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Biomedical subjects

B D Burleigh

Publications and source records attributed to B D Burleigh.

At least 19 recordsLinked to original sources
PubMed1998

Novel technetium (III)-Q complexes for functional imaging of multidrug resistance (MDR1) P-glycoprotein.

UNLABELLED: Overexpression of the multidrug resistance (MDR1) P-glycoprotein (Pgp) correlates with cancer chemotherapeutic failure. Lipophilic cationic radiopharmaceuticals such as 99mTc-sestamibi, 99mTc-tetrofosmin and 99Tc-furifosmin (Tc-Q12) have been validated as transport substrates for the MDR1 Pgp and may enable functional imaging of the MDR phenotype in cancer by observing enhanced washout rates of the tracers in those tumor areas expressing Pgp. To further explore and optimize the Pgp recognition properties of Schiff base phosphine mixed-ligand complexes of the Tc-Q series of nonreducible (Tc(III) cations, a variety of Tc-Q complexes were synthesized and tested in vitro for recognition as transport substrates by the human MDR1 Pgp. METHODS: Tracer assays with human drug-sensitive KB-3-1 epidermal carcinoma and MDR KB-8-5 cells expressing nonimmunodetectable and modest levels of MDR1 Pgp, respectively, were used to screen and pharmacologically characterize 37 novel 99mTc-Q analogs. RESULTS: The ideal agent should have low nonspecific binding, high distinction in net uptake between drug-sensitive cells and MDR tumor cells, and high enhancement of uptake in resistant cells after treatment with an MDR modulator, indicating selective blockade of Pgp-mediated efflux of the radiotracer. Three analogs, trans-[5,5'-(1,2-ethanediyldiimino)bis(2-OEt-2-Me-4-penten-3 -one)]bis[dimethyl(3-OMe-1-propyl)phosphine]99mTc(III) (99mTc-Q63) and two trans-[bis(methyl-bis(3-OMe-1-propyl)phosphine)] analogs (99mTc-Q57 and 99mTc-Q58) displayed transport distinctions between drug-sensitive and MDR cell lines that were equal to or greater than all previously available agents. Cyclosporin A, an MDR modulator, had no significant effect in KB-3-1 cells for these 99mTc-complexes but enhanced tracer accumulations in KB-8-5 cells with IC50 values of approximately 1 microM. In contrast, the non-MDR agents methotrexate and cisplatin had no effect on accumulation of 99mTc-Q complexes and 99mTc-sestamibi in KB-8-5 cells. CONCLUSION: Technetium-99m-Q57, 99mTc-Q58 and 99mTc-Q63 are avid transport substrates recognized by the human MDR1 Pgp, and have enhanced in vitro properties that may enable functional imaging of Pgp in vivo with improved signal-to-noise ratios and tissue contrast compared to currently available agents.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1989

Purification of the serum acid-stable insulin-like growth factor binding protein from the pig (Sus scrofa).

1. An acid-stable IGF binding protein was isolated and purified from porcine serum. 2. The protein comprised two major species with Mrs of 45 and 41 kDa determined using SDS-PAGE under reducing conditions. 3. The IGFBP preparation specifically bound both IGF-I and II. 4. Four distinct protein bands (Mrs of 23, 45, 50 and 75 kDa) in the porcine IGFBP preparation specifically bound radiolabelled IGF-I. 5. The porcine IGFBP exhibited sequence homology with IGFBPs from human plasma and rat serum. 6. This is the first report of the purification and characterization of the acid-stable IGFBP from porcine serum.

Amino Acid Sequence↗
PubMed1989

Administration of recombinant human insulin-like growth factor I to pigs: determination of circulating half-lives and chromatographic profiles.

Recombinant human insulin-like growth factor I (IGF-I) was injected daily (4 or 8 mg/pig) as an intra-arterial bolus into pigs for 3 consecutive days and the serum IGF-I concentration was measured to determine disappearance profiles. IGF-I partitioned to a fast component (half-life, t 1/2 = 5.7 min) and a slow component (t 1/2 = 253 min) in pig serum. Chromatography of serum revealed that the fast component comprised unbound IGF-I, whereas the slow component comprised IGF-I bound to 40- and 150-kD serum IGF-binding proteins. In addition, administration of exogenous IGF-I caused significant hypoglycemia.

