Background Hereditary ataxia (HA) represents a group of genetically heterogeneous neurodegenerati... more Background Hereditary ataxia (HA) represents a group of genetically heterogeneous neurodegenerative diseases caused by dysfunction of the cerebellum or disruption of the connection between the cerebellum and other areas of the central nervous system. Phenotypic manifestation of HA includes unsteadiness of stance and gait, dysarthria, nystagmus, dysmetria and complaints of clumsiness. There are no specific treatments for HA. Management strategies provide supportive treatment to reduce symptoms. Objectives This systematic review aimed to identify, evaluate and summarise the published literature on the therapeutic roles of natural remedies in the treatment of HA to provide evidence for clinical practice. Methods A systematic literature search was conducted using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). Web of Science, PubMed and Science Direct Scopus were thoroughly searched for relevant published articles from June 2007 to July 2020. Results Ten...
Stroke causes death and disability globally but no neuroprotectant is approved for post-stroke ne... more Stroke causes death and disability globally but no neuroprotectant is approved for post-stroke neuronal injury. Neuroprotective compounds can be identified using oxygen glucose deprivation (OGD) of neuronal cells as an in vitro stroke model. Nerve growth factor (NGF)-differentiated PC12 pheochromocytoma cells are frequently used. However, investigators often find their clonal variant undifferentiable and are uncertain of optimal culture conditions. Hence we studied 3 commonly used PC12 variants: PC12 Adh, PC12 from Riken Cell Bank (PC12 Riken) and Neuroscreen-1 (NS-1) cells. We found DMEM the optimal media for PC12 Riken and NS-1 cells. Using a novel serum-free media approach, we identified collagen IV as the preferred adhesive substrate for both cell lines. We found PC12 Adh cells cannot attach without serum and is unable to differentiate using NGF. NS-1 cells differentiated to a maximal 72.7 ± 5.2% %, with substantial basal differentiation. We optimised differentiated NS-1 cells f...
... Okamoto and Nishimoto (1992) identified a BBXB or BBXXB motif. In the case of the α 2A -AR i3... more ... Okamoto and Nishimoto (1992) identified a BBXB or BBXXB motif. In the case of the α 2A -AR i3c loop, Ikezu et al. ... Clarke WP. (1998) Effector pathway-dependent relative efficacy at serotonin type 2A and 2C receptors: Evidence for agonist-directed trafficking of receptor stimulus. ...
Green Fluorescent Protein (GFP) was first isolated from light-producing jellyfish in the 1960s. S... more Green Fluorescent Protein (GFP) was first isolated from light-producing jellyfish in the 1960s. Since the first reported expression of GFP into living organisms in 1994, it has become one of the most studied proteins in biochemistry and molecular biology. The ability of fluorescent proteins to fluoresce after expression in living cells has afforded researchers a tool to track the location and trafficking of proteins in cells and intact organisms. In 2008, the Nobel Prize in Chemistry was awarded to Shimomura, Chalfie and Tsien for their contribution to the discovery and development of GFP. This review traces the key events in that discovery process and highlights some significant applications of fluorescent proteins reported worldwide, including examples from Malaysia. Innovative applications of fluorescent proteins may help to answer many current biological questions and accelerate the development of tools to prevent or treat disease.
G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors in humans. Th... more G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors in humans. They convey extracellular signals into the cell interior by activating intracellular processes such as heterotrimeric G protein-dependent signaling pathways. They are widely distributed in the nervous system, and mediate key physiological processes including cognition, mood, appetite, pain and synaptic transmission. With at least 30% of marketed drugs being GPCR modulators, they are a major therapeutic target in the pharmaceutical industry's drug discovery programs. This review will survey recently patented ligands for GPCRs implicated in CNS disorders, in particular the metabotropic glutamate, adenosine and cannabinoid receptors. Metabotropic glutamate receptors regulate signaling by glutamate, the major excitatory brain neurotransmitter, while adenosine is a ubiquitous neuromodulater mediating diverse physiological effects. Recent patents for ligands of these receptors include mGluR5...
