This study investigated the role of peptidergic input in modulating respiratory rhythm and frequency in the preBötzinger Complex (preBötC), the hypothesized site of respiratory rhythm generation in mammals. The key findings were:
1) Application of NK1R and μ-opioid receptor agonists into the preBötC increased and decreased respiratory frequency, respectively, and these effects were independent of GABA and glycine transmission.
2) NK1R and μ-opioid receptor expression was concentrated in the preBötC region, defining its anatomical boundaries.
3) Subsets of preBötC neurons expressed both NK1Rs and μ-opioid
Cloning of the c dna for thyroid stimulating hormone subunit and changes in a...rubycharlie
1) The researchers cloned the cDNA for the thyroid stimulating hormone beta subunit (TSHβ) of the Japanese eel (Anguilla japonica) and investigated changes in the pituitary-thyroid axis during silvering.
2) They found the TSHβ gene contains two introns and three exons, and the protein has a signal peptide and mature peptide. The TSHβ sequence is highly similar to other fish species.
3) In vitro, thyrotropin-releasing hormone increased while thyroxine decreased TSHβ mRNA expression in cultured pituitaries. TSHβ mRNA and serum thyroxine levels both increased during silvering in wild female eels, supporting the
1) PSF, a pre-mRNA splicing factor, was identified as a constituent of PER complexes purified from mouse tissues.
2) PSF recruits the SIN3A-HDAC complex to the Per1 promoter in a circadian manner through interactions with PER complexes.
3) Recruitment of the SIN3A-HDAC complex by the PER complex leads to histone deacetylation at the Per1 promoter, providing a molecular mechanism for circadian clock negative feedback through rhythmic repression of Per1 transcription.
Aton et al., PNAS 2006 - GABA and Gi/o differentially control circadian rhyth...SaraAton
1) The study examined how GABA and Gi/o signaling differentially control circadian rhythms and synchrony in SCN clock neurons.
2) Blocking GABA receptors with antagonists increased the amplitude and precision of circadian rhythms in individual neurons but did not impair synchrony between neurons.
3) Inhibiting Gi/o proteins with pertussis toxin disrupted synchrony between neurons and abolished rhythms in many neurons.
This study identifies a loss-of-function single nucleotide polymorphism (SNP) in the human tryptophan hydroxylase-2 (hTPH2) gene in patients with unipolar major depression. hTPH2 encodes the rate-limiting enzyme for neuronal serotonin synthesis. The SNP replaces arginine with histidine at position 441, resulting in an 80% reduction in serotonin production when expressed in cells. Nine out of 87 patients with unipolar depression carried this mutation, compared to only three out of 219 controls. This suggests a defect in brain serotonin synthesis may represent an important genetic risk factor for unipolar major depression.
The document discusses research into the regulation and role of the transcription factor REST in neuronal differentiation of SH-SY5Y human neuroblastoma cells exposed to IGF-I and PMA. The key findings are: 1) IGF-I and PMA increase nuclear REST levels early in treatment but REST declines with longer exposure as neurite outgrowth increases, 2) Downregulating REST with antisense oligonucleotides enhances the effects of IGF-I and PMA on neuronal markers and neurite growth, 3) REST degradation involves the ubiquitin-proteasome system and contributes to later stage neuronal differentiation. The document also analyzes the O-glycosylation pattern of REST protein in these cells.
Determination of kinact / Ki for EGFR Irreversible Inhibitors Using Competiti...susanfoltin
This document discusses methods for determining the kinetic parameters (kinact and Ki) of irreversible inhibitors that target the epidermal growth factor receptor (EGFR) tyrosine kinase. It describes using a time-resolved fluorescence resonance energy transfer (TR-FRET) competition binding assay to measure kinact/Ki. For some EGFR inhibitors, the TR-FRET assay results did not show complete inhibition over time as expected. Further experiments revealed this may be due to a secondary probe binding site on EGFR. Varying probe concentration and using an enzymatic activity assay confirmed probe interaction could affect kinetic measurements for these EGFR inhibitors.
Glutamatergic signaling from the parabrachial nucleus (PB) plays a critical role in arousing from sleep in response to hypercapnia (high carbon dioxide levels). The researchers developed a mouse model of repetitive CO2-induced arousals and deleted the gene for vesicular glutamate transporter 2 (Vglut2) specifically in the PB using viral vectors. They found that deleting Vglut2 in the lateral PB more than doubled the latency to arousal from hypercapnia and resulted in failure to arouse in over 30% of trials. In contrast, deleting Vglut2 in the medial PB increased non-REM sleep and delta power but had minimal effects on hypercapnia-induced arousal. This suggests
The document describes research into identifying potent inhibitors of the SHP2 protein tyrosine phosphatase. Several small molecule inhibitors were discovered, including NSC-87877, which was shown to selectively inhibit SHP2 activity in vitro and in cell-based assays. It was the first evidence that SHP2 phosphatase is involved in growth factor-stimulated ERK activation. Further screening of a chemical library identified additional SHP2 inhibitors, such as HLM002903 and HLM019544, which were shown to inhibit SHP2-dependent cell growth and signaling. The goal of finding SHP2 inhibitors is to develop potential anti-cancer agents, as SHP2 mutations have been implicated in various cancers and
Cloning of the c dna for thyroid stimulating hormone subunit and changes in a...rubycharlie
1) The researchers cloned the cDNA for the thyroid stimulating hormone beta subunit (TSHβ) of the Japanese eel (Anguilla japonica) and investigated changes in the pituitary-thyroid axis during silvering.
2) They found the TSHβ gene contains two introns and three exons, and the protein has a signal peptide and mature peptide. The TSHβ sequence is highly similar to other fish species.
3) In vitro, thyrotropin-releasing hormone increased while thyroxine decreased TSHβ mRNA expression in cultured pituitaries. TSHβ mRNA and serum thyroxine levels both increased during silvering in wild female eels, supporting the
1) PSF, a pre-mRNA splicing factor, was identified as a constituent of PER complexes purified from mouse tissues.
2) PSF recruits the SIN3A-HDAC complex to the Per1 promoter in a circadian manner through interactions with PER complexes.
3) Recruitment of the SIN3A-HDAC complex by the PER complex leads to histone deacetylation at the Per1 promoter, providing a molecular mechanism for circadian clock negative feedback through rhythmic repression of Per1 transcription.
Aton et al., PNAS 2006 - GABA and Gi/o differentially control circadian rhyth...SaraAton
1) The study examined how GABA and Gi/o signaling differentially control circadian rhythms and synchrony in SCN clock neurons.
2) Blocking GABA receptors with antagonists increased the amplitude and precision of circadian rhythms in individual neurons but did not impair synchrony between neurons.
3) Inhibiting Gi/o proteins with pertussis toxin disrupted synchrony between neurons and abolished rhythms in many neurons.
This study identifies a loss-of-function single nucleotide polymorphism (SNP) in the human tryptophan hydroxylase-2 (hTPH2) gene in patients with unipolar major depression. hTPH2 encodes the rate-limiting enzyme for neuronal serotonin synthesis. The SNP replaces arginine with histidine at position 441, resulting in an 80% reduction in serotonin production when expressed in cells. Nine out of 87 patients with unipolar depression carried this mutation, compared to only three out of 219 controls. This suggests a defect in brain serotonin synthesis may represent an important genetic risk factor for unipolar major depression.
The document discusses research into the regulation and role of the transcription factor REST in neuronal differentiation of SH-SY5Y human neuroblastoma cells exposed to IGF-I and PMA. The key findings are: 1) IGF-I and PMA increase nuclear REST levels early in treatment but REST declines with longer exposure as neurite outgrowth increases, 2) Downregulating REST with antisense oligonucleotides enhances the effects of IGF-I and PMA on neuronal markers and neurite growth, 3) REST degradation involves the ubiquitin-proteasome system and contributes to later stage neuronal differentiation. The document also analyzes the O-glycosylation pattern of REST protein in these cells.
Determination of kinact / Ki for EGFR Irreversible Inhibitors Using Competiti...susanfoltin
This document discusses methods for determining the kinetic parameters (kinact and Ki) of irreversible inhibitors that target the epidermal growth factor receptor (EGFR) tyrosine kinase. It describes using a time-resolved fluorescence resonance energy transfer (TR-FRET) competition binding assay to measure kinact/Ki. For some EGFR inhibitors, the TR-FRET assay results did not show complete inhibition over time as expected. Further experiments revealed this may be due to a secondary probe binding site on EGFR. Varying probe concentration and using an enzymatic activity assay confirmed probe interaction could affect kinetic measurements for these EGFR inhibitors.
Glutamatergic signaling from the parabrachial nucleus (PB) plays a critical role in arousing from sleep in response to hypercapnia (high carbon dioxide levels). The researchers developed a mouse model of repetitive CO2-induced arousals and deleted the gene for vesicular glutamate transporter 2 (Vglut2) specifically in the PB using viral vectors. They found that deleting Vglut2 in the lateral PB more than doubled the latency to arousal from hypercapnia and resulted in failure to arouse in over 30% of trials. In contrast, deleting Vglut2 in the medial PB increased non-REM sleep and delta power but had minimal effects on hypercapnia-induced arousal. This suggests
The document describes research into identifying potent inhibitors of the SHP2 protein tyrosine phosphatase. Several small molecule inhibitors were discovered, including NSC-87877, which was shown to selectively inhibit SHP2 activity in vitro and in cell-based assays. It was the first evidence that SHP2 phosphatase is involved in growth factor-stimulated ERK activation. Further screening of a chemical library identified additional SHP2 inhibitors, such as HLM002903 and HLM019544, which were shown to inhibit SHP2-dependent cell growth and signaling. The goal of finding SHP2 inhibitors is to develop potential anti-cancer agents, as SHP2 mutations have been implicated in various cancers and
1) The document discusses various physiological mechanisms involved in levodopa-induced dyskinesias, including changes in signaling pathways in the striatum and alterations in gene expression.