Animals↗
PubMed1989

Comparison of the effects of administration of recombinant bovine growth hormone or N-Met insulin-like growth factor-I to lactating goats.

Five goats were injected with GH (15 mg/day), three goats received systemic infusions of insulin-like growth factor (IGF)-I (43 nmol/h) and four goats received systemic infusions of physiological saline (20 ml/h) on days 4-6 of a 10-day experimental period during mid-lactation. Milk yield increased by an average of 24% in GH-treated goats by the time of the third injection. In contrast, milk yield of IGF-I-infused goats did not differ from saline-infused animals although two of three goats did show a small increase (12%) after 36 h of IGF-I infusion. With GH and IGF-I treatments plasma IGF-I concentrations increased similarly, reaching maxima of 100-130 nmol/l within 24 h. Plasma IGF-I concentration was relatively constant in saline-infused goats at about 50 nmol/l throughout the experiment. Total IGF-I bound to 50 kDa and 150 kDa binding proteins in plasma was increased by GH and IGF-I treatments but, in contrast to IGF-I, GH increased the proportion of IGF-I bound to 150 kDa binding protein. In a second experiment, four goats received systemic infusion of IGF-I (43 nmol/h) and four goats received systemic infusion of physiological saline (20 ml/h). There was no evidence that milk yield was changed during IGF-I infusion. However, when those goats which had previously received IGF-I infusions were injected with GH, milk yield increased by 30%.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Reduction studies on bacterial recombinant somatomedin C/insulin-like growth factor-1.

N-Met-Somatomedin C/insulin-like growth factor-1 (rSmC) had been produced in recombinant Escherichia coli in monomeric form. The intact rSmC peptide is initially synthesized in E. coli cells in denatured form as inclusion bodies. The rSmC peptide in these inclusion bodies was found in reduced form. Isolation of this rSmC peptide was accomplished by separation and dissolution of the inclusion bodies, with dissociation of non-covalently aggregated species. The reduced rSmC was converted to a metastable state, termed un-refolded rSmC. Further processing of this rSmC generated two other isomers, termed refolded rSmC. The transitions of the peptide among these different states, reduced rSmC, un-refolded rSmC, and refolded rSmC can be readily monitored by reversed-phase high-performance liquid chromatography. By reduction and re-oxidation of the purified individual isomers we found that they are likely to be related to each other as conformation isomers which appear to be stabilized by disulfide bonds.

Animals↗
PubMed1988

Human recombinant insulin-like growth factor I. I. Development of a serum-free medium for clonal density assay of growth factors using BALB/c 3T3 mouse embryo fibroblasts.

A serum-free clonal density growth assay was developed for the quantification of the biological activity of human recombinant insulin-like growth factor I (IGF-I). The assay measures IGF-I stimulated growth of Balb/c 3T3 cells cultured over 4 d on poly-D-lysine-coated plastic surfaces in a serum-free medium formulation composed of a 1:1 (vol/vol) mixture of Ham's F12 and Dulbecco's modified Eagle's media, supplemented with 3.0 ng/ml bovine basic fibroblast growth factor (bFGF), 10 micrograms/ml human transferrin, 100 micrograms/ml ovalbumin, and 1.0 microM dexamethasone. Low-temperature trypsinization of serum-supplemented stock cultures combined with the use of poly-D-lysine-coated plates made it unnecessary to use serum or fibronectin to promote cell attachment and survival. Serum-free growth conditions were optimized with respect to the concentrations of the supplements. Addition of IGF-I resulted in 3.5-fold more cells than control cultures without IGF-I after 4 d. Deletion of BFGF resulted in no IGF-I stimulation of growth. The concentrations of various preparations of IGF-I required to achieve one-half maximal stimulation of cell number (ED50) ranged between 1.25 and 4.7 ng/ml. In parallel assays, IGF-I was 6.6 times more potent than human recombinant insulin-like growth factor II and 32 times more potent than insulin. When cells were seeded into medium containing IGF-I, transferrin, ovalbumin, and dexamethasone but no bFGF, growth was minimal. Dose-response addition of bFGF showed an ED50 of 0.9 ng/ml. The methods reported are useful to monitor the biological potency of recombinant and natural-source growth factors as well as providing a new means of studying the multiple growth factor requirements of Balb/c 3T3 cells in culture.