Green Fluorescent Protein (GFP) was first isolated from light-producing jellyfish in the 1960s. S... more Green Fluorescent Protein (GFP) was first isolated from light-producing jellyfish in the 1960s. Since the first reported expression of GFP into living organisms in 1994, it has become one of the most studied proteins in biochemistry and molecular biology. The ability of fluorescent proteins to fluoresce after expression in living cells has afforded researchers a tool to track the location and trafficking of proteins in cells and intact organisms. In 2008, the Nobel Prize in Chemistry was awarded to Shimomura, Chalfie and Tsien for their contribution to the discovery and development of GFP. This review traces the key events in that discovery process and highlights some significant applications of fluorescent proteins reported worldwide, including examples from Malaysia. Innovative applications of fluorescent proteins may help to answer many current biological questions and accelerate the development of tools to prevent or treat disease.
G protein-coupled receptors form a ternary complex of ligand, receptor, and G protein heterotrime... more G protein-coupled receptors form a ternary complex of ligand, receptor, and G protein heterotrimer (LRG) during signal transduction from the outside to the inside of a cell. Our goal was to develop a homogeneous, small-volume, bead-based approach compatible with high-throughput flow cytometry that would allow evaluation of G protein coupled receptor molecular assemblies. Dextran beads were derivatized to carry chelated nickel to bind hexahistidine-tagged green fluorescent protein (GFP) and hexahistidine-tagged G proteins. Ternary complexes were assembled on these beads using fluorescent ligand with wild-type receptor or a receptor-Gi␣2 fusion protein, and with a nonfluorescent ligand and receptor-GFP fusion protein.
G␥ dimers containing the ␥ 11 or ␥ 1 subunits are often less potent and effective in their abili... more G␥ dimers containing the ␥ 11 or ␥ 1 subunits are often less potent and effective in their ability to regulate effectors compared with dimers containing the ␥ 2 subunit. To explore the regions of the ␥ subunit that affect the activity of the ␥ dimer, we constructed eight chimeric ␥ subunits from the ␥ 1 and ␥ 2 subunits. Two chimeras were made in which the N-terminal regions of ␥ 1 and ␥ 2 were exchanged and two in which the C-terminal regions were transposed. Another set of chimeras was made in which the CAAX motifs of the chimeras were altered to direct modification with different prenyl groups. All eight ␥ chimeras were expressed in Sf9 cells with the  1 subunit, G␥ dimers were purified, and then they were assayed in vitro for their ability to bind to the G␣ i1 subunit, to couple G␣ i1 to the A1 adenosine receptor, to stimulate phospholipase C-, and to regulate type I or type II adenyl cyclases. Dimers containing the C-terminal sequence of the ␥ 2 subunit modified with the geranylgeranyl lipid had the highest affinity for G i1 ␣ (range, 0.5-1.2 nM) and were most effective at coupling the G i1 ␣ subunit to receptor. These dimers were most effective at stimulating the phosphatidylinositol-specific phospholipase C- isoform and inhibiting type I adenyl cyclase. In contrast, ␥ dimers containing the N-terminal sequence of the ␥ 2 subunit and a geranylgeranyl group are most effective at activating type II adenyl cyclase. The results indicate that both the N-and Cterminal regions of the ␥ subunit impart specificity to receptor and effector interactions.
Two constructs encoding the human -opioid receptor (hMOR) fused at its C terminus to either one o... more Two constructs encoding the human -opioid receptor (hMOR) fused at its C terminus to either one of two G␣ subunits, G␣ o1 (hMOR-G␣ o1 ) and G␣ i2 (hMOR-G␣ i2 ), were expressed in Escherichia coli at levels suitable for pharmacological studies (0.4 -0.5 pmol/mg). Receptors fused to G␣ o1 or to G␣ i2 maintained high-affinity binding of the antagonist diprenorphine. Affinities of the -selective agonists morphine, [D-Ala 2 ,N-Me-Phe 4 ,Gly 5ol]enkephalin (DAMGO), and endomorphins as well as their potencies and intrinsic activities in stimulating guanosine 5Ј-O-(3-[ 35 S]thiotriphosphate) ([ 35 S]GTP␥S) binding were assessed in the presence of added purified G␥ subunits. Both fusion proteins displayed high-affinity agonist binding and agonist-stimulated [ 35 S]GTP␥S binding. In the presence of G␥ dimers, the affinities of DAMGO and endomorphin-1 and -2 were higher at hMOR-G␣ i2 than at hMOR-G␣ o1 , whereas morphine displayed similar affinities at the two chimeras. Potencies of the four agonists in stimulating [ 35 S]GTP␥S binding at hMOR-G␣ o1 were similar, whereas at hMOR-G␣ i2 , endomorphin-1 and morphine were more potent than DAMGO and endomorphin-2. The intrinsic activities of the four agonists at the two fusion constructs were similar. The results confirm hMOR coupling to G␣ o1 and G␣ i2 and support the hypothesis of the existence of multiple receptor conformational states, depending on the nature of the G protein to which it is coupled. Key Words: G protein-coupled receptors-Receptor-G protein fusion--Opioid receptor-Escherichia coli-Pharmacology.
