PubMed Health⌕ Search

Biomedical subjects

D J Flint

Publications and source records attributed to D J Flint.

At least 37 records · Page 2Linked to original sources
PubMed1999

Identification of novel sites in the ovine growth hormone receptor involved in binding hormone and conferring species specificity.

Using site-directed mutagenesis we mutated the extracellular domain of the ovine growth hormone receptor (oGHR) to the corresponding amino acids in the rat GHR at two different sites: site A is between Thr28 and Leu34 and represents a major immunogenic epitope, while site B is between Ser121 and Asp124 and is involved in the interaction of the human GHR with growth hormone (GH). Native and mutant receptors were bacterially expressed and refolded, and then RIA and GH-binding assays were carried out on the purified recombinant proteins. Mutations at the N-terminal site A of oGHR led to greatly reduced binding to bovine GH and, in addition, to significant loss of recognition by a polyclonal antiserum to bovine GHR which recognizes site A as a major epitope. The crystal structure of human GH bound to human GHR did not resolve this extreme N-terminal region of the receptor but our data indicate that the N-terminal loop undertakes a 180 degrees turn bringing it into close proximity to the hormone-binding domain in a fashion analogous to the prolactin receptor. A fourfold decrease in affinity for binding bovine GH was also observed after mutation of site B. However, this change from the ovine sequence to the equivalent sequence in the rat GHR at site B caused a 2.4-fold increase in the affinity of binding to rat GH. Taken together, the changes in binding affinity of the site-B mutant for rat and bovine GH demonstrate that this site is involved in conferring species specificity for binding GH.

Amino Acid Sequence↗
PubMed1999

Generation of epitope-specific antibodies to rat GHBP in the sheep using an interspecies switching strategy involving site-directed mutagenesis of ovine GHBP.

Site-directed antibodies to the growth hormone receptor could be potentially useful as growth hormone mimics but, in previous attempts, we found that antisera generated using peptides derived from growth hormone receptor sequences failed to recognize the intact protein. As an alternative approach to this problem, we have now adopted a strategy of epitope-switching between rat and ovine growth hormone receptors to produce rat epitopes in the correct structural context. Using site-directed mutagenesis, we altered the two dominant linear epitopes in the ovine growth hormone binding protein to the analogous sequences in rat growth hormone binding protein. Site A, between Thr28 and Leu34, is equivalent to epitope 1 in ovine growth hormone binding protein and site B, between Ser121 and Asp124, corresponds to epitope 5. The wild-type ovine growth hormone binding protein and the two mutant proteins were bacterially expressed, refolded and, following purification by metal-chelate affinity chromatography, used to raise antisera in sheep. We showed using RIA, in which wild-type ovine growth hormone binding protein acted as a competitor for the binding of rat growth hormone binding protein, that only the site A mutant protein elicited a specific anti-rat growth hormone binding protein response. This was confirmed in subsequent RIA studies using the antiserum to the site A mutant protein in which only peptides corresponding to the site A sequences in mutant ovine growth hormone binding protein and rat growth hormone binding protein, but not that in wild-type ovine growth hormone binding protein, were able to act as competitors for rat growth hormone binding protein. Antibodies specific for rat growth hormone binding protein could be separated from the antiserum to the site A mutant protein by means of affinity chromatography using immobilized wild-type ovine growth hormone binding protein to remove antibodies which cross-reacted with the ovine protein. The work lays the foundations for further studies in which the biological effects of these antibody fractions will be investigated and demonstrates an approach with general applicability in the production of antibodies directed towards specific epitopes on protein molecules.

Amino Acid Sequence↗
PubMed1999

Immunological manipulation of body composition.

Hormonal growth promoters (e.g., growth hormone [GH], beta-adrenergic agonists, steroids), which improve growth rate and/or lean:fat ratios in the carcass, have received considerable adverse publicity and are either banned or have no licence for their use in the EC. This has led to the development of a number of techniques, involving the use of antibodies, aimed at regulating metabolic processes involved in determining growth and body composition. A different approach to the problem of excess fat deposition involves the use of antibodies directed against the plasma membranes of adipocytes in order to elicit their destruction and thereby limit the capacity for fat storage. This technique has been demonstrated in rats, sheep and pigs in both passive and active immunisation techniques. This promising approach is, however, limited by the lack of a commercially suitable vaccine. The identification of individual membrane proteins that are antigenic has been achieved and this affords the prospect of producing recombinant DNA-derived vaccines. Whether these new approaches will be perceived as acceptable to the general public remains a serious concern and a potential limitation to their development as many would-be sponsors withdraw their support for research in these areas.