Animals↗
PubMed1988

Human recombinant insulin-like growth factor I. II. Binding characterization and radioreceptor assay development using Balb/c 3T3 mouse embryo fibroblasts.

The binding of human recombinant insulin-like growth factor I (IGF-I) to BALB/c 3T3 mouse embryo fibroblasts has been characterized, resulting in the development of a radioreceptor assay. Binding of radioiodinated IGF-I (125I-IGF-I) to washed monolayers of BALB/c 3T3 cells was specific, time dependent, and stable, being maximal after a 10-h incubation at 15 degrees C with no loss of bound ligand or cells through 25 h. Scatchard analysis identified a class of high affinity binding sites with Kd = 59.6 pM and an estimated 1.57 X 10(5) receptors/cell. Half-maximal displacement of bound 125I-IGF-I occurred with 15 to 20 ng/ml unlabeled IGF-I competitor. Insulin-like growth factor II and insulin were far less effective competitors, providing half-maximal displacement at concentrations of 130 to 170 ng/ml and 2 to 3 micrograms/ml, respectively. Epidermal growth factor, transforming growth factor type alpha, and acidic and basic fibroblast growth factors did not compete for 125I-IGF-I binding at 1 microgram/ml. Cells fixed with glutaraldehyde before ligand binding did remain attached to culture dishes more tightly; however such pretreatment destroyed approximately 70% of ligand binding. Crosslinking data indicated that 125I-IGF-I binds specifically to a 330-kDalton receptor as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions. This receptor dissociated into 130-kDalton subunits when analyzed in the presence of dithiothreitol.

Animals↗
PubMed1987

Preparation and characterization of bovine growth hormones produced in recombinant Escherichia coli.

Two analogues of bovine growth hormone (BGH) have been produced in Escherichia coli by recombinant DNA techniques. In analogue Delta-1, the N-terminal alanine residue of the full-length bovine sequence is replaced by methionine. In analogue Delta-9, which is expressed at much higher levels than is Delta-1, the full-length bovine sequence is truncated at the N-terminus by eight residues and there is a serine-for-glycine substitution in the first position of the truncated protein. Both analogues, which were characterized by isoelectric focusing (i.e.f.), polyacrylamide-gel electrophoresis in the presence of SDS (SDS/PAGE), amino acid analysis and N-terminal amino acid sequence determination using combined g.l.c.-m.s., are compared with BGH isolated from pituitaries. In contrast with pituitary-derived BGH, the recombinant-derived proteins are homogeneous on SDS/PAGE and on i.e.f. In a radioimmunoassay, a radioreceptor assay and a bioassay in vivo (rat tibia), Delta-9 BGH showed very similar characteristics to the pituitary-derived hormone. Similar results have also been obtained with the Delta-1 analogue.

Amino Acids↗
PubMed1987

Metabolic clearance rate of insulin-like growth factor-I in fed and starved sheep.

The metabolic clearance of insulin-like growth factor-I (IGF-I) has been examined in sheep using a radioiodinated hormone preparation (131I-labelled IGF-I). Following i.v. administration, 131I-labelled IGF-I was distributed in a volume equivalent to plasma (60 ml whole blood/kg liveweight) and demonstrated a triphasic pattern of clearance with apparent half-lives (t 1/2) of 4.0 +/- 0.4 (S.E.M.), 52.4 +/- 3.4 and 792 +/- 26.5 min (n = 10). No significant differences in the t1/2 of the three phases were identified in fed compared with starved animals (fed, n = 4, phase 1 = 3.1 +/- 0.64, phase 2 = 46 +/- 5.9 and phase 3 = 756 +/- 27 min; starved, n = 6, phase 1 = 4.6 +/- 0.58, phase 2 = 57 +/- 3.2 and phase 3 = 816 +/- 38.5 min). Similarly, no significant differences in the distribution volume (fed, n = 4, 44 +/- 4 ml/kg live-weight; starved, n = 6, 39 +/- 2 ml/kg liveweight) or metabolic clearance rate (fed, n = 4, 2.9 +/- 0.15 ml/min; starved, n = 6, 3.2 +/- 0.5 ml/min) of the IGF-I were found in fed compared with starved animals. High-performance gel filtration chromatography of sequential plasma samples following injection of 131I-labelled IGF-I revealed three clear peaks of radioactivity which demonstrated markedly different patterns of clearance. These correspond to hormone complexed to binding proteins of 150,000 and 50,000 daltons and to 'free' hormone.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Specific immunoradiometric assay of insulin-like growth factor I with use of monoclonal antibodies.

