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Biomedical subjects

P J Mitchell

Publications and source records attributed to P J Mitchell.

At least 91 records · Page 5Linked to original sources
PubMed1991

Immobilisation of southern elephant seals and leopard seals with cyclohexamine anaesthetics and xylazine.

Ketamine and xylazine were given to 55 southern elephant seals (Mirounga leonina) for stomach lavaging, and to three leopard seals (Hydrurga leptonyx). The elephant seals showed prolonged apnoea and two of them died owing to aspiration of stomach contents. Two of the leopard seals died from unknown causes. Tiletamine and zolazepam were given to five elephant seals and one leopard seal. Two of the elephant seals and the leopard seal died from unknown causes. Xylazine alone was administered to 34 leopard seals. Sedation was poor at low dose rates (less than 1.7 mg/kg) but four of the seals given higher dose rates died owing to the aspiration of stomach contents.

Anesthetics↗
PubMed1991

Is antidepressant efficacy revealed by drug-induced changes in rat behaviour exhibited during social interaction?

Remission from depressive illness is associated with a modification in patients' behavioural reactions to environmental/social stimulation, and requires continuous drug treatment. We have examined the effects of antidepressant drug treatment and repeated electroconvulsive shock (ECS) on the behaviour of rats during social interaction (SI) to determine whether antidepressant treatments modify behavioural patterns of experimental animals that may be related to their ability to modify reactive human behaviour. Acute treatment of short-term isolated resident rats with nonsedative doses of antidepressant drugs, or sedative doses of haloperidol and diazepam, dose relatedly reduced aggressive behaviour exhibited during SI. Conversely, chronic antidepressant treatment (including ECS), but not chronic haloperidol or diazepam treatment, increased the aggressive behaviour of resident rats. These studies have revealed selective, diametrically opposite, effects of acute and chronic antidepressant treatment on rodent aggressive behaviour that may be indicative of antidepressant efficacy. The effects of chronic antidepressant treatment in particular may indicate a disinhibition of rodent social behaviour which may mirror the externalisation of emotions associated with the remission of depressive illness.

Aggression↗
PubMed1991

Transcription factor AP-2 is expressed in neural crest cell lineages during mouse embryogenesis.

We have analyzed the expression pattern of transcription factor AP-2 in mouse embryos to evaluate the potential of AP-2 as a regulator during vertebrate development. A partial cDNA encoding AP-2 was isolated from a mouse embryo cDNA library and used to prepare probes to measure AP-2 mRNA levels by RNase protection and RNA in situ hybridization. Between 10.5 and 15.5 days of embryogenesis, the relative abundance of AP-2 mRNA is greatest at 11.5 days and declines steadily thereafter. RNA in situ hybridization analysis of embryos between 8.5 and 12.5 days of gestation identified a novel expression pattern for AP-2. The principle part of this expression occurs in neural crest cells and their major derivatives, including cranial and spinal sensory ganglia and facial mesenchyme. AP-2 is also expressed in surface ectoderm and in a longitudinal column of the spinal cord and hindbrain that is contacted by neural crest-derived sensory ganglia. Additional expression of AP-2 occurs in limb bud mesenchyme and in meso-metanephric regions. This embryonic expression pattern is spatially and temporally consistent with a role for AP-2 in regulating transcription of genes involved in the morphogenesis of the peripheral nervous system, face, limbs, skin, and nephric tissues.

Amino Acid Sequence↗
PubMed1990

Transactivation by the hepatitis B virus X protein depends on AP-2 and other transcription factors.

The hepatitis B virus (HBV) X gene product (pX) could be important in disease pathogenesis because it is known to transactivate transcription from many viral and cellular gene promoters, including the HBV core gene promoter, the human immunodeficiency virus (HIV-1) long terminal repeat, and the c-myc promoter. We have previously shown that only a subset of the promoters that can be transactivated by pX is transactivated in any particular cell line, and have proposed that pX acts through multiple, cell type-specific transcription factors. We show here that pX acts through both AP-1 and AP-2 sites, and that pX has a transcription activation domain. We conclude that transactivation by pX depends on at least two distinct cellular DNA-binding transcription factors and we present a model for the action of pX.