hsp70 Heat shock protein G protein Receptor Stress Alpha 2a adrenergic receptor G protein-coupled... more hsp70 Heat shock protein G protein Receptor Stress Alpha 2a adrenergic receptor G protein-coupled receptors (GPCRs) transduce extracellular signals to the interior of the cell by activating membrane-bound guanine nucleotide-binding regulatory proteins (G proteins). An increasing number of proteins have been reported to bind to and regulate GPCRs. We report a novel regulation of the alpha 2A adrenergic receptor (α 2A -R) by the ubiquitous stress-inducible 70 kDa heat shock protein, hsp70. Hsp70, but not hsp90, attenuated G protein-dependent high affinity agonist binding to the α 2A -R in Sf9 membranes. Antagonist binding was unchanged, suggesting that hsp70 uncouples G proteins from the receptor. As hsp70 did not bind G proteins but complexed with the α 2A -R in intact cells, a direct interaction with the receptor seems likely. In the presence of hsp70, α 2A -R-catalyzed [ 35 S]GTPγS binding was reduced by approximately 70%. In contrast, approximately 50-fold higher concentrations of hsp70 were required to reduce agonist binding to the stress-inducible 5-hydroxytryptamine 1A receptor (5-HT 1A -R). In heat-stressed CHO cells, the α 2A -R was significantly uncoupled from G proteins, coincident with an increased localization of hsp70 at the membrane. The contrasting effect of hsp70 on the α 2A -R compared to the 5-HT 1A -R suggests that during stress, upregulation of hsp70 may attenuate signaling from specific GPCRs as part of the stress response to foster survival.
Background: Problem-based learning (PBL) has become the most significant innovation in medical ed... more Background: Problem-based learning (PBL) has become the most significant innovation in medical education of the past 40 years. In contrast to exam-centered, lecture-based conventional curricula, PBL is a comprehensive curricular strategy that fosters student-centred learning and the skills desired in physicians. The rapid spread of PBL has produced many variants. One of the most common is 'hybrid PBL' where conventional teaching methods are implemented alongside PBL. This paper contends that the mixing of these two opposing educational philosophies can undermine PBL and nullify its positive benefits. Schools using hybrid PBL and lacking medical education expertise may end up with a dysfunctional curriculum worse off than the traditional approach. Discussion: For hybrid PBL schools with a dysfunctional curriculum, standard PBL is a cost-feasible option that confers the benefits of the PBL approach. This paper describes the signs of a dysfunctional PBL curriculum to aid hybrid PBL schools in recognising curricular breakdown. Next it discusses alternative curricular strategies and costs associated with PBL. It then details the four critical factors for successful conversion to standard PBL: dealing with staff resistance, understanding the role of lectures, adequate time for preparation and support from the administrative leadership. Summary: Hybrid PBL curricula without oversight by staff with medical education expertise can degenerate into dysfunctional curricula inferior even to the traditional approach from which PBL emerged. Such schools should inspect their curriculum periodically for signs of dysfunction to enable timely corrective action. A decision to convert fully to standard PBL is cost feasible but will require time, expertise and commitment which is only sustainable with supportive leadership.
The dynamics of G protein heterotrimer complex formation and disassembly in response to nucleotid... more The dynamics of G protein heterotrimer complex formation and disassembly in response to nucleotide binding and receptor activation govern the rate of responses to external stimuli. We use a novel flow cytometry approach to study the effects of lipid modification, isoform specificity, lipid environment, and receptor stimulation on the affinity and kinetics of G protein subunit binding. Fluoresceinlabeled myristoylated GR i1 (F-R i1 ) was used as the ligand bound to G γ in competition binding studies with differently modified GR subunit isoforms. In detergent solutions, the binding affinity of GR i to γ was 2 orders of magnitude higher than for GR o and GR s (IC 50 of 0.2 nM vs 17 and 27 nM, respectively), while in reconstituted bovine brain lipid vesicles, binding was slightly weaker. The effects of receptor on the G protein complex were assessed in R 2A AR receptor expressing CHO cell membranes into which purified γ subunits and F-R i1 were reconstituted. These cell membrane studies led to the following observations: (1) binding of R subunit to the γ was not enhanced by receptor in the presence or absence of agonist, indicating that γ contributed essentially all of the binding energy for R i1 interaction with the membrane; (2) activation of the receptor facilitated GTPγS-stimulated detachment of F-R i1 from γ and the membrane. Thus flow cytometry permits quantiatitive and real-time assessments of protein-protein interactions in complex membrane environments.
