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V O'Connor

Publications and source records attributed to V O'Connor.

At least 19 recordsLinked to original sources
PubMed2004

Eml5, a novel WD40 domain protein expressed in rat brain.

We have isolated a novel transcript with homology to the major microtubule-associated protein in dividing sea urchin embryos, EMAP. The protein has a predicted MW of approximately 180 kDa and we have named it Eml5 (EMAP-like protein 5). Eml5 contains 11 putative WD40 domains and 3 hydrophobic stretches of 43 aa, HELP domains, which have been suggested to be involved in microtubule binding. Eml5 appears to consist of two tandem repeats of the complete EMAP protein separated by a putative dimerization domain. Eml5 mRNA and protein is expressed at high levels in the hippocampus, cerebellum and olfactory bulb, as determined by in situ hybridization and immunocytochemistry. Eml5 transcripts can be detected in fore- and hindbrain structures from embryonic day 13 onwards. Because other EMAP-like proteins are involved in regulating microtubule dynamics, it is likely that Eml5 plays a role in the regulation of cytoskeletal rearrangements during neuronal development and in adult brain

Alternative Splicing↗
PubMed2003

LTP but not seizure is associated with up-regulation of AKAP-150.

We have used differential display to profile and compare the mRNAs expressed in the hippocampus of freely moving animals after the induction of long-term potentiation (LTP) at the perforant path-dentate gyrus synapse with control rats receiving low-frequency stimulation. We have combined this with in situ hybridization and have identified A-kinase anchoring protein of 150 kDa (AKAP-150) as a gene selectively up-regulated during the maintenance phase of LTP. AKAP-150 mRNA has a biphasic modulation in the dentate gyrus following the induction of LTP. The expression of AKAP-150 was 29% lower than stimulated controls 1 h after the induction of LTP. Its expression was enhanced 3 (50%), 6 (239%) and 12 h (210%) after induction, returning to control levels by 24 h postinduction. The NMDA receptor antagonist CPP blocked the tetanus-induced modulation of AKAP-150 expression. Interestingly, strong generalized stimulation produced by electroconvulsive shock did not increase the expression of AKAP-150. This implies that the AKAP-150 harbours a novel property of selective responsiveness to the stimulation patterns that trigger NMDA-dependent LTP in vivo. Its selective up-regulation during LTP and its identified functions as a scaffold for protein kinase A, protein kinase C, calmodulin, calcineurin and ionotropic glutamate receptors suggest that AKAP-150 encodes is an important effector protein in the expression of late LTP.

A Kinase Anchor Proteins↗
PubMed2001

Mapping of calmodulin and Gbetagamma binding domains within the C-terminal region of the metabotropic glutamate receptor 7A.

Ca(2+)/calmodulin (Ca(2+)/CaM) and the betagamma subunits of heterotrimeric G-proteins (Gbetagamma) have recently been shown to interact in a mutually exclusive fashion with the intracellular C terminus of the presynaptic metabotropic glutamate receptor 7 (mGluR 7). Here, we further characterized the core CaM and Gbetagamma binding sequences. In contrast to a previous report, we find that the CaM binding motif localized in the N-terminal region of the cytoplasmic tail domain of mGluR 7 is conserved in the related group III mGluRs 4A and 8 and allows these receptors to also bind Ca(2+)/CaM. Mutational analysis of the Ca(2+)/CaM binding motif is consistent with group III receptors containing a conventional CaM binding site formed by an amphipathic alpha-helix. Substitutions adjacent to the core CaM target sequence selectively prevent Gbetagamma binding, suggesting that the CaM-dependent regulation of signal transduction involves determinants that overlap with but are different from those mediating Gbetagamma recruitment. In addition, we present evidence that Gbetagamma uses distinct nonoverlapping interfaces for interaction with the mGluR 7 C-terminal tail and the effector enzyme adenylyl cyclase II, respectively. Although Gbetagamma-mediated signaling is abolished in receptors lacking the core CaM binding sequence, alpha subunit activation, as assayed by agonist-dependent GTPgammaS binding, was not affected. This suggests that Ca(2+)/CaM may alter the mode of group III mGluR signaling from mono- (alpha) to bidirectional (alpha and betagamma) activation of downstream effector cascades.

Amino Acid Sequence↗
PubMed2001

Ntab, a novel non-coding RNA abundantly expressed in rat brain.