Animals↗
PubMed1990

Analysis of the mouse Dhfr/Rep-3 major promoter region by using linker-scanning and internal deletion mutations and DNase I footprinting.

The mouse dihydrofolate reductase (Dhfr) promoter region is buried within a CpG island (a region rich in unmethylated CpG dinucleotides), has a high G+C content, and lacks CAAT and TATA elements. The region contains four 48-bp repeats, each of which contains an Sp1-binding site. Another gene, Rep-3 (formerly designated Rep-1), shares the same general promoter region with Dhfr, being transcribed in the direction opposite that of Dhfr. Both genes appear to be housekeeping genes and are expressed at relatively low levels in all tissues. The 5' termini of the major Dhfr transcripts are separated from the 5' termini of the Rep-3 transcripts by approximately 140 bp. This curious structural arrangement suggested that the two genes might share common regulatory elements. To investigate the promoter sequences driving bidirectional transcription, a series of promoter mutations was constructed. These mutations were assayed by a replicating minigene system and by promoter fusions to the chloramphenicol acetyltransferase gene. Linker-scanning mutations that spanned the four repeats produced a variety of mRNA transcript phenotypes. The effects were primarily quantitative, generally reducing the abundance of transcripts for one or both genes. Some mutations affected Dhfr in a qualitative manner, such as by changing the startpoint of one of the major Dhfr transcripts or changing the relative abundance of the two major Dhfr transcripts. Additionally, protein transcription factors that bind to sequences in the mouse Dhfr/Rep-3 major promoter region, potentially affecting expression of either or both genes, were investigated by DNase I footprinting. The results indicate that multiple protein-DNA interactions occur in this region, reflecting potentially complex transcriptional control mechanisms that might modulate expression of either or both genes under different physiological conditions.

Animals↗
PubMed1989

Transcriptional regulation in mammalian cells by sequence-specific DNA binding proteins.

The cloning of genes encoding mammalian DNA binding transcription factors for RNA polymerase II has provided the opportunity to analyze the structure and function of these proteins. This review summarizes recent studies that define structural domains for DNA binding and transcriptional activation functions in sequence-specific transcription factors. The mechanisms by which these factors may activate transcriptional initiation and by which they may be regulated to achieve differential gene expression are also discussed.

Amino Acid Sequence↗
PubMed1989

Molecular cloning and localization to chromosome 6 of mouse INT1L1 gene.

The human INT1L1 gene, which exhibits homology to the protooncogene INT1 is very closely linked to the MET gene and cystic fibrosis locus on human chromosome 7. In the present study we have isolated overlapping genomic clones that correspond to the mouse homolog of the INT1L1 gene and have used the cloned DNA as probes to examine the distribution of the mouse INT1L1 gene within a series of 35 mouse-hamster somatic cell hybrids. These analyses have localized the INT1L1 gene to mouse chromosome 6. In addition, we demonstrate that the mouse INT1L1 and MET genes are coamplified in lines of spontaneously transformed mouse NIH3T3 cells, indicating that these genes may remain closely linked within the mouse genome.

Amino Acid Sequence↗
PubMed1989

Regulation of transcription factor AP-2 by the morphogen retinoic acid and by second messengers.

The expression of the transcription factor AP-2 recently has been shown to be enhanced during retinoic acid (RA)-induced differentiation of NT2 cells, a human teratocarcinoma cell line. Here we show that this induction of AP-2 mRNA is at the level of transcription and is transient, reaching a peak 48-72 hr after the addition of RA and declining thereafter, even in the continuous presence of RA. Increased levels of AP-2 mRNA are reflected in a similar elevation of AP-2 protein and accompanied by an increase in the AP-2-binding site-dependent transcriptional activity of a reporter gene. AP-2 also has been proposed to confer TPA and cAMP inducibility on promoters/enhancers containing AP-2-binding sites. We investigated the effect of these agents on the expression of AP-2 protein and mRNA. Our experiments demonstrate that expression of the AP-2 gene in HeLa cells is not elevated significantly by TPA or by a calcium ionophore and is not enhanced at all by agents that increase intracellular cAMP concentration. In fact, AP-2 mRNA is repressed by both TPA and the calcium ionophore A23187 through a delayed response. These data suggest that the AP-2-binding site-mediated cAMP and TPA responses are not regulated at the level of AP-2 expression but, rather, achieved either by post-translational changes in AP-2 or in conjunction with another protein.