G protein coupled receptors activate signal transducing guanine nucleotide-binding proteins (G pr... more G protein coupled receptors activate signal transducing guanine nucleotide-binding proteins (G proteins), which consist of an R subunit and a γ dimer. Whole cell studies have reported that receptors signal through specific γ subtypes. Membrane reconstitution studies with the adenosine A 1 and R 2A adrenergic receptors have reached a similar conclusion. We aimed to test the generality of this finding by comparing the γ subtype specificity for four G i -coupled receptors: R 2A adrenergic; A1 adenosine (A 1 -R); 5-hydroxytryptamine 1A (5-HT 1A -R); mu opioid. Membranes were reconstituted with GR i1 and five γ subtypes (dimerized to 1). Using a sensitive Rγ binding assay, we show that all recombinant γ (except 1γ1) had comparable affinity for R i1 . Using high affinity agonist binding as a measure of receptor-G protein coupling, γ-containing γ11 was the most potent for A 1 -R and 5-HT 1A -R (p < 0.05, one way ANOVA) while γ7 was most potent for the other two receptors. γ11 was 3-8-fold more potent for the A 1 -R than were the other γ subtypes. Also, γ11 was 2-8-fold more potent for A 1 -R than at the other receptors, suggesting a unique coupling specificity of the A 1 -R for γ11. In contrast, the discrimination by receptors for the other γ subtypes was limited (2-3-fold). Thus the exquisite γ specificity of individual receptors reported in whole cell studies may depend on in vivo mechanisms beyond direct receptor recognition of γ subtypes. † fluoride; DTT, dithiothreitol; buffer A, 50 mM Tris-HCl, pH 7.6, 5 mM MgCl 2, 1 mM EDTA; E. coli, Escherichia coli; FITC, F-R, fluorescein isothiocynate-labeled Ri1; bγ, biotinylated bovine brain γ; Gpp-(NH)p, guanyl-5′-yl imidodiphosphate; ANOVA, analysis of variance; PDZ, PSD-95, Dlg, ZO-1 homology.
G␥ dimers containing the ␥ 11 or ␥ 1 subunits are often less potent and effective in their abili... more G␥ dimers containing the ␥ 11 or ␥ 1 subunits are often less potent and effective in their ability to regulate effectors compared with dimers containing the ␥ 2 subunit. To explore the regions of the ␥ subunit that affect the activity of the ␥ dimer, we constructed eight chimeric ␥ subunits from the ␥ 1 and ␥ 2 subunits. Two chimeras were made in which the N-terminal regions of ␥ 1 and ␥ 2 were exchanged and two in which the C-terminal regions were transposed. Another set of chimeras was made in which the CAAX motifs of the chimeras were altered to direct modification with different prenyl groups. All eight ␥ chimeras were expressed in Sf9 cells with the  1 subunit, G␥ dimers were purified, and then they were assayed in vitro for their ability to bind to the G␣ i1 subunit, to couple G␣ i1 to the A1 adenosine receptor, to stimulate phospholipase C-, and to regulate type I or type II adenyl cyclases. Dimers containing the C-terminal sequence of the ␥ 2 subunit modified with the geranylgeranyl lipid had the highest affinity for G i1 ␣ (range, 0.5-1.2 nM) and were most effective at coupling the G i1 ␣ subunit to receptor. These dimers were most effective at stimulating the phosphatidylinositol-specific phospholipase C- isoform and inhibiting type I adenyl cyclase. In contrast, ␥ dimers containing the N-terminal sequence of the ␥ 2 subunit and a geranylgeranyl group are most effective at activating type II adenyl cyclase. The results indicate that both the N-and Cterminal regions of the ␥ subunit impart specificity to receptor and effector interactions.