Journal Article↗
PubMed1999

Amino acids within the extracellular matrix (ECM) binding region (201-218) of rat insulin-like growth factor binding protein (IGFBP)-5 are important determinants in binding IGF-I.

The highly conserved N-and C-terminal domains of IGFBPs are believed to participate in IGF binding, but only recently have some of the critical residues in the IGFBP sequence involved in ligand binding been identified. Here we describe two highly conserved amino acids in the C-terminal domain of rat IGFBP-5 that are involved in binding IGF-I. Site-directed mutagenesis was used to produce two mutants, G203K and Q209A, of rIGFBP-5. Relative to wild-type rIGFBP-5, an 8-fold reduction in affinity for human IGF-I was found for recombinant G203K protein in both IGF-I ligand blots and solution phase ligand binding assays, and a 7-and 6-fold reduction for Q209A respectively. This shows that Gly203 and Gln209 in IGFBP-5 are important determinants in binding IGF-I, and due to their complete conservation in all IGFBP sequences, we suggest that they are likely to be involved in binding IGF-I in all six binding proteins. In addition, these two non-basic residues lie within the ECM binding region (201-218) of IGFBP-5, demonstrating that the C-terminus contains partially overlapping IGF-I and ECM binding sites. We therefore propose that heparin binding to basic amino acids in IGFBP-5 between 201-218 may physically occlude subsequent interaction between IGF-I and Gly203/Gln209, and that this may explain previous work of others showing reduced affinity of ECM bound IGFBP-5 for IGF-I.

Amino Acid Sequence↗
PubMed1998

Effects of antibodies to adipocytes on body weight, food intake, and adipose tissue cellularity in obese rats.

Female Wistar rats were fed on a high fat diet for 18 weeks, during which their energy intake increased by 25% and body weight by 50% due to a doubling of adipose tissue tissue stores. Animals were then treated with increasing doses of a sheep polyclonal antiserum to rat adipocytes on days 1-4 and 7 after which they remained untreated for 14 weeks. Antibody treatment reduced body weight by 10% and the weight of parametrial and subcutaneous adipose tissue by 30-40%. This decrease was explicable entirely in terms of a decrease in the number of adipocytes presumably due to adipocyte lysis. These favourable changes in body fat mass were accompanied by improvement in at least one metabolic factor associated with obesity - serum leptin concentrations were significantly reduced in treated animals compared with high fat controls. Genetically obese Zucker rats also showed decreases in the number of adipocytes after treatment with antibodies but in contrast to diet-induced obese rats, they showed a compensatory increase in adipocyte volume which attenuated the effects on body fat mass. These results demonstrate for the first time, the potential to treat diet-induced obesity with antibodies to adipocytes by producing long-term reductions in the number of adipocytes, with minimal side-effects.

Adipocytes↗
PubMed1998

A diagnostic comparison of panoramic and intraoral radiographs.

OBJECTIVE: This study compared panoramic and intraoral radiographic surveys in the evaluation of specific dental pathoses in Air Force personnel. STUDY DESIGN: The radiographs of 30 subjects were read singly and in various combinations: panoramic survey only; periapicals plus bitewings; panoramic survey plus bitewings; and panoramic survey plus periapicals plus bitewings. Three independent, blinded examiners using standardized viewing conditions assessed the radiographs in random order for the presence of caries, periapical pathoses, bone loss, furcation involvement, impacted/unerupted teeth, internal/external root resorption, and retained roots. The consensus radiographic standard of true pathosis was the simultaneous interpretation by the three examiners of all radiographs for each subject. RESULTS AND CONCLUSIONS: The panoramic survey by itself was shown to have the lowest correlation with the consensus radiographic standard when basic military trainees with generalized dental pathoses were evaluated. The combination of panoramic survey plus bitewing radiographs exhibited a diagnostic yield for specific pathoses that was comparable to that of panoramic survey plus bitewings plus periapicals.

Adolescent↗
PubMed1998

Effects of food restriction on the responses of the mammary gland and adipose tissue to prolactin and growth hormone in the lactating rat.

