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Biomedical subjects

R R Bowsher

Publications and source records attributed to R R Bowsher.

At least 55 records · Page 3Linked to original sources
PubMed1992

Biosynthetic human proinsulin. Review of chemistry, in vitro and in vivo receptor binding, animal and human pharmacology studies, and clinical trial experience.

OBJECTIVE: To describe the rationale for the preclinical and clinical developmental course of human proinsulin (HPI), the second product after human insulin for the treatment of diabetes mellitus to be manufactured by DNA technology. RESEARCH DESIGN AND METHODS: The relevant and available published and unpublished preclinical and clinical information generated on pork proinsulin and human proinsulin has been integrated to demonstrate how certain clinically attractive features of pork proinsulin (a soluble intermediate-acting and possibly hepatospecific insulin agonist) led to the development of HPI. RESULTS: Clinical pharmacology studies demonstrated that HPI was definitely, although marginally, hepatospecific. More striking was the finding that the intrasubject/patient coefficient of variation of response to HPI was significantly less than that observed with NPH insulin. However, the fact that unique efficacy in controlled multicenter studies was not demonstrated suggested that these pharmacological features were not translated into clinical benefit. In one multicenter new patient study there were six myocardial infarctions, including two deaths, in patients treated for greater than or equal to 1 yr with HPI and none in the control group. CONCLUSIONS: To obtain an independent review of the risks and benefits of HPI, in February 1988, Lilly convened a consultant group that examined all relevant information on HPI available. These experts shared our concerns about the safety of HPI in light of the failure to demonstrate unique efficacy. Accordingly, clinical trials with HPI were suspended in February 1988. Experience with HPI demonstrates the challenge associated with the development of new drugs in general and insulin agonists in particular.

Amino Acid Sequence↗
PubMed1992

Sensitive, specific radioimmunoassay for quantifying pergolide in plasma.

Pergolide, a synthetic ergoline with potent dopaminergic activity, is used to treat Parkinson disease. The low plasma concentrations of pergolide achieved during therapy complicate the development of a method for its analysis. Because radioimmunoassay successfully measures other structurally related ergolines in physiological fluids, we undertook the development of a radioimmunoassay of pergolide. The detection limit of the radioimmunoassay is 21 ng/L with an optimal working range from 100 to 1000 ng/L. We maximized assay specificity by using a monoclonal antibody that displayed low cross-reactivity with pergolide sulfoxide, a major metabolite found in animals. The radioimmunoassay has performed acceptably for > 2 years during toxicology studies with rats and rhesus monkeys and in clinical studies involving patients with Parkinson disease. We consider the radioimmunoassay a valid method for quantifying therapeutic concentrations of pergolide in plasma.

Animals↗
PubMed1991

Conversion of p-tyrosine to p-tyramine in the isolated perfused rat kidney: modulation by perfusate concentrations of p-tyrosine.

We used the isolated perfused rat kidney to evaluate the role of renal decarboxylation of p-tyrosine as the source of urinary p-tyramine. Kidneys were perfused with concentrations of p-tyrosine ranging from 0.02 mM to 2.0 mM. p-Tyramine was measured by a sensitive and specific radioenzymatic assay. An increase in the perfusate concentration of p-tyrosine resulted in a significant increase in p-tyramine production that was blocked by the addition of NSD-1015, an inhibitor of aromatic-1-amino decarboxylase (AADC). We conclude p-tyrosine is the precursor for the renal production of p-tyramine, renal AADC catalyzes the formation of urinary p-tyramine, synthesized p-tyramine is predominantly excreted in the urine, and p-tyramine synthesis is modulated by the arterial delivery of p-tyrosine to the kidney.

Animals↗
PubMed1991

Measurement of insulin-like growth factor-II in physiological fluids and tissues. I. An improved extraction procedure and radioimmunoassay for human and rat fluids.

The measurement of serum insulin-like growth factors (IGFs) in serum is complicated by the presence of high affinity IGF-binding proteins. The accurate measurement of IGFs by radioligand binding assays requires that the interference from binding proteins be eliminated. Acid-gel chromatography, the standard method for removing binding proteins, is laborious and time consuming. Alternative methods for extracting serum IGFs include the use of HCl-ethanol treatment and reverse phase minicolumns. However, these methods are unsuitable for use with serum for some species, such as rat and sheep, due to incomplete removal of binding proteins. We developed a fast protein liquid chromatography size-exclusion chromatographic method for characterizing the presence of IGF-binding proteins in physiological fluids and used this method to systematically investigate different combinations of acids and organic solvents as potential extraction methods for IGFs. We developed and validated an improved extraction procedure that uses formic acid, Tween-20, and acetone. The new extraction method was used in conjunction with purified biosynthetic human IGF-II and a commercially available anti-IGF-II monoclonal antibody in the development of an improved RIA for IGF-II. The new RIA is sensitive (5.0 pg/tube), specific (IGF-I cross-reactivity, less than 1%), and reproducible [interassay precision (coefficient of variation), less than 9.2%). We measured the serum concentrations of IGF-II in adults and found a significant difference between normal subjects and individuals with insulin-dependent diabetes mellitus.

