Biomedical subjects

A J Clark

Publications and source records attributed to A J Clark.

At least 199 records · Page 11Linked to original sources

PubMed1992

Aspiration lesions of rat ventral hippocampus disinhibit responding in conditioned suppression or extinction, but spare latent inhibition and the partial reinforcement extinction effect.

Latent inhibition refers to a decrement in learning about a stimulus as a result of its prior non-reinforced presentation. There is evidence that lesions of nucleus accumbens and conventional hippocampal lesions both disrupt the development of latent inhibition. The partial reinforcement extinction effect reflects the observation that resistance to extinction is normally greater in animals that have been rewarded on a 50% random proportion of acquisition trials than in those rewarded on every trial. Conventional hippocampal lesions, excitotoxic lesions of hippocampus plus subiculum, or conventional lesions of nucleus accumbens abolish this effect. The present experiments examined the possibility that a projection originating in the ventral [temporal in the nomenclature proposed by Blackstad: (1956) J. comp. Neurol. 105, 417-537] subiculum and terminating in nucleus accumbens underlies the normal development of latent inhibition and the partial reinforcement extinction effect, by evaluating the performance on these two behaviours of rats with aspiration lesions in the ventral hippocampal region. There was equally clear evidence of latent inhibition and of a partial reinforcement extinction effect in controls and in rats with ventral hippocampal damage. However, superimposed on this, the hippocampal lesion induced a loss of behavioural inhibition in both paradigms. Subsequent anatomical analyses indicated that cell bodies in nearby retrohippocampal cortex had maintained intact projections to nucleus accumbens. We suggest that these extra-hippocampal projections may underlie the ability to learn to ignore irrelevant stimuli.

Analysis of Variance↗

PubMed1992

Rescuing transgene expression by co-integration.

To test whether foreign gene expression can be improved in transgenic mice by manipulating the site of integration, we co-integrated the efficiently expressed sheep beta-lactoglobulin gene with two poorly expressed beta-lactoglobulin-derived hybrid genes encoding human proteins. In each case, we observed a significant improvement in the frequency and level of expression of the hybrid gene. "Rescuing" transgene expression by co-integration may provide a general solution for improving the efficiency of heterologous gene expression in transgenic animals.

Animals↗

PubMed1992

Evidence for ATP binding and double-stranded DNA binding by Escherichia coli RecF protein.

RecF protein is one of the important proteins involved in DNA recombination and repair. RecF protein has been shown to bind single-stranded DNA (ssDNA) in the absence of ATP (T. J. Griffin IV and R. D. Kolodner, J. Bacteriol. 172:6291-6299, 1990; M. V. V. S. Madiraju and A. J. Clark, Nucleic Acids Res. 19:6295-6300, 1991). In the present study, using 8-azido-ATP, a photo-affinity analog of ATP, we show that RecF protein binds ATP and that the binding is specific in the presence of DNA. 8-Azido-ATP photo-cross-linking is stimulated in the presence of DNA (both ssDNA and double-stranded DNA [dsDNA]), suggesting that DNA enhances the affinity of RecF protein for ATP. These data suggest that RecF protein possesses independent ATP- and DNA-binding sites. Further, we find that stable RecF protein-dsDNA complexes are obtained in the presence of ATP or ATP-gamma-S [adenosine-5'-O-(3-thio-triphosphate)]. No other nucleoside triphosphates served as necessary cofactors for dsDNA binding, indicating that RecF is an ATP-dependent dsDNA-binding protein. Since a mutation in a putative phosphate-binding motif of RecF protein results in a recF mutant phenotype (S. J. Sandler, B. Chackerian, J. T. Li, and A. J. Clark, Nucleic Acids Res. 20:839-845, 1992), we suggest on the basis of our data that the interactions of RecF protein with ATP, with dsDNA, or with both are physiologically important for understanding RecF protein function in vivo.

Adenosine Triphosphate↗

PubMed1992

Bromocriptine inhibits pro-opiomelanocortin mRNA and ACTH precursor secretion in small cell lung cancer cell lines.