Background Hereditary ataxia (HA) represents a group of genetically heterogeneous neurodegenerati... more Background Hereditary ataxia (HA) represents a group of genetically heterogeneous neurodegenerative diseases caused by dysfunction of the cerebellum or disruption of the connection between the cerebellum and other areas of the central nervous system. Phenotypic manifestation of HA includes unsteadiness of stance and gait, dysarthria, nystagmus, dysmetria and complaints of clumsiness. There are no specific treatments for HA. Management strategies provide supportive treatment to reduce symptoms. Objectives This systematic review aimed to identify, evaluate and summarise the published literature on the therapeutic roles of natural remedies in the treatment of HA to provide evidence for clinical practice. Methods A systematic literature search was conducted using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA). Web of Science, PubMed and Science Direct Scopus were thoroughly searched for relevant published articles from June 2007 to July 2020. Results Ten...
Stroke causes death and disability globally but no neuroprotectant is approved for post-stroke ne... more Stroke causes death and disability globally but no neuroprotectant is approved for post-stroke neuronal injury. Neuroprotective compounds can be identified using oxygen glucose deprivation (OGD) of neuronal cells as an in vitro stroke model. Nerve growth factor (NGF)-differentiated PC12 pheochromocytoma cells are frequently used. However, investigators often find their clonal variant undifferentiable and are uncertain of optimal culture conditions. Hence we studied 3 commonly used PC12 variants: PC12 Adh, PC12 from Riken Cell Bank (PC12 Riken) and Neuroscreen-1 (NS-1) cells. We found DMEM the optimal media for PC12 Riken and NS-1 cells. Using a novel serum-free media approach, we identified collagen IV as the preferred adhesive substrate for both cell lines. We found PC12 Adh cells cannot attach without serum and is unable to differentiate using NGF. NS-1 cells differentiated to a maximal 72.7 ± 5.2% %, with substantial basal differentiation. We optimised differentiated NS-1 cells f...
... Okamoto and Nishimoto (1992) identified a BBXB or BBXXB motif. In the case of the α 2A -AR i3... more ... Okamoto and Nishimoto (1992) identified a BBXB or BBXXB motif. In the case of the α 2A -AR i3c loop, Ikezu et al. ... Clarke WP. (1998) Effector pathway-dependent relative efficacy at serotonin type 2A and 2C receptors: Evidence for agonist-directed trafficking of receptor stimulus. ...
Green Fluorescent Protein (GFP) was first isolated from light-producing jellyfish in the 1960s. S... more Green Fluorescent Protein (GFP) was first isolated from light-producing jellyfish in the 1960s. Since the first reported expression of GFP into living organisms in 1994, it has become one of the most studied proteins in biochemistry and molecular biology. The ability of fluorescent proteins to fluoresce after expression in living cells has afforded researchers a tool to track the location and trafficking of proteins in cells and intact organisms. In 2008, the Nobel Prize in Chemistry was awarded to Shimomura, Chalfie and Tsien for their contribution to the discovery and development of GFP. This review traces the key events in that discovery process and highlights some significant applications of fluorescent proteins reported worldwide, including examples from Malaysia. Innovative applications of fluorescent proteins may help to answer many current biological questions and accelerate the development of tools to prevent or treat disease.
G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors in humans. Th... more G protein-coupled receptors (GPCRs) are the largest class of cell surface receptors in humans. They convey extracellular signals into the cell interior by activating intracellular processes such as heterotrimeric G protein-dependent signaling pathways. They are widely distributed in the nervous system, and mediate key physiological processes including cognition, mood, appetite, pain and synaptic transmission. With at least 30% of marketed drugs being GPCR modulators, they are a major therapeutic target in the pharmaceutical industry's drug discovery programs. This review will survey recently patented ligands for GPCRs implicated in CNS disorders, in particular the metabotropic glutamate, adenosine and cannabinoid receptors. Metabotropic glutamate receptors regulate signaling by glutamate, the major excitatory brain neurotransmitter, while adenosine is a ubiquitous neuromodulater mediating diverse physiological effects. Recent patents for ligands of these receptors include mGluR5...
