transporters. Growth at cold temperatures altered expression of genes for lipid metabolism, gas vesicles, and cold shock proteins. Heat shock induced several chaperone genes. The study provides insights into Halobacterium's responses to environmental stresses at the gene expression level.
This lab report details an experiment to construct knockouts of the FAD2 gene in the model plant Thlaspi arvense using RNA interference to reduce unsaturated fatty acid content and improve the quality of seed oil for biodiesel production. The FAD2 gene was amplified from pennycress cDNA using PCR. The amplified FAD2 fragments were inserted into an entry vector, which was then transformed into E. coli cells. Successful transformants were identified through DNA sequencing. The purified FAD2 constructs are now ready to be transformed into a binary vector for plant transformation experiments to knockout the FAD2 gene in pennycress seeds and analyze effects on seed oil composition.
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
International Journal of Clinical Pharmacology & Toxicology (IJCPT) ISSN:2167-910X is an Open Access journal and a peer-reviewed journal. Clinical Pharmacology & Toxicology is the all-encompassing and becoming an increasingly important discipline for the identification of disease targets and drug designing with their toxicological effects and means to eradicate diseases.
This document provides information on the kokum fruit tree (Garcinia indica). It describes the tree's origin in southern India and distribution in tropical forests. It outlines the tree's taxonomy and classification. The document discusses the culinary, pharmaceutical and industrial uses of kokum fruit, rind and butter. It also describes the tree's sex types, varieties including the high yielding 'Konkan Amrit' variety, challenges around scattered production and short harvesting periods, and opportunities for popularizing kokum-based products.
This study aimed to evaluate the efficacy of oral montelukast in acute asthma exacerbation. It was a randomized, double-blind, placebo-controlled trial conducted in a tertiary care hospital over 2 years. Patients presenting with acute asthma exacerbation were randomized to receive standard therapy with either montelukast or placebo. The primary outcomes of lung function, duration of hospital stay, and secondary outcomes showed no significant differences between the montelukast and placebo groups. The study concluded that montelukast provided no added benefit over standard therapy alone for acute asthma exacerbation. Larger multicenter trials are still needed.
This study compared the effects of three Toll-like receptor (TLR) agonists - P3CSK4 (TLR2 agonist), Lipid A (TLR4 agonist), and Poly I:C (TLR3 agonist) - on human monocytes and dendritic cells. It found that all three TLR agonists enhanced the expansion of IFN-γ producing CD4+ T cells when primed with dendritic cells, but only P3CSK4 and Lipid A enhanced T cell proliferation when primed with monocytes. Distinct molecular signatures induced in monocytes and dendritic cells by each TLR agonist correlated with their differential effects on T cell responses. TNF-α and CXCL
This study aimed to identify the site of ubiquitination on Activation-Induced Cytidine Deaminase (AID) by the ubiquitin ligase RING Finger Protein 126 (RNF126). Using site-directed mutagenesis, the researchers found that mutating all lysine residues (K0 mutant) prevented ubiquitination, suggesting ubiquitination occurs on a lysine. Analysis of single lysine mutants revealed the K22 mutant still showed ubiquitination, indicating K22 is the main site. However, RNF126 can ubiquitinate AID at other residues as well. Together, this suggests RNF126 favors K22 as the primary ubiquitination site on AID, though it can modify AID at additional
The document discusses cannabinoids and their effects on the brain. It describes how the main psychoactive compound in cannabis, tetrahydrocannabinol (THC), acts through the CB1 cannabinoid receptor. It notes that GABAergic neurons highly express CB1 receptors. Activation of these receptors inhibits the release of neurotransmitters. The document also discusses endogenous cannabinoids like anandamide and their role as retrograde synaptic signals. Finally, it reviews some central effects of cannabinoids like impaired memory and their medical applications.
This study tested the hypothesis that a base-pairing interaction between nucleotide A79 in the Hepatitis Delta Virus (HDV) ribozyme and nucleotide U(-1) in its substrate is necessary for catalytic activity. Mutant ribozymes and substrates with variations at these positions were created and their kinetic activity analyzed. While mutation of A79 significantly reduced activity, further experiments found the hypothesis was incorrect. Additional nucleotides like A78 may interact with the substrate and warrant further investigation.
The document summarizes the central dogma of biology and the discovery of DNA as the genetic material. It describes key experiments that showed DNA replicates in a semiconservative manner, with each parental strand serving as a template for a new complementary daughter strand. The process of DNA replication requires several enzymes including DNA polymerase, helicase, ligase and primase to unwind, copy and join new DNA strands.
This document describes a technique for bioassaying histamine in the presence of prostaglandins using an Amberlite XAD-2 column. The column removes prostaglandins, rabbit aorta contracting substance, and possibly slow reacting substance of anaphylaxis, but allows histamine to pass through. Two banks of bioassay organs (rabbit aorta strip, rat stomach strip, guinea pig ileum) are used, with one bank above the column and one below. This allows accurate measurement of histamine levels in samples also containing prostaglandins and other substances that interfere with histamine bioassay. The method was tested using effluent from shocked, perfused guinea pig lungs and showed improved accuracy
DNA damage repair Neil3 gene Knockout in MOLT-4iosrjce
RNAi is superannuated cellular mechanism that protect organism against viruses that replicate
through double- stranded RNA. RNAi can be used to diminish gene expression from plasmid expressing and
inserted sequence repeat. A stable harpin would be expressed after the vector was integrated into the genome.
In this paper a shiRNA expressing vector for Neil3 was designed and developed which is capable of replication
in MOLT-4. This shiRNA vector had the ability to arose the RNAi pathway, and reduce the gene expression of
Neil3. This was assessed by using pSilence 4.1CMV as a vector, and Gapdh as positive control.
This study compared the cytotoxicity and induction of genes encoding metabolic enzymes in cells exposed to different alkylated polycyclic aromatic hydrocarbons (PAHs). An SRB assay showed little cell death between exposures of 1uM naphthalene, 1uM dimethyl naphthalene, 1uM BAP, and the vehicle control, except for 5uM BAP. RT-PCR analysis found that 1uM BAP induced expression of CYP1A1 and CYP1B1 genes, but 1uM naphthalene and dimethyl naphthalene did not induce gene expression. Comparing responses to the parent naphthalene and dimethylated naphthalene,
This study aims to clone and analyze GAPDH genes from various plant species to compare amino acid sequences related to catalytic function. Previous work successfully cloned GAPDH from Oxalis corniculata and Plectranthus amboinicus but not Myrtaceae psidium. The current study will continue work on the cloned samples through midipreps, restriction enzyme digestion and sequencing. Sequence data will undergo bioinformatics analysis to compare conserved amino acids of the GAPDH protein between plant species. Results could provide new genetic information published in GenBank and further the understanding of energy production pathways in plants.
The document discusses DNA barcoding of Impatiens balsamina using chloroplast and nuclear markers. Genomic DNA was successfully extracted from I. balsamina leaves. The psbA-trnH marker was found to be the best barcode, amplifying a 337 bp fragment that showed 100% sequence identity to I. balsamina voucher sequences in GenBank. RbcL and ITS2 were also successfully amplified and sequenced, while matK amplification was unsuccessful. The sequences were verified through bioinformatics analysis, demonstrating DNA barcoding can authenticate I. balsamina.
Introduction to Genetic Material, Physical and Chemical properties of the same and various types of coiling mechanisms as well as information about chromosomal and extra-chromosomal DNA.
This document is a thesis presented by Archie Lovatt for the degree of Doctor of Philosophy at the University of Leicester in 1994. The thesis describes molecular analysis of a P-N-acetyl-hexosaminidase gene from the oral pathogen Porphyromonas gingivalis W83. Lovatt cloned the nahA gene encoding P-N-acetyl-hexosaminidase from P. gingivalis and characterized the gene and its protein product. Sequence analysis revealed that nahA is 2331 base pairs long and encodes a 111 amino acid protein with homology to hexosaminidases from other sources. The nahA gene appears to be conserved in P. gingivalis strains
Identification of perfect housekeeping genes for gene expression studies in p...ICRISAT
Gene expression analysis using quantitative real-time PCR (qRTPCR) is a very sensitive technique which completely depends on stable performance of reference genes used in the study.
3 July 2014
1. DREB genes play an important role in improving crop tolerance to stresses like drought, salt, and cold. DREB transcription factors regulate stress-responsive genes allowing plants to adapt.
2. Case studies showed overexpression of DREB genes enhanced stress tolerance in crops like rice and sugarcane. Co-transformation with DREB and other stress genes improved tolerance more than single DREB genes.
3. DREB genes respond differently to various stresses. DREB1 genes respond mainly to cold while DREB2 genes respond to dehydration and heat. Proper expression analysis of DREB genes is important for abiotic stress tolerance in transgenic crops.
This study examined the genetic attributes associated with the enhanced sensitivity of an HIV-1 clade C envelope protein (HVTR-PG80v2.eJ7) to autologous broadly neutralizing plasma antibodies from an elite neutralizer. The researchers found that mutations in the V3/C3 region of the envelope protein were associated with its increased sensitivity to neutralization by autologous plasma antibodies. Depletion experiments showed that these mutations altered the envelope conformation to better expose epitopes targeted by both neutralizing and non-neutralizing antibodies in the plasma. Therefore, distinct vulnerabilities associated with antibody evasion could be linked to mutations in the V3/C3 region of the HIV envelope.
Transcriptional profiling of Halobacterium sp. NRC-1 showed changes in gene expression in response to changes in salinity and temperature. Growth under high salt stress resulted in modulation of genes for ion transporters like potassium and phosphate transporters. Growth at cold temperatures altered expression of genes for lipid metabolism, gas vesicles, and cold shock proteins. Heat shock induced several chaperone genes. The study provides insights into Halobacterium's responses to environmental stresses at the gene expression level.