1-Methyl-3-isobutylxanthine↗
PubMed1989

Nonsense mutations in the dihydrofolate reductase gene affect RNA processing.

Steady-state dihydrofolate reductase (dhfr) mRNA levels were decreased as a result of nonsense mutations in the dhfr gene. Thirteen DHFR-deficient mutants were isolated after treatment of Chinese hamster ovary cells with UV irradiation. The positions of most point mutations were localized by RNA heteroduplex mapping, the mutated regions were isolated by cloning or by enzymatic amplification, and base changes were determined by DNA sequencing. Two of the mutants suffered large deletions that spanned the entire dhfr gene. The remaining 11 mutations consisted of nine single-base substitutions, one double-base substitution, and one single-base insertion. All of the single-base substitutions took place at the 3' position of a pyrimidine dinucleotide, supporting the idea that UV mutagenesis proceeds through the formation of pyrimidine dimers in mammalian cells. Of the 11 point mutations, 10 resulted in nonsense codons, either directly or by a frameshift, suggesting that the selection method favored a null phenotype. An examination of steady-state RNA levels in cells carrying these mutations and a comparison with similar data from other dhfr mutants (A. M. Carothers, R. W. Steigerwalt, G. Urlaub, L. A. Chasin, and D. Grunberger, J. Mol. Biol., in press) showed that translation termination mutations in any of the internal exons of the gene gave rise to a low-RNA phenotype, whereas missense mutations in these exons or terminations in exon 6 (the final exon) did not affect dhfr mRNA levels. Nuclear run-on experiments showed that transcription of the mutant genes was normal. The stability of mature dhfr mRNA also was not affected, since (i) decay rates were the same in wild-type and mutant cells after inhibition of RNA synthesis with actinomycin D and (ii) intronless minigene versions of cloned wild-type and nonsense mutant genes were expressed equally after stable transfection. We conclude that RNA processing has been affected by these nonsense mutations and present a model in which both splicing and nuclear transport of an RNA molecule are coupled to its translation. Curiously, the low-RNA mutant phenotype was not exhibited after transfer of the mutant genes, suggesting that the transcripts of transfected genes may be processed differently than are those of their endogenous counterparts.

Animals↗
PubMed1987

Positive and negative regulation of transcription in vitro: enhancer-binding protein AP-2 is inhibited by SV40 T antigen.

We have purified a 52 kd protein, AP-2, that binds to enhancer regions of SV40 and human metallothionein IIA (hMT IIA) and stimulates RNA synthesis from these promoters in vitro. Surprisingly, AP-2 also binds to two SV40 early promoter regions recognized by Sp1 and T antigen. Juxtaposed binding sites for AP-2 and Sp1 in the 21 bp repeats may facilitate productive interactions between the two factors. In contrast, sequence-specific binding of AP-2 to SV40 and hMT IIA DNA is inhibited by the viral repressor protein T antigen. Furthermore, T antigen inhibits AP-2-dependent transcriptional activation of the hMT IIA promoter in vitro. The inhibition is neither a direct nor an indirect result of T antigen binding to DNA, because the hMT IIA promoter lacks T antigen binding sites. Instead, sedimentation studies suggest that protein-protein interactions between AP-2 and T antigen block AP-2 binding to DNA. These findings suggest novel mechanisms for mediating positive and negative regulation of transcription.

Antigens, Polyomavirus Transforming↗
PubMed1987

Epidemiology of Mycobacterium ulcerans infection in koalas (Phascolarctos cinereus) on Raymond Island, southeastern Australia.