2) Studies in monkeys found that high doses of levodopa can induce dyskinesias, and that dyskinesias arise more quickly in severely dopamine-depleted monkeys.
3) Research has shown changes in activity of areas of the motor cortex and basal ganglia with levodopa treatment, as well as differential loss of striatal projection systems in Parkinson's disease.
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.
This study investigated how oxidative stress activates the PI3K pathway in neurons affected by neurodegenerative diseases. The researchers found that ingestion of the oxidative stress inducer Paraquat in Drosophila larvae caused axonal transport defects and neuronal cell death. Expressing active PI3K suppressed Paraquat-mediated cell death but not axonal blocks, indicating PI3K acts downstream of transport defects. Expression of active PI3K also suppressed cell death from polyQ protein expression but did not affect associated transport defects. Dominant negative PI3K disrupted normal transport of huntingtin protein, linking PI3K directly to transport. Together, the findings suggest axonal transport defects activate the PI3K pathway to decrease oxidative stress-induced
A planar surface antibody array was configured to detect and quantify Receptor Tyrosine Kinase (RTK) protein-protein interactions. Two sets of antibody pairs were identified that are able to distinguish between two combinations of EGFR-c-Met heterodimers, which differ in their joint conformation. The antibody array sandwich immunoassay allowed monitoring of the disruption of receptor heterodimers using small-molecule inhibitors and revealed differential sensitivity of EGFR-HER2 and EGFR-c-Met complexes to the EGFR kinase inhibitor gefitinib.
Albu_et_al-2014-Journal_of_Sleep_ResearchStefana Albu
This study examined the effects of deleting the FK506-binding protein 51 (FKBP51) gene on sleep architecture and stress responses in mice. Polysomnography recordings showed that FKBP51 knockout (KO) mice had increased wakefulness and suppressed non-rapid eye movement sleep and rapid eye movement sleep at baseline compared to wild-type mice. After 6 hours of sleep deprivation or 1 hour of restraint stress, FKBP51 KO mice exhibited less recovery sleep than wild-type mice, although slow-wave activity during non-rapid eye movement sleep was higher in KO mice, especially after sleep deprivation. Microdialysis experiments revealed lower levels of free corticosterone in the hippocampus of FKBP51 KO mice,
Marie-Laure Rives Allostery Functional Selectivity KOR Columbia University Marie-Laure Rives, PhD
The document discusses G protein-coupled receptors (GPCRs) and their modulation by signaling molecules and allosteric modulators. It focuses on the dopamine D2 and adenosine A2A receptors, describing how their heterodimerization was detected ex vivo in the striatum using proximity ligation assays. The document also discusses the therapeutic potential of kappa opioid receptor ligands and developing functionally selective ligands that activate signaling pathways for therapeutic effects but not adverse effects. Specifically, it describes developing G protein-biased kappa agonists like 6'GNTI that do not recruit arrestin in order to achieve pain relief without dysphoric effects.
Esta es la nota del Dr Beaudet que publicó ayer la revista Nature en la que detalla los resultados del ensayo preclinico (con ratones) de activacion del gen paterno con ASOS.
This study identifies novel transcriptional targets of the protein PGC-1α in the brain. The researchers:
1) Conducted a microarray on cells overexpressing PGC-1α to identify upregulated genes.
2) Mined the microarray results for genes related to inhibitory neurons and measured their expression in mice with altered PGC-1α levels.
3) Found that PGC-1α regulates genes involved in synaptic function (Syt2, Cplx1), structure (Nefh), and metabolism. Conditional deletion of PGC-1α in inhibitory neurons decreased expression of these genes and impaired neuronal function and memory.
This document summarizes several research publications on neuroscience topics:
1. One study found that acetaminophen affects central monoaminergic neurotransmission in rats, increasing serotonin levels in various brain regions and decreasing dopamine metabolites, suggesting monoamines may be involved in its analgesic action.
2. Another study showed that the benzodiazepine triazolam suppresses the induction of long-term potentiation in hippocampal neurons, consistent with its positive modulation of GABA-A receptor function.
3. A third study found that chronic treatment with either a substance P receptor antagonist or conventional antidepressant increases burst firing of neurons in the locus coeruleus, similarly to how imipramine works.
Using c-fos-like Immunoreactivity as a Neural Marker for Nociceptor ActivityMegan Atkins
This document summarizes a study examining the effects of morphine and pregabalin on c-fos expression induced by formalin injection in rats. The study optimized immunohistochemistry techniques to detect c-fos protein expression after noxious stimulation. Rats were injected with formalin to induce pain behavior, and then treated with morphine or pregabalin. C-fos expression and pain behavior were assessed. Both drugs reduced formalin-induced c-fos expression and pain behavior in a dose-dependent manner, demonstrating that c-fos detection can be used to evaluate how analgesics alter neural processing of nociceptive stimuli. This study provides a novel analysis of pregabalin's effects on c-fos expression.
This document summarizes Angela Sanchez's research on how temperature-regulated genes affect aging in C. elegans. Key findings include:
- Many proteostasis and protein synthesis genes are differentially expressed between 15°C and 25°C.
- Inhibition of certain temperature-regulated proteostasis genes, like cpr-7 and egl-45, causes earlier onset of paralysis at 15°C but not 25°C.
- A neuromuscular exam showed locomotion defects, especially in backward movement, precede paralysis upon proteostasis gene inhibition. This suggests the dysfunction starts in motor neurons.
- Motor neurons innervating the posterior half have fewer synaptic connections, which
The role of vasopressin in light-induced c-Fos expression in the SCNJane Chapman
- Light exposure at night causes rapid induction of the immediate early gene c-Fos in the suprachiasmatic nucleus (SCN), which is implicated in shifting circadian rhythms.
- A population of retinal projections to the SCN express the neuropeptide vasopressin (VP), but the physiological significance is unknown.
- This study investigated whether VP released from retinal neurons mediates light-induced c-Fos expression in the SCN. Rats pre-treated with a VP antagonist before light exposure still showed c-Fos expression in the SCN, with no significant decrease. This suggests VP is not essential for generating light-induced c-Fos expression in the SCN.
1) Dopamine transmission in the basal ganglia controls motor behavior through two pathways - the direct pathway which stimulates motion, and the indirect pathway which inhibits motion.
2) CK1δ over-expression disrupts this system, possibly through dopamine deficiency caused by downregulation of D1 and D2 receptors.
3) This study found an increase in calretinin-containing neurons in the striatum of CK1δ over-expressing mice, suggesting this imbalance contributes to their ADHD-like behaviors.
1) TDP-43 is a splicing factor that plays roles in various aspects of RNA metabolism and whose dysfunction is central to ALS and FTLD pathogenesis.
2) Deletion of the Drosophila homologue of TDP-43 leads to locomotive defects in flies, and expression of human TDP-43 can rescue this.
3) The C-terminal tail of TDP-43 contains a Q/N domain that mediates its interaction with other hnRNP proteins and is essential for TDP-43 aggregation. Tandem repeats of this domain induce TDP-43 aggregation.
GABAB receptor-mediated selective peripheral analgesia by the non-proteinogen...Igor Putrenko
1) The document examines the hypothesis that the amino acid isovaline produces peripheral analgesia through GABAB receptors without affecting the central nervous system.
2) Experiments in mice found that isovaline, like the GABAB agonists baclofen and GABA, reduced pain-related behavior induced by prostaglandin E2 injection into the paw in a GABAB receptor-dependent manner, but unlike baclofen, isovaline did not produce sedation or hypothermia indicating a lack of central effects.
3) Immunohistochemistry revealed co-localization of GABAB1 and GABAB2 receptor subunits on fine nerve endings and keratinocytes in skin
Neuroprotective Agents To Reduce Neuronal Loss Following SciAnnabel De Coster
The document summarizes research into investigating the neuroprotective effects of the peptide PRARIY in models of spinal cord injury in vitro. The research aimed to characterize neuronal cell death induced by excitotoxic and inflammatory stimuli in cell culture models, study the uptake and localization of fluorescently-labelled PRARIY peptide in neurons, and determine if PRARIY provides neuroprotection compared to a scrambled control peptide. Preliminary results found PRARIY entered neurons and reduced cell death induced by glutamate, though results were inconclusive due to variability between experiments. Future work proposed improving the cell death model and further investigating PRARIY's effects and localization.
Expression of Glutaminase and Vesicular Glutamate Transporter Type 2 Immunore...Heith Crosby
This study examined the effects of a surgical tail incision on glutaminase (GLS) and vesicular glutamate transporter type 2 (VGluT2) expression in sacral dorsal root ganglia (DRG) neurons in rats. The researchers found that sacral DRG neurons innervate the area of surgical incision using retrograde tracing. Both GLS and VGluT2 immunoreactivity were elevated in sacral DRG neurons for up to 72 hours post-incision, while GLS mRNA levels rapidly decreased and remained depressed for at least 96 hours. The rapid depletion of GLS mRNA and subsequent elevation of GLS and VGluT2 proteins in sacral DRG neurons following surgical incision
Cox2002-Automated_selection_of_aptamers_against_protein_targets_translated_in...J. Colin Cox
This document describes an experiment to develop a method for selecting aptamers against protein targets generated through in vitro transcription and translation of genes. Specifically, they attempt to select aptamers against the human U1A protein, a component of the nuclear spliceosome, where the U1A protein was produced through in vitro transcription and translation of its gene, and was also biotinylated to allow for immobilization during aptamer selection. The results showed that the selected aptamer sequences closely mimicked the natural RNA binding sequences and structures of U1A, demonstrating the potential of this method for high-throughput aptamer generation against proteomes.