Exogenous GH is used extensively in the USA to stimulate milk production in dairy cattle but its effectiveness is reduced in undernourished animals. It has been proposed that GH increases milk yield by stimulating IGF-I secretion and that this IGF-I-response is nutritionally sensitive and thus acts as a 'sensor' of energy balance. To investigate this possibility, we placed lactating rats on three planes of nutrition, ad libitum, 50% or 25% of ad libitum for 48 h. Subgroups of these animals were treated for 48 h with bromocriptine, to suppress prolactin secretion, and anti-rat GH, to neutralize GH action. From 24 to 48 h some of the treated animals were assessed for their milk yield response to prolactin or GH. Food restriction reduced milk yield in control rats by approximately 50% and was accompanied by a catabolic state, as judged by lipid mobilization from adipose tissue and by low concentrations of serum insulin, IGF-I, triiodothyronine and thyroxine, and increased serum nonesterified fatty acid concentrations. In animals fed ad libitum, anti-rat GH plus bromocriptine treatment produced an 80% decrease in milk yield and a dramatic fall in the activity of acetyl-CoA carboxylase in mammary tissue. GH was able to stimulate milk yield when given from 24 to 48 h; however, its effectiveness decreased progressively as food intake was reduced. The milk yield response to GH was accompanied by an increase in serum IGF-I concentrations and this response also decreased progressively with reduction of food intake, consistent with the hypothesis that IGF-I determines the milk yield response to GH and thus regulates GH action on the mammary gland in a nutritionally dependent fashion. However, the milk yield response to prolactin and the milk yield of control rats decreased in line with food intake without any changes in serum IGF-I concentrations. This clearly indicates that factors other than IGF-I are responsible for restricting milk yield. In order to assess other possible candidates for this role, we monitored serum glucose, non-esterified fatty acids, insulin triiodothyronine and thyroxine concentrations, but found no evidence for any simple relationship between these parameters and the milk yield response to prolactin and GH. Surprisingly we found that the ability of GH or prolactin to prevent epithelial cell loss in in the mammary gland was completely insensitive to nutrient intake, despite the fact that IGF-I is considered to be an important survival factor for mammary epithelial cells. Finally, we also demonstrated that, at least during short-term food restriction, the lactating rat is capable of mobilizing significant amounts of lipid from adipose tissue, such that it could provide the total output of triglyceride in milk, which is much greater than has previously been proposed.

Acetyl-CoA Hydrolase↗
PubMed1997

Interactions of prolactin and growth hormone (GH) in the regulation of mammary gland function and epithelial cell survival.

The relative importance of GH and prolactin in mammary gland function varies between species with prolactin playing a major role in rodents and GH taking lead role in ruminants. In rodents, however, GH appears to play a vital role in maintaining a high-fat/low volume milk in the absence of prolactin and a similar finding has been demonstrated in goats where prolactin deficiency causes a more modest (15%) decrease in milk yield. Surprisingly GH-deficiency in goats induced no further decline in milk yield whereas exogenous GH or prolactin both stimulated milk output considerably. Although direct effects of prolactin on mammary epithelial cells are well-documented effects of GH are believed to be mediated indirectly via IGF-1 production from the liver. We have been unable to confirm this hypothesis in rats and believe this to be because it is too simplistic. By considering prolactin and GH to be survival factors for the mammary gland we now propose a mechanism by which they interact through the IGF system. Involution of the mammary gland involves apoptosis and, in rats, it is induced by prolactin-deficiency or milk accumulation. Coincidentally with this process mammary epithelial cells synthesize and secrete and IGF binding protein, IGFBP-5. We hypothesize that GH stimulates IGF-1 production, possibly from the mammary parenchyma. IGF-1 then acts as a survival factor for the mammary gland. Prolactin plays an essential role since it suppresses the secretion of IGFBP-5 which would otherwise inhibit IGF-1 action and lead to the induction of cell death.

Animals↗
PubMed1997

Hormonal control of insulin-like growth factor-binding protein-5 production in the involuting mammary gland of the rat.