Mycobacterium ulcerans infections were found in 11 koalas (Phascolarctos cinereus) between 1980 and 1985, in a population of approximately 200 koalas on Raymond Island in southeastern Australia. Ulcers caused by the infection occurred on the face, forearm, rump, groin and footpads. Seven koalas had multiple ulcers. All the infected animals were mature (age classes 4, 5 and 6), and eight were male. The distribution of ulcers corresponded with the distribution of wounds in a sample of 87 koalas. Many of these wounds were associated with social behaviour.

Animals↗
PubMed1986

A geriatric service in East London.

The background and establishment of a geriatric service in East London are described. Data relating to 37 admissions for geriatric assessment to the long-stay wards at Frere Hospital and 250 patients dealt with by the District Surgeon's community geriatric service staff are analysed to indicate the multifactorial nature of the assessments and care provided. Taken in conjunction with previous experience elsewhere, the indications are that effective services for care of the aged can be developed without special facilities, by re-allocation of some duties of existing staff and without vast capital expenditure. Certain recommendations are made for the development of an effective network of services for hospital-based care of the aged throughout the RSA.

Black or African American↗
PubMed1986

BRL 20596, a novel anilide with central dopamine antagonist activity.

BRL 20596 (N-(4-amino-5-chloro-2-methoxyphenyl)-1-phenylmethyl-4-piperidine-carbox amide) is a novel anilide related to clebopride (a gastric prokinetic benzamide) in which the sole change is reversal of the amide bond. Previous studies have shown conformational and electronic differences between these molecules which result in the anilide losing its gastric prokinetic activity, whilst retaining its central nervous system activity. Pharmacological and biochemical properties of BRL 20596 are compared here in animals with chlorpromazine, clebopride, haloperidol and sulpiride. BRL 20596 potently inhibited a number of behaviours, such as conditioned avoidance, amphetamine-induced stereotypy and turning, and apomorphine-induced climbing. Homovanillic acid (HVA) levels in the striatum and nucleus accumbens were raised at similar dose levels to those which inhibited these behaviours, whilst sedative activity was only exhibited at much higher dose levels. Haemodynamic changes were only observed with high IV doses of BRL 20596. Much lower doses of sulpiride were needed to raise prolactin levels than to raise HVA levels. This was not the case with BRL 20596 and the other drugs, where the doses needed for the two effects were similar. The results suggest that BRL 20596 is a central dopamine antagonist, with low sedative and haemodynamic activity.

Animals↗
PubMed1986

Effect of gamma rays at the dihydrofolate reductase locus: deletions and inversions.

A series 11 gamma-ray-induced mutants at the dihydrofolate reductase (dhfr) locus in Chinese hamster ovary cells has been examined for the types of DNA sequence change brought about by this form of ionizing radiation. All 11 mutants were found to have suffered major structural changes affecting the dhfr gene. In eight of the mutants, all or part of the dhfr gene has been deleted. The extent of these deletions was examined in seven of these mutants and, for comparison, in two deletion mutants that were induced by UV irradiation. For this purpose, probes from an overlapping set of cosmids that span 210 kb of DNA in this region were used. Three of seven gamma-ray-induced mutants and one UV-induced mutant were shown to have deleted the entire 210-kb region. In the remaining mutants, endpoints ranging from within the dhfr gene to 100 kb downstream were observed. No upstream endpoints were detected, so that an upper limit on the size of these large deletions could not be assigned. Three of the 11 gamma-ray-induced mutants contained an interruption in the dhfr gene without any detectable loss of sequence. Restriction analysis of these interrupted mutants showed that at least 8-14 kb of "foreign" DNA sequence became joined to the gene at the point of disruption. Cytogenetic analysis of these mutants showed that in two cases an inversion of the banding pattern on chromosome Z-2 had taken place. The inverted dhfr mutants contain very low amounts of dhfr RNA sequences, and the 5' end of an inversion mutant gene exhibits the same pattern of DNA methylation and DNase I-hypersensitivity as the wild-type gene. Our results suggest that ionizing radiation causes primarily, if not exclusively, large deletions and inversions in mammalian cells.