Green Fluorescent Protein (GFP) was first isolated from light-producing jellyfish in the 1960s. S... more Green Fluorescent Protein (GFP) was first isolated from light-producing jellyfish in the 1960s. Since the first reported expression of GFP into living organisms in 1994, it has become one of the most studied proteins in biochemistry and molecular biology. The ability of fluorescent proteins to fluoresce after expression in living cells has afforded researchers a tool to track the location and trafficking of proteins in cells and intact organisms. In 2008, the Nobel Prize in Chemistry was awarded to Shimomura, Chalfie and Tsien for their contribution to the discovery and development of GFP. This review traces the key events in that discovery process and highlights some significant applications of fluorescent proteins reported worldwide, including examples from Malaysia. Innovative applications of fluorescent proteins may help to answer many current biological questions and accelerate the development of tools to prevent or treat disease.
G protein-coupled receptors form a ternary complex of ligand, receptor, and G protein heterotrime... more G protein-coupled receptors form a ternary complex of ligand, receptor, and G protein heterotrimer (LRG) during signal transduction from the outside to the inside of a cell. Our goal was to develop a homogeneous, small-volume, bead-based approach compatible with high-throughput flow cytometry that would allow evaluation of G protein coupled receptor molecular assemblies. Dextran beads were derivatized to carry chelated nickel to bind hexahistidine-tagged green fluorescent protein (GFP) and hexahistidine-tagged G proteins. Ternary complexes were assembled on these beads using fluorescent ligand with wild-type receptor or a receptor-Gi␣2 fusion protein, and with a nonfluorescent ligand and receptor-GFP fusion protein.
G␥ dimers containing the ␥ 11 or ␥ 1 subunits are often less potent and effective in their abili... more G␥ dimers containing the ␥ 11 or ␥ 1 subunits are often less potent and effective in their ability to regulate effectors compared with dimers containing the ␥ 2 subunit. To explore the regions of the ␥ subunit that affect the activity of the ␥ dimer, we constructed eight chimeric ␥ subunits from the ␥ 1 and ␥ 2 subunits. Two chimeras were made in which the N-terminal regions of ␥ 1 and ␥ 2 were exchanged and two in which the C-terminal regions were transposed. Another set of chimeras was made in which the CAAX motifs of the chimeras were altered to direct modification with different prenyl groups. All eight ␥ chimeras were expressed in Sf9 cells with the  1 subunit, G␥ dimers were purified, and then they were assayed in vitro for their ability to bind to the G␣ i1 subunit, to couple G␣ i1 to the A1 adenosine receptor, to stimulate phospholipase C-, and to regulate type I or type II adenyl cyclases. Dimers containing the C-terminal sequence of the ␥ 2 subunit modified with the geranylgeranyl lipid had the highest affinity for G i1 ␣ (range, 0.5-1.2 nM) and were most effective at coupling the G i1 ␣ subunit to receptor. These dimers were most effective at stimulating the phosphatidylinositol-specific phospholipase C- isoform and inhibiting type I adenyl cyclase. In contrast, ␥ dimers containing the N-terminal sequence of the ␥ 2 subunit and a geranylgeranyl group are most effective at activating type II adenyl cyclase. The results indicate that both the N-and Cterminal regions of the ␥ subunit impart specificity to receptor and effector interactions.
Two constructs encoding the human -opioid receptor (hMOR) fused at its C terminus to either one o... more Two constructs encoding the human -opioid receptor (hMOR) fused at its C terminus to either one of two G␣ subunits, G␣ o1 (hMOR-G␣ o1 ) and G␣ i2 (hMOR-G␣ i2 ), were expressed in Escherichia coli at levels suitable for pharmacological studies (0.4 -0.5 pmol/mg). Receptors fused to G␣ o1 or to G␣ i2 maintained high-affinity binding of the antagonist diprenorphine. Affinities of the -selective agonists morphine, [D-Ala 2 ,N-Me-Phe 4 ,Gly 5ol]enkephalin (DAMGO), and endomorphins as well as their potencies and intrinsic activities in stimulating guanosine 5Ј-O-(3-[ 35 S]thiotriphosphate) ([ 35 S]GTP␥S) binding were assessed in the presence of added purified G␥ subunits. Both fusion proteins displayed high-affinity agonist binding and agonist-stimulated [ 35 S]GTP␥S binding. In the presence of G␥ dimers, the affinities of DAMGO and endomorphin-1 and -2 were higher at hMOR-G␣ i2 than at hMOR-G␣ o1 , whereas morphine displayed similar affinities at the two chimeras. Potencies of the four agonists in stimulating [ 35 S]GTP␥S binding at hMOR-G␣ o1 were similar, whereas at hMOR-G␣ i2 , endomorphin-1 and morphine were more potent than DAMGO and endomorphin-2. The intrinsic activities of the four agonists at the two fusion constructs were similar. The results confirm hMOR coupling to G␣ o1 and G␣ i2 and support the hypothesis of the existence of multiple receptor conformational states, depending on the nature of the G protein to which it is coupled. Key Words: G protein-coupled receptors-Receptor-G protein fusion--Opioid receptor-Escherichia coli-Pharmacology.
