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J Beattie

Publications and source records attributed to J Beattie.

At least 19 recordsLinked to original sources
PubMed1995

Localisation of metallothionein isoform mRNAs in rat hepatoma (H4) cells.

The localisation of metallothionein isoform mRNAs in rat hepatoma (H4) cells was investigated using two approaches, namely Northern hybridisation of total RNA extracted from free, cytoskeletal-bound and membrane-bound polysomes isolated by a sequential detergent/salt extraction procedure and in situ hybridisation. The cytoskeletal-bound polysomes were enriched in metallothionein-I (MT-I) and c-myc mRNAs but showed a significantly lower enrichment in MT-II mRNA. These findings indicate that the MT-I mRNA is localised to the cytoskeleton during translation. In situ hybridisation using a biotin-labelled oligonucleotide probe revealed a predominantly perinuclear localisation for the MT-I mRNA.

Actins↗
PubMed1995

Glucocorticoids regulate the secretion of a 21kDa-IGF-binding protein by sheep adipose tissue explants.

Using a solution phase assay we have demonstrated that sheep adipose tissue explants secrete insulin-like growth factor binding proteins (IGFBPs) when cultured in serum-free medium over a 24 h period. Further, we demonstrate that secretion of IGFBP(s) is inhibited (up to 50%) by incubation of the cultures in the presence of 10(-8) M dexamethasone. This inhibitory effect is overcome when insulin (10 ng/ml) and ovine growth hormone (100 ng/ml) are incubated together (but not separately) with glucocorticoid. Further characterisation of this IGF binding activity by high performance size exclusion chromatography and Western ligand blot analysis indicated that under our culture conditions sheep adipose tissue explants secrete one predominant 21 kDa IGFBP and it is this BP which is hormonally regulated as described above. We discuss our results in the context of endocrine/paracrine/autocrine control of adipose tissue metabolism and differentiation.

Adipose Tissue↗
PubMed1995

Identification and characterization of the type-IVA cyclic AMP-specific phosphodiesterase RD1 as a membrane-bound protein expressed in cerebellum.

An antiserum was generated against a dodecapeptide whose sequence is found at the C-terminus of a cyclic AMP (cAMP)-specific, type-IVA phosphodiesterase encoded by the rat 'dunc-like' cyclic AMP phosphodiesterase (RD1) cDNA. This antiserum identified a single approximately 73 kDa protein species upon immunoblotting of cerebellum homogenates. This species co-migrated upon SDS/PAGE with a single immunoreactive species observed in COS cells transfected with the cDNA for RD1. Native RD1 in cerebellum was found to be predominantly (approximately 93%) membrane-associated and could be found in isolated synaptosome populations, in particular those enriched in post-synaptic densities. Fractionation of lysed synaptosomes on sucrose density gradients identified RD1 as co-migrating with the plasma membrane marker 5'-nucleotidase. Laser scanning confocal and digital deconvolution immunofluorescence studies done on intact COS cells transfected with RD1 cDNA showed RD1 to be predominantly localized to plasma membranes but also associated with the Golgi apparatus and intracellular vesicles. RD1-specific antisera immunoprecipitated phosphodiesterase activity from solubilized cerebellum membranes. This activity had the characteristics expected of the type-IV cAMP phosphodiesterase RD1 in that it was cAMP specific, exhibited a low Km cAMP of 2.3 microM, high sensitivity to inhibition by 4-[3-(cyclopentoxyl)-4-methoxyphenyl]-2-pyrrolidone (rolipram) (Ki approximately 0.7 microM) and was unaffected by Ca2+/calmodulin and low concentrations of cyclic GMP. The phosphodiesterase activities of RD1 solubilized from both cerebellum and transfected COS cell membranes showed identical first-order thermal denaturation kinetics at 50 degrees C. Native RD1 from cerebellum was shown to be an integral protein in that it was solubilized using the non-ionic detergent Triton X-100 but not by either re-homogenization or high NaCl concentrations. The observation that hydroxylamine was unable to cause the release of RD1 from either cerebellum or COS membranes and that [3H]palmitate was not incorporated into the RD1 protein immunoprecipitated from COS cells transfected with RD1 cDNA, indicated that RD1 was not anchored by N-terminal acylation. The engineered deletion of the 25 residues forming the unique N-terminal domain of RD1 caused both a profound increase in its activity (approximately 2-fold increase in Vmax) and a profound change in intracellular distribution. Thus, immunofluorescence studies identified the N-terminal truncated species as occurring exclusively ion the cytosol of transfected COS cells. The cDNA for RD1 thus appears to encode a native full-length type-IVA phosphodiesterase that is expressed in cerebellum.(ABSTRACT TRUNCATED AT 400 WORDS)