This lab report details an experiment to construct knockouts of the FAD2 gene in the model plant Thlaspi arvense using RNA interference to reduce unsaturated fatty acid content and improve the quality of seed oil for biodiesel production. The FAD2 gene was amplified from pennycress cDNA using PCR. The amplified FAD2 fragments were inserted into an entry vector, which was then transformed into E. coli cells. Successful transformants were identified through DNA sequencing. The purified FAD2 constructs are now ready to be transformed into a binary vector for plant transformation experiments to knockout the FAD2 gene in pennycress seeds and analyze effects on seed oil composition.
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
International Journal of Clinical Pharmacology & Toxicology (IJCPT) ISSN:2167-910X is an Open Access journal and a peer-reviewed journal. Clinical Pharmacology & Toxicology is the all-encompassing and becoming an increasingly important discipline for the identification of disease targets and drug designing with their toxicological effects and means to eradicate diseases.
This document provides information on the kokum fruit tree (Garcinia indica). It describes the tree's origin in southern India and distribution in tropical forests. It outlines the tree's taxonomy and classification. The document discusses the culinary, pharmaceutical and industrial uses of kokum fruit, rind and butter. It also describes the tree's sex types, varieties including the high yielding 'Konkan Amrit' variety, challenges around scattered production and short harvesting periods, and opportunities for popularizing kokum-based products.
This study aimed to evaluate the efficacy of oral montelukast in acute asthma exacerbation. It was a randomized, double-blind, placebo-controlled trial conducted in a tertiary care hospital over 2 years. Patients presenting with acute asthma exacerbation were randomized to receive standard therapy with either montelukast or placebo. The primary outcomes of lung function, duration of hospital stay, and secondary outcomes showed no significant differences between the montelukast and placebo groups. The study concluded that montelukast provided no added benefit over standard therapy alone for acute asthma exacerbation. Larger multicenter trials are still needed.
Formulating coherent science and technology policies in nigeriaAlexander Decker
This document summarizes the history of science and technology policy in Nigeria since independence in 1960. It finds that policies have been ad-hoc and fragmented, lacking coherence and sustained focus. The first attempt at a national S&T policy was in 1966 but did not function due to civil war. Subsequent agencies and ministries in the 1970s-1980s focused on S&T but lacked longevity due to reorganizations and mergers. The document argues this is due to a lack of political will and underfunding for research. Universities expanded rapidly but without proper funding and facilities for research. Researchers face challenges of low pay and lack of maintenance that discourage retaining talent.
This document summarizes a project focused on in-situ conservation of tropical fruit tree diversity in South and Southeast Asia. The project showcases an integrated community biodiversity management approach used to conserve native Garcinia and Mangifera species on farms in Western Ghats, India. The approach documents crop diversity, creates awareness, establishes local nurseries, provides capacity building, and supports added-value activities. Key outcomes include assessing and documenting 48 mango varieties and 3 Garcinia species, establishing grafting experts to conserve knowledge, empowering local institutions, and generating income through new food products developed by women's groups. The community biodiversity management approach is found to holistically conserve biodiversity while improving livelihoods.
Este documento describe los principales documentos institucionales requeridos por la LOMCE, incluyendo un Proyecto Educativo, Normas de Organización y Convivencia, una Programación General Anual, y un Proyecto de Gestión. Cada documento tiene un propósito específico como orientar la autonomía pedagógica, establecer normas de conducta, planificar el curso escolar, y concretar la autonomía de gestión del centro respectivamente.
Sacred groves are small patches of forests in India dedicated to local deities and managed by communities. They serve as important reservoirs of biodiversity and medicinal plants. The coastal districts of Udupi and Dakshina Kannada in Karnataka contain many small sacred groves collectively covering a large area. Studies show they are rich in plant diversity including many endemic and rare species. However, these sacred groves now face threats such as encroachment, loss of traditional beliefs, and fragmentation due to modernization that could destroy this unique conservation model.
Sanjay Yadav presented on evaluating the neuroprotective effects of piracetam and vinpocetine in a rat model of Parkinsonism induced by rotenone. Motor functions, biochemical markers, and histopathology were assessed. Rats treated with rotenone showed motor deficits, decreased dopamine, increased oxidative stress, and neuronal loss compared to controls. Piracetam and vinpocetine treatment attenuated motor deficits, normalized biochemical alterations, and reduced neuronal loss compared to rotenone-treated rats. The results suggest piracetam and vinpocetine may have neuroprotective effects in Parkinsonism by reducing inflammation and oxidative stress.
Comparative evaluation of 2g single dose versus conventional dose azithromycin in uncomplicated skin and skin structure infections. Indian Journal Of Pharmacology. August 2015;Vol. 47; Issue 4
This document summarizes a randomized open-labeled phase IV clinical trial that evaluated the efficacy and safety of adding bromocriptine to metformin therapy in Indian patients with type 2 diabetes mellitus. The trial involved 74 patients randomized into three groups: metformin alone, metformin with 0.8 mg bromocriptine, or metformin with 1.6 mg bromocriptine. HbA1c levels were significantly reduced in all three groups over the course of the trial. Intergroup analysis found no significant differences in HbA1c reduction between the groups. Three patients reported adverse effects in the lower bromocriptine dose group. The study concluded that adding bromocriptine to metformin therapy is more effective in glycemic control compared
This document provides information on referencing and citation styles. It discusses the purpose of referencing, which is to avoid plagiarism and allow readers to identify sources. Different citation styles are covered, including Vancouver, Harvard and APA systems. Guidance is given on citing various sources like journal articles, books, book chapters, websites and more. Standard formats are outlined for structuring citations for each source type.
This document provides guidance on how to present a journal club. It discusses the definition and history of journal clubs, their aims to keep participants up to date on current literature and teach critical appraisal skills. Journal clubs can cover a range of topics and formats. The document outlines best practices for selecting articles, presenting critically on the content, and facilitating discussion. It emphasizes the benefits of journal clubs for improving knowledge, skills, and evidence-based practice.
This document summarizes several case studies related to pharmacology. It discusses appropriate antibiotic treatment for various infections, potential drug interactions, side effects of medications, and important counseling points for patients. Key drugs mentioned include amoxicillin, cephalosporins, ciprofloxacin, metronidazole, and various antidepressants and benzodiazepines. The case studies provide examples to illustrate proper medication use and management of side effects or risks.
This document provides instructions on how to write references in the Harvard and Vancouver styles. It explains that references are important to avoid plagiarism, show the breadth of research, acknowledge direct quotes, and provide evidence to support arguments. It then outlines the key elements to include for different types of references such as books, e-books, journal articles, and works with no author. Finally, it describes how to format in-text citations and structure a reference list in the Vancouver style.
The document summarizes different types of receptors and their classification. It discusses four main types of receptors: ligand gated ion channel receptors (inotropic), G-protein coupled receptors (metabotropic), kinase linked receptors, and nuclear receptors. It provides details about their molecular structure, signaling mechanisms, examples, and comparisons between receptor types. In summary, the document provides an overview of receptor pharmacology, classification of receptors, and their role in drug action and signaling pathways.
This study examined the sensitivity of six neuronal nicotinic acetylcholine receptor (nAChR) combinations to various nicotinic agonists when expressed in Xenopus oocytes. Each receptor combination displayed a distinct sensitivity profile. For example, the α2β2 combination was 10-fold more sensitive to nicotine than acetylcholine, while α3β2 was less sensitive to nicotine. Both the α and β subunits contributed to the pharmacological properties of neuronal nAChRs.
In vivo modulation of dopaminergic nigrostriatal pathways by cytisine derivat...Georgi Daskalov
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Robert J. Lefkowitz and Brian K. Kobilka won the 2012 Nobel Prize in Chemistry for their groundbreaking work studying G-protein–coupled receptors (GPCRs). Their research revealed key insights into how GPCRs function at the molecular level to transmit signals from outside to inside of cells. Specifically, Lefkowitz and Kobilka were able to clone and sequence the first GPCR, detect their binding properties, determine their three-dimensional structure, and elucidate the allosteric mechanism of signal transduction, establishing GPCRs as a family of related receptors. Their seminal findings provided a deeper understanding of the intricate signaling mechanisms of GPCRs and laid the foundation for advancing research and drug development targeting
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3) Nicotine displaced [3H]DBE binding in a stereospecific manner, with (-)-nicotine being approximately 6 times more potent
Diversity and distribution of nicotinic acetylcholine receptors in the locus ...Georgi Daskalov
This document summarizes a study investigating the diversity and distribution of nicotinic acetylcholine receptors (nAChRs) in neurons of the locus ceruleus (LC). The key findings are:
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2) One population (type A) consistently expressed α3 and β4 subunits and responded strongly to cytisine. The other (type B) lacked α3/β4 but expressed α6 and β3, responding more to nicotine.
3) Nicotine modulated noradrenaline release in the hippocampus with an order of potency of nicotine > cytisine
Its a brief ppt describing about the type of neurotansmitters in insect synapse and their respective receptors. It also sketches about the synaptic transmission in insect nervous system
This experiment studied the effects of stress during puberty using rat models. Rats were subjected to stressful events known as juvenile social subjugation to model stress during puberty. Autoradiography was used to visualize CRF1 and CRF2 receptors in the brain which are involved in the stress response. The results found that the basolateral nucleus of the amygdala primarily expressed CRF1 receptors, while the medial preoptic nucleus secondarily expressed these receptors. The medial preoptic nucleus also expressed CRF2 receptors, but not as intensely as CRF1 receptors. Expression of both receptors increased with age and was generally higher in females compared to males. This supported the idea that females have a more plastic
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This study examined the interaction of silver nanoparticles (Ag NPs) with human endothelial cells. Transmission electron microscopy and dynamic light scattering were used to characterize the 40nm and 80nm BPEI- and lipoic acid-coated Ag NPs. The nanoparticles were then incubated with human umbilical vein endothelial cells (HUVECs) for 24 hours. Microscopy showed intracellular uptake of Ag NPs in HUVECs and changes to cellular morphology at 10 μg/mL concentrations. Cell viability assays also demonstrated dose-dependent toxicity of Ag NPs to HUVECs regardless of size, surface charge, or chemistry. The results indicate the physicochemical properties of Ag NPs influence their interaction and toxicity in human
1) Rats treated with 3-nitropropionic acid (3-NP), a model of Huntington's disease, exhibited weight loss, gait abnormalities, and striatal lesions.
