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Biomedical subjects

A Chambers

Publications and source records attributed to A Chambers.

At least 37 records · Page 2Linked to original sources
PubMed1995

Pharmacological evidence for an involvement of group II and group III mGluRs in the presynaptic regulation of excitatory synaptic responses in the CA1 region of rat hippocampal slices.

The actions of four mGluR antagonists, (+)-MCPG, MAP4, MCCG and (S)-4CPG, were evaluated against agonist-induced depressions of synaptic transmission at the Schaffer collateral-commissural pathway in rat hippocampal slices. (+)-MCPG (1 mM) reversed very effectively depressions of field EPSPs induced by (1S,3R)-ACPD and (1S,3S)-ACPD but had weak and variable effects on depressions induced by L-AP4. It had no effect on depressions induced by either (-)-baclofen or carbachol. In contrast, MAP4 (500 microM) reversed very effectively depressions induced by L-AP4 without affecting depressions induced by (1S,3S)-ACPD. MCCG (1 mM) had the opposite activity; it antagonized depressions induced by (1S,3S)-ACPD but not those induced by L-AP4. Finally, (S)-4CPG (1 mM) reversed small depressions of field EPSPs induced by high concentrations (50-100 microM) of (1S,3R)- and (1S,3S)-ACPD, but not L-AP4, whilst having no effect on large depressions induced by 10 microM (1S,3S)-ACPD in voltage-clamped cells. These results confirm and extend the effectiveness and selectivity of (+)-MCPG as an mGluR antagonist. The divergent effects of the group I antagonist, (S)-4CPG, can be explained by an indirect action on postsynaptic receptors which is manifest when high agonist concentrations are used in non-voltage-clamp experiments. The action of MCCG and MAP4 indicates that two pharmacologically-distinct mGluRs, belonging to classes II and III, can regulate synaptic transmission in the CA1 region via presynaptic mechanisms.

Animals↗
PubMed1994

The yeast protein Gcr1p binds to the PGK UAS and contributes to the activation of transcription of the PGK gene.

Analysis of the upstream activation sequence (UAS) of the yeast phosphoglycerate kinase gene (PGK) has demonstrated that a number of sequence elements are involved in its activity and two of these sequences are bound by the multifunctional factors Rap1p and Abf1p. In this report we show by in vivo footprinting that the regulatory factor encoded by GCR1 binds to two elements in the 3' half of the PGK UAS. These elements contain the sequence CTTCC, which was previously suggested to be important for the activity of the PGK UAS and has been shown to be able to bind Gcr1p in vitro. Furthermore, we find that Gcr1p positively influences PGK transcription, although it is not responsible for the carbon source dependent regulation of PGK mRNA synthesis. In order to mediate its transcriptional influence we find that Gcr1p requires the Rap1p binding site, in addition to its own, but not the Abf1p site. As neither a Rap1p nor a Gcr1p binding site alone is able to activate transcription, we propose that Gcr1p and Rap1p interact in an interdependent fashion to activate PGK transcription.

Base Sequence↗
PubMed1994

Use of a selection technique to identify the diversity of binding sites for the yeast RAP1 transcription factor.

We have used the technique known as selected and amplified binding (SAAB) to isolate binding sites for the yeast transcription factor RAP1 from a degenerate pool of oligonucleotides. A total of 47 sequences were isolated, of which two were shown to be contaminating non-RAP1 binding sites. After excluding these two sequences the remainder of the sequences were used to derive a new consensus binding site for RAP1. The new consensus 5' A/G T A/G C A C C C A N N C C/A C C 3' is a significant extension of the existing consensus (4). It is longer by two base pairs at the 5' end and is significantly more constrained at the 3' end. An analysis of the combinations of mis-matches in individual SAAB sequences, compared to the consensus RAP1 binding site, has allowed us to analyse the structure of the RAP1 binding site in some detail. The binding site can be sub-divided into three regions; a core binding site, a 5' flanking region and a 3' flanking region. The core binding site, consisting of the sequence 5'CACCCA3', is critical for recognition by RAP1. The less conserved flanking regions are not as important. Interactions between RAP1 and these regions probably stabilise the interaction between RAP1 and the core binding site. Each of the sequences isolated in the SAAB analysis was used to search release 78 of the EMBL+GenBank DNA data base. The searches identified 102 potential binding sites for RAP1 within promoters of yeast genes.