The document is a log of running routes and events around New York City in 2015 that were documented by Nike for promotional purposes. It mentions specific runs and runners in various neighborhoods of NYC, including the Lower East Side, Williamsburg, Central Park, along the East River, under the Manhattan Bridge, Prospect Park, and the West Side Highway. It also references several social media accounts and campaigns run by Nike in NYC that year, including spotlighting different running groups and the release of new shoe models.
1) The document discusses various physiological mechanisms involved in levodopa-induced dyskinesias, including changes in signaling pathways in the striatum and alterations in gene expression.
2) Studies in monkeys found that high doses of levodopa can induce dyskinesias, and that dyskinesias arise more quickly in severely dopamine-depleted monkeys.
3) Research has shown changes in activity of areas of the motor cortex and basal ganglia with levodopa treatment, as well as differential loss of striatal projection systems in Parkinson's disease.
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.
This study investigated how oxidative stress activates the PI3K pathway in neurons affected by neurodegenerative diseases. The researchers found that ingestion of the oxidative stress inducer Paraquat in Drosophila larvae caused axonal transport defects and neuronal cell death. Expressing active PI3K suppressed Paraquat-mediated cell death but not axonal blocks, indicating PI3K acts downstream of transport defects. Expression of active PI3K also suppressed cell death from polyQ protein expression but did not affect associated transport defects. Dominant negative PI3K disrupted normal transport of huntingtin protein, linking PI3K directly to transport. Together, the findings suggest axonal transport defects activate the PI3K pathway to decrease oxidative stress-induced
A planar surface antibody array was configured to detect and quantify Receptor Tyrosine Kinase (RTK) protein-protein interactions. Two sets of antibody pairs were identified that are able to distinguish between two combinations of EGFR-c-Met heterodimers, which differ in their joint conformation. The antibody array sandwich immunoassay allowed monitoring of the disruption of receptor heterodimers using small-molecule inhibitors and revealed differential sensitivity of EGFR-HER2 and EGFR-c-Met complexes to the EGFR kinase inhibitor gefitinib.
Albu_et_al-2014-Journal_of_Sleep_ResearchStefana Albu
This study examined the effects of deleting the FK506-binding protein 51 (FKBP51) gene on sleep architecture and stress responses in mice. Polysomnography recordings showed that FKBP51 knockout (KO) mice had increased wakefulness and suppressed non-rapid eye movement sleep and rapid eye movement sleep at baseline compared to wild-type mice. After 6 hours of sleep deprivation or 1 hour of restraint stress, FKBP51 KO mice exhibited less recovery sleep than wild-type mice, although slow-wave activity during non-rapid eye movement sleep was higher in KO mice, especially after sleep deprivation. Microdialysis experiments revealed lower levels of free corticosterone in the hippocampus of FKBP51 KO mice,
Marie-Laure Rives Allostery Functional Selectivity KOR Columbia University Marie-Laure Rives, PhD
The document discusses G protein-coupled receptors (GPCRs) and their modulation by signaling molecules and allosteric modulators. It focuses on the dopamine D2 and adenosine A2A receptors, describing how their heterodimerization was detected ex vivo in the striatum using proximity ligation assays. The document also discusses the therapeutic potential of kappa opioid receptor ligands and developing functionally selective ligands that activate signaling pathways for therapeutic effects but not adverse effects. Specifically, it describes developing G protein-biased kappa agonists like 6'GNTI that do not recruit arrestin in order to achieve pain relief without dysphoric effects.
Esta es la nota del Dr Beaudet que publicó ayer la revista Nature en la que detalla los resultados del ensayo preclinico (con ratones) de activacion del gen paterno con ASOS.
This study identifies novel transcriptional targets of the protein PGC-1α in the brain. The researchers:
1) Conducted a microarray on cells overexpressing PGC-1α to identify upregulated genes.
2) Mined the microarray results for genes related to inhibitory neurons and measured their expression in mice with altered PGC-1α levels.
3) Found that PGC-1α regulates genes involved in synaptic function (Syt2, Cplx1), structure (Nefh), and metabolism. Conditional deletion of PGC-1α in inhibitory neurons decreased expression of these genes and impaired neuronal function and memory.
This document summarizes several research publications on neuroscience topics:
1. One study found that acetaminophen affects central monoaminergic neurotransmission in rats, increasing serotonin levels in various brain regions and decreasing dopamine metabolites, suggesting monoamines may be involved in its analgesic action.
2. Another study showed that the benzodiazepine triazolam suppresses the induction of long-term potentiation in hippocampal neurons, consistent with its positive modulation of GABA-A receptor function.
3. A third study found that chronic treatment with either a substance P receptor antagonist or conventional antidepressant increases burst firing of neurons in the locus coeruleus, similarly to how imipramine works.
Using c-fos-like Immunoreactivity as a Neural Marker for Nociceptor ActivityMegan Atkins
This document summarizes a study examining the effects of morphine and pregabalin on c-fos expression induced by formalin injection in rats. The study optimized immunohistochemistry techniques to detect c-fos protein expression after noxious stimulation. Rats were injected with formalin to induce pain behavior, and then treated with morphine or pregabalin. C-fos expression and pain behavior were assessed. Both drugs reduced formalin-induced c-fos expression and pain behavior in a dose-dependent manner, demonstrating that c-fos detection can be used to evaluate how analgesics alter neural processing of nociceptive stimuli. This study provides a novel analysis of pregabalin's effects on c-fos expression.
This document summarizes Angela Sanchez's research on how temperature-regulated genes affect aging in C. elegans. Key findings include:
- Many proteostasis and protein synthesis genes are differentially expressed between 15°C and 25°C.
- Inhibition of certain temperature-regulated proteostasis genes, like cpr-7 and egl-45, causes earlier onset of paralysis at 15°C but not 25°C.
- A neuromuscular exam showed locomotion defects, especially in backward movement, precede paralysis upon proteostasis gene inhibition. This suggests the dysfunction starts in motor neurons.
- Motor neurons innervating the posterior half have fewer synaptic connections, which
The role of vasopressin in light-induced c-Fos expression in the SCNJane Chapman
- Light exposure at night causes rapid induction of the immediate early gene c-Fos in the suprachiasmatic nucleus (SCN), which is implicated in shifting circadian rhythms.
- A population of retinal projections to the SCN express the neuropeptide vasopressin (VP), but the physiological significance is unknown.
- This study investigated whether VP released from retinal neurons mediates light-induced c-Fos expression in the SCN. Rats pre-treated with a VP antagonist before light exposure still showed c-Fos expression in the SCN, with no significant decrease. This suggests VP is not essential for generating light-induced c-Fos expression in the SCN.
1) Dopamine transmission in the basal ganglia controls motor behavior through two pathways - the direct pathway which stimulates motion, and the indirect pathway which inhibits motion.
2) CK1δ over-expression disrupts this system, possibly through dopamine deficiency caused by downregulation of D1 and D2 receptors.
3) This study found an increase in calretinin-containing neurons in the striatum of CK1δ over-expressing mice, suggesting this imbalance contributes to their ADHD-like behaviors.
1) TDP-43 is a splicing factor that plays roles in various aspects of RNA metabolism and whose dysfunction is central to ALS and FTLD pathogenesis.
2) Deletion of the Drosophila homologue of TDP-43 leads to locomotive defects in flies, and expression of human TDP-43 can rescue this.
3) The C-terminal tail of TDP-43 contains a Q/N domain that mediates its interaction with other hnRNP proteins and is essential for TDP-43 aggregation. Tandem repeats of this domain induce TDP-43 aggregation.
GABAB receptor-mediated selective peripheral analgesia by the non-proteinogen...Igor Putrenko
1) The document examines the hypothesis that the amino acid isovaline produces peripheral analgesia through GABAB receptors without affecting the central nervous system.
2) Experiments in mice found that isovaline, like the GABAB agonists baclofen and GABA, reduced pain-related behavior induced by prostaglandin E2 injection into the paw in a GABAB receptor-dependent manner, but unlike baclofen, isovaline did not produce sedation or hypothermia indicating a lack of central effects.
3) Immunohistochemistry revealed co-localization of GABAB1 and GABAB2 receptor subunits on fine nerve endings and keratinocytes in skin
Neuroprotective Agents To Reduce Neuronal Loss Following SciAnnabel De Coster
The document summarizes research into investigating the neuroprotective effects of the peptide PRARIY in models of spinal cord injury in vitro. The research aimed to characterize neuronal cell death induced by excitotoxic and inflammatory stimuli in cell culture models, study the uptake and localization of fluorescently-labelled PRARIY peptide in neurons, and determine if PRARIY provides neuroprotection compared to a scrambled control peptide. Preliminary results found PRARIY entered neurons and reduced cell death induced by glutamate, though results were inconclusive due to variability between experiments. Future work proposed improving the cell death model and further investigating PRARIY's effects and localization.
Expression of Glutaminase and Vesicular Glutamate Transporter Type 2 Immunore...Heith Crosby
This study examined the effects of a surgical tail incision on glutaminase (GLS) and vesicular glutamate transporter type 2 (VGluT2) expression in sacral dorsal root ganglia (DRG) neurons in rats. The researchers found that sacral DRG neurons innervate the area of surgical incision using retrograde tracing. Both GLS and VGluT2 immunoreactivity were elevated in sacral DRG neurons for up to 72 hours post-incision, while GLS mRNA levels rapidly decreased and remained depressed for at least 96 hours. The rapid depletion of GLS mRNA and subsequent elevation of GLS and VGluT2 proteins in sacral DRG neurons following surgical incision
Cox2002-Automated_selection_of_aptamers_against_protein_targets_translated_in...J. Colin Cox
This document describes an experiment to develop a method for selecting aptamers against protein targets generated through in vitro transcription and translation of genes. Specifically, they attempt to select aptamers against the human U1A protein, a component of the nuclear spliceosome, where the U1A protein was produced through in vitro transcription and translation of its gene, and was also biotinylated to allow for immobilization during aptamer selection. The results showed that the selected aptamer sequences closely mimicked the natural RNA binding sequences and structures of U1A, demonstrating the potential of this method for high-throughput aptamer generation against proteomes.