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This study investigated how genetic variation in the serotonin transporter gene (Slc6a4) and integrin beta 3 gene (Itgb3) interact to modulate serotonin uptake and transporter expression in mouse brain synapses. The researchers prepared synaptoneurosomes (preserved pre- and post-synaptic structures) from mouse midbrain, hippocampus and cortex with different genotypes of Slc6a4 and Itgb3. They found reduced serotonin transporter expression and uptake activity in midbrain synaptoneurosomes of mice with both genes heterozygous, revealing an interaction between the two genes. In contrast, changes were driven mostly by Slc6a4 in the hippocampus. The study provides evidence that
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This document discusses using bromophenols as potential therapeutics for treating type 2 diabetes mellitus (T2DM). Bromophenols have been identified as inhibitors of the enzyme tyrosine phosphatase 1B (PTP1B), which is involved in insulin signaling. A series of new bromophenol analogs will be synthesized and tested for PTP1B inhibitory activity using in vitro enzyme assays to elucidate their mechanism of action. Bromophenols occur naturally in marine organisms and have attracted interest as anti-diabetic agents due to their suspected PTP1B inhibitory activity. However, quinone species formed from bromophenols could be toxic, so further study is needed to
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This document summarizes national trends in prescription drug expenditures in the United States for 2015 and provides projections for 2016. Key findings include:
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This document discusses how pharmaceutical companies can improve clinical development and manufacturing processes through product lifecycle management (PLM). It identifies 7 key business processes for transforming R&D operations: 1) drug development program management, 2) regulatory archive management, 3) clinical trial management, 4) scale-up and commercial manufacturing, 5) quality management, 6) packaging and marketing asset management, and 7) global product registration. Implementing PLM using Oracle's solutions can deliver ROI by improving productivity, reducing time to market, and lowering development costs.
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Health-care interventions to promote and assist tobacco cessation: a review o...Georgi Daskalov
This document reviews the efficacy, effectiveness, and affordability of healthcare interventions for tobacco cessation. It finds that brief advice from healthcare workers can promote smoking cessation and is affordable globally. Telephone and text support programs and printed materials can assist with quit attempts and are globally affordable. Face-to-face behavioral support increases quit rates for cigarettes and smokeless tobacco and is affordable in middle- and high-income countries. Several medications can aid quitting when combined with behavioral support, with cytisine and nortriptyline being globally affordable. Brief advice, telephone/text support, self-help materials, cytisine, and nortriptyline are identified as globally affordable tobacco cessation interventions.
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Travel Clinic Cardiff: Health Advice for International TravelersNX Healthcare
Travel Clinic Cardiff offers comprehensive travel health services, including vaccinations, travel advice, and preventive care for international travelers. Our expert team ensures you are well-prepared and protected for your journey, providing personalized consultations tailored to your destination. Conveniently located in Cardiff, we help you travel with confidence and peace of mind. Visit us: www.nxhealthcare.co.uk
These lecture slides, by Dr Sidra Arshad, offer a simplified look into the mechanisms involved in the regulation of respiration:
Learning objectives:
1. Describe the organisation of respiratory center
2. Describe the nervous control of inspiration and respiratory rhythm
3. Describe the functions of the dorsal and respiratory groups of neurons
4. Describe the influences of the Pneumotaxic and Apneustic centers
5. Explain the role of Hering-Breur inflation reflex in regulation of inspiration
6. Explain the role of central chemoreceptors in regulation of respiration
7. Explain the role of peripheral chemoreceptors in regulation of respiration
8. Explain the regulation of respiration during exercise
9. Integrate the respiratory regulatory mechanisms
10. Describe the Cheyne-Stokes breathing
Study Resources:
1. Chapter 42, Guyton and Hall Textbook of Medical Physiology, 14th edition
2. Chapter 36, Ganong’s Review of Medical Physiology, 26th edition
3. Chapter 13, Human Physiology by Lauralee Sherwood, 9th edition
Histololgy of Female Reproductive System.pptxAyeshaZaid1
Dive into an in-depth exploration of the histological structure of female reproductive system with this comprehensive lecture. Presented by Dr. Ayesha Irfan, Assistant Professor of Anatomy, this presentation covers the Gross anatomy and functional histology of the female reproductive organs. Ideal for students, educators, and anyone interested in medical science, this lecture provides clear explanations, detailed diagrams, and valuable insights into female reproductive system. Enhance your knowledge and understanding of this essential aspect of human biology.
Travel vaccination in Manchester offers comprehensive immunization services for individuals planning international trips. Expert healthcare providers administer vaccines tailored to your destination, ensuring you stay protected against various diseases. Conveniently located clinics and flexible appointment options make it easy to get the necessary shots before your journey. Stay healthy and travel with confidence by getting vaccinated in Manchester. Visit us: www.nxhealthcare.co.uk
share - Lions, tigers, AI and health misinformation, oh my!.pptxTina Purnat
• Pitfalls and pivots needed to use AI effectively in public health
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• Building trust with communities online and offline
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Medical Quiz ( Online Quiz for API Meet 2024 ).pdf
cytisine
1. Ž .European Journal of Pharmacology 393 2000 123–135
www.elsevier.nlrlocaterejphar
Pharmacological and null mutation approaches reveal nicotinic receptor
diversity
Paul Whiteaker a,)
, Michael J. Marks a
, Sharon R. Grady a
, Ying Lu a
, Marina R. Picciotto b
,
Jean-Pierre Changeux c
, Allan C. Collins a
a
Institute for BehaÕioral Genetics, UniÕersity of Colorado, Campus Box 447, Boulder, CO 80303-0447, USA
b
Department of Psychiatry, Yale UniÕersity School of Medicine, New HaÕen, CT, USA
c
Laboratory of Molecular Neurobiology, Institute Pasteur, Paris, France
Accepted 21 January 2000
Abstract
w125
x Ž . w3
xWe have developed an array of assays for nicotinic acetylcholine receptor binding and function. I a-Bungarotoxin-, y - H nico-
w3
xtine-, and H epibatidine-binding nicotinic acetylcholine receptors were assayed in mouse brain membranes and sections. Nicotinic
w3
x w3
x Žw3
x . 86 q
acetylcholine receptor function was quantified using synaptosomal H dopamine, H g-aminobutyric acid H GABA , and Rb
efflux techniques. Additionally, the effects of b2 subunit deletion on each of the measures were assessed. Detailed pharmacological
w125
xcomparison revealed minimally six nicotinic binding subtypes: I a-bungarotoxin-binding nicotinic acetylcholine receptors; b2-sub-
Ž . w3
x w3
xunit-dependent and -independent high-affinity y - H nicotine-binding sites; b2-dependent and -independent cytisine-resistant H epi-
w3
xbatidine-binding sites; and a b2-dependent low-affinity H epibatidine binding site. Comparative pharmacology suggested that
w3
x Ž . 86 q Ž .H GABA and dihydro-b-erythroidine DHbE -sensitive Rb efflux are mediated by the same probably a4b2 nicotinic acetyl-
w3
x 86 q
choline receptor subtype, while other nicotinic acetylcholine receptor subtypes evoke H dopamine and DHbE-resistant Rb efflux. In
whole-brain preparations, each measure of nicotinic acetylcholine receptor function was b2 dependent. The majority of b2-independent
w3
xH epibatidine binding was located in small, scattered brain nuclei, suggesting that individual nuclei may prove suitable for identification
of novel, native nicotinic acetylcholine receptors. q 2000 Elsevier Science B.V. All rights reserved.
Keywords: Nicotinic acetylcholine receptor; Pharmacological comparison; Subunit null mutation; Binding; Activation
1. Introduction
Molecular cloning techniques have identified nine nico-
Ž .tinic acetylcholine receptor subunits a2–7, b2–4 , the
mRNAs of which are expressed in varying patterns and
Žquantities throughout the mammalian brain Lindstrom et
.al., 1996 . Mature nicotinic acetylcholine receptors appear
to be pentameric assemblies of these subunits, and because
different combinations of subunits produce receptors of
different subtypes, the potential for nicotinic acetylcholine
receptor diversity in mammalian brain is vast.
Identifying which nicotinic acetylcholine receptor sub-
types are expressed in mammalian central nervous system
)
Corresponding author. Tel.: q1-303-492-8752; fax: q1-303-492-
8063.
Ž .CNS has been a difficult task due to a paucity both of
truly subtype specific nicotinic compounds, and of well-
characterized assays for nicotinic receptor binding and
activation. These problems exacerbate each other, and
progress in one area is likely to have positive repercus-
sions in the other. To date, extensive biochemical data
have only been collected for two native nicotinic acetyl-
choline receptor subtypes: the ‘‘high-affinity agonist bind-
Ž w3
x Ž .ing’’ a4b2 subtype labeled by H cytisine and y -
w3
xH nicotine; Whiting and Lindstrom, 1987; Flores et al.,
.1992; Picciotto et al., 1995; Marubio et al., 1998 and the
Ž w125
xpredominantly or entirely a7 subtype labeled by I a-
.bungarotoxin, Schoepfer et al., 1990; Seguela et al., 1992 .