We have previously reported that a human small cell lung cancer (SCLC) cell line (COR L103) that expresses the proopiomelanocortin (POMC) gene and secretes ACTH precursor peptides is relatively resistant to glucocorticoid regulation. Using this model, we have now examined alternative regulatory mechanisms of the POMC gene and found that both the mRNA and ACTH precursor peptides were stimulated four- and two-fold, respectively, after 48 h incubation with db-cAMP. Next, we examined the dopamine agonist, bromocriptine, which acts predominantly through D2 receptors linked to adenyl cyclase to cause a reduction in intracellular cAMP. Bromocriptine suppressed cAMP levels and inhibited precursor peptide secretion within 24 h in a dose-dependent manner (0.15-15 microM). At the highest dose, peptide secretion was inhibited from 95 to 53 pmol/mg protein, and POMC mRNA was reduced by 50%, while beta-actin mRNA remained unchanged. This effect could not be mimicked by incubation of cells with the alpha-adrenergic antagonist, phenoxybenzamine, suggesting that the alpha-adrenergic effects of bromocriptine were not responsible for this observation. These cells also secrete estradiol, but the secretory rate was unaffected by bromocriptine, suggesting, with the beta-actin data, that the POMC inhibition was not a cytotoxic effect. No recovery in precursor peptide secretion was seen in a 48-h period after the removal of bromocriptine. However, when the postchallenge incubation was extended to 8 d, there was a recovery in secretory potential between day 3 and day 8 and normal growth kinetics in the 4 d after removal of the drug. In contrast to these findings, the mouse corticotroph cell line, AtT20, showed no response to bromocriptine, in keeping with reports that this agonist has no effect on anterior lobe corticotrophs. We conclude that bromocriptine effectively inhibits POMC expression in SCLC cells, and that this phenomenon might be of useful clinical application.

Adrenocorticotropic Hormone↗

PubMed1992

Stimulation of early gene expression by angiotensin II in bovine adrenal glomerulosa cells: roles of calcium and protein kinase C.

The adrenal glomerulosa cell is a major site of action of angiotensin II (AII), which binds to AT1 receptors to stimulate phosphoinositide hydrolysis and Ca2+ mobilization, and the subsequent production of aldosterone. All also influences adrenal growth and proliferation and promotes thymidine incorporation in adrenocortical cells. In primary cultures of bovine glomerulosa cells, AII was found to induce the expression of several early growth response genes (c-fos, c-jun, JunB, and Krox 24). This effect of AII was dose-dependent and was blocked by [Sar1,IIe8] AII and the nonpeptide antagonist DuP 753, indicating that it is mediated by the AT1 subtype of the AII receptor. ACTH, which elevates cAMP in glomerulosa cells, was a relatively weak inducer of c-fos expression but was as potent as AII in stimulating the expression of JunB. ACTH did not further enhance the maximal effect of AII on c-fos expression. The role of the AII-induced cytoplasmic Ca2+ increase in generating the c-fos response was suggested by the ability of the Ca2+ ionophore ionomycin to induce c-fos expression. However, mobilization of intracellular Ca2+ by the Ca2+ ATPase inhibitor thapsigargin, as well as the stimulation of Ca2+ influx by depolarization with potassium, were less potent stimuli of c-fos expression. Omission of Ca2+ from the extracellular medium, which abolishes the plateau phase of the AII-induced Ca2+ signal without affecting the early increase due to Ca2+ mobilization, enhanced the early phase of the AII-induced c-fos response, indicating that Ca2+ also has an inhibitory effect on the early gene response. Activation of protein kinase C by phorbol 12-myristate, 13-acetate (PMA) also stimulated c-fos expression, but the combination of PMA and ionomycin did not further increase the c-fos response. Inhibition of protein kinase C by staurosporine, or its depletion by prolonged exposure to PMA, prevented the c-fos response to PMA but only partially inhibited the response to AII, suggesting the involvement of other factors in stimulus-transcription coupling from the AT1 receptor.

1-Sarcosine-8-Isoleucine Angiotensin II↗

PubMed1991

Interaction of DNA-binding proteins with a milk protein gene promoter in vitro: identification of a mammary gland-specific factor.

