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Biomedical subjects

J Beattie

Publications and source records attributed to J Beattie.

At least 19 recordsLinked to original sources
PubMed2001

Differential inhibition of recombinant bovine GH (rbGH) activity in vitro by in vivo enhancing monoclonal antibodies.

We have previously described the effects of complexing recombinant bovine growth hormone (rbGH) with the in vivo enhancing monoclonal antibodies (Mabs) OA11 and OA15 and the non-enhancing Mab OA14 on the subsequent activity of GH in different tissue culture models. We reported that all of these Mabs caused the inhibition of GH-stimulated Jak-2 tyrosine kinase phosphorylation in the GH responsive pre-adipocyte cell line 3T3-F442A. However, using the mouse myeloid cell line FDC-P1 transfected with the full length ovine GH receptor (GHR), we subsequently found that OA11 and OA14 remained inhibitory with respect to the end point measurement of GH stimulated mitogenesis but that OA15 had no inhibitory effect on GH stimulated mitogenesis in this cell line. In order to correlate longer term mitogenic effects of Mab-GH complexes with signalling events in this transfected cell line model, we now report on the effects of complexing with Mab on the subsequent GH stimulated phosphorylation of Stat5b (signal transducer and activator of transcription). In agreement with our data for the mitogenic activity of GH-Mab complexes, we found that OA11 and OA14 inhibit GH activation of Stat5b but that OA15 is not inhibitory. Further to this, the dose-response effect of both OA11 and OA14 on the GH stimulation of Stat5b in the FDC-P1-oGHR transfected cells correlates with the previously described dose-response effects for both Mabs in the context of GH stimulation of mitogenic effects. We conclude that in this oGHR transfected cell line model, Mab effects on short and long term GH signalling events are tightly correlated. The observation that neither of the in vivo enhancing Mabs--OA11 or OA15--amplifies the response to GH in our transfected cell line model, coupled with the differential nature of Mab effects on GH activity (OA11--inhibition; OA15--no effect) may argue for an in vivo mechanism for enhancement of GH activity.

Animals↗
PubMed2001

Evaluation of an acute point-of-care system screening for respiratory syncytial virus infection.

There continues to be a significant risk of children contracting hospital-acquired infections caused by respiratory syncytial virus (RSV). In order to provide 24 h screening, we examined a point-of-care system (near-patient testing) for use by non-laboratory healthcare workers (HCWs) in a short stay unit adjoining the accident and emergency department of a large paediatric hospital. Three studies were conducted over consecutive winter epidemics, in which 2193 nasopharyngeal aspirates were obtained from children < 2 years old. An average of 23 trained HCWs tested aspirates with the Abbott TESTPACK(R) RSV assay. Material was sent to the virology laboratory for examination for RSV and other respiratory viruses by direct immunofluorescence. The mean performance characteristics of near patient testing were sensitivity 90%, specificity 92%, positive predictive value 92% and negative predictive value 92%. This was acceptable for clinical purposes. The near-patient testing provided a rapid answer and ensured that infants could be segregated according to infection status. Early antiviral treatment could be commenced and needless antibiotics avoided. During the study the hospital-acquired infection rate was the lowest recorded, although this may have been influenced by national trends and lower rates of inpatient care for infants with bronchiolitis.

Acute Disease↗
PubMed2001

Nephrocalcinosis in preterm babies.

OBJECTIVES: To determine prospectively the incidence and cause of nephrocalcinosis in preterm infants. STUDY DESIGN: Inborn babies of gestation less than 32 weeks or birth weight less than 1500 g were eligible to be entered into a prospective observational study. Two renal ultrasound scans were performed, the first at 1 month postnatal age and the second at term or discharge. Data were collected on gestation, birth weight, sex, race, family history of renal calculi, oliguria on first day, respiratory support (ventilation, steroid, and oxygen dependency), and use of nephrotoxic drugs (gentamicin, vancomycin, and frusemide). Intake of fluid, calcium, and phosphate and plasma urea, creatinine, calcium, and phosphate were recorded for the first 6 weeks of life. Random urinary calcium/creatinine, oxalate/creatinine, and urate/creatinine ratios and tubular absorption of phosphate were measured once at term. RESULTS: A total of 101 preterm infants were studied. Twenty three (23%) had abnormal ultrasound scans. Sixteen (16%) had nephrocalcinosis. On univariate analysis, gestational age, male sex, duration of ventilation, oxygen dependency, duration and frequency of gentamicin treatment, toxic gentamicin/vancomycin levels, and postnatal dexamethasone were significantly associated with nephrocalcinosis. In addition, babies with nephrocalcinosis had a lower intake of fluid, calcium, and phosphate, longer duration of total parenteral nutrition, and higher urinary oxalate/creatinine and urate/creatinine ratios than infants who did not have the condition. There was also a significant association with plasma urea and creatinine but not with plasma calcium or phosphate or urinary calcium. Multivariate analysis showed that the strongest predictors of nephrocalcinosis were duration of ventilation, toxic gentamicin/vancomycin levels, low fluid intake, and male sex. CONCLUSION: 16% of babies born at less than 32 weeks gestation developed nephrocalcinosis. The multifactorial origin, in particular, the association with extreme prematurity and severity of respiratory disease, is confirmed. In addition, an association with male sex, frequency and duration of gentamicin use, and high urinary oxalate and urate excretion is shown.