hsp70 Heat shock protein G protein Receptor Stress Alpha 2a adrenergic receptor G protein-coupled... more hsp70 Heat shock protein G protein Receptor Stress Alpha 2a adrenergic receptor G protein-coupled receptors (GPCRs) transduce extracellular signals to the interior of the cell by activating membrane-bound guanine nucleotide-binding regulatory proteins (G proteins). An increasing number of proteins have been reported to bind to and regulate GPCRs. We report a novel regulation of the alpha 2A adrenergic receptor (α 2A -R) by the ubiquitous stress-inducible 70 kDa heat shock protein, hsp70. Hsp70, but not hsp90, attenuated G protein-dependent high affinity agonist binding to the α 2A -R in Sf9 membranes. Antagonist binding was unchanged, suggesting that hsp70 uncouples G proteins from the receptor. As hsp70 did not bind G proteins but complexed with the α 2A -R in intact cells, a direct interaction with the receptor seems likely. In the presence of hsp70, α 2A -R-catalyzed [ 35 S]GTPγS binding was reduced by approximately 70%. In contrast, approximately 50-fold higher concentrations of hsp70 were required to reduce agonist binding to the stress-inducible 5-hydroxytryptamine 1A receptor (5-HT 1A -R). In heat-stressed CHO cells, the α 2A -R was significantly uncoupled from G proteins, coincident with an increased localization of hsp70 at the membrane. The contrasting effect of hsp70 on the α 2A -R compared to the 5-HT 1A -R suggests that during stress, upregulation of hsp70 may attenuate signaling from specific GPCRs as part of the stress response to foster survival.
Background: Problem-based learning (PBL) has become the most significant innovation in medical ed... more Background: Problem-based learning (PBL) has become the most significant innovation in medical education of the past 40 years. In contrast to exam-centered, lecture-based conventional curricula, PBL is a comprehensive curricular strategy that fosters student-centred learning and the skills desired in physicians. The rapid spread of PBL has produced many variants. One of the most common is 'hybrid PBL' where conventional teaching methods are implemented alongside PBL. This paper contends that the mixing of these two opposing educational philosophies can undermine PBL and nullify its positive benefits. Schools using hybrid PBL and lacking medical education expertise may end up with a dysfunctional curriculum worse off than the traditional approach. Discussion: For hybrid PBL schools with a dysfunctional curriculum, standard PBL is a cost-feasible option that confers the benefits of the PBL approach. This paper describes the signs of a dysfunctional PBL curriculum to aid hybrid PBL schools in recognising curricular breakdown. Next it discusses alternative curricular strategies and costs associated with PBL. It then details the four critical factors for successful conversion to standard PBL: dealing with staff resistance, understanding the role of lectures, adequate time for preparation and support from the administrative leadership. Summary: Hybrid PBL curricula without oversight by staff with medical education expertise can degenerate into dysfunctional curricula inferior even to the traditional approach from which PBL emerged. Such schools should inspect their curriculum periodically for signs of dysfunction to enable timely corrective action. A decision to convert fully to standard PBL is cost feasible but will require time, expertise and commitment which is only sustainable with supportive leadership.
The dynamics of G protein heterotrimer complex formation and disassembly in response to nucleotid... more The dynamics of G protein heterotrimer complex formation and disassembly in response to nucleotide binding and receptor activation govern the rate of responses to external stimuli. We use a novel flow cytometry approach to study the effects of lipid modification, isoform specificity, lipid environment, and receptor stimulation on the affinity and kinetics of G protein subunit binding. Fluoresceinlabeled myristoylated GR i1 (F-R i1 ) was used as the ligand bound to G γ in competition binding studies with differently modified GR subunit isoforms. In detergent solutions, the binding affinity of GR i to γ was 2 orders of magnitude higher than for GR o and GR s (IC 50 of 0.2 nM vs 17 and 27 nM, respectively), while in reconstituted bovine brain lipid vesicles, binding was slightly weaker. The effects of receptor on the G protein complex were assessed in R 2A AR receptor expressing CHO cell membranes into which purified γ subunits and F-R i1 were reconstituted. These cell membrane studies led to the following observations: (1) binding of R subunit to the γ was not enhanced by receptor in the presence or absence of agonist, indicating that γ contributed essentially all of the binding energy for R i1 interaction with the membrane; (2) activation of the receptor facilitated GTPγS-stimulated detachment of F-R i1 from γ and the membrane. Thus flow cytometry permits quantiatitive and real-time assessments of protein-protein interactions in complex membrane environments.