The document is a log of running routes and events around New York City in 2015 that were documented by Nike for promotional purposes. It mentions specific runs and runners in various neighborhoods of NYC, including the Lower East Side, Williamsburg, Central Park, along the East River, under the Manhattan Bridge, Prospect Park, and the West Side Highway. It also references several social media accounts and campaigns run by Nike in NYC that year, including spotlighting different running groups and the release of new shoe models.
Strategia integrata de Dezvoltare Urbana iasiCIVICA
Va invitam sa consultati lista cu proiecte propuse de Primaria Iasi pentru perioada 2015-2030.
Ne puteti trimite sugestii la contact@asociatiacivica.ro.
Fond de investitii si sustenabilitate financiaraCIVICA
Propunere privind constituirea unui fond public de investitii care sa contribuie la cresterea sustenabilitatii financiare a municipiului Iasi si finantarea multi-anuala a unor domenii prioritare precum educatia sau proiecte culturale strategice.
Ericka Green is seeking an administrative assistant position with over 10 years of experience in various business environments. She has strong skills in medical terminology, insurance billing, organization, communication, and customer service. Her most recent role was as a registrar for Parallon Business Solutions where she interviewed patients, ensured accurate charts, verified insurance, and processed patient paperwork. Prior to that she was a scheduler where she scheduled patients for testing through the Meditech system. She is skilled in Microsoft Office, data entry, typing 60 wpm, and has a background in retail, customer service, and administrative roles.
PON (Passive Optical Network) is a fiber optic network architecture that uses unpowered optical splitters to enable a single fiber to serve multiple premises. It provides higher bandwidth compared to traditional copper networks. The key components of a PON include an Optical Line Termination device at the service provider's central office, Optical Network Units/Terminals near end users, and an Optical Distribution Network containing splitters, fibers, and connectors to transmit data between components.
El documento habla sobre el acoso sexual en las redes sociales. Explica que a pesar de que las redes sociales facilitan la comunicación, también se han convertido en un medio para el acoso. Describe formas de acoso como el acoso entre iguales y el "grooming". También ofrece consejos sobre medidas de prevención para protegerse del acoso en las redes.
Este documento presenta una agenda para una sesión de aprendizaje sobre Dropbox. La agenda incluye introducir el tema en 5-10 minutos, explorar Dropbox durante 15 minutos, concretar objetivos en 20 minutos, y concluir en 5 minutos. Luego se enumeran varios pasos para aprender a crear una cuenta de Dropbox, ampliar el espacio de almacenamiento gratuito, descargar el programa, subir archivos, realizar modificaciones generales, crear carpetas en la nube, respaldar archivos, encontrar archivos eliminados, y compartir archivos y carpet
This document discusses barriers to the promotion of women teachers and the impact on their job satisfaction and attitudes towards work. It begins by introducing the topic and noting traditional beliefs that have prevented women from being seen as leaders. It then discusses intrinsic and extrinsic barriers that can hinder women's professional advancement. The objective and significance of studying this issue is presented. The research problem, hypotheses, scope, conceptual framework and literature review are also summarized. The methodology section outlines the research design, subjects, instruments, data collection procedures and statistical analysis that will be used in the study.
An Introduction to The Work of Byron KatieByron Katie
A graphic introduction to The Work of Byron Katie. The Work is a way of identifying and questioning the thoughts that cause all the suffering in the world.
Byron Katie founded The Work after experiencing severe depression during her early thirties in 1986. After experiencing a life-altering realization in which she accepted that it was her beliefs about the world that had caused her suffering rather than the surrounding world. She created a process which led her to question the thoughts she had about the world. This process of self-inquiry woke up with Katie, as her, the morning of her life-changing realization. Ever since, Katie has been teaching others her method of self-inquiry at free public events,in prisons, hospitals, schools and at workshops.
This document describes a study that identified 1445 putative transcription factor genes in the mouse genome and mapped the expression patterns of over 1000 of these genes and associated co-regulators in developing mouse brains. The study found that 349 genes showed restricted expression patterns that adequately described the anatomical organization of the brain. A searchable brain atlas database was created containing a comprehensive inventory of murine transcription factors and their expression patterns.
Bilateral but not unilateral lesions of preBötzinger complex (preBötC) neurons that express neurokinin-1 receptors (NK1R neurons) in rats profoundly disrupted normal breathing patterns and responses to challenges. Rats with near complete bilateral destruction of preBötC NK1R neurons (preBötC- rats) showed an ataxic breathing pattern with irregular breathing periods and amplitude. They also exhibited abnormal blood gas levels and pathological responses to changes in oxygen and carbon dioxide levels, including fatal apneas with 100% oxygen. Thus, the approximately 600 preBötC NK1R neurons in rats appear critical for generating normal breathing rhythms and regulating responses to physiological challenges
The (Ugly) Truth of the Loyalty Point Program: What Marketers Say VS What Cus...Digital Alchemy Limited
The loyalty point program is largely adopted by marketers and customers, but are you sure your customers see it the same way as you do? Come and find out how “what marketers say” could be different from “what customers hear” as well as the truth behind it!
Visit our website for more details and to subscribe to our eNewsletter
http://www.digitalalchemy.asia
Let’s connect…
http://www.facebook.com/DigitalAlchemyLimited
http://twitter.com/Digitl_Alchemy
http://www.youtube.com/user/DigitalAlchemyPtyLtd
http://www.linkedin.com/company/digital-alchemy
http://plus.google.com/109695231036450612270/posts
http://instagram.com/Digitl_Alchemy
Don’t forget to follow our SlideShare channel for more content in the world of database marketing!
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.
hemichannel makes it a major contributor toionic dysregulaSusanaFurman449
hemichannel makes it a major contributor to
ionic dysregulation in ischemia. Second, Px1
hemichannel opening may result in efflux of
glucose and adenosine triphosphate (ATP),
further compromising the neuron_s recovery
from an ischemic insult. Consistent with this
was our observation that fluorescent dyes
became membrane-permeable only during
OGD. Hemichannels are putative conduits for
ATP release from astrocytes (21) and in the
cochlea (22). Third, the large amplitude of
the Px1 hemichannel current at holding po-
tentials near the neuron_s resting membrane
potential (È –60 mV) indicates that these
currents likely contribute substantially to
Banoxic depolarization,[ a poorly understood
but well-recognized and key component of
ischemic neuronal death (2, 23, 24). There-
fore, hemichannel opening may be an impor-
tant new pharmacological target to prevent
neuronal death in stroke.
References and Notes
1. A. J. Hansen, Physiol. Rev. 65, 101 (1985).
2. P. Lipton, Physiol. Rev. 79, 1431 (1999).
3. M. Kamermans et al., Science 292, 1178 (2001).
4. J. E. Contreras et al., Proc. Natl. Acad. Sci. U.S.A. 99, 495
(2002).
5. R. P. Kondo, S. Y. Wang, S. A. John, J. N. Weiss,
J. I. Goldhaber, J. Mol. Cell. Cardiol. 32, 1859 (2000).
6. H. Li et al., J. Cell Biol. 134, 1019 (1996).
7. L. Bao, S. Locovei, G. Dahl, FEBS Lett. 572, 65
(2004).
8. R. Bruzzone, M. T. Barbe, N. J. Jakob, H. Monyer,
J. Neurochem. 92, 1033 (2005).
9. R. Bruzzone, S. G. Hormuzdi, M. T. Barbe, A. Herb,
H. Monyer, Proc. Natl. Acad. Sci. U.S.A. 100, 13644
(2003).
10. See supporting material on Science Online.
11. J. Gao et al., Neuron 48, 635 (2005).
12. M. Aarts et al., Cell 115, 863 (2003).
13. C. Tomasetto, M. J. Neveu, J. Daley, P. K. Horan, R. Sager,
J. Cell Biol. 122, 157 (1993).
14. G. Feng et al., Neuron 28, 41 (2000).
15. A. Nimmerjahn, F. Kirchhoff, J. N. Kerr, F. Helmchen,
Nat. Methods 1, 31 (2004).
16. G. Sohl, S. Maxeiner, K. Willecke, Nat. Rev. Neurosci. 6,
191 (2005).
17. J. C. Saez, M. A. Retamal, D. Basilio, F. F. Bukauskas,
M. V. Bennett, Biochim. Biophys. Acta 1711, 215 (2005).
18. R. J. Thompson, M. H. Nordeen, K. E. Howell,
J. H. Caldwell, Biophys. J. 83, 278 (2002).
19. M. L. Fung, G. G. Haddad, Brain Res. 762, 97 (1997).
20. H. Benveniste, J. Drejer, A. Schousboe, N. H. Diemer,
J. Neurochem. 43, 1369 (1984).
21. C. E. Stout, J. L. Costantin, C. C. Naus, A. C. Charles,
J. Biol. Chem. 277, 10482 (2002).
22. H. B. Zhao, N. Yu, C. R. Fleming, Proc. Natl. Acad. Sci.
U.S.A. 102, 18724 (2005).