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1995

Production of insulin-like growth factor binding protein (IGFBP), IGFBP-3 protease, and expression of IGF-I receptors by cells of the sheep immune system.

Single-cell suspensions of sheep thymus cells were cultured in serum-free medium with or without polyclonal activators (phytohaemagglutinin or concanavalin A) and the resultant conditioned medium was assayed for insulin-like growth factor binding protein (IGFBP) activity by binding of [125I]IGF-I, using charcoal to separate free from bound. All cultures produced IGFBP but mitogen stimulation significantly increased IGFBP concentrations, indicating production by lymphoid cells. Conditioned medium also degraded recombinant human [125I]IGFBP-3, suggesting IGFBP-3 protease production within the thymus. This degradation was inhibited by several protease inhibitors (phenylmethylsulphonyl fluoride, aprotinin, N-alpha-p-tosyl-L-lysine chloromethyl ketone), suggesting the presence of a serine protease. Cell surface [125I]IGF-I binding was demonstrated on cells from thymus, mesenteric lymph node, peripheral blood mononuclear cells and platelets. The [125I]IGF-I binding to platelets could be inhibited by unlabelled peptides, with relative potencies IGF-I > IGF-II >> insulin. Scatchard analysis of IGF-I competitive binding revealed a Kd of 266 pmol/l and approximately 40 receptor sites per cell. The high-affinity binding of IGF-I and competition by insulin suggested that the [125I]IGF-I binding was to an IGF-I receptor rather than to a membrane-associated IGFBP, to which insulin does not bind. These data provide further support for the role of the IGF-IGFBP axis in the immune system, particularly in relation to the thymus.

Animals↗
PubMed1994

Identification of two splice variant forms of type-IVB cyclic AMP phosphodiesterase, DPD (rPDE-IVB1) and PDE-4 (rPDE-IVB2) in brain: selective localization in membrane and cytosolic compartments and differential expression in various brain regions.

In order to detect the two splice variant forms of type-IVB cyclic AMP phosphodiesterase (PDE) activity, DPD (type-IVB1) and PDE-4 (type-IVB2), anti-peptide antisera were generated. One set ('DPD/PDE-4-common'), generated against a peptide sequence found at the common C-terminus of these two PDEs, detected both PDEs. A second set was PDE-4 specific, being directed against a peptide sequence found within the unique N-terminal region of PDE-4. In brain, DPD was found exclusively in the cytosol and PDE-4 exclusively associated with membranes. Both brain DPD and PDE-4 activities, isolated by immunoprecipitation, were cyclic AMP-specific (KmcyclicAMP: approximately 5 microM for DPD; approximately 4 microM for PDE-4) and were inhibited by low rolipram concentrations (K1rolipram approximately 1 microM for both). Transient expression of DPD in COS-1 cells allowed identification of an approx. 64 kDa species which co-migrated on SDS/PAGE with the immunoreactive species identified in both brain cytosol and membrane fractions using the DPD/PDE-4-common antisera. The subunit size observed for PDE-4 (approx. 64 kDa) in brain membranes was similar to that predicted from the cDNA sequence, but that observed for DPD was approx. 4 kDa greater. Type-IV, rolipram-inhibited PDE activity was found in all brain regions except the pituitary, where it formed between 30 and 70% of the PDE activity in membrane and cytosolic fractions when assayed with 1 microM cyclic AMP, PDE-4 formed 40-50% of the membrane type-IV activity in all brain regions save the midbrain (approx. 20%). DPD distribution was highly restricted to certain regions, providing approx. 35% of the type-IV cytosolic activity in hippocampus and 13-21% in cortex, hypothalamus and striatum with no presence in brain stem, cerebellum, midbrain and pituitary. The combined type-IVB PDE activities of DPD and PDE-4 contributed approx. 10% of the total PDE activity in most brain regions except for the pituitary (zero) and the mid-brain (approx. 3%. The isolated cDNAs for DPD and PDE-4 appear to reflect transcription products which are expressed in vivo in brain. The unique N-terminal domain of PDE-4 is suggested to target this PDE to membranes in brain. Type-IVB PDEs are differentially expressed in various brain regions, indicating that there are tissue-specific controls on both the expression of the gene and the splicing of its products.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1994