Base Sequence↗
PubMed1994

The type III phosphodiesterase inhibitor milrinone and type V PDE inhibitor dipyridamole individually and synergistically reduce elevated pulmonary vascular resistance.

The effects of the phosphodiesterase (PDE) inhibitors milrinone and dipyridamole were studied in an in situ perfused rabbit lung model in which the pulmonary vascular resistance (PVR) was elevated by infusion of the thromboxane-A2 mimetic U46619. Dose-response curves for reduction of elevated PVR were generated for each of these drugs. The EC50 for milrinone was approximately 2 microM. The EC50 for dipyridamole was approximately 0.2 microM. In separate experiments, 0.1 microM milrinone was found to reduce elevated PVR by 4.6 +/- 2.4%, 0.06 microM dipyridamole reduced elevated PVR by 8.2 +/- 2.8%, whereas the combination of 0.1 microM milrinone and 0.06 microM dipyridamole reduced elevated PVR by 41.9 +/- 7.3%. In more limited experiments, it was determined that the PDE type V inhibitor zaprinast also caused a synergistic reduction of PVR when used with milrinone. We concluded that both the type III PDE inhibitor milrinone and the type V PDE inhibitors dipyridamole or zaprinast are effectively able to reduce elevated PVR and that the combination of PDE type III and type V inhibitors is synergistic in the ability to reduce elevated PVR. We speculate that type V PDE may play a more important role than type III PDE in the regulation of pulmonary vascular tone. It is proposed that the combination of milrinone and dipyridamole has the potential to be useful in the clinical treatment of elevated PVR.

Animals↗
PubMed1994

A Reb1p-binding site is required for efficient activation of the yeast RAP1 gene, but multiple binding sites for Rap1p are not essential.

The Saccharomyces cerevisiae RAP1 protein (Rap1p) is a key multifunctional transcription factor. Using gel retardation analysis, four binding sites for Rap1p have been identified within the promoter of the RAP1 gene. These sites are located downstream of a binding site for the transcription factor Reb1p. The Reb1p site and an associated AT-rich region are important for transcriptional activation, but deletion of three of the Rap1p-binding sites had little effect on promoter activity. The activity of the RAP1 promoter has been analysed in a yeast strain (YDS410) that contains a temperature-sensitive mutation in the RAP1 gene. This mutation renders the DNA-binding activity of Rap1p temperature dependent. When YDS410 was grown at a semi-permissive temperature (30 degrees C), the activity of the RAP1 promoter increased by approximately 170%, compared with the same strain grown at the permissive temperature (25 degrees C). A RAP1 promoter in which three of the four Rap1p-binding sites had been deleted, showed only a small increase in activity in the same experiment. These data confirm that Rap1p is not required for activation of the RAP1 gene, and suggest a role for Rap1p in negative autoregulation.

Base Sequence↗
PubMed1993

Sexual practices, sexually transmitted diseases and other risk factors for HIV among injecting drug users.

Data were collected from 814 clients attending anonymously for counselling before tests for human immunodeficiency virus (HIV) infection at the Burnett Clinic in Auckland. Just over 10 per cent of clients (n = 83) reported ever having injected drugs. This group was matched according to age, gender, ethnicity and sexual orientation with an equal sized control group drawn from clients who had not injected drugs. The groups were compared in terms of their sexual practices, sexual partnerships, other drug use, current and past health status and their perception of HIV risk. The injecting drug users reported more sex partners than the nonusers, they were less likely to be in a relationship and they were less likely to be monogamous. Vaginal and oral sex were the most common practices in both groups but anal sex was more common among users than the nonusers. Regular condom use was reported by less than a quarter of each group. The health status of drug users was generally poorer than that of the nonusers. They reported greater use of alcohol and other drugs, more mental health problems, and more symptoms of ill health. They also had experienced more sexually-transmitted diseases. Only 1 per cent of users and 2 per cent of nonusers tested positively for HIV, although users saw themselves and their partners as at greater risk of HIV infection than the nonusers.