The minimal 5' regulatory region of the sheep beta-lactoglobulin gene (BLG), as defined in transgenic mice, was used to identify nuclear factors which may be involved in milk protein gene expression in the lactating mammary gland. This 406bp promoter region was dissected into short, overlapping, double-stranded oligonucleotides to facilitate identification of the bound proteins. A variety of sites, for both known and previously undescribed DNA-binding proteins, are occupied in vitro. Some of these factors were investigated in detail. Two forms of nuclear factor I (NFI), which have different recognition site affinities, are present in nuclear extracts from lactating mammary gland and bind to at least 5 sites in this BLG control element. In addition, a factor (milk protein binding factor, MPBF) which is specific to extracts from both mouse and sheep lactating mammary gland binds to 3 BLG promoter sites and may be a milk protein gene transcription factor.

Animals↗

PubMed1991

The mas oncogene enhances angiotensin-induced [Ca2+]i responses in cells with pre-existing angiotensin II receptors.

The proposal that the mas oncogene is an angiotensin receptor was evaluated in Xenopus oocytes injected with human and rat mas RNA transcripts, and during transient expression of mas in several cell lines. No evidence of mas-induced angiotensin II (AII) receptors or [Ca2+]i responses was observed in Xenopus oocytes or in most of the transfected cells. However, Cos-1 cells, which showed a small endogenous [Ca2+]i response to AII, exhibited a modest but reproducible enhancement of this response after mas transfection. Such responses were inhibited by [Sar1, Ala8]AII and [Sar1, Ile8]AII, but not by [D-Arg1, D-Pro2, D-Trp7,9, Leu11] substance P, an antagonist reported to inhibit mas-induced responses to AII in oocytes. These findings are not compatible with the proposal that the mas oncogene is an angiotensin receptor, but suggest that expression of mas leads to increased responsiveness of the endogenous AII signaling system.

1-Sarcosine-8-Isoleucine Angiotensin II↗

PubMed1991

Effect of RecF protein on reactions catalyzed by RecA protein.

RecF protein is one of at least three single strand DNA (ssDNA) binding proteins which act in recombination and repair in Escherichia coli. In this paper we show that our RecF protein preparation complexes with ssDNA so as to retard its electrophoretic movement in an agarose gel. The apparent stoichiometry of RecF-ssDNA-binding measured in this way is one RecF molecule for every 15 nucleotides and the binding appears to be cooperative. Interaction of the other two ssDNA-binding proteins, RecA and Ssb proteins, has been studied extensively; so in this paper we begin the study of the interaction of RecF and RecA proteins. We found that the RecF protein preparation inhibits the activity of RecA protein in the formation of joint molecules whether added before or after addition of RecA protein to ssDNA. It, therefore, differs from Ssb protein which stimulates joint molecule formation when added to ssDNA after RecA protein. We found that our RecF protein preparation inhibits two steps prior to joint molecule formation: RecA protein binding to ssDNA and coaggregate formation between ssDNA-RecA complexes and dsDNA. We found that it required a much higher ratio of RecF to RecA protein than normally occurs in vivo to inhibit joint molecule formation. The insight that these data give to the normal functioning of RecF protein is discussed.

Adenosine Triphosphatases↗

PubMed1991

Production of pharmaceutical proteins in milk.

There is every reason to expect that it will be possible within the next few years to begin to use farm animals to produce large quantities of some of the human proteins that are needed for the treatment of disease. Revolutionary new opportunities for the production of novel proteins in milk have been created by the development of methods for gene transfer. Exploitation of these opportunities depends upon selection and cloning of milk protein genes and identification of the sequences that govern tissue specific hormonally induced expression in the mammary gland. Studies with three genes, ovine beta-lactoglobulin, rat beta-casein and whey acidic protein of rat and mouse, suggest that they may all meet this requirement. Fragments of the ovine beta-lactoglobulin, murine whey acidic protein and rabbit beta-casein genes have directed production of novel proteins in the milk of transgenic mice, sheep, rabbits and pigs. The proteins were biologically active and usually co-migrated with authentic proteins. In early experiments, protein concentration was low, but our recent observations suggest that fusion genes containing genomic clones direct production of concentrations of protein that are suitable for commercial exploitation. In the longer term, two approaches may offer the potential of more reliable expression. Control elements capable of directing expression that is independent of site of insertion of the gene, but dependent on the number of copies of the gene, have been identified for a small number of genes. The availability of such elements for the milk protein genes would increase the reliability of gene expression considerably. Alternatively, targeted mutation of genes may allow the insertion of coding sequences within an existing gene so avoiding position effects.