G protein coupled receptors activate signal transducing guanine nucleotide-binding proteins (G pr... more G protein coupled receptors activate signal transducing guanine nucleotide-binding proteins (G proteins), which consist of an R subunit and a γ dimer. Whole cell studies have reported that receptors signal through specific γ subtypes. Membrane reconstitution studies with the adenosine A 1 and R 2A adrenergic receptors have reached a similar conclusion. We aimed to test the generality of this finding by comparing the γ subtype specificity for four G i -coupled receptors: R 2A adrenergic; A1 adenosine (A 1 -R); 5-hydroxytryptamine 1A (5-HT 1A -R); mu opioid. Membranes were reconstituted with GR i1 and five γ subtypes (dimerized to 1). Using a sensitive Rγ binding assay, we show that all recombinant γ (except 1γ1) had comparable affinity for R i1 . Using high affinity agonist binding as a measure of receptor-G protein coupling, γ-containing γ11 was the most potent for A 1 -R and 5-HT 1A -R (p < 0.05, one way ANOVA) while γ7 was most potent for the other two receptors. γ11 was 3-8-fold more potent for the A 1 -R than were the other γ subtypes. Also, γ11 was 2-8-fold more potent for A 1 -R than at the other receptors, suggesting a unique coupling specificity of the A 1 -R for γ11. In contrast, the discrimination by receptors for the other γ subtypes was limited (2-3-fold). Thus the exquisite γ specificity of individual receptors reported in whole cell studies may depend on in vivo mechanisms beyond direct receptor recognition of γ subtypes. † fluoride; DTT, dithiothreitol; buffer A, 50 mM Tris-HCl, pH 7.6, 5 mM MgCl 2, 1 mM EDTA; E. coli, Escherichia coli; FITC, F-R, fluorescein isothiocynate-labeled Ri1; bγ, biotinylated bovine brain γ; Gpp-(NH)p, guanyl-5′-yl imidodiphosphate; ANOVA, analysis of variance; PDZ, PSD-95, Dlg, ZO-1 homology.
G␥ dimers containing the ␥ 11 or ␥ 1 subunits are often less potent and effective in their abili... more G␥ dimers containing the ␥ 11 or ␥ 1 subunits are often less potent and effective in their ability to regulate effectors compared with dimers containing the ␥ 2 subunit. To explore the regions of the ␥ subunit that affect the activity of the ␥ dimer, we constructed eight chimeric ␥ subunits from the ␥ 1 and ␥ 2 subunits. Two chimeras were made in which the N-terminal regions of ␥ 1 and ␥ 2 were exchanged and two in which the C-terminal regions were transposed. Another set of chimeras was made in which the CAAX motifs of the chimeras were altered to direct modification with different prenyl groups. All eight ␥ chimeras were expressed in Sf9 cells with the  1 subunit, G␥ dimers were purified, and then they were assayed in vitro for their ability to bind to the G␣ i1 subunit, to couple G␣ i1 to the A1 adenosine receptor, to stimulate phospholipase C-, and to regulate type I or type II adenyl cyclases. Dimers containing the C-terminal sequence of the ␥ 2 subunit modified with the geranylgeranyl lipid had the highest affinity for G i1 ␣ (range, 0.5-1.2 nM) and were most effective at coupling the G i1 ␣ subunit to receptor. These dimers were most effective at stimulating the phosphatidylinositol-specific phospholipase C- isoform and inhibiting type I adenyl cyclase. In contrast, ␥ dimers containing the N-terminal sequence of the ␥ 2 subunit and a geranylgeranyl group are most effective at activating type II adenyl cyclase. The results indicate that both the N-and Cterminal regions of the ␥ subunit impart specificity to receptor and effector interactions.
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Papers by William Lim