Identification of additional naturally expressed nicotinic
acetylcholine receptor subtypes is a priority as it will yield
insights into the rules governing assembly of subunit pep-
tides into receptor proteins, facilitate generation and isola-
0014-2999r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.
Ž .PII: S0014-2999 00 00052-2
2. ( )P. Whiteaker et al.rEuropean Journal of Pharmacology 393 2000 123–135124
tion of subtype specific compounds, and enhance under-
standing of the physiological roles of individual neuronal
nicotinic acetylcholine receptor subtypes in normal andror
pathological states.
One of our laboratory’s main priorities has been to
develop and characterize binding and functional assays of
nicotinic receptors. The primary motivation in this effort
has been to identify discrepancies between different mea-
sures of nicotinic acetylcholine receptor binding and func-
tion, which would indicate heterogeneity in the nicotinic
acetylcholine receptor populations responsible.
w3
xH Epibatidine has been shown to bind to multiple
nicotinic acetylcholine receptor subtypes with high-affinity
ŽPerry and Kellar, 1995; Marks et al., 1998; Parker et al.,
. Ž . Ž1998 . In rat Perry and Kellar, 1995 and mouse Marks et
. w3
xal., 1998 CNS, the majority of high-affinity H epibati-
dine binding occurs at the a4b2 subtype nicotinic acetyl-
Žcholine receptor, but additional sites distinguished by their
.relatively low cytisine affinity are also expressed in small
nuclei, dispersed across the brain. This information pro-
w3
xvided the impetus to use H epibatidine binding as a tool
to identify novel nicotinic acetylcholine receptor subtypes
in mouse brain.
A growing consensus that many nicotinic acetylcholine
receptors are located presynaptically, where they modulate
Ž .neurotransmitter release Wonnacott, 1997 , has encour-
aged efforts to study nicotinic acetylcholine receptor func-
tion in preparations of isolated nerve termini, or synapto-
somes. Accordingly, we have concentrated our efforts on
developing novel functional assays of nicotinic acetyl-
choline receptor mediated synaptosomal release. Such as-
says may be divided into those that monitor neurotrans-
mitter release, an indirect measure of nicotinic acetyl-
choline receptor activation, and those that measure ion flux
through the nicotinic acetylcholine receptor directly. In the
first category, we have described nicotinic acetylcholine
w3
xreceptor-mediated mouse brain synaptosomal H dopa-
Ž . w3
xmine Grady et al., 1992 and H g-aminobutyric acid
Žw3
x . Ž .H GABA Lu et al., 1998 efflux assays. In order to
directly measure nicotinic acetylcholine receptor-mediated
ion flux, we have characterized 86
Rbq
efflux assays, using
Ž .both discrete sampling Marks et al., 1993 and continuous
flow monitoring detection, which offers considerable in-
Ž .creases in temporal resolution Marks et al., 1999 . Ion
flux assays offer a means to measure activation of all of
the nicotinic acetylcholine receptors in a given preparation,
while neurotransmitter release assays will only measure
activation of nicotinic acetylcholine receptors associated
with synaptosomes containing the transmitter in question.
Whether this added selectivity is an advantage depends on
the particular experimental application. Detailed pharmaco-
logical comparisons among these biochemical assays indi-
cate considerable heterogeneity in the nicotinic acetyl-
choline receptor-mediated responses, suggesting mediation
by a number of different nicotinic acetylcholine receptor
subtypes.
As an adjunct to the conventional pharmacological ap-
proach, we have begun to use subunit null mutant animals,
as a further tool to provide insights as to the identities of
these putative nicotinic acetylcholine receptor subtypes.
Changes in, or losses of, nicotinic measures upon alter-
ations in nicotinic acetylcholine receptor subunit gene
expression powerfully implicate that gene’s product as a
component of the nicotinic acetylcholine receptor mediat-
ing the tested measure. The results reported here demon-
strate the utility of b2-null mutant mice in establishing the
role of the b2 nicotinic acetylcholine receptor subunit in
binding and functional measures of mouse brain nicotinic
acetylcholine receptors.
2. Materials and methods
2.1. Materials
w 3
x Ž . Ž . w7,8- H Dopamine 40–60 Cirmmol , y - N-methyl-
3
x Ž . w125
x ŽH nicotine 75 Cirmmol , I a-bungarotoxin initial
. w3
x Žspecific activity 200 Cirmmol and H GABA 84–90
.Cirmmol were obtained from Amersham, Arlington
w3
x Ž .Heights, IL. H Epibatidine 33.8 Cirmmol , and carrier
free 86
RbCl were bought from DuPont-NEN, Boston, MA.
The following compounds were purchased from Research
Ž .Biochemicals International, Natick, MA: q -epibatidine
Ž . Ž .hydrochloride, y -epibatidine hydrochloride, q -anato-
Ž .xin-a, epiboxidine, dihydro-b-erythroidine DHbE and
Ž Ž . .3- 2- S -azetidinylmethoxy pyridine dihydrochloride
Ž .A85380 . Sucrose and HEPES hemisodium salt were
Ž .bought from Boehringer-Mannheim Indianapolis, IN .
Econosafe scintillation cocktail was a product of Research
Products International, Arlington Heights, IL. Mecamy-
lamine was a gift from Merck Sharp and Dohme Research
Laboratory, Rahway, NJ. All other chemicals were sourced
from Sigma, St. Louis, MO.
2.2. Mice
C57BLr6J and b2 nicotinic acetylcholine receptor null
Ž .mutant mice Picciotto et al., 1995 were bred at the
ŽInstitute for Behavioral Genetics University of Colorado,
.Boulder, CO . C57BLr6J mice were housed five per cage,
and b2 nicotinic acetylcholine receptor null mutant mice
Žwere housed with like-sex littermates two to five per
.cage . Mice were maintained in a vivarium at 228C with a
Ž .12-h lightrdark cycle lights on from 7 AM to 7 PM . The
Žmice were allowed free access to food Harlan Tekland
.Rodent Diet and water. Animals of both sexes were used
between 60 and 90 days of age. All animal care and
experimental procedures were performed in accordance
with the guidelines and with the approval of the Animal
Care and Utilization Committee of the University of Col-
orado, Boulder.
3. ( )P. Whiteaker et al.rEuropean Journal of Pharmacology 393 2000 123–135 125
2.3. Autoradiography: preparation of sections
Autoradiography procedures were similar to those de-
Ž .scribed previously Pauly et al., 1989; Marks et al., 1998 .
ŽMice C57BLr6J or wild-type, heterozygous, or homozy-
.gous b2-null mutants were killed by cervical dislocation,
the brains were removed from the skull and rapidly frozen
Ž .by immersion in isopentane y358C, 10 s . The frozen
brains were wrapped in aluminum foil, packed in ice, and
Žstored at y708C until sectioning. Tissue sections 14 mm
.thick prepared using an IEC Minotome Cryostat refriger-
ated to y168C were thaw-mounted onto subbed micro-
Ž .scope slides Richard Allen, Richland, MI . Slides were
Ž .subbed by incubation with gelatin 1% wrv rchromium
Ž .aluminum sulfate 0.1% wrv for 2 min at 378C, drying
overnight at 378C, incubation at 378C for 30 min in 0.1%
Ž . Ž .wrv poly-L-lysine in 25 mM Tris pHs8.0 , and drying
at 378C overnight. Mounted sections were stored, desic-
cated, at y708C until use. Between 6 and 10 series of
sections were collected from each mouse brain.
( ) [3
]2.4. Autoradiography: y - H nicotine binding
Series of mouse brain sections were incubated in bind-
Žing buffer NaCl, 144 mM; KCl, 1.5 mM; CaCl , 2 mM;2
.MgSO , 1 mM; HEPES, 20 mM; pHs7.5 at 228C for 104
Ž . w3
xmin, prior to y - H nicotine binding. The samples were
Ž . w3
xthen incubated with 20 nM y - H nicotine for 1 h at
228C. An adjacent series of sections from each mouse was
Ž . w3
xused to determine non-specific y - H nicotine binding
Ž Ž . .in the presence of 1 mM y -nicotine bitartrate . The
Ž .slides were then washed as follows all washes at 08C : 5 s
Ž .in binding buffer twice , 5 s in 0.1= binding buffer
Ž . Ž .twice , 5 s in 5 mM HEPES pHs7.5 , twice.
[125
]2.5. Autoradiography: I a-bungarotoxin binding
A series of sections from each mouse was incubated in
Ž .binding buffer q0.1% wrv bovine serum albumin at
228C for 10 min. The samples were then incubated with 2
w125
x Ž .nM I a-bungarotoxin in binding buffer q0.1% wrv
bovine serum albumin for 4 h at 228C. An adjacent series
of sections from each mouse was used to determine non-
w125
x Žspecific I a-bungarotoxin binding in the presence of 1
Ž . .mM y -nicotine bitartrate . The slides were then washed
Ž .as follows all washes at 08C : 10 min in binding buffer
Ž . Ž .twice , 5 s in 0.1=binding buffer twice , 5 s in 5 mM
Ž .HEPES pHs7.5 , twice.
[3
]2.6. Autoradiography: high-affinity H epibatidine bind-
ing
w3
xSections for use in H epibatidine binding were incu-
bated in binding buffer at 228C for 10 min, followed by
w3
xincubation with 500 pM H epibatidine for 2 h at 228C.
Three series of adjacent sections were used from each
w3
x Žmouse to measure total H epibatidine binding no com-
. w3
xpeting ligand , H epibatidine binding in the presence of
Ž .50 nM unlabeled cytisine cytisine-resistant binding , and
w3
x Žnon-specific H epibatidine binding in the presence of 1
Ž . .mM unlabeled y -nicotine . The concentration of unla-
beled cytisine was chosen on the basis of results obtained
Ž .by Marks et al. 1998 . Slides were washed by sequential
Ž .incubation in the following buffers all steps at 08C : 5 s in
Ž . Ž .binding buffer twice , 5 s in 0.1= binding buffer twice ,
Ž .and twice for 5 s in 5 mM HEPES pHs7.5 .