Aminoglycosides↗
PubMed2001

The carboxy-terminal domain of IGF-binding protein-5 inhibits heparin binding to a site in the central domain.

The IGF-binding protein (IGFBP)-5 protein contains consensus heparin binding motifs in both its carboxy (C)-terminal and central domains, although only the C-terminal site has previously been shown to be functional. We have made two chimeric IGFBP proteins by switching domains between rat IGFBP-5 and -2, named BP552 and BP522 to reflect the domains present, and a truncated rat IGFBP-5 mutant (1-168), named BP550. The ability of these proteins and wild-type (wt) IGFBPs-5 and -2 to bind to either IGFs or heparin was determined using biosensor real-time analysis and heparin ligand blotting respectively. We report that the chimeric molecules have IGF binding affinities comparable to those of the native IGFBPs from which they were derived and, as expected, the binding of BP550 to IGFs was greatly compromised. More surprising was the finding that the ability of BP552 and BP550 to bind to heparin was equivalent to that of wtIGFBP-5, whereas wtIGFBP-2 and BP522 failed to bind. These results demonstrate that the active heparin binding site in BP552 and BP550 is contained within the central domain of IGFBP-5, and that this site is active only in the absence of the C-terminal domain. We subsequently mutated two basic amino acids (R136A:R137A) in the central consensus binding sites between residues 132-140. This resulted in the loss of heparin binding for BP550, confirming the importance of these two basic amino acids in the central domain heparin binding activity. In light of these findings, we suggest that C-terminally truncated fragments of IGFBP-5 generated in vivo by proteolysis could retain heparin/extracellular matrix binding properties.

Animals↗
PubMed2000

Overlap of IGF- and heparin-binding sites in rat IGF-binding protein-5.

Using site-directed mutagenesis, we have undertaken a study of a potential IGF-binding site in the C-terminal domain of rat IGFBP-5, lying close to or within a previously described heparin-binding domain (residues 201-218) in this protein. After analysis of binding activity using three different methods - ligand blotting, solution phase equilibrium binding and biosensor measurement of real-time on- and off-rates - we report that the mutation of two highly conserved residues within this region (glycine 203 and glutamine 209) reduces the affinity of the binding protein for both IGF-I and IGF-II, while having no effect on heparin binding. In addition, we confirm that mutation of basic residues within the heparin-binding domain (R201L, K202E, K206Q and R214A) results in a protein that has attenuated heparin binding but shows only a small reduction in affinity for IGF-I and -II. Previous findings have described the reduction in affinity of IGFBP-5 for IGFs that occurs after complexation of the binding protein with heparin or other components of the extracellular matrix (ECM) and have postulated that such an interaction may result in conformational changes in protein structure, affecting subsequent IGF interaction. Our data suggesting potential overlap of heparin- and IGF-binding domains argue for a more direct effect of ECM modulation of the affinity of IGFBP-5 for ligand by partial occlusion of the IGF-binding site after interaction with ECM.

Amino Acid Sequence↗
PubMed1999

Workforce dilemmas: a comparison of staffing in a generalist and a specialist intensive care unit.

Intensive care units are arguably one of the most costly resources a hospital has to maintain in terms of nursing staff, skills and technology. Given that the Government's agenda on quality remains one of obtaining cost-effective healthcare, it is imperative that nursing managers consider the implications of the new policy shift for how they currently provide services. The purpose in this paper is to compare the different staffing levels adopted by managers in generalized and neurosurgical intensive care in an acute hospital trust. The dilemmas facing managers making staffing decisions without any definitive guidelines for resourcing these specialized units are examined.

Cost-Benefit Analysis↗
PubMed1999

Binding and signalling properties of a growth hormone enhancing monoclonal antibody.