AIDS Serodiagnosis↗
PubMed1992

Sexually transmitted diseases amongst a sample of people seeking HIV testing.

AIMS: To examine the incidence of sexually transmitted diseases according to gender, age, sexual orientation, sexual behaviour and drug use. METHODS: Data were collected from 814 clients attending anonymously for HIV testing at the Burnett Centre in Auckland. During pretest counselling clients were asked questions designed to assess their risk of HIV infection including a detailed history of sexually transmitted diseases (STDs). RESULTS: Amongst attenders 44% reported at least one lifetime STD including 16% reporting more than one. Multiple STDs were not associated with gender or sexual orientation but they were associated with the practice of anal sex and with a history of multiple sex partners. Alcohol and drug use were also related to multiple STDs. For men the most common STD was NSU with 20% of them reporting it whereas for women genital warts was the most common reported by 18%. Amongst those under 20 years 9% reported genital warts, 7% NSU and 6% chlamydia. CONCLUSIONS: The findings confirm that it is sexual practices which put people at risk of infection from STDs rather than their sexual orientation. They also draw attention to the role that drugs and alcohol play in the practice of unsafe sex and the consequent transmission of STDs. Finally they suggest that screening for STDs as well as HIV in AIDS clinics should be encouraged.

Adult↗
PubMed1992

Condom use in anal intercourse amongst people who identify as homosexual, heterosexual or bisexual.

AIMS: to study condom use amongst those who practice anal intercourse. METHODS: a sample of 814 clients attending anonymously for pretest counselling for HIV infection at the Burnett Clinic, Auckland, gave detailed information about their sexual behaviour. RESULTS: insertive or receptive anal intercourse was reported in 39% of clients. This included 89% among homosexual men, 78% among bisexual men, 17% among heterosexual men and 21% among heterosexual women. In those practising anal intercourse concomitant condom use was reported by 71% of homosexual men, 53% of bisexual men, 16% of heterosexual men and 7% of heterosexual women. Those most likely to use condoms were homosexual men, prostitutes, those with multiple partners and those with an HIV infected partner. Those least likely to use condoms were heterosexual men or women. CONCLUSIONS: whilst substantial changes in sexual practices appear to have been made in the homosexual community, heterosexuals practising anal intercourse have made few such changes. More explicit AIDS education may be necessary which acknowledges that anal intercourse is practised by heterosexuals and advises condom use accordingly.

Ambulatory Care Facilities↗
PubMed1991

Aging, sleep disorders, and male sexual function.

The relation between age, sleep disorders, nocturnal penile tumescence, and sexual behavior was investigated in 70 healthy married men aged 45-75 years. They had an extensive psychosexual interview, a medical and psychiatric evaluation, and were studied in the sleep laboratory for four nights. Electroencephalogram (EEG), eye movements, muscle tone, and penile tumescence were monitored continuously, and respiratory airflow and bilateral anterior tibialis recordings were obtained during the first sleep session. There was a marked age-related increase in sleep-disordered breathing, but no significant changes in periodic leg movements with age. Respiratory distress and periodic leg movement (PLM) indices were mostly unrelated with nocturnal penile tumescence (NPT) measures as well as with all sexual behavior dimensions when age was taken into account in the analysis of results. Men with a respiratory distress disorder did not differ from a nondisordered group of similar age in NPT and behavioral parameters. Men with PLM disorder differed from their comparison group in only two variables: fewer number of maximum tumescent episodes and less frequent sexual thoughts. Men who met criteria for erectile impotence did not differ significantly in degree of respiratory or PLM disturbances or in the prevalence of sleep disorders when compared to an aged-matched sexually nondysfunctional group. The overall results did not support the notion that sleep disorders are involved in the increased prevalence of erectile impotence in healthy older individuals. Assessment of blood oxygenation and respiratory effort, in addition to airflow, need to be carried out before final conclusions can be drawn on the significance of sleep-disordered breathing in male sexuality.

Age Factors↗
PubMed1990

Phosphorylation influences the binding of the yeast RAP1 protein to the upstream activating sequence of the PGK gene.