2.7. Autoradiography: image collection
Labeled sections were initially dried with a stream of
Ž .air, then by overnight storage 228C under vacuum.
ŽMounted, desiccated sections were apposed to film 4–7
w125
xdays, Amersham Hyperfilm b-Max film for I a-
bungarotoxin-labeled sections; 8–12 weeks, Amersham
3 3
.Hyperfilm- H for H-labeled sections . After the films had
been exposed to the sections for an appropriate length of
time, they were developed, the films were illuminated
using a Northern Light light box, and autoradiographic
images of the sections were captured using a CCD imager
camera.
2.8. Membrane preparation
ŽEach mouse C57BLr6J or wild-type, heterozygous, or
.homozygous b2-null mutant was killed by cervical dislo-
cation. The brain was removed from the skull and placed
on an ice-cold platform. The hindbrain, cerebellum and
olfactory bulbs were discarded without further dissection
Ž .‘‘whole-brain’’ preparation . Samples were homogenized
Žin ice-cold hypotonic buffer NaCl, 14.4 mM; KCl, 0.2
mM; CaCl , 0.2 mM; MgSO , 0.1 mM; HEPES 2 mM;2 4
.pHs7.5 using a Teflon-glass tissue grinder. The particu-
late fractions were obtained by centrifugation at 20000=g
Ž .15 min, 48C; Sorval RC-2B centrifuge . The pellets were
resuspended in fresh homogenization buffer, incubated at
228C for 10 min, then harvested by centrifugation as
before. Each pellet was washed twice more by resuspen-
Žsionrcentrifugation, then stored in pellet form under ho-
.mogenization buffer at y708C until used. Protein concen-
trations in the membrane preparations were measured ac-
Ž .cording to the method of Lowry et al. 1951 , using bovine
serum albumin as the standard.
2.9. Ligand binding to membranes
w3
x ŽHigh-affinity H epibatidine binding at low ligand
.concentrations was quantified as described previously
Ž .Marks et al., 1998 . Incubations were performed in 1-ml
polypropylene tubes in a 96-well format, using 200 mg of
whole-brain membrane protein per tube. A 500-ml reaction
volume was used to minimize problems of ligand deple-
tion, and all incubations progressed for 2 h at 228C. The
4. ( )P. Whiteaker et al.rEuropean Journal of Pharmacology 393 2000 123–135126
w3
x Ž .concentration of H epibatidine 500 pM used in inhibi-
tion binding experiments was chosen to maintain binding
of ligand to the tissue at 5% or less of total ligand added.
w3
xCytisine-resistant H epibatidine binding was determined
by including 50 nM cytisine in the incubation conditions.
Non-specific binding was determined in all experiments by
Ž .the addition of 1 mM y -nicotine. Filter counts were
determined by liquid scintillation counting.
w3
xBinding of H epibatidine at high ligand concentrations
Ž .low plus high-affinity binding was measured as described
Ž .by Marks et al. 1999 , using a 100-ml incubation volume.
w3
xTotal H epibatidine binding was determined in the pres-
w3
xence of 10 nM H epibatidine at 228C for 60 min. Non-
specific binding was measured by the inclusion of 1 mM
Ž .y -nicotine in the incubation. Low-affinity binding was
w3
xcalculated by subtracting H epibatidine binding at 500
pM from that measured at 10-nM-labeled ligand, or as the
amount of low-affinity binding inhibited by incubation
with 300 mM dTC.
Ž . w3
xTotal y - H nicotine binding to membrane prepara-
tions was performed using the same protocol as for high
Ž . w3
xconcentration lowqhigh affinity H epibatidine bind-
Ž . w3
x Žing, but samples were incubated with y - H nicotine 20
.nM at 228C for 30 min.
2.10. Synaptosome preparation
ŽMice C57BLr6J or wild-type, heterozygous, or ho-
.mozygous b2-null mutants were killed by cervical dislo-
cation. Brains were removed from the skulls and placed on
86 q w3
xan ice-cold platform. For Rb and H GABA release
experiments the hindbrain, cerebellum and olfactory bulbs
were discarded without further dissection, resulting in a
‘‘whole-brain’’ tissue sample. Striatal tissue alone was
w3
xcollected for use in H dopamine release experiments.
Tissue was resuspended in 10 volumes of isotonic sucrose
Ž .solution 0.32 M sucrose, 5 mM HEPES, pHs7.5 . Crude
synaptosomal preparations were made by homogenization
Ž .in a hand-held glassrPTFE tissue grinder 20 strokes . The
homogenate was centrifuged at 500=g for 10 min, and
the resulting supernatant was then centrifuged at 12,000=g
for 20 min to yield the synaptosomal P2 pellet.
86 q (2.11. Rb superfusion protocol continuous flow moni-
)toring, fraction collection
86
Rbq
efflux superfusion using continuous flow moni-
toring was performed according to the protocol of Marks
Ž .et al. 1999 . Synaptosomal P2 pellets were resuspended
Žinto uptake buffer NaCl, 140 mM; KCl, 1.5 mM; CaCl ,2
2 mM; MgSO , 1 mM; HEPES hemisodium salt 25 mM;4
. 86 q
glucose 20 mM; pHs7.5 and then loaded with Rb .
Loading was achieved by incubation with 4 mCi of carrier
free 86
Rbq
at 228C for 30 min in a final volume of 35 ml.
Samples to be used with acetylcholine were incubated
Ž .during loading with diisopropylfluorophosphate 10 mM ,
an irreversible inhibitor of cholinesterase, for the least 10
min of loading. Uptake was terminated and unincorporated
86
Rbq
removed by filtration of the sample under gentle
Ž .vacuum y0.2 atm onto a 6-mm-diameter glass fiber
Ž .filter Type ArE; Gelman, Ann Arbor, MI , followed by
two washes with 0.5 ml uptake buffer.
Following filtration and wash, glass fiber filters contain-
ing 86
Rbq
loaded synaptosomes were transferred to
polypropylene superfusion supports. 86
Rbq
perfusion
Žbuffer NaCl, 135 mM; CsCl, 5 mM; KCl, 1.5 mM;
CaCl , 2 mM; MgSO , 1 mM; HEPES hemisodium salt 252 4
mM; glucose 20 mM; tetrodotoxin 50 nM; bovine serum
Ž . .albumin fraction V , 0.1%; pHs7.5 was delivered to the
filters at the rate of 2.5 mlrmin using a peristaltic pump
Ž .Gilson Minpuls 3; Gilson, Middleton, WI . Buffer was
removed from the platforms at using a pump running at a
Ž .higher rate 3.2 mlrmin , preventing the accumulation of
perfusion buffer on top of the filters. Efflux of 86
Rbq
from
the samples was achieved by pumping the superfusate
through a 200-ml volume flow-through Cherenkov count-
ing cell in a b-RAM radioactivity high-pressure liquid
Ž .chromatography detector INrUS systems, Tampa, FL .
Stimulation of the samples was performed by diverting
perfusion buffer through a 200-ml test loop containing the
test solution by means of a four-way PTFE injection valve
Ž .Alltech associates, Deerfield, IL , producing a stimulation
time of 5 s. DHbE-sensitive 86
Rbq
efflux was measured
Ž .following stimulation with 10 mM y -nicotine, while
DHbE-resistant 86
Rbq
efflux was evoked using 10 mM
Žepibatidineq2 mM DHbE present throughout the super-
.fusion process where used . When samples were to be
stimulated with acetylcholine or carbachol, the perfusion
Ž .buffer was supplemented with atropine 1 mM . Each
synaptosomal sample was stimulated only once, and re-
lease of 86
Rbq
from the samples was monitored for a total
of 4 min, with the stimulating pulse arriving at 2 min. This
timing permitted the definition of basal efflux before and
after agonist application.
Where 86
Rbq
efflux was monitored by fraction collec-
tion, the procedures were identical to those described
above with the following modifications: buffer perfusion
rate was 2 mlrmin, buffer was removed using a pump
Ž .running at a higher rate 2.5 mlrmin , fractions of eluate
were collected every 30 s, and their radioactive contents
were assessed using a Packard Tricarb 1600 gamma
counter.
[3
]2.12. H Dopamine superfusion protocol
w3
xH Dopamine superfusion was performed using a mod-
Ž .ification of the protocol of Grady et al. 1997 . The P2
synaptosomal pellet was resuspended into 800 ml of
Ždopamine perfusion buffer NaCl, 128 mM; KCl, 2.4 mM;
5. ( )P. Whiteaker et al.rEuropean Journal of Pharmacology 393 2000 123–135 127
CaCl , 3.2 mM; KH PO , 1.2 mM; MgSO , 1.2 mM;2 2 4 4
HEPES hemisodium salt 25 mM; glucose 10 mM; ascorbic
.acid, 1 mM; pargyline, 10 mM; pHs7.5 , and incubated
w3
x Žat 378C for 10 min. H Dopamine was added 4 mCi,
.yielding a final concentration of approximately 0.1 mM ,
and incubation was continued for a further 5 min. Samples
to be used with acetylcholine were incubated during load-
Ž .ing with diisopropylfluorophosphate 10 mM , an irre-
Ž .versible inhibitor of cholinesterase. Samples 80 ml were
collected and loading terminated by collection onto 6-
mm-diameter glass fiber filters and washing with superfu-
sion buffer, as for 86
Rbq
-loaded synaptosomes.