23. T. R. Anderson, C. R. Jarvis, A. J. Biedermann, C. Molnar,
R. D. Andrew, J. Neurophysiol. 93, 963 (2005).
24. G. G. Somjen, Physiol. Rev. 81, 1065 (2001).
25. Supported by the Canadian Institutes for Health Research
and the Canadian Stroke Network. B.A.M. has a Tier 1
Canada Research Chair in Neuroscience and a Michael
Smith Foundation for Health Research distinguished
scholar award. We thank Y.-T. Wang, C. C. Naus, and
T. Snutch for critical re ...
This study investigated how endoplasmic reticulum (ER) stress induced by glucosamine (GlcN) is involved in pancreatic beta-cell dysfunction. The researchers found that GlcN treatment increased levels of the ER stress markers BiP/GRP78 and CHOP/GADD153 in mouse pancreatic islets and INS-1E beta cells. GlcN also decreased mRNA levels of genes important for beta-cell function like Glut2, glucokinase, and Insulin1. Pretreating cells with a chemical chaperone partially prevented GlcN-induced ER stress and gene expression changes. However, an antioxidant did not prevent ER stress, indicating oxidative stress is not involved. The results suggest
A typical Realization of the process with linear recovery of AldosteroneIJERA Editor
Hypercortisolism as a sign of hypothamamus-pituitary-adrenocortical (HPA) axis overactivity and sleep EEG
changes are frequently observed in depression. Closely related to the
HPA axis is the renin-angiotensin-aldosterone system (RAAS) as 1. adrenocorticotropic hormone (ACTH) is a
common stimulus for cortisol and aldosterone, 2. cortisol release is suppressed by mineralocorticoid receptor
(MR) agonists 3. angiotensin II (ATII) releases CRH and vasopressin from the hypothalamus. The first passage
time and the bounds of the survival functions for the application are also obtained
Using Pathway Studio in Neurodegenerative diseaseAnn-Marie Roche
Dr. Gabor Juhasz of ELTE University in Budapest discusses use of Pathway Studio in the study of neurodegenerative diseases such as Alzheimer’s Disease.
The researchers engineered a genetic toolset called pB-Tet-GOI for flexible control of transgene expression in stem and progenitor-derived cell lineages. The system incorporates the latest tetracycline transactivator and reverse transactivator variants to provide regulated transgene expression upon addition or removal of doxycycline. It allows for doxycycline-induced, doxycycline-suppressed, doxycycline-resistant (constitutive), and doxycycline-induced/constitutive regulation of transgenes. Initial tests showed the system provides inducible transgene expression with minimal leakiness and can be used to bidirectionally express reporters and genes of interest to direct cell differentiation.
The document discusses two-component systems in bacteria. A two-component system consists of a sensor histidine kinase that autophosphorylates upon sensing a signal and transfers the phosphoryl group to a cognate response regulator. The response regulator then undergoes a conformational change and activates or represses target genes. The system allows bacteria to sense and adapt to various stressors like oxidative stress and nutrient starvation.
Insights into the epigenetic mechanisms involving histone lysineashutosh mahale
1. The study investigated the role of histone lysine methylation and demethylation in an internal carotid artery occlusion (ICAO) mouse model of mild to moderate stroke.
2. The ICAO model resulted in neuronal damage to the striatum. Epigenetic markers like H3K9me2 were decreased after ischemia but recovered with time.
3. Inhibiting jmjD2 demethylases after ischemia prevented the decrease in H3K9me2, reduced neuronal cell death, and improved neurological outcomes, suggesting epigenetic mechanisms are implicated in stroke pathology and recovery.
This study identified an excitatory neural circuit from the paraventricular nucleus (PVH) of the hypothalamus that drives hunger. Using rabies virus tracing and optogenetics, the researchers found that subsets of PVH neurons that express thyrotropin-releasing hormone (TRH) and pituitary adenylate cyclase-activating polypeptide (PACAP) provide strong excitatory input to agouti-related peptide (AgRP) neurons in the arcuate nucleus. Stimulating these PVH neurons increased hunger-related behaviors, while inhibiting them decreased feeding. Further experiments showed that PACAP can directly activate AgRP neurons through PAC1 receptors, in addition to glutamate transmission.
and lifelong absence of FXR as occurs inthe FXR-null model, .docxjustine1simpson78276
and lifelong absence of FXR as occurs in
the FXR-null model, can result in
abnormal metabolic effects that are quite
different from those caused by acute,
transient antagonism of this receptor.
Because the FXR-null mouse was
produced using Cre–loxP technology,
conditional disruption of this allele after
normal development has occurred can
now be used to help resolve this issue. An
alternative explanation is that the site(s)
of pharmacological action of
guggulsterone do not include all of the
tissues in which FXR is functional, such as
the liver and gut (i.e. although FXR
synthesis is uniformly absent from all
tissues of the FXR-null mouse model,
guggulsterone might antagonize FXR only
within a subset of these sites). In the
absence of in vivo data regarding the
modulation of FXR target gene expression
by guggulsterone, this is difficult to judge.
Thus, it remains a possibility that the
effects of orally-administered
guggulsterone occur primarily at the level
of the gut (i.e. versus gut and liver), for
instance, by affecting cholesterol
absorption and bile-acid reuptake
processes regulated by FXR, rather than
the hepatic biosynthesis and transport of
bile acids. Again, the conditional nature of
the strategy used to create the FXR-null
mouse model allows for tissue-specific
deletion of the FXR gene and might help
resolve this issue.
As reinforced by the recent work of
Urizar et al. [3], as well as by the present
therapeutic use of bile-acid binding
resins for hypercholesterolemia, there
exists an intimate linkage between bile
acid and cholesterol metabolism. Recent
demonstrations that FXR is also involved
in the regulation of genes (e.g. encoding
apolipoprotein A-I, apolipoprotein C-II
and phospholipids transfer protein) [4–6]
more closely linked with lipid rather
than bile-acid homeostasis, presents
additional avenues by which FXR
ligands could be beneficial for the
treatment of disorders of lipid
metabolism. As suggested by the work of
Urizar et al. [3] and others (e.g. [7]),
careful and comprehensive study of the
effects of natural products, such as
guggulsterone, on the function of nuclear
hormone receptors, is likely to yield
additional agents with desirable
therapeutic effects.
References
1 Sinal, C.J. et al. (2000) Targeted disruption of the
nuclear receptor FXR/BAR impairs bile acid and
lipid homeostasis. Cell 102, 731–744
2 Singh, R.B. et al. (1994) Hypolipidemic and
antioxidant effects of Commiphora mukul as an
adjunct to dietary therapy in patients with
hypercholesterolemia. Cardiovasc. Drugs Ther. 8,
659–664
3 Urizar, N.L. et al. (2002) A natural product that
lowers cholesterol as an antagonist ligand for
FXR. Science 296, 1703–1706
4 Claudel, T. et al. (2002) Bile acid-activated nuclear
receptor FXR suppresses apolipoprotein A-I
transcription via a negative FXR response
element. J. Clin. Invest. 109, 961–971
5 Kast, H.R. et al. (2001) Farnesoid X-activated
receptor induces apolipoprotein C-II
transcription: a molecular mech.
1) The study examines the role of tissue plasminogen activator (tPA) and plasmin in promoting axonal regrowth after spinal cord injury (SCI) via degradation of chondroitin sulfate proteoglycans (CSPGs).
2) It finds that tPA and plasmin are upregulated after SCI and can degrade CSPG core proteins. Mice lacking tPA show attenuated neurite outgrowth and recovery after SCI, even with chondroitinase ABC (ChABC) treatment to degrade CSPG sugar chains.
3) Coadministration of ChABC and plasmin enhanced recovery in tPA-deficient mice and further supported recovery in wild-type mice with S
Eeg time series data analysis in focal cerebral ischemic rat modelijbesjournal
The mammalian brain exists in a number of attractors. In order to characterize these attractors we have collected the time series data from the EEG recording of rat models. The time series was obtained by recording of the frontoparietal, occipital and temporal regions of the rat brain. Significant changes have
been observed in the dimensionalities of these brain attractors between the normal state, focal ischemic
state and the drug induced state. Thus, these three states were characterized by unique lyapunov exponents,
correlation dimensions and embedding dimensions. The inverse of the lyapunov exponent gave us the long
term coherence of the rat brain and was found to differ for the three states. The autocorrelation function
measured the mean similarity of the EEG signal with itself after a time t. The degree of decay was high indicating that there was maximum correlation in the time series. Thus, the autocorrelation functions clearly indicate the effect of focal cerebral ischemia and drugs induced on the rat brain.
This document summarizes research on molecular mechanisms of pain conducted by Dr. Nana Voitenko and colleagues. It discusses 4 parts: 1) classification and structure of glutamate receptors including AMPA receptors, 2) involvement of spinal AMPA receptors in inflammatory pain transmission, 3) role of extrasynaptic AMPA receptors in maintaining persistent pain, and 4) targeting of protein kinase C alpha using antisense oligonucleotides to treat inflammatory pain. The research finds that peripheral inflammation induces internalization of GluR2-containing AMPA receptors from synapses and insertion of GluR1-containing receptors at extrasynaptic sites, contributing to persistent pain states.
- STEP levels are elevated in Fragile X mice and inhibit long-term potentiation.
- Researchers tested the STEP inhibitor TC-2153 in wild type and Fragile X mice.
- TC-2153 treatment led to increased phosphorylation of STEP substrates like NR2B, Pyk2 and ERK1/2 in both wild type and Fragile X mice by inhibiting STEP.
- This suggests that TC-2153 may be an effective treatment for cognitive deficits in Fragile X Syndrome by blocking the elevated STEP activity.
This document summarizes a mathematical model of circannual prolactin cycles in sheep. It introduces biological rhythms and circadian rhythms controlled by photoperiod. The model uses differential equations to model melatonin, tachykinin, and prolactin levels over time, with parameters for production, degradation, thresholds, and time delays between factors. Linear stability analysis shows the equilibrium is a stable spiral. In short photoperiod, the model loses its cyclic prolactin pattern, possibly due to changes in pituitary responsiveness over time.