Yeast repressor activator protein 1 (RAP1) binds in vitro to specific DNA sequences that are found in diverse genetic elements. Expression of the yeast phosphoglycerate kinase gene (PGK) requires the binding of RAP1 to the activator core sequence within the upstream activating sequence (UAS) of PGK. A DNA fragment Z+ which contains the activator core sequence of the PGK(UAS) has been shown to bind RAP1. Here we report that phosphatase treatment of RAP1 affected its binding to the PGK(UAS) but that this depended on the nature of the sequence flanking the 5' end of the activator core sequence. When the sequence flanking the 5' end of the activator core sequence was different from the PGK RAP1-binding site, phosphatase treatment of RAP1 decreased its binding to the DNA. When the 5' end of the binding site was a match to the PGK RAP1-binding site dephosphorylation of RAP1 increased RAP1 binding to the DNA. These observations were reproduced when the minimal functional DNA-binding domain of the RAP1 protein was used, implicating a phosphorylation-dependent binding of RAP1. This is the first evidence for phosphorylation-dependent binding of RAP1.

Amino Acid Sequence↗
PubMed1990

ARS binding factor 1 binds adjacent to RAP1 at the UASs of the yeast glycolytic genes PGK and PYK1.

The UAS of the yeast gene encoding the glycolytic enzyme phosphoglycerate kinase (PGK) contains several different sequence elements involved in transcriptional activation. These elements include the activator core sequence, which is bound by the RAP1 protein, and three copies of the pentamer sequence 5' CTTCC 3'. Upstream of the activator core sequence is a region (Yfp), identified as the site of a strong DNA-protein interaction. The Yfp region contains the consensus binding site for the factor ABF1. We have purified the Y protein, which binds to the Yfp region, to homogeneity. The Y protein migrates as a doublet on SDS-polyacrylamide gel electrophoresis with an apparent molecular weight of 125 KDa. These properties are similar to those of ABF1. ABF1 synthesised in vitro bound strongly to the Yfp region and formed a gel retardation complex of identical mobility to the complex formed by the Y protein. UAS1 of the pyruvate kinase gene (PYK1) promoter contains a RAP1 binding site and single copy of the CTTCC sequence. We have now identified an ABF1 binding site close to the RAP1 binding site and CTTCC sequence in the PYK1 promoter. This site is strongly bound by ABF1 in vitro. The organisation of the PGK and PYK1 UASs is thus similar to each other and to the transcriptional silencer HMR(E) which also contains these sequences.

Base Sequence↗
PubMed1990

A nuclear translational block imposed by the HIV-1 U3 region is relieved by the Tat-TAR interaction.

Replication of HIV-1 depends on the viral Tat protein, which functions via a target sequence, TAR, present in the proviral long terminal repeat (LTR) and at the 5' end of viral mRNAs. We have shown that Tat potentiates the expression of TAR-containing RNAs, but only when Tat and the TAR-containing RNA are present in the nucleus. We now show that a small change in the TAR loop abolishes nuclear potentiation by Tat. Furthermore, the HIV-1 U3 region induces expression incompetence in mRNA synthesized by this promoter. RNAs of identical structure are, however, translated efficiently when produced from the CMV-IE promoter. The Tat-TAR system appears, therefore, to rescue the expression potential of HIV-1 LTR-directed RNA.

Animals↗
PubMed1990

Characterisation of the DNA binding domain of the yeast RAP1 protein.

The 827 amino acid yeast RAP1 protein interacts with DNA to regulate gene expression at numerous unrelated loci in the yeast genome. By a combination of amino, carboxy and internal deletions, we have defined an internal 235 amino acid fragment of the yeast RAP1 protein that can bind efficiently to the RAP1 binding site of the PGK Upstream Activation Sequence (UAS). This domain spans residues 361 to 596 of the full length protein and lacks any homology to the DNA binding 'zinc finger' or 'helix-turn-helix' structural motifs. All the RAP1 binding sites we have tested bind domain 361-596, arguing that RAP1 binds all its chromosomal sites via this domain. The domain could not be further reduced in size suggesting that it represents the minimal functional DNA binding domain. The relevance of potential regions of secondary structure within the minimal binding domain is discussed.

Base Sequence↗