We have used a sequential, qualitative biosensor based assay to demonstrate that OA15, a monoclonal antibody which enhances in vivo the activity of bovine growth hormone (bGH) does not disrupt the interaction between bGH and its cognate receptor (as represented by recombinant bovine GH binding protein -rbGHBP). We have confirmed this using a classical cell-based radio-receptor assay with the GH-responsive mouse pre-adipocyte cell line 3T3-F442A. The fact that OA 15 binding to bGH still allows hormone to interact with its receptor, allows us to test the hypothesis that there is any amplification of signalling events following hormone-MAb treatment of 3T3-F442A cells. We have used as a reporter of GH activity the rapid stimulation of JAK-2 tyrosine phosphorylation which is a critical first step in GH signalling events. We demonstrate that binding of rbGH by OA15 attenuates hormone stimulation of JAK-2 tyrosine phosphorylation. We conclude that although OA15 does not disrupt GH-GH receptor (GHR) interactions it does interfere with subsequent GH activity at the molecular and cellular level. We further speculate therefore that the biological enhancing activity of this antibody is most likely due to an in vivo effect as presentation of antibody-hormone complexes to a GH-target cell inhibits hormone activity.

3T3 Cells↗
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

Characterization of the IGF axis in a rat liver-derived epithelial cell line.

We have used the techniques of chemical cross-linking, Western blotting and immunoprecipitation-ligand blotting to demonstrate that insulin-like growth factor binding protein-2 (IGFBP-2) is associated with plasma membranes of an epithelial cell line derived from rat liver as well as being secreted into the medium by these cells. We demonstrate that these cells secrete IGF-I, but not IGF-II into serum free medium. Evidence from signalling, cell proliferation and cross-linking experiments indicate that these cells also express cell surface IGF-I receptors. Dose-response experiments indicate an enhanced biological activity of the IGF-I analogue des (1-3) IGF-I compared to wild-type IGF-I in both acute signalling experiments and longer-term (24 h) mitogenic assays. As this IGF-I analogue has lower affinity for IGFBPs, we believe that in this cell culture system, activity of IGF-I may be attenuated in the long and short term by the accumulation of IGFBP-2 in conditioned medium and by the presence of IGFBP-2 associated with the cell membrane and/or ECM.

Animals↗
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↗
PubMed1999

Effects of complexation with in vivo enhancing monoclonal antibodies on activity of growth hormone in two responsive cell culture systems.

We describe the properties of three monoclonal antibodies (MAbs) to ovine GH, two of which have previously been shown to enhance, in vivo, the biological activity of bovine and ovine growth hormone. We have examined the effects of these MAbs on GH activity in two appropriate GH-responsive cell culture systems, investigating both acute signalling effects (Janus-activated kinase (Jak)-2 tyrosine phosphorylation -5 min) and longer-term (MTT-formazan production -24 h) effects of hormone-antibody complexes. In the 3T3-F442A pre-adipocyte cell line (which has been demonstrated to be GH responsive), we show that complexation of recombinant bovine (rb) GH with either of the two enhancing anti-ovine GH MAbs (OA11 and OA15) and the non-enhancing MAb, OA14, attenuates the ability of GH to stimulate tyrosine phosphorylation of Jak-2 at a 5-min time point. Using the mouse myeloid cell line, FDC-P1, stably transfected with the full-length ovine GH receptor (oGHR), we demonstrate that rbGH causes a dose-dependent increase in MTT-formazan production by these cells. Further, we demonstrate that OA11 and OA14, but not OA15, cause a decrease in this stimulatory activity of rbGH over a hormone concentration range of 5-50 ng/ml at both 24 and 48 h. We conclude that the different in vitro activities of the two in vivo enhancing MAbs are most probably related to the time-courses over which these two assays are performed, and also to the relative affinities between antibody, hormone and receptor. In addition, the in vitro inhibitory activity of the enhancing MAb OA11 in both short- and long-term bioassay lends further support to an exclusively in vivo model for MAb-mediated enhancement of GH action.

Adipocytes↗
PubMed1997

A case of hyperzincemia with functional zinc depletion: a new disorder?

We report the case of an 1l-y-old boy with a plasma Zn concentration greater than 200 micromol/L, but with symptoms consistent with Zn deficiency. He has had hepatosplenomegaly, rashes, stunted growth (<3rd centile), anemia, and impaired immune function since infancy. He also has vasculitis and osteoporosis. A plasma Zn-binding protein has been separated and characterized by a combination of size exclusion and ion exchange chromatography and electrophoretic studies and by immunologic methods. Antibodies to the partially purified protein have been raised in rabbits. Size exclusion chromatography shows that Zn is bound to a protein with a mass 110000-300000 kD. Electrophoretic and mass spectrometry studies suggest that the protein may be composed of several subunits. One component of the isolated protein reacts with antiserum to alpha2-macroglobulin; immunoprecipitation studies confirm that the protein is not alpha2-macroglobulin or a histidine-rich glycoprotein. Kinetic studies of zinc metabolism in the patient and his mother with stable Zn isotopes show the presence of increased exchangeable Zn, with a rapid flux from plasma to a stable pool. Liver and muscle Zn and Cu concentrations are raised, but with no abnormal liver histology. Immunoreactive metallothionein in the liver is increased. We suggest that this boy may suffer from a previously unrecognized inborn error of Zn metabolism causing symptomatic zinc deficiency.

Biopsy↗