This study investigated the effects of various anabolic steroids and progesterone on progesterone production and STARD1 mRNA levels in human ovarian cells. The results showed that:
1) Dihydrotestosterone increased basal progesterone levels at some time points and increased STARD1 mRNA levels in the presence of a cAMP stimulant.
2) Progesterone increased STARD1 mRNA levels in the presence of cAMP at some time points.
3) Mesterolone and trenbolone increased basal progesterone levels but did not affect levels stimulated by cAMP, and had mixed effects on STARD1 mRNA levels.
4) Superdrol had little effect on progesterone levels but tended to reduce cAMP-stim
Role of pro-brain-derived neurotrophic factor (proBDNF)to ma.docxjoellemurphey
Role of pro-brain-derived neurotrophic factor (proBDNF)
to mature BDNF conversion in activity-dependent
competition at developing neuromuscular synapses
H. Shawn Jea,b,c,1, Feng Yanga,b,d,1, Yuanyuan Jia,e, Guhan Nagappana,e, Barbara L. Hempsteadf, and Bai Lua,b,e,2
aSection on Neural Development and Plasticity, National Institute of Child Health and Human Development, Bethesda, MD 20892-3714; bGenes, Cognition,
and Psychosis Program (GCAP), National Institute of Mental Health, Bethesda, MD 20892-3714; cProgram in Neuroscience and Behavioral Disorders, Duke–
National University of Singapore (Duke-NUS) Graduate Medical School, 169857, Singapore; dLieber Institute for Brain Development, The Johns Hopkins
University Medical Campus, Baltimore, MD 21205; eR&D China, GlaxoSmithKline, Pudong, Shanghai 201203, China; and fDivision of Hematology, Department
of Medicine, Weill Medical College, Cornell University, New York, NY 10021
Edited* by Richard L. Huganir, The Johns Hopkins University School of Medicine, Baltimore, MD, and approved August 20, 2012 (received for review May
10, 2012)
Formation of specific neuronal connections often involves compe-
tition between adjacent axons, leading to stabilization of the active
terminal, while retraction of the less active ones. The underlying
molecular mechanisms remain unknown. We show that activity-
dependent conversion of pro–brain-derived neurotrophic factor
(proBDNF) to mature (m)BDNF mediates synaptic competition. Stim-
ulation of motoneurons triggers proteolytic conversion of proBDNF
to mBDNF at nerve terminals. In Xenopus nerve–muscle cocultures,
in which two motoneurons innervate one myocyte, proBDNF-
p75NTR signaling promotes retraction of the less active terminal,
whereas mBDNF–tyrosine-related kinase B (TrkB) p75NTR (p75 neu-
rotrophin receptor) facilitates stabilization of the active one. Thus,
proBDNF and mBDNF may serve as potential “punishment” and “re-
ward” signals for inactive and active terminals, respectively, and
activity-dependent conversion of proBDNF to mBDNF may regulate
synapse elimination.
neuromuscular junction | pro-neurotrophin | synapse competition
The nervous system responds to experience by altering thenumber and strength of synaptic connections (1). Activity-
dependent synaptic competition, a general process seen in many
parts of the developing nervous system, plays a critical role in
shaping patterns of neuronal connections (2–7). At the neuro-
muscular junction (NMJ), for example, multiple axons compete
for the same postsynaptic muscle cell during early postnatal life
until all but one is eliminated (8–10). Extensive experimental
data support the view that the more active terminal or “cartel”
gets stabilized, whereas less active ones withdraw, resulting in
canonical elimination of polyneuronal innervation (8, 11). It is
generally believed that this synaptic competition is mediated by
a “punishment” or “elimination” signal, produced by the post-
synaptic cell, that cau ...
1) Mice lacking the inhibitory synapse cell adhesion molecule neuroligin 2 (NL2) were found to exhibit increased anxiety-like behavior.
2) While these NL2-deficient mice appeared to have a decrease in the density of inhibitory synaptic puncta, electron microscopy revealed no actual change in inhibitory synapse numbers.
3) This suggests that NL2 deletion impairs the function of inhibitory synapses without decreasing their numbers, and this decrease in inhibitory synaptic function correlates with increased anxiety in the mice.
The document discusses the functional interaction between mGlu1a and GABAB receptors. It first provides background on G protein-coupled receptors and their importance as drug targets. It then reviews evidence that mGlu1a and GABAB receptors physically interact in cortical neurons and Purkinje cells based on co-localization and co-immunoprecipitation studies. The study aims to further investigate the physical interaction between the receptors and the mechanism underlying their functional cross-talk using biophysical techniques like BRET, TR-FRET, and cell-surface co-immunoprecipitation. Preliminary data suggests mGlu1a and GABAB do not form complexes at the cell surface but may oligomerize intracellularly.
1. Modulation of Respiratory
Frequency by Peptidergic Input
to Rhythmogenic Neurons in
the PreBo¨tzinger Complex
Paul A. Gray,1,3
* Jens C. Rekling,1
* Christopher M. Bocchiaro,2
Jack L. Feldman1,2
†
Neurokinin-1 receptor (NK1R) and -opioid receptor (OR) agonists affected
respiratory rhythm when injected directly into the preBo¨tzinger Complex (pre-
Bo¨tC), the hypothesized site for respiratory rhythmogenesis in mammals. These
effects were mediated by actions on preBo¨tC rhythmogenic neurons. The
distribution of NK1Rϩ
neurons anatomically defined the preBo¨tC. Type 1 neu-
rons in the preBo¨tC, which have rhythmogenic properties, expressed both
NK1Rs and ORs, whereas type 2 neurons expressed only NK1Rs. These findings
suggest that the preBo¨tC is a definable anatomic structure with unique phys-
iological function and that a subpopulation of neurons expressing both NK1Rs
and ORs generate respiratory rhythm and modulate respiratory frequency.
Respiratory rhythm is generated by neurons
in the brainstem. Surprisingly, given the vital
role and the apparent simplicity of breathing
movements, the identity of these neurons and
the mechanisms of respiratory rhythmogen-
esis and frequency modulation are unknown.
Respiratory neurons are found in a narrow
column in the ventrolateral medulla extend-
ing from the facial nucleus to the spinal cord
(1, 2). The preBo¨tC is the limited portion of
this column necessary and sufficient for in
vitro neonatal rodent medullary slice prepa-
rations to generate respiratory-related motor
nerve output (2, 3). Perturbations of neuronal
function within the preBo¨tC in adult rats and
cats severely disrupt breathing (4), consistent
with the hypothesis that this region contains
the neurons responsible for generating respi-
ratory rhythm. Unfortunately, these and relat-
ed experiments only circumscribe the approx-
imate boundaries of the preBo¨tC. In the ab-
sence of a precise anatomic delineation of the
preBo¨tC, defining its functional role or the
roles of individual neurons it contains is
problematic.
Local application of the NK1R agonist, sub-
stance P (SP), into the preBo¨tC in vitro increas-
es the frequency of endogenous respiratory-
related output; similar application of the OR
agonist [D-Ala2
,N-Me-Phe4
,Gly5
-ol]enkephalin
acetate (DAMGO) or the ␥-aminobutyric acid
type B receptor (GABABR) agonist baclofen
decreases respiratory frequency (5–7). Similar
responses are seen in vivo (8). Because these
drugs affect frequency, they must act either
directly on preBo¨tC rhythmogenic neurons or
indirectly on other preBo¨tC neurons that pro-
vide modulatory signals to the rhythm genera-
tor. In vitro respiratory-related output does not
require chlorine-mediated postsynaptic inhibi-
tion (2, 3, 5, 9). If peptide neuromodulators
affect respiratory frequency by direct actions on
only inhibitory neurons, as has been suggested
for opioid actions in the midbrain (10), then
peptide receptor-expressing neurons could not
be responsible for rhythm generation. Thus, we
examined whether the effects of SP and
DAMGO on respiratory rhythm in vitro were
affected by blocking GABA type A receptor
(GABAAR) and glycine receptor activation
(11). Pressure injection of SP (2 pmol) into the
preBo¨tC of rhythmically active brainstem slices
from rats increased respiratory frequency to
192% Ϯ 5% of control, whereas injection of
DAMGO (1.5 pmol) decreased frequency to
64% Ϯ 1% of control (Fig. 1). GABABR acti-
vation has similar effects (5, 7). Bath applica-
tion of the GABAAR antagonist bicuculline
and the glycine receptor antagonist strych-
nine to these slices did not change the base-
line respiratory frequency or the effects on
respiratory frequency of SP or DAMGO (Fig.
1B). Thus, the preBo¨tC NK1R-, GABABR-,
and OR-expressing neurons that mediate
agonist frequency-modulating effects are
likely excitatory and are either directly in-
volved in respiratory rhythm generation or
are nonrespiratory neurons that modulate
rhythm-generating circuits.
We examined the immunohistochemical
expression pattern of NK1R, OR, and
GABABR in the ventrolateral medulla of
adult rats (12). Within the ventrolateral respi-
ratory column, GABABR expression was
widespread. However, both NK1R and OR
expression were concentrated within the pre-
Bo¨tC and absent from the adjacent Bo¨tzinger
complex (Bo¨tC) (1) and rostral ventral respi-
ratory group (1) (Fig. 2); NK1R expression in
adult mouse brainstem was similar (13). The
preBo¨tC NK1Rϩ
neurons were segregated
from both motoneurons and catecholaminer-
gic sympathetic neurons as revealed by dou-
ble immunohistochemistry for choline acetyl-
transferase (ChAT) or tyrosine hydroxylase
(TH) (13, 14).