Animals↗

PubMed1991

An embryo-specific protein of barley (Hordeum vulgare).

An immunological approach has been used to identify embryo-specific products that can be used as molecular markers of embryogenesis. Immunoadsorption of antisera to remove antigens common to embryos, meristematic cells and callus, revealed one major embryo-specific antigen, a polypeptide of 17 kDa. The antigen appeared at mid-stages of zygotic embryo formation and remained at similar levels up to six days post-germination of the seedling. The polypeptide could not be detected by protein staining, suggesting it is a non-abundant product. Appearance of the antigen could be induced by culture of zygotic embryos in vitro on abscisic acid (1 microM) or mannitol (9% mass/vol.). Cross-reactive products of near-identical molecular mass were observed in embryos of wheat, rye and oats but not distantly related cereals, nor embryos from dicotyledonous species. The timing of the appearance of the antigen was different in embryos formed from microspores during anther culture in vitro. In the cultured material, the 17-kDa polypeptide preceded the appearance of morphologically distinct embryonic structure.

Abscisic Acid↗

PubMed1991

Developmental regulation of the sheep beta-lactoglobulin gene in the mammary gland of transgenic mice.

beta-Lactoglobulin (BLG) is the most abundant whey protein in sheep milk but it is not present in mouse milk. We have previously shown that transgenic mice carrying the BLG gene express it specifically in the mammary gland and secrete BLG into milk at high concentrations. Here we demonstrate that BLG transcription is correctly initiated in mice and that BLG synthesis is restricted to the secretory epithelial cells of the mammary gland. We have also determined the temporal pattern of milk protein gene expression and find that the BLG transgene is regulated coordinately with mouse beta-casein and that the patterns of regulation of BLG in mouse and sheep share some similarities.

Animals↗

PubMed1991

Electrolytic lesions of the nucleus accumbens in rats which abolish the PREE enhance the locomotor response to amphetamine.

The partial reinforcement extinction effect (PREE) refers to the increased resistance to extinction observed in animals trained on a partial reinforcement (PR) schedule compared with those trained on a schedule of continuous reinforcement (CR). It has been suggested that the PREE is dependent upon the integrity of the septo-hippocampal system, but recent evidence has indicated that the role originally proposed for the lateral septal nucleus may in fact be subserved by the nucleus accumbens. Experiment I therefore tested the effects of electrolytic lesions of the nucleus accumbens on the PREE. These lesions abolished the PREE, the abolition resulting from a decreased rate of extinction in the lesion CR rats coupled with an increased rate of extinction in the PR rats. These results clearly implicate the nucleus accumbens in the development of the PREE, and suggest that theoretical models of the PREE based simply upon consideration of septohippocampal interactions need radical revision. The lesion also enhanced running speeds in acquisition in both the CR and the PR groups. Experiment II therefore assessed spontaneous locomotor activity and the locomotor response to amphetamine challenge at two doses. The lesion produced no increase in spontaneous locomotion; an enhanced increase in response to 1 mg/kg amphetamine; and no changes in the stereotyped behaviours induced by 10 mg/kg amphetamine.

Amphetamine↗

PubMed1991

Evidence for functional separation of alpha-1 and alpha-2 noradrenaline receptors by pre-synaptic terminal re-uptake mechanisms.