Location of an epitope defined by an enhancing monoclonal antibody to growth hormone: some structural details and biological implications.

We have previously shown that a murine monoclonal antibody (MAb OA15) prepared against ovine GH (oGH) can enhance the somatogenic activity of bovine GH (bGH), as determined by increased incorporation of 35SO4(2-) into costal cartilage of hypopituitary Snell dwarf mice in vivo We have now established that MAb OA15 can also enhance the biological activity of oGH and porcine GH (pGH) in vivo. Using multiple pin peptide synthesis techniques a set of overlapping immobilized octamers representing the entire bGH sequence were synthesized and tested for their ability to bind MAb OA15 using an enzyme-linked immunosorbent assay. The pattern of binding showed that OA15 defined a functionally continuous epitope comprising residues 91-102. This region includes the C-terminal end of helix 2 plus a portion of the adjacent loop linking helices 2 and 3. Polyclonal antisera to a synthetic peptide representing this epitope mimicked the ability of MAb OA15 to enhance oGH, bGH, and pGH. Window size analysis showed that the heptapeptide 94-100 (SRVFTNS) represents the minimum unit to retain full recognition of MAb OA15. The fact that pGH, bGH, and oGH have identical sequences in this region also explains the ability of OA15 to both cross-react with and enhance the biological activity of each of these GHs. Replacement net analysis (where each residue in the heptamer is substituted with each of the 19 naturally occurring L-amino acids) demonstrated that residues R95 and T98 are critical for antibody binding and also indicated that the substitution of V96 with I, as found in rat GH, would permit the observed binding of OA15 to this hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1994

Several insulin-like growth factor-I analogues and complexes of insulin-like growth factors-I and -II with insulin-like growth factor-binding protein-3 fail to mimic the effect of growth hormone upon lactation in the rat.

Lactation was suppressed in rats using a combined treatment of bromocriptine (to reduce prolactin concentrations) and a specific antiserum to rat GH administered twice daily for 2 days. When milk production had ceased, as determined by litter weight loss and the absence of milk in the stomachs of pups, attempts were made to reinitiate lactation using prolactin, GH, insulin-like growth factor-I (IGF-I) precomplexed to recombinant human IGF-binding protein-3 (hIGFBP-3) or IGF-I plus IGF-II precomplexed to hIGFBP-3. Despite the fact that all treatments except prolactin led to increases in serum IGFs and IGFBP-3, only prolactin and GH provoked the reinitiation of milk production as determined by increased litter weight gain, milk in the stomach of pups and a significant increase in the weight of the mammary glands. Since the mammary gland has been shown to produce IGFBPs which may inhibit IGF action we also tested three IGF-I analogues, R3-IGF-I, Long-IGF-I and Long-R3-IGF-I. R3-IGF-I has a single amino acid substitution (Glu to Arg) at position 3 whereas Long-IGF-I has a 13 amino acid N-terminal extension. These modifications dramatically reduce the ability of these analogues to bind to IGFBPs although they remain active at the IGF-I receptor. Such IGF analogues would therefore be expected to be active irrespective of the production of inhibitory IGFBPs. However, none was effective in reinitiating lactation, even at doses which have been shown to be biologically effective in terms of nitrogen retention.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

The use of multiple-pin peptide synthesis in an analysis of the continuous epitopes recognised by various anti-(recombinant bovine growth hormone) sera. Comparison with predicted regions of immunogenicity and location within the three-dimensional structure of the molecule.