We have identified a novel transcript that is abundantly and specifically expressed in both the adult and developing rat CNS. Within the full-length cDNA sequence we were unable to identify a clear open reading frame. Moreover, we were unable to detect any protein product derived from the full-length cDNA sequence using an in vitro translation assay. Therefore, we suggest this gene is one of a growing number of non-coding mRNA-like RNA transcripts that exert their cellular functions directly as an RNA. We have named this novel gene Ntab for non-coding transcript abundantly expressed in brain (accession number AY035551). In addition, in some regions of the brain we find evidence for RNA accumulation in cellular processes at some distance from the soma. These findings suggest that Ntab is actively transported and may function within cellular processes. Since Ntab is a targeted non-coding RNA, such cellular functions could include the targeting and/or regulation of localised translation of other mRNA species.

Animals↗
PubMed2001

Subfield-specific immediate early gene expression associated with hippocampal long-term potentiation in vivo.

It is not known whether NMDA receptor-dependent long-term potentiation (LTP) is mediated by similar molecular mechanisms in different hippocampal areas. To address this question we have investigated changes in immediate early gene and protein expression in two hippocampal subfields following the induction of LTP in vivo and in vitro. In granule cells of the dentate gyrus, LTP induced in vivo by tetanic stimulation of the perforant path was followed by strong induction of the immediate early genes (IEGs) Zif268, Arc and Homer. The increase in Zif268 mRNA was accompanied by an increase in protein expression. In contrast, we were unable to detect modulation of the IEGs Zif268, Arc, Homer and HB-GAM following induction of LTP by high-frequency stimulation of the commissural projection to CA1 pyramidal cells in vivo. In this pathway, we also failed to detect modulation of Zif268 protein levels. Zif268, Arc and Homer can be modulated in CA1 pyramidal cells approximately twofold after electroshock-induced maximal seizure, which demonstrates potential responsiveness to electrical stimuli. When LTP was induced in vitro neither CA1 pyramidal cells nor granule cells showed an increase in Zif268, Arc or Homer mRNA. However, in the slice preparation, granule cells have a different transcriptional state as basal IEG levels are elevated. These results establish the existence of subfield-specific transcriptional responses to LTP-inducing stimulation in the hippocampus of the intact animal, and demonstrate that in area CA1-enhanced transcription of Zif268, Arc and Homer is not required for the induction of late LTP.

Animals↗
PubMed2001

Seizure-induced gene expression in area CA1 of the mouse hippocampus.

Synaptic plasticity in the hippocampus requires activity-dependent gene expression. We have therefore profiled gene expression in area CA1 following the induction of an electroshock-evoked maximal seizure. Using cDNA microarrays, the differential expression of approximately 9000 cDNAs was examined. In situ hybridization on 14 transcripts that showed strongest modulation in the microarray screen (1.8-2-fold) confirmed the differential expression of a single gene that encodes for the nuclear hormone receptor NGFI-B (Nur77, N10). Although this gene is only modestly up-regulated (approximately 2-fold) in area CA1, in situ hybridization revealed that maximal seizures induce a marked (approximately 12-fold) up-regulation of NGFI-B in the dentate gyrus. These data support the notion [French et al. (2001) Eur. J. Neurosci., 13, 968-976] that CA1 pyramidal neurons are more refractory than granule cells of the dentate gyrus with respect to activity-dependent gene transcription. Furthermore, our results argue against a large cohort of activity-dependent genes in area CA1.

Animals↗
PubMed1999

Calmodulin dependence of presynaptic metabotropic glutamate receptor signaling.

Glutamatergic neurotransmission is controlled by presynaptic metabotropic glutamate receptors (mGluRs). A subdomain in the intracellular carboxyl-terminal tail of group III mGluRs binds calmodulin and heterotrimeric guanosine triphosphate-binding protein (G protein) betagamma subunits in a mutually exclusive manner. Mutations interfering with calmodulin binding and calmodulin antagonists inhibit G protein-mediated modulation of ionic currents by mGluR 7. Calmodulin antagonists also prevent inhibition of excitatory neurotransmission via presynaptic mGluRs. These results reveal a novel mechanism of presynaptic modulation in which Ca(2+)-calmodulin is required to release G protein betagamma subunits from the C-tail of group III mGluRs in order to mediate glutamatergic autoinhibition.

Amino Acid Sequence↗
PubMed1999

Women's knowledge about the physical and emotional changes associated with menopause.