Information transfer within the central nervous system is predominantly chemical in nature, and occurs both through synaptic specialisations and non-specific diffuse release. The localisation and description of receptors for these two types of neurotransmission is currently a contentious issue. In the present study, the noradrenaline reuptake inhibitor cocaine has been shown to overcome the inhibitory effects of idazoxan, a selective alpha-2 receptor antagonist, but not phentolamine, a non-selective alpha receptor antagonist, on eating following injection of noradrenaline into the rat hypothalamic paraventricular nucleus. Similarly, lesion by 6-hydroxydopamine of noradrenaline terminals in the paraventricular nucleus also reduced the efficacy of idazoxan in blocking eating induced by noradrenaline. These data confirm that postsynaptic alpha-2 receptors are involved in the feeding response to exogenous noradrenaline, but in addition, when taken in conjunction with previously published data, are used to suggest a differential distribution of NA receptors within the hypothalamic paraventricular nucleus. It is proposed that the alpha-2 subtype may be extrasynaptic, the alpha-1 subtype intrasynaptic.

Adrenergic alpha-Antagonists↗

PubMed1991

Targeting expression to the mammary gland: intronic sequences can enhance the efficiency of gene expression in transgenic mice.

We are studying the tissue-specific expression of the sheep milk-whey protein gene, beta-lactoglobulin. We have used sequences derived from this gene to target the expression of biomedical proteins into milk with the intention to exploit this technology in transgenic sheep as a means of protein production. In the present study, a series of beta-lactoglobulin hybrid genes and beta-lactoglobulin minigenes were evaluated for expression in the mammary gland of transgenic mice. In particular, we have assessed whether there is a requirement for introns for efficient transgene expression in the mammary gland, since the coding sequences of many candidate proteins are available only as cDNAs. The results suggest that the inclusion of natural introns in constructs can enhance the efficiency of transgene expression. Thus, a hybrid construct comprising 4.3 kb of the immediate 5' flanking sequences of beta-lactoglobulin fused to a genomic minigene encoding human alpha-antitrypsin (alpha 1AT) was expressed much more efficiently than an alpha 1AT-cDNA construct containing the same beta-lactoglobulin segment. Similarly, the intact beta-lactoglobulin gene was expressed more efficiently than the corresponding intronless beta-lactoglobulin minigene. This effect was not seen in transient expression experiments in baby hamster kidney cells when beta-lactoglobulin-alpha 1AT constructs were driven by SV40 enhancer sequences. The effect cannot be explained by a simple requirement for splicing, since the inclusion of the first beta-lactoglobulin intron into cDNA constructs encoding human alpha 1AT or beta-lactoglobulin itself failed to enhance the efficiency of transgene expression. It is concluded that sequence elements within introns may interact with the upstream 5' flanking sequences of beta-lactoglobulin and enable the latter to function efficiently in the mammary gland of transgenic mice.

Animals↗

PubMed1991

rec genes and homologous recombination proteins in Escherichia coli.

The twenty-five years since the first published report of recA mutants in Escherichia coli has seen the identification of more than 12 other recombination genes. The genes are usually grouped into three pathways named RecBCD, RecE and RecF for prominent genes which function in each. A proposal is made here that there are two RecF pathways, one sensitive and one resistant to exonuclease I, the SbcB enzyme. Five methods of grouping the genes functionally are discussed: 1) by enzyme activity, 2) by common indirect suppressor, 3) by common phenotype, 4) by common regulation and 5) by epistasis. Five classes of enzyme activities implicated in recombination are discussed according to their involvement in presynapsis, synapsis or postsynapsis: 1) nucleases 2) helicases 3) DNA-binding proteins 4) topoisomerases and 5) ligases. Plausible presynaptic steps for the RecBCD, RecF (SbcBS) and RecE pathways show the common feature of generating 3'-terminated single-stranded DNA (ssDNA). On this ssDNA it is proposed that a RecA protein filament is generated discontinuously. This implies the existence of nucleation and possibly measurement and 3' end protection proteins. Specific proposals are made for which recombination genes might encode such products. Finally the generality of the RecA-ssDNA-filament mechanism of synapsis in the cellular biological world is discussed.

Escherichia coli↗

PubMed1991

Neuroendocrine and behavioural responses to hyperosmolality in rats with lesions of the lateral hypothalamus made by N-methyl-D-aspartate.