Washed filters bearing loaded synaptosomes were trans-
ferred onto 13-mm Gelman type ArE filters on polypropy-
Žlene platforms, and perfused with buffer dopamine perfu-
sion buffer supplemented with 10 mM nomifensine and
Ž .0.1% wrv bovine serum albumin; atropine 1 mM was
added when samples were to be stimulated with acetyl-
.choline or carbachol at a rate of 0.6 mlrmin for 10 min
before fraction collection was started. Fractions were col-
lected every 30 s, and buffer was actively pumped away
from the platforms at a rate of 1 mlrmin. Agonists and
antagonists were added to the perfusion buffer for 30 s
Ž .one fraction .
[3
]2.13. H GABA superfusion protocol
w3
xH GABA superfusion was performed according to the
Ž .protocol of Lu et al. 1998 . Synaptosomal P2 pellets were
Žresuspended into GABA perfusion buffer NaCl, 128 mM;
KCl, 2.4 mM; CaCl , 3.2 mM; KH PO , 1.2 mM; MgSO ,2 2 4 4
1.2 mM; HEPES hemisodium salt 25 mM; glucose 10
.mM; pHs7.5 , then incubated for 10 min at 378C with 1
ŽmM aminooxyacetic acid an inhibitor of GABA amino-
. w3
xtransferase , prior to H GABA loading. Loading was
achieved by incubation for a further 10 min at 378C in the
w3
x Žpresence of H GABA and unlabeled GABA to final
.concentrations of 0.1 and 0.25 mM, respectively . Samples
to be used with acetylcholine were incubated during load-
Ž .ing with diisopropylfluorophosphate 10 mM , an irre-
versible inhibitor of cholinesterase. Loading was termi-
nated by collection of samples onto glass fiber filters and
washing with superfusion buffer, as for 86
Rbq
-loaded
synaptosomes.
w3
xAs for H dopamine perfusion experiments, washed
w3
xfilters bearing H GABA-loaded synaptosomes were
transferred onto 13-mm Gelman type ArE filters on
Žpolypropylene platforms, and perfused with buffer perfu-
sion buffer supplemented with 0.1% wrv bovine serum
Ž .albumin; atropine 1 mM was added when samples were
.to be stimulated with acetylcholine or carbachol . Buffer
was pumped on at a rate of 1.8 mlrmin for 10 min before
fraction collection was started. Fractions were collected
every 12 s, and buffer was actively pumped away from the
platforms at a rate of 2.4 mlrmin. Agonists and antago-
Žnists were added to the perfusion buffer for 12 s one
.fraction .
2.14. Data analysis
86 q w3
x w3
xAgonist stimulated Rb , H dopamine and H -
GABAefflux were determined as follows. The fractions
preceding and following stimulation represent basal re-
lease, and were fit as the first-order process E sE eyk T
,t o
where E is the efflux at time t, E is the initial basalt o
efflux, and k is the rate of decline of efflux. This allowed
calculation of theoretical basal efflux in each fraction.
Agonist stimulated release was then quantified by subtract-
ing theoretical basal release from the number of counts
measured during agonist exposure. For 86
Rbq
efflux, filter
counts were assessed at the end of each experiment, and
86
Rbq
efflux was normalized as percent of tissue 86
Rbq
w3
xcontents released by agonist exposure. For H dopamine
w3
xand H GABA efflux experiments, agonist stimulated re-
lease was normalized as a multiple of theoretical baseline
release.
Dose–response curves were fitted using either the
Michaelis–Menten equation, the Hill equation, or two
Michaelis–Menten equations simultaneously. Curve fitting
was performed using the nonlinear curve fitting algorithm
Ž .in SigmaPlot 5.0 Jandel Scientific, San Rafael, CA .
3. Results
3.1. Autoradiography
Ž . w3
x w3
xThe nicotinic ligands y - H nicotine, H epibatidine,
w125
xand I a-bungarotoxin exhibited considerable variation
in their binding patterns at the levels of the superior and
Ž . w3
xinferior colliculi, as illustrated in Fig. 1. y - H Nicotine
binding was widespread at the level of the superior collicu-
lus in wild-type animals. Particularly high densities of
Ž . w3
xy - H nicotine binding were seen in the whole of the
interpeduncular nucleus, and the superior colliculus. The
thalamus and some layers of the cortex were also relatively
w3
xdensely labeled. The pattern of H epibatidine binding
Ž . w3
xclosely resembled that of y - H nicotine binding, but
binding densities in the superficial layers of the superior
colliculus and in the interpeduncular nucleus were much
w3
xgreater, making H epibatidine binding in the rest of the
section appear fainter by comparison. Cytisine-resistant
w3
xH epibatidine binding at this level of the brain was
restricted to the interpeduncular nucleus and the superficial
layers of the superior colliculus, the regions that differed
Ž . w3
x w3
xbetween the y - H nicotine and H epibatidine binding
w125
xpatterns. In contrast, the highest levels of I a-
bungarotoxin binding were seen in the outer shell of the
interpeduncular nucleus, the superficial layers of the supe-
rior colliculus, the subiculum, and the red nucleus.
6. ( )P. Whiteaker et al.rEuropean Journal of Pharmacology 393 2000 123–135128
Ž . w3
x w125
x w3
x Ž .Fig. 1. Autoradiographic representation of y - H nicotine, I a-bungarotoxin, and H epibatidine total, and with 50 nM cytisine binding in
Ž .wild-type and b2 nicotinic acetylcholine receptor subunit-null mouse brain. Sections 14 mm were collected at the level of the superior and inferior
Ž . w3
x Ž . w125
x Ž . w3
x Ž .colliculi, then incubated with 20 nM y - H nicotine top row , 2 nM I a-bungarotoxin second row , 500 pM H epibatidine alone third row , or
w3
x Ž .500 pM H epibatidineq50 nM cytisine last row as described in the Methods section. The panels are digital images of autoradiograms. The
Ž .abbreviations used to identify brain regions are: Cx, cortex; DTN, dorsal tegmental nucleus; ICCN, inferior colliculus central nucleus ; ICDC, inferior
Ž . Ž . Ž .colliculus dorsal cortex ; ICEC, inferior colliculus external cortex ; IPN, interpeduncular nucleus; IPNC, interpeduncular nucleus caudal nucleus ; PN,
pontine nuclei; RN, red nucleus; SC, superior colliculus; Sub, subiculum; Thal, thalamus.
Ž . w3
xAt the level of the inferior colliculus, y - H nicotine
binding was largely restricted to the external cortex of the
inferior colliculus, the pontine nuclei, and to the area
w125
xsurrounding the dorsal tegmental area, while I a-
bungarotoxin labeling was particularly intense in the cen-
tral nucleus and dorsal cortex of the inferior colliculus, and
in the dorsal tegmental nucleus itself. At the level of the
w3
xsuperior colliculus, H epibatidine binding was similar to
Ž . w3
xthat of y - H nicotine, but with a striking increase in
binding in the dorsal cortex compared to the rest of the
inferior colliculus. In these sections, cytisine-resistant
w3
xH epibatidine binding was almost exclusively located in
the dorsal cortex of the inferior colliculus.
Deletion of the nicotinic acetylcholine receptor b2 sub-
Ž .unit had a dramatic effect on the expression of y -
w3
xH nicotine binding, eliminating it in all regions studied,
apart from the caudal subnucleus of the interpeduncular
Ž .nucleus where binding was greatly reduced . In contrast,
w125
xb2 subunit deletion had no discernable effect on I
w3
xa-bungarotoxin binding. Overall levels of H epibatidine
binding were greatly reduced by b2 subunit deletion, but
binding remained in the interpeduncular nucleus, central
nucleus and dorsal cortex of the inferior colliculus, and a
small subset of the pontine nuclei. Cytisine-resistant
w3
xH epibatidine binding in the interpeduncular nucleus was
unaffected by loss of b2 subunit expression, which also
w3
xhad little effect on cytisine-resistant H epibatidine bind-
ing in the inferior colliculus. In contrast, cytisine-resistant
w3
xH epibatidine binding in the superficial layers of the
superior colliculus and pontine nuclei was eliminated in
the absence of the b2 subunit.
[3
]3.2. Membrane binding: low and high H epibatidine
concentrations
Ž . w3
xAs shown by Marks et al. 1999 , H epibatidine binds
to two classes of nicotinic sites: those with K values ind
7. ( )P. Whiteaker et al.rEuropean Journal of Pharmacology 393 2000 123–135 129
the picomolar range, and those with nanomolar affinity for
the ligand. The effects of b2 genotype on both sets of
binding sites are illustrated in Fig. 2.
Ž . w3
xAs shown in Fig. 2 top row , cytisine inhibited H epi-
batidine binding to mouse whole-brain membrane prepara-
w3
xtions in a biphasic manner. Labeling with 500 pM H epi-
batidine saturated the high-affinity binding sites, of which
Žapproximately 15–20% were cytisine-resistant Marks et
.al., 1999 . As predicted by the autoradiography experi-
ments, loss of the b2 nicotinic acetylcholine receptor
subunit reduced the number of whole-brain high-affinity
w3
xH epibatidine binding sites by approximately 95%. In
w3
xb2-heterozygous animals, high-affinity H epibatidine
binding sites were reduced by approximately 50%, demon-
strating a clear gene dosage effect. Cytisine-resistant
w3
x ŽH epibatidine binding sites defined in the presence of 50
.nM cytisine were proportionately less affected by loss of
b2 gene expression: heterozygous b2-null mutants re-
tained approximately 75% of wild-type cytisine-resistant
w3
xH epibatidine binding, while homozygous b2-null mu-
tants expressed 5 fmolrmg protein of cytisine-resistant
w3
xH epibatidine binding, or approximately 25% of that seen
in wild-type animals. Whole-brain cytisine-sensitive
w3
x Ž Ž . w3
xH epibatidine binding corresponding to y - H nico-
.tine binding sites; Marks et al., 1998 fell to undetectable
levels in the absence of b2 subunit expression. Thus, all
w3
x Ž . Ž . w3
xFig. 2. H Epibatidine binding at low 500 pM and high 10 nM ligand concentrations: effect of b2 genotype. H Epibatidine binding to whole brain
w3
xmouse particulate fractions was measured as described in the Methods section. The top left panel represents inhibition of H epibatidine binding by
Ž . Ž .cytisine at low- ` and high- I labeled ligand concentrations, while the bottom left panel displays inhibition by D-tubocurarine at the same labeled
w3
xligand concentrations. The lines represent two site Michaelis–Menten fits to the data. The inset panels show the difference in H epibatidine binding
w3
xinhibition observed between assays using high and low concentrations, and represent calculated inhibition profiles for low-affinity H epibatidine binding.
w3
xThe remaining panels in the upper row show the effects of b2 genotype on total high-affinity H epibatidine binding, cytisine resistant high-affinity
w3
x w3
xH epibatidine binding, and cytisine sensitive high-affinity H epibatidine binding. The remaining panels in the bottom row illustrate the effect of b2
Ž . w3
x w3
xgenotype on total lowqhigh affinity H epibatidine binding at 10 nM labeled ligand, binding of 10 nM H epibatidineq300 mM D-tubocurarine
Ž w3
x . Ž . w3
x Ž w3
xhigh-affinity H epibatidine binding only , and D-tubocurarine 300 mM sensitive binding of 10 nM H epibatidine high-affinity H epibatidine
.binding only . Each bar represents the mean"SEM of at least four independent determinations.