In 1992, a survey was undertaken to study women's knowledge of the physical and emotional changes associated with menopause. Two groups of midlife women: a random sample (n = 381) and a sample of women who attended menopause seminars (n = 95) were asked to select from a list of 39 changes those they thought were directly caused by menopause. Information from readily available books written for a general audience was used to construct the marking guide for a "commonly available knowledge" score. A template for a "biomedical" knowledge score based on an estrogen deficiency perspective of menopause was derived from recently published academic medical literature. The mean score for the commonly available knowledge items was 27 out of 39 (69.2% correct). The mean biomedical knowledge score was lower, 19.3 out of 35 (55.1% correct). While the two groups of women were significantly different in terms of current and past hormone replacement therapy (HRT) use, we found no differences between them in the mean commonly available knowledge or biomedical knowledge scores. This finding challenges the widely held assumption that active information-seekers are more interested and have a better level of knowledge than the general population. The findings also suggest that women are less likely to accept a biomedical model of menopause and that they are using HRT for a multiplicity of reasons, not just those related to estrogen deficiency.

Estrogen Replacement Therapy↗
PubMed1998

A neuronal Sec1 homolog regulates neurotransmitter release at the squid giant synapse.

Sec1-related proteins are essential for membrane fusion at distinct stages of the constitutive and regulated secretory pathways in eukaryotic cells. Studies of neuronal isoforms of the Sec1 protein family have yielded evidence for both positive and negative regulatory functions of these proteins in neurotransmitter release. Here, we have identified a squid neuronal homolog (s-Sec1) of Sec1 proteins and examined its function in neurotransmitter release at the squid giant synapse. Microinjection of s-Sec1 into the presynaptic terminal of the giant synapse inhibited evoked neurotransmitter release, but this effect was prevented by coinjecting the cytoplasmic domain of squid syntaxin (s-syntaxin), one of the binding partners of s-Sec1. A 24 amino acid peptide fragment of s-Sec1, which inhibited the binding of s-Sec1 to s-syntaxin in vitro, completely blocked release, suggesting an essential function of the s-Sec1/s-syntaxin interaction in transmitter release. Electron microscopy showed that injection of s-Sec1 did not change the spatial distribution of synaptic vesicles at presynaptic release sites ("active zones"), whereas the inhibitory peptide increased the number of docked vesicles. These distinct morphological effects lead us to conclude that Sec1 proteins function at different stages of synaptic vesicle exocytosis, and that an interaction of s-Sec1 with syntaxin-at a stage blocked by the peptide-is necessary for docked vesicles to fuse.

Animals↗
PubMed1998

Regulation of neurotransmitter release kinetics by NSF.

NSF (N-ethylmaleimide-sensitive factor) is an adenosine triphosphatase (ATPase) that contributes to a protein complex essential for membrane fusion. The synaptic function of this protein was investigated by injecting, into the giant presynaptic terminal of squid, peptides that inhibit the ATPase activity of NSF stimulated by the soluble NSF attachment protein (SNAP). These peptides reduced the amount and slowed the kinetics of neurotransmitter release as a result of actions that required vesicle turnover and occurred at a step subsequent to vesicle docking. These results define NSF as an essential participant in synaptic vesicle exocytosis that regulates the kinetics of neurotransmitter release and, thereby, the integrative properties of synapses.

Adenosine Triphosphatases↗
PubMed1998

Bleeding pattern and endometrial changes during continuous combined hormone replacement therapy. The Ogen/Provera Study Group.

OBJECTIVE: To establish the optimum oral daily dose of micronized medroxyprogesterone acetate, given in combination with a fixed oral dose of estrone (E1) sulfate as hormone replacement therapy, that provides endometrial protection and induces cessation of vaginal bleeding. METHODS: This multicenter, randomized, double-blind study was conducted for 2 years. Five hundred sixty-eight postmenopausal women were randomized to take E1 sulfate 1.25 mg daily and one of three doses of medroxyprogesterone acetate (2.5, 5, or 10 mg) daily. Any vaginal bleeding was recorded by patients in a daily diary, and endometrial biopsies were performed at entry into the study and at 3, 12, and 24 months. RESULTS: Forty-two percent of all women reported some bleeding at month 3 of therapy. However, by month 6, 76.5, 80.1, and 80.9% of women were amenorrheic in the 2.5-, 5-, and 10-mg medroxyprogesterone acetate groups, respectively. Over time, the percentage of women with no bleeding increased in each group, and by 24 months 91.5, 89.9, and 94.3% were amenorrheic in the 2.5- and 10-mg medroxyprogesterone acetate groups, respectively. Approximately 10% of women continue to have some bleeding, regardless of the dose of medroxyprogesterone acetate. There were no statistically significant differences in the number of women with bleeding at any time point between the three groups. There were no cases of endometrial hyperplasia reported in the study population over the 2 years. CONCLUSION: All three studied doses of medroxyprogesterone acetate, given in combination with 1.25 mg of E1 sulfate, provide adequate endometrial protection and render approximately 80% of women amenorrheic by 6 months of therapy.