A recently developed technology called epitope scanning permits the rapid and accurate delineation of continuous stretches of amino acids in a protein which constitute the sequential epitopes recognised by an antiserum raised to that protein. In the present report, we describe the use of this technique to identify the epitopes in the recombinant bovine growth-hormone (rbGH) molecule recognised by three polyclonal guinea-pig antisera and two polyclonal rabbit antisera. The results obtained show that, for guinea-pig antisera, 3 or 4, very-well-defined major continuous epitopes are present. As would be expected given the intrinsic genetic factors (major histocompatibility restriction, antigen processing and presentation) controlling the immune response in individual animals, subtle differences are evident in the precise location and relative reactivities of these epitopes in different guinea-pig antisera. Nevertheless, there is a large degree of overlap in these epitopes, such that immunodominant regions of the antigen can be clearly delineated. In a structural sense, these epitopes share a common motif in that they are sited in areas of the protein antigen with little secondary structure (loop/coil), although there is some contribution by neighbouring alpha-helices. For the two rabbit antisera, the response tends to be rather more heterogeneous, with recognition of more peptides and less clearly defined epitopes than was the case with the guinea-pig antiserum. Comparison of the four guinea-pig epitopes, identified by our experimental methods with computer predictions for this molecule (Jameson-Wolf antigenic index), indicate that two are strongly predicted, one is weakly predicted and one is not predicted. These observations, together with the displayed intraspecies and interspecies variation clearly indicate the limitations of these predictive methods. In conclusion, we have demonstrated that, despite the expected variation in the exact location of continuous epitopes defined by different anti-rbGH sera, there are large regions of overlap defining immunogenic core regions within the molecule. We believe that studies of this nature, together with further understanding of antigen processing and peptide presentation to immune cells, may have a role to play in the development of candidate peptide vaccines.

Amino Acid Sequence↗
PubMed1992

Critical evaluation of a theory of molecular recognition using human insulin-like-growth-factor-I fragment 21-40 and its complementary peptide.

Using solid-phase methods we have synthesized human insulin-like-growth-factor-I (IGF-I) fragment 21-40 (IGF-I 21-40) and the peptide derived from the 5'----3' translation of the complementary nucleic acid of this peptide, 'I-FGI 20-40' (the complementary peptide). According to a recently proposed theory of molecular recognition, these two peptides should bind specifically to each other. We have tested this theory by using both solid- and solution-phase direct-binding assays for this complementary-peptide pair. We have also investigated the ability of I-FGI 20-40 to interfere with native IGF-I binding during radioimmunoassay (r.i.a.), radio-receptor (r.r.a.) assay and ligand-blot analysis of IGF-binding proteins. We have obtained no evidence of any interaction between IGF-I 21-40 and I-FGI 20-40 in either solid- or solution-phase assays. In addition, I-FGI 20-40 does not interfere in the assays used to detect IGF-I binding antibodies (r.i.a.), receptors (r.r.a.) or binding proteins (ligand blots). Our data therefore question the universality of this particular theory of molecular recognition.

Amino Acid Sequence↗
PubMed1992

Investigation of the solid- and solution-phase binding reactivities of continuous epitopes recognized by polyclonal guinea-pig anti-recombinant bovine growth hormone antisera.