8. ( )P. Whiteaker et al.rEuropean Journal of Pharmacology 393 2000 123–135130
w3
xwhole-brain H epibatidine binding detectable in homozy-
gous b2-null mice was cytisine resistant.
Ž .As shown in Fig. 2 left column, insets , low-affinity
w3
x ŽH epibatidine binding sites defined as the difference
w3
xbetween sites detected using 500 pM and 10 nM H epi-
.batidine; Marks et al., 1999 comprised approximately 50
fmolrmg protein in wild-type animals. Again, most
w3
xH epibatidine binding at 10 nM ligand was dependent on
b2 subunit expression: in the absence of b2 subunit
expression, only 20 fmolrmg protein of sites were re-
tained, from a wild-type population of 150 fmolrmg pro-
tein. Heterozygous b2-null mutants retained an intermedi-
w3
xate amount of H epibatidine binding. High-affinity
w3
x w3
xH epibatidine binding sites defined using 10 nM H epi-
Žbatidine in the presence of 300 mM dTC Marks et al.,
.1999 were indistinguishable from those defined with 500
w3
x w3
xpM H epibatidine. Low-affinity H epibatidine binding
sites were considerably less sensitive to b2 genotype than
high-affinity sites, with approximately 40% being retained
in homozygous b2-null mutant animals. Again, a clear
gene dosage effect was seen: heterozygous b2-null mutant
animals expressed an intermediate density of low-affinity
w3
xH epibatidine binding sites.
3.3. Synaptosomal efflux assays: inter-assay comparisons
In order to compare the pharmacological properties of
the various nicotinic acetylcholine receptor activation as-
says developed in this laboratory, the EC and maximum50
Ž .efflux E values for a core group of nine nicotinicmax
Žreceptor agonists acetylcholine, anatoxin-a, carbamyl-
choline, cytisine, epibatidine, methylcarbamylcholine,
Ž . Ž . .y -nicotine, q -nicotine, and tetramethylammonium
were determined in each assay. In some cases, other
agonists were also assessed.
86 q w3
x w3
xFig. 3. Comparisons of agonist potencies in synaptosomal Rb -, H dopamine-, and H GABA-efflux assays. EC values were determined for a panel50
86 q w3
x w3
xof nicotinic receptor agonists in the continuous flow monitoring Rb -efflux, H dopamine-, and H GABA-release synaptosomal nicotinic acetylcholine
receptor activation assays, as described in the Materials and Methods section. Points represent the means of at least three independent determinations, error
bars were omitted for clarity.
9. ( )P. Whiteaker et al.rEuropean Journal of Pharmacology 393 2000 123–135 131
Pairwise comparisons of agonist potency are shown in
Fig. 3. Dramatic differences in relative agonist potency
between assays were not observed, as shown by the statis-
Žtically significant correlations in EC values the lowest r50
value measured was 0.70, the highest was 0.95, between
w3
x 86 q
the H GABA and DHbE-resistant Rb efflux and the
w3
x 86 q
H GABA and DHbE-sensitive Rb efflux assays, re-
.spectively . However, EC values at the DHbE-resistant50
response were typically two orders of magnitude higher
than those measured in the other assays.
Larger differences were noted between assays in com-
parisons of relative agonist efficacies, as shown in Fig. 4.
w3
xAgonist efficacies in H GABA and DHbE-sensitive
86 q Ž .Rb efflux assays were highly correlated rs0.91 ,
echoing the high correlation in agonist potencies between
these two measures of nicotinic acetylcholine receptor
activation. Each of the other pairs of assays showed notice-
ably lower correlation between agonist efficacies than
were noted for agonist potencies, with the poorest match of
w3
xagonist potencies observed between the H dopamine and
86 q Ž .DHbE-resistant Rb efflux assays rs0.12 .
3.4. Synaptosomal efflux assays: effect of b2 genotype
The effects of b2 genotype on each of the nicotinic
acetylcholine receptor activation assays were studied, and
the results are summarized in Fig. 5. Previous work identi-
Ž . 86 q
fied the standard fraction collecting synaptosomal Rb
efflux response as being mediated by nicotinic acetyl-
Žcholine receptors of the a4b2 subtype Marks et al.,
.1996 . As might be expected, loss of b2 subunit expres-
sion resulted in loss of standard 86
Rbq
efflux stimulated
Ž . Žby 10 mM y -nicotine 1.55"0.16% of tissue contents
in wild type animals, 0.13"0.03% in homozygous b2-null
.mutants . Although some loss of response was seen in
heterozygous b2-null mutants, the majority of the response
Žwas retained 1.15"0.06% of tissue contents, or 74% of
.wild-type activity . In contrast, the effect of b2 genotype
Ž . w3
xon y - H nicotine binding, which measures a4b2 nico-
tinic acetylcholine receptor density, was more noticeable in
heterozygous b2-null mutant animals than in the func-
tional assay: whole-brain binding dropped to 24.4"0.8
fmolrmg protein, or 55% of wild-type levels of 44.4"7.0
86 q w3
x w3
xFig. 4. Comparisons of agonist efficacies in synaptosomal Rb -, H dopamine-, and H GABA-efflux assays. Maximum activation elicited by a panel
86 q w3
x w3
xof nicotinic receptor agonists was measured in the continuous flow monitoring Rb -efflux, H dopamine-, and H GABA-release synaptosomal
nicotinic acetylcholine receptor activation assays, as described in the Methods section. Points represent the means of at least three independent
determinations, error bars were omitted for clarity.
10. ( )P. Whiteaker et al.rEuropean Journal of Pharmacology 393 2000 123–135132
86 q w3
xFig. 5. Comparison of the effects of b2 nicotinic acetylcholine receptor subunit-null mutation on agonist induced synaptosomal Rb , H dopamine, and
w3
x Ž .H GABA efflux. Synaptosomes were prepared from mice of each b2-null genotype wild-type, heterozygous, and homozygous . The magnitude of
86 q w3
x w3
x Ž .Rb -efflux, H dopamine, and H GABA release evoked by a maximally stimulating dose of y -nicotine was determined for each genotype, in each
Ž . w3
x Ž .assay. For comparison, the effect of b2 genotype on y - H nicotine binding to whole-brain membranes is also shown bottom right panel . Values are
the means"SEM of at least three independent determinations.
Ž . w3
xfmolrmg protein. Whole-brain y - H nicotine binding
was undetectable in homozygous b2-null mutants. The
effects of b2-null mutation on each of the other functional
responses were qualitatively similar to those measured on
the standard 86
Rbq
efflux response, with almost complete
elimination of nicotinic acetylcholine receptor mediated
function in the homozygous b2-null mutants, and )50%
retention of function in the heterozygotes.
4. Discussion
The assays described in this study represent the results
of an effort to develop and characterize a variety of
nicotinic acetylcholine receptor-mediated binding and
functional measures. The main motivation for this effort
was to attempt to detect differences among the assays,
pointing to underlying nicotinic acetylcholine receptor di-
versity. Success in this endeavor would be an important
step towards identifying the expression and physiological
roles of the potentially wide variety of mammalian neu-
ronal nicotinic acetylcholine receptor subtypes.
In the autoradiography experiments, the pattern of
w125
x Ž .I a-bungarotoxin 2 nM binding was distinctively dif-
w3
x Ž . w3
xferent from that of H epibatidine and y - H nicotine.
w125
xThis fits with the consensus that I a-bungarotoxin bind-
ing sites in mammalian brain differ from high-affinity
Žagonist binding sites Clarke et al., 1985; Pauly et al.,
.1989 , and largely or entirely correspond to a single class
of a7 containing nicotinic acetylcholine receptors. The
w125
xlack of effect of b2 genotype on I a-bungarotoxin
Ž .binding reinforces this identification. In contrast, y -
w3
x Ž .H nicotine 20 nM binding was heavily dependent upon
the expression of the b2 nicotinic acetylcholine receptor
subunit, as would be expected for a population thought to
be almost exclusively composed of a4b2 subtype nico-
tinic acetylcholine receptors. A small population of high-
Ž . w3
xaffinity y - H nicotine binding sites was retained in the
caudal nucleus of the interpeduncular nucleus in b2-null
homozygous animals. This confirms the findings of Zoli et
11. ( )P. Whiteaker et al.rEuropean Journal of Pharmacology 393 2000 123–135 133
Ž .al. 1998 and demonstrates that non-b2-containing nico-
Ž . w3
xtinic acetylcholine receptors can bind y - H nicotine
with detectable affinity. As shown in Fig. 5, this novel
population, while potentially important in the interpedun-
cular nucleus, represents a vanishingly small portion of the
Ž . w3
xwhole-brain y - H nicotine binding population.