We identified two monoclonal antibodies that bind spatially distinct epitopes on insulin-like growth factor I (IGF-I). Using these two antibodies, we developed a simultaneous, two-site immunoradiometric assay (IRMA) specific for IGF-I. This IRMA has no detectable cross reactivity with insulin, proinsulin, prolactin, or somatotropin, and less than 2% crossreactivity with IGF-II. The assay response varies linearly with IGF-I concentrations of 0-800 micrograms/L in serum; the detection limit is about 10 micrograms/L. A comparison of 26 IGF-I serum values from the IRMA and from a previously reported IGF-I specific RIA gave a correlation coefficient of 0.96 with no substantial bias (slope = 1.10). IGF-I values for serum, as an aid in assessing growth abnormalities, are easily (only three pipetting steps) obtained in less than 4 h.

Animals↗
PubMed1987

Natural and recombinant DNA-derived human insulin-like growth factor-I compared for use in radioligand assays.

We compared two preparations of human insulin-like growth factor (IGF)-I, one isolated from human plasma and the other prepared by recombinant DNA technology, for use in three radioimmunoassays and a radioreceptor assay for IGF-I, two radioreceptor assays for IGF-II, and a protein-binding assay for both IGFs. In the IGF-I assays, iodinated natural IGF-I consistently showed higher binding than did iodinated biosynthetic IGF-I, whether or not the iodopeptides were purified before use by hydrophobic interaction chromatography. Comparing the potency of the unlabeled peptides in the eight assay systems, we found the natural and the biosynthetic IGF-I to be equipotent in every case except for a radioimmunoassay involving a monoclonal IGF-I antibody (the synthetic peptide had 2.4 times greater activity), and an IGF-II radioreceptor assay involving ovine placental membrane receptors (the natural peptide had 10-fold greater activity). We conclude that recombinant DNA-derived IGF-I is suitable for use, both as a standard and as a radioligand, in a wide variety of IGF assays.

Animals↗
PubMed1981

Preparation, specific binding, and biological activity of a monosubstituted fluorescein derivative of prolactin.

A monosubstituted fluorescein derivative of ovine prolactin has been prepared by the reaction of fluorescein isothiocyanate with the hormone at pH 8.0. Reaction under the specified conditions results in a derivative incorporating one fluorescein moiety per molecule of prolactin as judged by coordinated UV spectroscopy and amino acid analysis. The absence of NH2-terminal threonine in the derivative indicates that the point of fluorescein attachment is the NH2-terminal amino group, whose substitution is expected to be favored at pH 8.0. The derivative was found to retain significant specific binding on both rabbit mammary and rat ventral prostate membrane preparations in competition with 125I-labeled prolactin, and to show saturable stimulation of progesterone production in primary cultures of rat granulosa cells. It is anticipated that this derivative will be useful in studies of prolactin-target cell interactions. This study shows that NH2-terminal monosubstituted derivatives of ovine prolactin are readily formed under specified conditions at pH 8.0; these conditions may be useful in the preparation of other derivatives of this type.

Animals↗
PubMed1981

Steroid and plasminogen activator production by cultured rat granulosa cells in response to hormone treatment.

Granulosa cells isolated from immature, DES-primed female rats were incubated in medium-199 plus 10% chicken serum with addition of FSH, or testosterone, or both. Cultures were incubated at 37 degree C for 7 days; medium samples were taken daily and analyzed for steroids and plasminogen-activator production. Only cultures containing FSH + testosterone produced significant amounts of both estradiol and progesterone after 2 days of incubation. The rate of estradiol production increased steadily up to the 4th day and then leveled off; the production of progesterone reached a maximum around the 3rd day, and then declined rapidly afterward. FSH alone was able to stimulate plasminogen activator production at the first day. Cultures with FSH + testosterone produced an additional peak of plasminogen activator activity at the 4th day. Plasminogen-activator production is thus not correlated with steroidogenesis in a simple way. We conclude that the granulosa cell require the presence of both FSH and testosterone at the beginning of incubation for normal response. Delayed addition of either hormone, or both, to the culture causes damage to the cells ability to produce normal responses to hormone treatment.