We have demonstrated a 50-fold increase in the concentration of insulin-like growth factor-binding protein-5 (IGFBP-5) in milk after 2 days of mammary involution induced by removal of the suckling young. IGFBP-5 was identified by its immunoreactivity with an antiserum to IGFBP-5 and was shown by in situ hybridization to be synthesized by the secretory epithelial cells undergoing apoptosis. Smaller increases in IGFBP-2 and -4 messenger RNAs (mRNAs) were also evident, but neither protein could be detected on Western ligand blots of milk. Preliminary evidence failed to detect mRNAs for IGFBP-1, -3, or -6. The large increase in IGFBP-5 concentrations in milk from involuting mammary glands was inhibited by 90% if the dams received concurrent PRL injections for 2 days, but was unaffected by GH, progesterone, corticosterone, or an antiserum to insulin-like growth factor I (IGF-I). In lactating rats allowed to continue nursing their young, 17beta-estradiol failed to affect IGFBP-5 concentrations, whereas in animals that had half the teats sealed to prevent milk removal, IGFBP-5 concentrations increased 5- to 10-fold in the sealed gland compared with those in the contralateral gland where milk removal continued. The changes in IGFBP-5 concentrations in milk were accompanied by similar changes in steady state mRNA levels of IGFBP-5 in mammary tissue. We have previously shown that PRL inhibits apoptosis and involution of the mammary gland, whereas teat sealing has the opposite effect. We, therefore, propose that IGFBP-5 serves to inhibit IGF-I-mediated cell survival, but that it is normally suppressed by PRL and milk removal. Although IGFBP-5, when bound to extracellular matrix, augments the action of IGF, we believe that in the involuting mammary gland IGFBP-5 inhibits IGF action by interacting with casein micelles, which contain calcium phosphate nanoclusters, thereby preventing IGF interaction with IGF receptors. This is analogous to the interaction of IGFBP-5 with hydroxyapatite, which serves to sequester IGFs in bone. IGFBP-5 may, in fact, play a central role in inducing apoptosis, as it is also up-regulated in involuting prostate and thyroid glands as well as in atretic ovarian follicles.

Animals↗
PubMed1997

Effects of growth hormone on cholesterol metabolism in the lactating rat mammary gland.

Lactating rats were treated for 48 h with bromocriptine (to inhibit prolactin release) or bromocriptine together with an antiserum to rat GH. Animals given the combined treatment were also supplemented concurrently with bovine GH (bGH) or human insulin-like growth factor-I (hIGF-I). The effects of these treatments on the activities of 3-methyl-3-glutaryl-CoA reductase (HMG-CoA reductase), acyl-CoA:cholesterol acyltransferase (ACAT) and neutral cholesteryl ester hydrolase (CEH) and on the microsomal concentrations of non-esterified and esterified cholesterol were measured. Lack of prolactin decreased HMG-CoA reductase but did not affect ACAT, neutral CEH or the concentrations of microsomal cholesterol or cholesteryl esters. In the absence of both hormones, an even greater reduction in HMG-CoA reductase together with increases in ACAT, neutral CEH and both of the microsomal sterols were observed. Concurrent supplementation with either bGH or hIGF-I wholly or partially prevented the effects on HMG-CoA reductase but only bGH was active against the increase in ACAT. Neither bGH nor hIGF-I could prevent the effects of the anti-hormone treatment on neutral CEH, and the changes in ACAT and CEH activities were broadly reflected in the microsomal sterol concentrations. The results indicate that the cessation of lactation brings about rapid changes in the activities of the enzymes involved in cholesterol metabolism within the mammary gland with a definite switch from synthesis to storage. Supplementation with bGH alone was sufficient to maintain cholesterol synthesis at control levels and could also significantly inhibit storage of the sterol as its ester. In the absence of GH, hIGF-I partially supported cholesterol synthesis but had no effect on its conversion to the ester. On a whole-tissue basis, enzyme activities could be correlated with the physiological effects of the anti-hormone treatments.

Animals↗
PubMed1997

Hormonal regulation of microsomal cytochrome P450 2C11 in rat liver and kidney.

The current study was conducted to investigate hormonal regulation of cytochrome P450 2C11 (CYP2C11) in rat liver and kidney of adult male rats. In two experiments, hypophysectomy (Hx) resulted in decreased (P < .05) hepatic CYP2C11 apoprotein and mRNA levels. Growth hormone (GH) replacement of Hx rats prevented the decline in hepatic CYP2C11 apoprotein and mRNA levels, whereas, subcutaneous injection of testosterone had no effect. Rat pituitary extract is equally effective in intact or castrated Hx rats in preventing the decline in hepatic CYP2C11 apoprotein and mRNA levels. Specific neutralization of rat GH by sheep anti-rat GH serum reduced (P < . 05) serum IGF-I concentrations, hepatic CYP2C11 apoprotein and mRNA levels. Hx of male rat resulted in decreased (P < .05) renal CYP2C11 apoprotein and mRNA levels, and treatment with GH failed to prevent these effects; however, supplementation of Hx rats with testosterone or rat pituitary extract prevented the Hx-induced decrease of renal CYP2C11 apoprotein and mRNA levels, and the effects of rat pituitary extract occurred only in intact rats. Neutralization of rat GH by anti-rGH significantly reduced (P < .05) CYP2C11 mRNA levels and serum T concentrations but not serum LH concentrations. These results indicate that although hepatic CYP2C11 is regulated by GH, rat renal CYP2C11 is regulated primarily by gonadal steroids.