Previous workers demonstrated that the majority of
w3
x Ž .high-affinity H epibatidine binding occurs at y -
w3
xH nicotine binding, a4b2 nicotinic acetylcholine recep-
Ž .tors Perry and Kellar, 1995; Marks et al., 1998 . In
Ž . w3
xaddition to y - H nicotine binding sites, these workers
w3
xalso showed that H epibatidine binds with high affinity to
a second population of nicotinic acetylcholine receptors,
distinguished by a relatively low affinity for the nicotinic
receptor agonist cytisine. As illustrated in Fig. 1, the
Ž . w3
xdifferences in binding between y - H nicotine and
w3
xH epibatidine can be explained by the presence of addi-
w3
xtional, cytisine-resistant H epibatidine binding sites. In
Ž .agreement with Marks et al. 1998 , the majority of high-
w3
xaffinity H epibatidine binding was abolished in animals
lacking the b2 nicotinic acetylcholine receptor subunit.
However, even in homozygous b2-null mutant animals, a
w3
xsmall amount of high-affinity H epibatidine binding was
Ž .detectable Fig. 1 . As shown in Fig. 1, some cytisine-re-
w3
xsistant H epibatidine binding sites are retained in ho-
mozygous b2-null mutant mice. As noted by Marks et al.
Ž . w3
x1998 , the pattern of cytisine-resistant H epibatidine
binding site expression largely coincides with that of the
a3 nicotinic acetylcholine receptor subunit mRNA, sug-
gesting a role for this subunit in the cytisine-resistant
w3
xH epibatidine binding sites. This hypothesis is supported
Ž .by evidence from Parker et al. 1998 and Xiao et al.
Ž .1998 , who show that heterologously expressed a3-con-
taining nicotinic acetylcholine receptors have relatively
w3
xlow affinities for cytisine, but bind H epibatidine with
Ž .high-affinity. Membrane binding experiments Fig. 2 show
w3
xthat H epibatidine binding in homozygous b2-null mu-
tant mice is exclusively cytisine-resistant. While cytisine-
w3
xresistant H epibatidine binding sites in the interpeduncu-
lar nucleus and inferior colliculus are retained in the
absence of b2 subunit expression, cytisine-resistant sites
in the superficial layers of superior colliulus are lost in
mice that do not express the b2 subunit. In homozygous
w3
xb2-null mice, cytisine-resistant H epibatidine binding was
found in regions expressing high levels of the b4 nicotinic
Žacetylcholine receptor subunit Dinelly-Miller and Patrick,
.1992 . Thus, it is possible that, in mouse brain, a3 and b4
w3
xsubunits may combine to form cytisine-resistant H epi-
batidine binding nicotinic acetylcholine receptors.
In addition to labeling a variety of nicotinic acetyl-
Ž .choline receptor subtypes at low concentrations 500 pM ,
w3
xH epibatidine binds to lower affinity sites when used at
Ž .concentrations 10 nM similar to those employed for other
ligands. As detailed in Fig. 2, these low-affinity sites are
distinguished by a relatively high sensitivity to the antago-
nist D-tubocurarine. This identification is reinforced by the
extremely close match between the amounts of high-affin-
w3
xity H epibatidine binding sites, and those detected using
w3
x Ž10 nM H epibatidineq300 mM D-tubocurarine Fig. 2,
.Table 1 . In a similar manner to their high-affinity counter-
w3
xparts, low-affinity H epibatidine binding sites may be
divided into two groups, those that are lost in the absence
of b2 subunit expression, and those that are retained. The
w3
xidentity of these low-affinity H epibatidine binding sites
is not known, although it is possible that the b2 subunit-in-
dependent sites may correspond to a7 nicotinic acetyl-
choline receptors, which have a nanomolar affinity for
w3
x Ž .H epibatidine Gerzanich et al., 1995 .
Thus, autoradiography and membrane binding studies
demonstrate the existence of at least six nicotinic acetyl-
Ž . w125
xcholine receptor subtypes: 1 I a-bungarotoxin bind-
Žing, a7 containing nicotinic acetylcholine receptors; 2 and
. Ž . w3
x3 high-affinity y - H nicotine binding sites dependent
Ž .on, or independent of, b2 subunit expression; 4 and 5 b2
subunit-dependent and -independent cytisine-resistant
w3
x Ž . w3
xH epibatidine binding sites; and 6 low-affinity H epi-
batidine binding sites that require b2 expression. In addi-
w3
xtion, if low-affinity H epibatidine binding sites, which
are independent of b2 expression are not composed of a7
Žcontaining nicotinic acetylcholine receptors see preceding
.paragraph , these may represent a seventh pharmacological
subtype. The ligand binding studies illustrate an important
point: the majority of the novel nicotinic acetylcholine
receptor subtypes are concentrated in small, dispersed
brain nuclei. Consequently, attempts to characterize these
sites will be greatly assisted by concentrating on individual
nuclei, rather than using whole-brain preparations. Further,
subunit-null mutant mice may prove very useful in such
attempts, by eliminating expression of nicotinic acetyl-
Ž .choline receptor subtypes for instance, the a4b2 , which
mask the presence of the novel receptors.
As for the results of binding assay comparisons, activa-
tion pharmacologies vary between the synaptosomal re-
Ž .lease assays. Marks et al. 1999 demonstrated striking
Žsimilarities between the properties of the standard fraction
. 86 q
collecting Rb efflux assay, and the DHbE-sensitive
continuous flow 86
Rbq
efflux response, indicating that
both were probably measures of activation at the same
a4b2-subtype nicotinic acetylcholine receptor. This identi-
fication is reinforced by the abolition of both responses in
Ž . Ž .b2-null homozygotes Fig. 5 . Interestingly, although y -
w3
xH nicotine binding at a4b2-subtype nicotinic acetyl-
choline receptors is approximately halved in b2-null het-
Ž .erozygotes Fig. 5 , the effect on function in each of the
assays is much less dramatic. This suggests that either a
degree of functional compensation occurs in the heterozy-
gous b2-null animals, whereby the remaining receptors are
more functionally efficient, or wild-type animals express a
substantial population of ‘spare’, or unused receptors.
As indicated by the high correlations in pairwise com-
parisons, no dramatic differences were observed in rank
order of potency between functional assays, although it
12. ( )P. Whiteaker et al.rEuropean Journal of Pharmacology 393 2000 123–135134
should be noted that the absolute EC values of drugs for50
DHbE-resistant 86
Rbq
efflux were approximately 100-fold
higher than for the other responses, indicating mediation
by a different nicotinic acetylcholine receptor population.
When agonist efficacies are considered, however, differ-
ences between the assays become much more apparent.
For instance, the agonist potencies between the
w3
x 86 q
H dopamine release and DHbE-resistant Rb efflux
Ž .assays are highly correlated rs0.90 , but when agonist
efficacies are compared between assays this similarity
Ž .disappears rs0.12 . Only one pair of assays retained a
high correlation between agonist potencies and efficacies
Ž . w3
xrs0.95 and 0.91, respectively : H GABA release and
DHbE-sensitive 86
Rbq
efflux. Together with the ex-
tremely similar absolute drug EC values between the two50
assays, this is strong evidence that whole-brain synaptoso-
w3
xmal H GABA release is also mediated by a4b2 nicotinic
acetylcholine receptors. Consequently, a4b2 nicotinic
acetylcholine receptors may be assigned responsibility for
w3
xat least the majority of nicotinically mediated H GABA
release, and standard and DHbE-sensitive continuous flow
86
Rbq
efflux responses in whole-brain synaptosomal
w3
xpreparations. Conversely, H dopamine release and
DHbE-resistant 86
Rbq
efflux are presumably evoked
through another two, distinct nicotinic acetylcholine recep-
tor subtypes. These additional nicotinic acetylcholine re-
ceptor subtypes are also dependent on expression of the b2
nicotinic acetylcholine receptor subunit. The results pre-
sented here show that the approach of comparing phar-
macological properties across functional assays can be
highly successful in illuminating underlying nicotinic
acetylcholine receptor diversity. However, it is important
to note that the common practice of comparing agonist
potencies is relatively ineffectual in this regard. Given the
generally poor subtype selectivity of existing nicotinic
compounds, the full potential of this approach is only
Žrealized when other assay parameters such as absolute
.potencies, and especially agonist efficacies are considered
simultaneously.
In conclusion, the studies described here have provided
evidence for a wealth of pharmacologically distinct nico-
tinic acetylcholine receptor subtypes, responsible for both
nicotinic activation and binding in mammalian brain. Some
progress has been made in assigning particular measures to
individual nicotinic acetylcholine receptor subtypes, but
much work remains to be done in this regard. As the
preliminary findings presented here for b2-null mutant
mice demonstrate, the growing number of nicotinic acetyl-
choline receptor subunit mutants becoming available will
provide invaluable assistance, as will the existence of a
battery of reliable, pharmacologically distinct nicotinic
assays. Interestingly, the b2-null mutant mice have so far
proven to be of more use in discriminating between the
nicotinic binding sites than the functional assays character-
Žized to date all of which are abolished by b2 subunit
.deletion . However, the ligand binding results indicate that
individual brain nuclei may prove to be rich sources of
novel nicotinic acetylcholine receptor subtypes, and would
make excellent candidates for study using both ligand
binding and functional approaches.
Acknowledgements
This work was supported by grants DA-03194 and
DA-11156 from the National Institute on Drug Abuse.
ACC is supported, in part, by Research Scientist Award
DA-00197 from the National Institute on Drug Abuse.
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