Adult↗
PubMed1998

Protein interactions implicated in neurotransmitter release.

Biochemical evidence indicates that the exocytotic release of neurotransmitters involves both evolutionary conserved membrane proteins, the SNAREs, as well as ubiquitous cytosolic fusion proteins, NSF and SNAPs. We have analyzed the biochemical properties and the physiological effects of these proteins. Our data suggest models how NSF, SNAPs and SNAREs may function in neurotransmitter exocytosis.

Adenosine Triphosphatases↗
PubMed1997

Disruption of syntaxin-mediated protein interactions blocks neurotransmitter secretion.

The membrane protein syntaxin participates in several protein-protein interactions that have been implicated in neurotransmitter release. To probe the physiological importance of these interactions, we microinjected into the squid giant presynaptic terminal botulinum toxin C1, which cleaves syntaxin, and the H3 domain of syntaxin, which mediates binding to other proteins. Both reagents inhibited synaptic transmission yet did not affect the number or distribution of synaptic vesicles at the presynaptic active zone. Recombinant H3 domain inhibited the interactions between syntaxin and SNAP-25 that underlie the formation of stable SNARE complexes in vitro. These data support the notion that syntaxin-mediated SNARE complexes are necessary for docked synaptic vesicles to fuse.

Amino Acid Sequence↗
PubMed1997

Ca2+ or Sr2+ partially rescues synaptic transmission in hippocampal cultures treated with botulinum toxin A and C, but not tetanus toxin.

Botulinum (BoNT/A-G) and tetanus toxins (TeNT) are zinc endopeptidases that cleave proteins associated with presynaptic terminals (SNAP-25, syntaxin, or VAMP/synaptobrevin) and block neurotransmitter release. Treatment of hippocampal slice cultures with BoNT/A, BoNT/C, BoNT/E, or TeNT prevented the occurrence of spontaneous or miniature EPSCs (sEPSCs or mEPSCs) as well as the [Ca2+]o-independent increase in their frequency induced by phorbol ester, 0.5 nM alpha-latrotoxin, or sucrose. [Ca2+]o-independent and -dependent release thus requires that the target proteins of clostridial neurotoxins be uncleaved. In contrast, significant increases in mEPSC frequency were produced in BoNT-treated, but not TeNT-treated, cultures by application of the Ca2+ ionophore ionomycin in the presence of 10 mM [Ca2+]o. The frequency of sEPSCs was increased in BoNT-treated, but not TeNT-treated, cultures by increasing [Ca2+]o from 2.8 to 5-10 mM or by applying 5 mM Sr2+. Large Ca2+ and Sr2+ influxes thus can rescue release after BoNT treatment, albeit less than in control cultures. The nature of the toxin-induced modification of Ca2+-dependent release was assessed by recordings from monosynaptically coupled CA3 cell pairs. The paired-pulse ratio of unitary EPSCs evoked by two presynaptic action potentials in close succession was 0.5 in control cultures, but it was 1.4 and 1.2 in BoNT/A- or BoNT/C-treated cultures when recorded in 10 mM [Ca2+]o. Log-log plots of unitary EPSC amplitude versus [Ca2+]o were shifted toward higher [Ca2+]o in BoNT/A- or BoNT/C-treated cultures, but their slope was unchanged and the maximal EPSC amplitudes were reduced. We conclude that BoNTs reduce the Ca2+ sensitivity of the exocytotic machinery and the number of quanta released.

Animals↗
PubMed1996

Interactions of glycine and strychnine with their receptor recognition sites in mouse spinal cord.