To determine whether GABAergic and
peptidergic effects on breathing are due to
convergent inputs onto individual neurons,
we looked for OR or GABABR colocaliza-
tion in NK1Rϩ
neurons. We found NK1Rϩ
/
ORϩ
neurons and processes (Fig. 3A) and
NK1Rϩ
/ORϪ
neurons and processes (of 88
preBo¨tC NK1Rϩ
neurons examined in detail,
Departments of 1
Neurobiology and 2
Physiological Sci-
ence and 3
Interdepartmental Ph.D. Program in Neu-
roscience, University of California Los Angeles, Box
951763, Los Angeles, CA 90095–1763, USA.
*These authors contributed equally to this work.
†To whom correspondence should be addressed. E-
mail: feldman@ucla.edu
Fig. 1. Effects of peptides injected into preBo¨tC
are independent of GABAA and glycinergic
transmission. (A) Microinjection into preBo¨tC
of DAMGO (2 pmol) slows and SP (1.5 pmol)
speeds up endogenous respiratory-related
rhythm. Traces are integrated XII motor nerve
output; bars under each trace represent injec-
tion period with total amount indicated. (B)
Frequency response to microinjection of
DAMGO and SP into preBo¨tC is not affected by
blocking chlorine-mediated postsynaptic trans-
mission with bath application of bicuculline (20
M) and strychnine (1 M) sufficient to block
the effects of the combined microinjection of
the GABAA agonist, THIP (1 nmol), and glycine
(1 nmol). Histograms of grouped data across
multiple experiments are shown; number of
epochs (20 cycles per epoch) are indicated
within bars; error bars are SEM. Statistical com-
parisons are between individual pairs of epochs
in the same experiment: *, significant differ-
ence (P Ͻ 0.05; Student’s t test) between
conditions in all pairs; ns, no significant differ-
ence between conditions in all pairs; §, signifi-
cant difference (P Ͻ 0.05) relative to control in
all pairs; ns, no significant difference relative to
control in all pairs. Bic/Str, bath application of
bicuculline and strychnine.
R E P O R T S
19 NOVEMBER 1999 VOL 286 SCIENCE www.sciencemag.org1566
2. 53 coexpressed OR) (Fig. 3B). We found
NK1Rϩ
/GABABRϩ
(Fig. 3C) as well as
NK1Rϩ
/GABABRϪ
neurons (of 160 pre-
Bo¨tC NK1Rϩ
neurons examined in detail,
118 coexpressed GABABR). We also found
NK1RϪ
/GABABRϩ
neurons (Fig. 3C) and a
few ORϩ
/NK1RϪ
processes (Fig. 3B) and
somas (13). Thus, GABAergic and peptider-
gic synaptic inputs converge on subsets of
preBo¨tC neurons. Moreover, in the ventrolat-
eral respiratory column, neurons that are tar-
gets of these convergent modulatory inputs
that affect respiratory frequency are present
only in the preBo¨tC. We propose that the
expression of NK1Rϩ
neuronal somata in
the ventrolateral respiratory column defines
the anatomic extent of the preBo¨tC.
These results do not differentiate whether
these preBo¨tC neurons generate or simply
modulate respiratory rhythm. We tested the
responsiveness of behaviorally identified re-
spiratory neurons to neurokinin and opioid
agonists (15). In the preBo¨tC of rhythmically
active slices from mice, inspiratory neurons
fall into two categories (types 1 and 2) based
on their electroresponsive properties and
membrane potential trajectories (16, 17) (Fig.
4A). After blockade of action potential driven
synaptic transmission by 2 M tetrodotoxin
(TTX), bath application of SP depolarized
type 1 neurons 7.7 Ϯ 1.5 mV (n ϭ 4) (Fig.
4B), whereas type 2 neurons depolarized
much less [2.7 Ϯ 0.8 mV (n ϭ 4) (Fig. 4B)].
Input resistance was not significantly
changed by SP in type 1 or type 2 neurons
(Wilcoxon signed rank test). Bath application
of DAMGO (in TTX) hyperpolarized all type
1 neurons tested (7.9 Ϯ 1.8 mV; n ϭ 5) and
reduced their input resistance to 67% of con-
trol value but had no effect on either the
membrane potential or input resistance of
type 2 neurons (n ϭ 6) (Fig. 4C). The cur-
rent-voltage (I-V) curves of type 1 neurons
before and after DAMGO (under voltage
clamp in the presence of TTX) intersected at
Ϫ102 mV with normal [Kϩ
]o concentration
(n ϭ 5), whereas when [Kϩ
]o was increased
by 10 mM, the intersection point shifted to
Ϫ66 mV (n ϭ 3; P Ͻ 0.05, Mann-Whitney
rank sum test) (Fig. 4D). Taken together,
these results suggest that the effect of
DAMGO was mediated by activation of a Kϩ
conductance.
Most models for respiratory rhythm gen-
eration predict that depolarization of rhythm-
generating neurons will speed up rhythm and
that their hyperpolarization will slow it down
(2, 3, 18). Type 1 neurons, which are rhyth-
mogenic (2, 16, 17), are the only preBo¨tC
inspiratory neurons depolarized by both SP
and thyrotropin-releasing hormone (16), pep-
tides that increase respiratory frequency, and
hyperpolarized by DAMGO, a peptide that
decreases respiratory frequency; this is con-
sistent with these models and the hypothesis
(2) that type 1 neurons are responsible for
both rhythmogenesis and frequency control.
The preBo¨tC appears to have an important
function in generating respiratory rhythm and
in processing signals affecting respiratory
frequency (2). We propose that the kernel for
Fig. 2. NK1R- and OR-expressing neurons are
limited to and define the preBo¨tC. (A) Cartoon
indicating approximate location and boundaries
of ventral respiratory column regions and ad-
jacent ambiguual motoneuron pools in sagittal
section. (B) NK1R immunohistochemical ex-
pression in a single sagittal section through the
respiratory column corresponding to box in (A).
Higher magnification images showing cell soma
staining from preBo¨tC (C) and relative lack of
staining in Bo¨tC (D). Confocal, negative com-
posite images of OR immunohistochemical
staining indicating different expression levels in
the preBo¨tC (E) (blue arrow) and Bo¨tC (F) (red
arrow) in transverse section. Note high levels of
expression of these peptide receptors in the
cNA, a cranial motoneuron pool not related to
respiration. Scale in micrometers. cNA, com-
pact formation of the nucleus ambiguus; scNA,
subcompact formation of NA; rVRG, rostral
ventral respiratory group; VII, facial motor nu-
cleus; D, dorsal; L, lateral.
Fig. 3. G-protein–coupled receptors are coex-
pressed on individual preBo¨tC neurons. Confo-
cal projections of NK1Rϩ
somas (A to C) (left)
and processes (B) (left), ORϩ
somas (A)
(right) and processes (A and B) (right), and
GABABRϩ
somas (C) (right). Arrows indicate
coexpression and arrowheads indicate absence
of coexpression. Bars ϭ 25 m.
Fig. 4. Postsynaptic membrane responses of
inspiratory neurons in the preBo¨tC to SP and
DAMGO. (A) Characteristic membrane poten-
tial trajectory between inspiratory discharges
of type 1 and type 2 inspiratory neurons. Note
the long-lasting hyperpolarization characteris-
tic of type 1 neurons. (B) Membrane responses
of type 1 and type 2 neurons to bath-applied SP
(1 M) after blockade of action potential driven
synaptic transmission by TTX (2 M). Hyper-
polarizing current pulses (1-s duration every
5 s) were injected to measure input resistance
throughout the application and the membrane
potential was brought back to control value by
injecting bias current at the indicated time
(bias I). (C) Membrane responses of type 1 and
type 2 neurons to bath-applied 5 M DAMGO
( TTX ). (D) ( Top) I-V curves (in the presence of
TTX ) before and after DAMGO application (5
M) in normal [Kϩ
]o (6.2 mM). Arrow indicates
potential at which the two curves intersect.
(Bottom) I-V curves (in the presence of TTX )
before and after DAMGO application (5 M) in
high [Kϩ
]o (16.2 mM). Note intersection points
of the curves have shifted in the depolarizing
direction.
R E P O R T S
www.sciencemag.org SCIENCE VOL 286 19 NOVEMBER 1999 1567
3. the basic respiratory rhythm is a small class of
preBo¨tC rhythmogenic neurons (type 1) onto
which several modulatory systems converge.
These neurons may overlap or even be identical
to other preBo¨tC neurons with intrinsic oscilla-
tory bursting properties—for example, pace-
maker neurons (3, 19) and preinspiratory neu-
rons (20)—because 50% of type 1 neurons
have bursting properties (16) and at least some
project to the midline (21). However, whether
endogenous burst activity is critical for gener-
ation of respiratory rhythm per se (3, 18–20)
remains to be determined. Alterations in pepti-
dergic transmission in the preBo¨tC may play a
role in respiratory disorders such as sleep apnea
(22) and sudden infant death syndrome (23).
The possibility that multiple peptide systems
affecting respiration converge on a particular
identifiable class of neurons represents an inter-
esting locus for ventilatory control. The coex-
pression within the ventrolateral respiratory col-
umn of NK1R and OR in preBo¨tC neurons
provides an opportunity to exploit targeted mo-
lecular manipulation of breathing in animal
models and a basis for analysis of human respi-
ratory disorders.
References and Notes
1. A. L. Bianchi, M. Denavit-Saubie, J. Champagnat,
Physiol. Rev. 75, 1 (1995); J. L. Feldman, in Handbooks
of Physiology: The Nervous System. Intrinsic Regula-
tory System in the Brain, F. Bloom, Ed. (American
Physiological Society, Bethesda, MD, 1986), pp. 463–
524; E. G. Dobbins and J. L. Feldman, J. Comp. Neurol.