We have used the technique of multiple pin peptide synthesis to identify three major continuous epitopes in the recombinant bovine (rb) GH molecule. We have synthesized these peptides, residues 24-40, 139-152 and 179-189, as N-terminally acetylated, C-terminal amides and confirmed their reactivity in a standard solid-phase ELISA. Subsequently, for epitope 139-152, we have synthesized a peptide affinity column and used this to isolate antibodies with this epitope specificity from whole antiserum. In addition, we demonstrate that under native conditions in a liquid phase RIA, these antibodies will precipitate [125I]rbGH. Further, peptide 139-152 itself also cross-reacts in an rbGH RIA inhibiting binding by up to 20%. Our data suggest that during the immune response to rbGH in guinea-pigs a substantial part of the B-cell response is directed to the 139-152 region and that this part of the protein is a native epitope.

Amino Acid Sequence↗
PubMed1992

Investigation of the mechanism of action of growth hormone in stimulating lactation in the rat.

The role of GH was examined using an antiserum to rat GH (anti-rGH). When administered to lactating rats on day 2 of lactation it was without effect, whereas bromocriptine markedly suppressed milk production, with no additional effect of combined treatment. On day 6 of lactation, treatment with anti-rGH was also without effect, whilst bromocriptine again suppressed milk production. Combined treatment, however, suppressed milk synthesis completely, suggesting that GH was capable of maintaining about 50% of normal milk yield in the absence of prolactin at day 6 of lactation. By day 14 of lactation, anti-rGH treatment alone was capable of decreasing milk yield by about 20%, and again milk secretion only stopped completely when GH and prolactin were suppressed. These data suggest that the role of GH in supporting lactation increases as lactation progresses. The effects of GH in stimulating growth and in increasing milk yield in ruminants have been proposed to be mediated via insulin-like growth factor-I (IGF-I). In rats treated with anti-rGH, both IGF-I and IGF-II were decreased in serum. The concentration of the major IGF-binding protein (IGFBP-3) was not, however, affected by inhibition of GH or prolactin individually, but was decreased in animals treated with bromocriptine and anti-rGH. In animals given both bromocriptine and anti-rGH, concurrent treatment with recombinant bovine GH maintained milk yield at 50% of control values and normalized serum IGF-I, IGF-II and IGFBP-3 concentrations. By contrast, concurrent treatment with IGF-I or IGF-II, despite normalizing their respective concentrations in serum, failed to affect milk yield.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1991

Investigating the effect of stimulus range on attribute weight.

Classic economic theories of choice assume that trade-offs are made consistently and are not swayed by irrelevant contextual variables. One possible source of trade-off inconsistency is investigated by asking whether attribute weights are affected by variations in the range of the stimuli presented. Ss should be influenced by the range of the stimuli only when distributional characteristics provide information about the value of the stimuli. In six experiments, Ss made judgments when the range (a) did not provide information about the value of the scores on the scales, (b) might provide such information, and (c) certainly provided this information. In Experiments 1-5, Ss were not influenced by the range of the stimuli. The only exception was Experiment 6, which required Ss to judge hypothetical job candidates on the basis of stimulus scales with which the Ss had no previous experience. In this experiment the meaning of the scales was determined entirely by the location of each stimulus within the presented distribution. In general, the results support the classical view of decision making: that attribute weights exist independent of situational demands, and that Ss' judgments are not influenced by irrelevant stimuli.

Adult↗
PubMed1988

A radiochemical microassay for aspartate aminotransferase activity in the nervous system.

A radiochemical procedure for measuring aspartate aminotransferase activity in the nervous system is described. The method is based on the exchange of tritium atoms at positions 2 and 3 of L-2,3-[3H]aspartate with water when this amino acid is transaminated in the presence of alpha-ketoglutarate to form oxaloacetate. The tritiated water is separated from the radiolabeled aspartate by passing the reaction mixture over a cation exchange column. Confirmation that the radioactivity in the product is associated with water was obtained by separating it by anion exchange HPLC and by evaporation. The product formation is linear with time up to 120 min and with tissue in the 0.05- to 10-micrograms range. The apparent Km for aspartate in the rat brain homogenate is found to be 0.83 mM and that for alpha-ketoglutarate to be 0.12 mM. Methods that further improve the sensitivity of the assay are also discussed.

Animals↗