Interactions between the inhibitory neurotransmitter glycine and its receptor antagonist strychnine have been studied in mouse spinal cord membranes and both agents employed to protect against residue selective protein modifying reagents in order to identify contact residues for ligand binding. Glycine was found to behave as a full competitive inhibitor of [3H]-strychnine binding, provided that precautions were taken to prevent radioligand binding to the glass-fibre filters used to terminate the assays. Hill coefficients for the glycine inhibition of [3H]-strychnine binding were not significantly different from one, indicating a lack of cooperative interactions. For the protection experiments, N-bromosuccinimide, tetranitromethane, diethylpyrocarbonate and 2,3-butanedione were used under conditions selective for tryptophan, tyrosine, histidine and arginine residues, respectively. Of these reagents, N-bromosuccinimide, tetranitromethane and diethylpyrocarbonate caused a decrease in total [3H]-strychnine binding without affecting the ability of unlabelled strychnine to compete. In contrast, the same reagents disrupted the ability of glycine to inhibit [3H]-strychnine binding. The presence of either excess glycine (10(-2) M) or strychnine (10(-4) M) during the above treatments was found to prevent the decrease in total and strychnine-specific [3H]-strychnine binding. However, only in the case of diethylpyrocarbonate treatment were both agonist and antagonist able to protect against the loss of glycine-specific [3H]-strychnine binding. The reagent 2,3-butanedione caused an increase in total and strychnine-specific [3H]-strychnine binding (which we have shown elsewhere to be at a site unrelated to the inhibitory glycine receptor). When the above protein modifying reagents were applied under the same conditions to specific strychnine binding antibodies, all four caused significant decreases in subsequent [3H]-strychnine binding. Strychnine was found to afford significant protection of the antibodies against N-bromosuccinimide, tetranitromethane and 2,3-butanedione, but not against diethylpyrocarbonate. Our results suggest that glycine and strychnine compete at overlapping but conformationally distinct sites on the receptor. Tyrosine, tryptophan, histidine and arginine residues are implicated as strychnine contact residues with a shared role for histidine in the recognition of glycine.

Animals↗
PubMed1996

The menopause and hormone replacement therapy: Australian general practitioners' self-reported opinions, attitudes and behaviour.

OBJECTIVES: We describe the attitude and views of general practitioners towards the menopause and hormone replacement therapy (HRT) in metropolitan Brisbane, Australia. METHODS: A total of 216 general practitioners were nominated by a random sample of urband-welling women aged 45-54 years who formed the Brisbane Women's health study. A 20-30 minute face-to-face questionnaire with the general practitioners was administered and analysed by demographic characteristics. RESULTS: There was a 93% response rate. Management of the menopause and HRT was routinely undertaken by general practitioners for their own patients. After deciding to initiate HRT, > 60% of general practitioners ordered five investigations or more. They may have confused the risk of thrombo-embolism from oestrogens used in the post-menopause with that for contraception. There were differences between male and female practitioners in some areas. Male general practitioners, in particular, reported more difficulty with tailoring and adjusting regimes. CONCLUSIONS: Specific areas for further education are explored to meet the educational needs of general practitioners.

Adult↗
PubMed1995

Clostridial neurotoxins compromise the stability of a low energy SNARE complex mediating NSF activation of synaptic vesicle fusion.

A 20S complex composed of the cytosolic fusion proteins NSF and SNAP and the synaptosomal SNAP receptors (SNAREs) synaptobrevin, syntaxin and SNAP-25 is essential for synaptic vesicle exocytosis. Formation of this complex is thought to be regulated by synaptotagmin, the putative calcium sensor of neurotransmitter release. Here we have examined how different inhibitors of neurotransmitter release, e.g. clostridial neurotoxins and a synaptotagmin peptide, affect the properties of the 20S complex. Cleavage of synaptobrevin and SNAP-25 by the neurotoxic clostridial proteases tetanus toxin and botulinum toxin A had no effect on assembly and disassembly of the 20S complex; however, the stability of its SDS-resistant SNARE core was compromised. This SDS-resistant low energy conformation of the SNAREs constitutes the physiological target of NSF, as indicated by its ATP-dependent disassembly in the presence of SNAP and NSF. Synaptotagmin peptides caused inhibition of in vitro binding of this protein to the SNAREs, a result that is inconsistent with synaptotagmin's proposed role as a regulator of SNAP binding. Our data can be reconciled by the idea that NSF and SNAP generate synaptotagmin-containing intermediates in synaptic vesicle fusion, which catalyse neurotransmitter release.

Adenosine Triphosphate↗