347, 64 (1994).
2. J. C. Rekling and J. L. Feldman, Annu. Rev. Physiol. 60,
385 (1998).
3. J. C. Smith, H. H. Ellenberger, K. Ballanyi, D. W.
Richter, J. L. Feldman, Science 254, 726 (1991).
4. A. Monnier, F. Hayashi, D. R. McCrimmon, Soc. Neu-
rosci. Abstr. 346, 5 (1998); N. Koshiya and P. G.
Guyenet, J. Physiol. London 491, 859 (1996); J. M.
Ramirez, S. W. Schwarzacher, O. Pierrefiche, B. M.
Olivera, D. W. Richter, J. Physiol. London 507, 895
(1998); J. H. Hsieh et al., J. Autom. Nerv. Sys. 73, 7
(1998).
5. J. L. Feldman and J. C. Smith, Ann. NY Acad. Sci. 563,
114 (1989).
6. S. M. Johnson, J. C. Smith, J. L. Feldman, J. Appl.
Physiol. 80, 2120 (1996).
7. J. Brockhaus and K. Ballanyi, Eur. J. Neurosci. 10, 3823
(1998).
8. A. C. Bonham, Resp. Physiol. 101, 219 (1995); D. R.
McCrimmon and G. S. Mitchell, in Regulation of
Breathing, J. A. Dempsey and A. I. Pack, Eds. (Dekker,
New York, 1995), vol. 79, pp. 151–218.
9. G. D. Funk, J. C. Smith, J. L. Feldman, J. Neurophysiol.
70, 1497 (1993); X. M. Shao and J. L. Feldman,
J. Neurophysiol. 77, 1853 (1997).
10. H. L. Fields and M. M. Heinricher, Philos. Trans. R. Soc.
London Ser. B 308, 361 (1985).
11. Transverse 400- or 800-m-thick slices of the brain-
stem from BALB/c mice (P0–P3) and Sprague-Dawley
rats (P0–P4) were cut at the level of the caudal and
rostral preBo¨tC, respectively, as reported (3) and
perfused in artificial cerebrospinal fluid (aCSF) con-
taining 128 mM NaCl, 9 mM KCl, 0.5 mM NaH2PO4,
1.5 mM CaCl2, 1.0 mM MgSO4, 23.5 mM NaHCO3, 30
mM glucose (rat), or 130 mM NaCl, 5.4 mM KCl, 0.8
mM KH2PO4, 26 mM NaHCO3, 30 mM glucose, 1 mM
MgCl2, 0.8 mM CaCl2 (mouse). Rhythmic respiratory-
related motor output was recorded by a suction
electrode placed on a XII nerve rootlet. Nerve activity
was amplified, filtered at 3 Hz to 1 kHz, and rectified
nerve activity was integrated. SP, DAMGO, 4,5,6,7-
tetrahydroisoxazolo[5,4-c]pyridin3-ol (THIP) hydro-
chloride, glycine, and TTX were obtained from RBI
and dissolved in aCSF. Drugs were microinjected with
glass micropipettes (tip diameter 6 to 12 m) low-
ered (100 to 300 m) into the preBo¨tC by microma-
nipulator. Injections were made with a controlled
pressure source for 5 s. Ejection volume was moni-
tored with a calibrated eyepiece reticule.
12. Female adult Sprague-Dawley rats (n ϭ 17) and
BALB/c mice (n ϭ 2) were transcardially perfused
with 4% paraformaldehyde in phosphate-buffered
saline (PBS) with or without picric acid, cryoprotected
in 25% sucrose PBS, embedded in OCT, sectioned at
25 to 40 m on a cryostat, and processed free
floating. Sections were incubated in primary antibody
diluted in serum at 4°C for 24 to 48 hours, placed in
fluorescent (Jackson Immunoresearch) or biotin
(Vector Laboratories) conjugated species-specific
secondary antibody overnight, and mounted on gel-
atin-subbed slides. In several rats, OR immunohis-
tochemistry was amplified with tyramine signal am-
plification–direct green amplification (NEN Life-
sciences) and bright-field NK1R staining was visualized
with a Vectastain Elite ABC kit and diaminobenzidine.
Antibodies: rabbit antibody to NK1R (anti-NK1R)
(1:2500 fluorescent, 1:25,000 bright field; Chemicon),
guinea pig anti-GABABR (1:3000; Chemicon), goat anti-
ChAT (1:100; Chemicon), mouse anti-TH (1:1000;
Boehringer Mannheim), rabbit anti-OR (1:75,000 TSA
or 1:7000 fluorescent; Instar). Bright-field images were
digitally acquired (Polaroid DMC) into Photoshop
(Adobe Systems) and background subtracted to re-
move chip nonlinearities. Confocal images were ac-
quired with a Zeiss 410 SCM and maximal intensity
projections were generated in NIH Image. All images
were filtered and adjusted for contrast and light
levels in Photoshop for clarity. Cell counts for coex-
pression were obtained by visual inspection of con-
focal images (n ϭ 2).
13. P. A. Gray and J. L. Feldman, data not shown.
14. J. L. Feldman and H. H. Ellenberger, Annu. Rev.
Physiol. 50, 593 (1988); H. H. Ellenberger, J. L. Feld-
man, W. Z. Zhan, J. Comp. Neurol. 294, 212 (1990);
H. H. Ellenberger and J. L. Feldman, J. Comp. Neurol.
294, 202 (1990).
15. Neurons located at the caudal or rostral part of the
preBo¨tC in mice were visualized with differential inter-
ference contrast and infrared video microscopy. Glass
micropipettes (resistances typically were 2 to 3 MW )
were filled with a solution containing 115 mM
HMeSO3, 115 mM KOH, 5 mM NaCl, 1 mM MgCl2, 0.01
mM CaCl2, 0.1 mM BAPTA [1,2-bis(2-aminophen-
oxy)ethane-N,N,NЈ,NЈ-tetraacetic acid, tetra-Kϩ
salt],
10 mM Hepes, 3 mM 2-ATP(Mg2ϩ
) (pH 7.3). Current- or
voltage-clamp recordings were made with an Axoc-
lamp-2A (Axon Instruments) amplifier in bridge or sin-
gle electrode voltage clamp mode. The I-V relationship
was determined by injecting a slow (7-s cycle) ramp-
shaped current and plotting the rising phase of the I-V.
Signals were recorded on videocassette (pulse code
modulation; Vetter Instruments, model 3000A), digi-
tized at 1 to 20 kHz with a Digidata 1200 A/D board
(Axon Instruments) and analyzed with Axoscope soft-
ware (Axon Instruments). Statistical values are given as
mean Ϯ SEM.
16. J. C. Rekling, J. Champagnat, M. Denavit-Saubie´,
J. Neurophysiol. 75, 811 (1996).
17. , J. Neurophysiol. 75, 795 (1996).
18. R. J. Butera Jr., J. Rinzel, J. C. Smith, J. Neurophysiol.
82, 382 (1999); J. Neurophysiol. 82, 398 (1999).
19. N. Koshiya and J. C. Smith, Nature 400, 360 (1999).
20. H. Onimaru, A. Arata, I. Homma, Jpn. J. Physiol. 47,
385 (1997).
21. J. C. Rekling and J. L. Feldman, data not shown.
22. E. Fletcher and J. Schaff, in Abnormalities of Respira-
tion During Sleep, E. Fletcher, Ed. (Grune & Stratton,
Orlando, FL, 1986), pp. 203–228; E. C. Fletcher, Car-
diologia 42, 469 (1997); S. Thalhofer and P. Dorow,
Respiration 64, 2 (1997).
23. J. P. Orlowski, Pediatrics 78, 233 (1986); V. Carpen-
tier, H. Vaudry, E. Mallet, A. Laquerrie´re, P. Leroux,
Neuroscience 86, 159 (1998); A. Coquerel et al.,
Neurochem. Int. 20, 97 (1992).
24. Supported by a Ford Foundation Pre-Doctoral Fellow-
ship for Minorities and a predoctoral fellowship from
the Porter Physiology Development Program of the
American Physiological Society to P.A.G., a Parker B.
Francis Fellowship to J.C.R., and NIH grants HL40959
and HL37941. We thank N. Brecha, D. Buonomono, L.
Kruger, and F. Schweizer for assistance with this
manuscript.
12 July 1999; accepted 8 October 1999
Identification of a Conserved
Receptor-Binding Site on the
Fiber Proteins of
CAR-Recognizing Adenoviridae
Peter W. Roelvink,* Gai Mi Lee, David A. Einfeld, Imre Kovesdi,
Thomas J. Wickham
The human adenovirus serotype 5 (Ad5) is used widely for applications in
human gene therapy. Cellular attachment of Ad5 is mediated by binding of the
carboxyl-terminal knob of its fiber coat protein to the Coxsackie adenovirus
receptor (CAR) protein. However, Ad5 binding to CAR hampers the development
of adenovirus vectors capable of specifically targeting (diseased) tissues or
organs. Through sequence analysis and mutagenesis, a conserved receptor-
binding region was identified on the side of three divergent CAR-binding knobs.
The feasibility of simultaneous CAR ablation and redirection of an adenovirus
to a new receptor is demonstrated.
The human adenovirus family has 49 viral
serotypes that are associated with a wide
range of pathologies and tissue tropisms (1,
2). The serotype Ad5 has been studied inten-
sively and is currently used as a vector in
human gene therapy (3). Cellular attachment
is mediated by binding of the COOH-termi-
nal knob of the adenovirus fiber coat protein
R E P O R T S
19 NOVEMBER 1999 VOL 286 SCIENCE www.sciencemag.org1568