Excitotoxic lesions of rat lateral hypothalamus produce impairments in eating and drinking, but not motor deficits. However, it has not been established what causes these eating and drinking impairments. In the present experiments, drinking, plasma osmolality and arginine-vasopressin concentration were measured in lateral hypothalamic-lesioned and control rats following systemic injection of hypertonic saline. In response to hyperosmolality, N-methyl-D-aspartate-lesioned rats drank significantly less than controls but showed normal increases in plasma osmolality and arginine-vasopressin concentration. This dissociation of neuroendocrine and behavioural responses suggests that the impairment of rats with excitotoxic lesions of the lateral hypothalamus is unrelated to physiological (as opposed to behavioural) mechanisms of homeostasis.

Animals↗

PubMed1991

The regulation of feeding and drinking in rats with lesions of the lateral hypothalamus made by N-methyl-D-aspartate.

Rats bearing excitotoxic lesions of the lateral hypothalamus are hypodipsic and hypophagic, but responses to 24 h food or water deprivation are normal, as are responses to different taste stimuli. The most striking deficit present in lateral hypothalamic-lesioned rats is an inability to respond as controls to dehydrating, dipsogenic or glucoprivic challenges. The present experiments examined the ability of rats bearing bilateral N-methyl-D-aspartate-induced lesions of the lateral hypothalamus to recognize and respond to changes in their internal environments. All of the lesioned rats tested showed mild to moderate hypophagia and hypodipsia, and none responded properly by drinking over 1 h after i.p. injection of hypertonic saline. However, the addition of glucose to the water supply promoted an increase in drinking and a decrease in lab chow consumption to maintain a constant energy intake; the addition of salt to the diet promoted an increase in drinking and no change in eating; 24 h water deprivation induced the same amount of drinking in lateral hypothalamic-lesioned rats as in controls; and injection i.p. of water (but not physiological saline) before drinking water was returned to rats which were 24 h water deprived suppressed drinking. These data suggest that lateral hypothalamic-lesioned rats are in receipt of normal information from their peripheries, and that they can adjust their behaviour over a period of days or minutes to changes in the internal milieu. The most consistent deficit is in responding actively and rapidly to challenging stimuli; the nature of this and the mechanisms which might produce it are discussed. We suggest that the consequences of excitotoxic lesion are better explained by disruption of input to the cortex from the lateral hypothalamus rather than by interference with metabolic processes.

Adaptation, Physiological↗

PubMed1991

A comparison of excitotoxic lesions of the basal forebrain by kainate, quinolinate, ibotenate, N-methyl-D-aspartate or quisqualate, and the effects on toxicity of 2-amino-5-phosphonovaleric acid and kynurenic acid in the rat.

1. It has been suggested that an NMDA1 receptor subtype might be activated by N-methyl-D-aspartate (NMDA) and ibotenate and an NMDA2 subtype by NMDA or quinolinate, and that the NMDA2 site might be more susceptible to blockade by kynurenic acid. 2. Experiments were carried out to examine the ability of 2-amino-5-phosphonovaleric acid (AP5) and kynurenic acid to antagonize the neurotoxic properties of kainate, ibotenate, NMDA, quinolinate and quisqualate injected into the rat basal forebrain. 3. Following histological analysis of the injection sites, lesion volume was assessed parametrically. Each of the toxins except quisqualate was found to make lesions of parvocellular neurones within the basal forebrain with a relative order of potency: kainate much greater than quinolinate greater than ibotenate = NMDA. 4. Equimolar doses of AP5 abolished the toxicity produced by quinolinate and NMDA; toxicity to kainate and ibotenate was attenuated to approximately 40% of the toxin-alone condition. 5. The antagonistic properties of kynurenate were dose-dependent: equimolar kynurenate had no effect on quinolinate but attenuated the actions of ibotenate, kainate and NMDA; 2 x equimolar kynurenate had no effect on quinolinate or ibotenate but attenuated the toxicity of kainate and NMDA; and 3 x equimolar kynurenate had no effect on the toxicity of kainate or ibotenate, attenuated the actions of NMDA and abolished the toxic action of quinolinate. 6. The results are discussed in terms of the actions of the various toxins at different receptors, differentially sensitive to AP5 and kynurenate.

2-Amino-5-phosphonovalerate↗