Animals↗
PubMed1986

Multiple transcription start sites, DNase I-hypersensitive sites, and an opposite-strand exon in the 5' region of the CHO dhfr gene.

Transcription of the 26-kilobase (kb) dihydrofolate reductase (dhfr) gene in CHO cells is initiated at two sites: a major site (approximately 85% of the dhfr mRNA) at -63 relative to the translation start and a minor site (approximately 15%) at -107. Transcription also occurs from the opposite DNA strand in the dhfr 5' region, with a probable initiation site at approximately -195 relative to the dhfr translation start. A 4-kb polyadenylated RNA that is derived from the opposite-strand transcription increases threefold in abundance after serum starvation of CHO cells for 24 h. dhfr mRNA levels do not change during this time. The first dhfr exon lies within a 1-kb genomic region marked by exceptionally high G + C content and lack of DNA methylation. This region also includes a 214-base-pair (bp) exon for the opposite-strand transcript and five of the six DNase I-hypersensitive sites identified at the dhfr locus. Analysis of the DNA sequences of hamster, human (M. Chen, T. Shimada, A. D. Moulton, A. Cline, R. K. Humphries, J. Maizel, and A. W. Nienhuis, J. Biol. Chem. 259:3933-3943, 1984), and mouse (M. McGrogan, C. C. Simonsen, D. T. Smouse, P. J. Farnham, and R. T. Schimke, J. Biol. Chem. 260:2307-2314, 1985) dhfr genes reveals the presence of a 29-bp unit that is conserved 45 to 49 bp upstream of major and minor dhfr transcription start sites. This unit follows the consensus: GRGGCGGTGGCCTNNNNTGTCRCAARTRGGTR. The 5' part of the 29-bp unit contains a GC box that agrees with the GGGCGG consensus-binding site for the RNA polymerase II transcription factor Sp1 (D. Gidoni, W. A. Dynan, and R. Tjian, Nature (London) 312:409-413, 1984). Each of the three mammalian dhfr genes has several G-rich GC boxes proximal to the major dhfr transcription start site and several GC boxes of the opposite orientation (C rich) in a distal region about 500 bp upstream.

Animals↗
PubMed1986

Spontaneous splicing mutations at the dihydrofolate reductase locus in Chinese hamster ovary cells.

We isolated and characterized three spontaneous mutants of Chinese hamster ovary cells that were deficient in dihydrofolate reductase activity. All three mutants contained no detectable enzyme activity and produced dihydrofolate reductase mRNA species that were shorter than those of the wild type by about 120 bases. Six exons are normally represented in this mRNA; exon 5 was missing in all three mutant mRNAs. Nuclease S1 analysis of the three mutants indicated that during the processing of the mutant RNA, exon 4 was spliced to exon 6. The three mutant genes were cloned, and the regions around exons 4 and 5 were sequenced. In one mutant, the GT dinucleotide at the 5' end of intron 5 had changed to CT. In a second mutant, the first base in exon 5 had changed from G to T. In a revertant of this mutant, this base was further mutated to A, a return to a purine. Approximately 25% of the mRNA molecules in the revertant were spliced correctly to produce an enzyme with one presumed amino acid change. In the third mutant, the AG at the 3' end of intron 4 had changed to AA. A mutation that partially reversed the mutant phenotype had changed the dinucleotide at the 5' end of intron 4 from GT to AT. The splicing pattern in this revertant was consistent with the use of cryptic donor and acceptor splice sites close to the original sites to produce an mRNA with three base changes and a protein with two amino acid changes. These mutations argue against a scanning model for the selection of splice site pairs and suggest that only a single splice site need be inactivated to bring about efficient exon skipping (a regulatory mechanism for some genes). The fact that all three mutants analyzed exhibited exon 5 splicing mutations indicates that these splice sites are hot spots for spontaneous mutation.

Animals↗