Animals↗
PubMed1980

The culture of hormone-dependent epithelial cells from the rat ventral prostate.

This paper describes a method for obtaining cultures of rat ventral prostate epithelial cells. The prostate is first perfused with a collagenase solution before removal from the animal; subsequent mincing and incubation in vitro produces a suspension of alveolar cell clumps. Upon incubation, these clumps attach to the surface of the culture dish and spread into discrete epithelial cell colonies, which both retain differentiated morphology, and secrete a species of plasminogen activator that is characteristic of prostatic tissue. These properties were not observed in cultures prepared from single cell suspensions of the same organ. Maintenance of epithelial colony integrity and secretory activity specifically required the continued presence of stromal cells, glucocorticoids and insulin. Androgenic steroids were much less effective than glucocorticoids in stimulating plasminogen activator secretion and in maintaining colony integrity, in spite of the well-established androgen dependence of prostatic tissue morphology in vivo and in organ culture. Furthermore, no effects of prolactin were observed, either when this hormone was tested alone or in conjunction with steroid hormones. Of 3 retinoids tested, retinal was highly cytotoxic at concentrations in the range of 1 microM, whereas retinol and retinoic acid were without detectable effect.

Animals↗
PubMed1979

Synthesis, characterization and fluorescence studies on N-alpha-dansylalanyllysyl trypsino (HIS-46)-methane.

The 1-dimethylamino-5-naphthalene sulfonyl (dansyl) derivative of alanyllsyl chloromethane was synthesized and employed to introduce the fluorescent dansyl moiety specifically into the active site of trypsin via affinity labelling. The potential of dansylalanyllysyl chloromethane lies in its high degree of selectivity and markedly faster rate of enzyme inactivation when compared to previously synthesized, single residue affinity label chromophores. This permits the practical utilization of stoichiometric amounts of the inhibitor to achieve 100% inactivation of trypsin, even at high dilutions. The transfer of energy between the four tryptophan residues of trypsin and the bound dansyl group has been investigated in the fluorescent inhibitor-enzyme conjugate. From transfer efficiency measurements mean distances of 19.0 A and 19.3 A between the point of attachment of the dansyl group and the four tryptophan residues of trypsin have been calculated. These compare well with the mean value of 18.8 A derived from calculations based on crystallographic model studies.

Amino Acids↗
PubMed1977

Studies of disulfide bond location in ovine lutropin beta subunit.

By combinations of selective chemical cleavage (cyanogen bromide), selective enzymatic cleavage (trypsin, thermolysin), and random cleavage (partial acid hydrolysis) a series of disulfide-containing peptides have been isolated from ovine lutropin beta subunit. These peptides suggest six disulfide linkages between half-cystine residues in positions 23-72, 26-110, 93-100, 34-88, 9-90, and 38-57. The latter pair was placed by elimination of other possibilities. The first three pairs are in agreement with a report by Chung, D., Sairam, M. R. and Li, C. H. (1975) Int. J. Peptide Protein Res. 7, 487-493; the pair 93-100 has also been detected by Reeve, J. R., Cheng, K. W. and Pierce, J. G. (1975) Biochem. Biophys. Res. Commun. 67, 149-155, using partial reduction and alkylation. In an attempt to improve the efficiency of enzymatic attack, a preliminary partial reduction as per Reeve et al. [16] was done. In this instance a peptide suggesting an additional disulfide linkage between half-cystines 23-26 was obtained as well as peptides consistent with the 23-72 and 26-110 placements. This was interpreted as an artifactual opening and recombining during partial reduction-reoxidation to produce the 23-26 linkage. The placement of three disulfide bonds (34-88, 9-90, and 38-57) is in disagreement with the pairings Chung et al. [15] suggest for these six half-cystine residues. Six reasons for uncertainty in the placement of disulfide bonds are discussed. It is concluded the definitive placement of the disputed three disulfide bonds in ovine lutropin beta subunit remains an open question.

Amino Acid Sequence↗