Animals↗
PubMed1996

An immobilised peptide array identifies antibodies to a discontinuous epitope in the extracellular domain of the bovine growth hormone receptor.

Using an array of overlapping decapeptides representing the extracellular domain of the bovine (b) growth-hormone receptor (GHR) we have mapped the continuous, dominant epitopes defined by five rabbit and one guinea pig polyclonal antisera to recombinant bovine growth-hormone-binding protein (rbGHBP). We report that six major epitopes are identified by these antisera and that these largely occur in areas of non-ordered secondary structure, although there is some contribution from the extensive beta-sheet structure of GHBP. Similar to our previously described studies for growth hormone (GH), we have again found slight differences between animals in the exact location of these epitopes. Using peptide-affinity chromatography we have isolated a population of antibodies reactive with epitope 1 (the N-terminal epitope:GHBP residues 21-38). Analysis of these antibodies by further peptide affinity chromatography and competitive radioimmunoassay experiments indicated cross-reactivity of epitope-1-specific antibodies with epitope 4 (in the interdomain hinge region of the GHBP:residues 111-126). We suggest that, although separate in the primary structure of the molecule, the tertiary fold exhibited by GHBP may bring into close proximity areas of sequence representing epitope 1 and epitope 4 such that they represent a conformational epitope. Under these conditions our experiments indicate that peptides 1 and 4 may represent partial functional epitopes for this antibody population and consequently demonstrate that this approach may be useful in describing discontinuous epitopes.

Amino Acid Sequence↗
PubMed1996

The role of prolactin and growth hormone in the regulation of casein gene expression and mammary cell survival: relationships to milk synthesis and secretion.

We have compared involution of the rat mammary gland, induced by litter removal, where milk accumulation occurs, with involution induced in the presence of the suckling young by combined PRL and GH deficiency. Both treatments induced involutionary processes involving apoptosis, as judged by DNA ladders and resulted in significant decreases in the DNA content of the gland. Surprisingly, the effects of hormone deprivation on protein output in milk were principally explained by the loss of secretory cells, as there were only modest decreases in casein messenger RNA (mRNA) expression and protein synthesis rates per U DNA in vitro. The association of casein mRNA with the polysome fraction was also unaffected by hormone deprivation, whereas involution induced by litter removal resulted in much greater decreases in steady state levels of casein mRNA and an increased association of the mRNAs with the monosome fraction. In PRL- and GH-deficient rats, PRL treatment could prevent all of these effects, GH was partially effective, whereas putative mediators of GH action, insulin-like growth factor I (IGF-I), IGF-II, and IGF-binding protein-3, were ineffective. This lack of effect of IGFs may be due to an inhibitory IGFBP, which we demonstrate to be present in increased amounts in the involuting mammary gland.

Animals↗
PubMed1996

Repartitioning of maternal muscle protein towards the foetus induced by a polyclonal antiserum to rat GH.

Growth and protein accretion were studied in maternal muscle and liver and in foetuses of rats on day 20 of pregnancy. In young rats, weighing 120 g at mating, muscle mass and protein content of three hind-limb muscles, soleus, plantaris and gastrocnemius, increased on average by 7% compared with non-pregnant controls although the rate of muscle protein synthesis was decreased. In mature rats, rates of muscle protein synthesis were also reduced on day 20 of pregnancy but no change in muscle mass was observed. Rates of liver protein synthesis and accretion were increased in the pregnant animals; the effect was larger in the young pregnant rat. Administration of an antibody to rat GH (anti-rGH) for 10 days to young pregnant rats reversed the effect on the same three maternal muscles and resulted in a 9-11% lower muscle mass and protein content, compared with control pregnant animals. In both young and mature dams serum IGF-I concentrations were halved on day 20 of pregnancy, a further small reduction was observed in response to anti-rGH. No significant change in serum insulin or corticosterone levels was observed. Anti-rGH treatment also reduced food intake but foetal weight at 20 days was significantly increased (14%). The effects on maternal muscle were not the result of loss of appetite associated with anti-GH administration as, in rats pair-fed to the intake of the anti-rGH group, maternal muscle and foetal weights were the same as in animals with food available ad libitum. The data suggest that the GH/IGF axis is involved in the partitioning of nutrients between the dam and the foetus.

Animals↗