Adult↗
PubMed1991

Measurement of insulin-like growth factor-II in physiological fluids and tissues. II. Extraction quantification in rat tissues.

The tissue distribution and developmental patterns of insulin-like growth factor-II (IGF-II) have not been investigated in rat tissues, primarily because of the lack of an efficient extraction method for IGF-II and a sensitive RIA. IGF-II was extracted from rat tissues by formic acid, and the extract was heated at an acidic pH and treated with acetone. The removal of binding proteins was demonstrated by fast protein liquid chromatography size exclusion column and the elimination of a dilutional bias in the RIA. Using rat IGF-II as standard, we optimized a RIA for the quantification of IGF-II in rat tissues. In adult rats, IGF-II was found in all 15 tissues examined, with the highest concentration in the pituitary, followed by kidney, seminal vesicles, intestine, and serum. This distribution is not only different from that of IGF-I, but also differs from that reported for IGF-II mRNA and IGF-II receptors, suggesting that the rates of synthesis and/or metabolism of IGF-II are tissue dependent. Developmentally, IGF-II levels fell postnatally in most tissues, a pattern similar to that of IGF-II mRNA and IGF-II receptor. This developmental pattern supports the hypothesis that IGF-II is important in early growth and development. A relatively homogeneous distribution was observed in the adult rat brain, a distribution also different from that reported for IGF-II mRNA. In the pituitary, the highest concentration was found in the posterior pituitary, followed by the intermediate and anterior pituitary. In conclusion, IGF-II is found in many tissues of adult rats. This observation supports an autocrine and/or paracrine roles for IGF-II.

Animals↗
PubMed1991

Sustained pulsatile insulin secretion from adenomatous human beta-cells. Synchronous cycling of insulin, C-peptide, and proinsulin.

The endocrine pancreas secretes insulin in a pulsatile fashion. This rhythm is generated at a site within the pancreas, although its precise location has not been determined. With an in vitro system, we tested the possibility that beta-cells might generate spontaneous pulsatile insulin secretion in the absence of any external influence. Human insulinoma tissue from five patients was perifused for 7-10 h with RPMI-1640 medium and constant concentrations of glucose (5.5 mM). Insulin, C-peptide, and proinsulin were measured in the effluent collected at 3.3-min intervals. All three peptides demonstrated pulsatility of secretion in a similar, synchronous fashion that was sustained throughout each study. The Clifton cycle detection program demonstrated cycling in all five tumors, with an average period for all tumors of 28, 29, and 26 min for insulin, C-peptide, and proinsulin, respectively. Spectral analysis confirmed the regularity and consistency of the hormonal secretory patterns. Mean hormone concentrations secreted by different tumors varied, but insulin and C-peptide were secreted in a nearly 1:1 ratio. This study demonstrates 1) that beta-cells are able to generate spontaneous pulsatile insulin secretory activity, which is independent of innervation or the presence of other islet cells, and 2) proinsulin secretion from the beta-cell also has an inherent pulsatility. The synchrony observed in the cycles of proinsulin and its peptide products confirms their common secretory pathway in the beta-cell. We conclude that the beta-cell may be the originator of insulin cycling.

Adenoma↗
PubMed1991

Radioenzymatic assay for histamine: development and validation.

Radioenzymatic assays are sensitive analytic tools that use an enzyme to quantify a substrate for that enzyme. Purified histamine N-methyltransferase has been used as the basis for an assay for histamine. The sensitivity of the procedure is less than 10 fmol. The specificity of the assay is increased when the transferase reaction is carried out at 0-3 degrees C. Data documenting the precision of the assay, the stability of histamine in human plasma, and the gastric secretory rate of histamine are presented along with a chronologic description of the development of the technique.

Adult↗
PubMed1991

Competitive particle concentration fluorescence immunoassay for measuring 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (lometrexol) in serum.

A competitive particle concentration fluorescence immunoassay (PCFIA) is described for measuring 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (lometrexol; Lilly) in human serum. b-Phycoerythrin-labeled lometrexol competes with free lometrexol for binding to a limiting concentration of lometrexol-specific antibodies immobilized by a second antibody to submicrometer-diameter polystyrene particles in specially designed 96-well plates. Reaction particles are washed and concentrated onto filter membranes in the wells of the plates and the fluorescence is measured at 575 nm. The method, including sample preparation and data reduction, is automated and can be completed in less than 2 h. The assay has a standard curve maximum measurable concentration of 1000 micrograms/L and a minimum detectable concentration of 0.1 microgram/L. Analytical recovery of lometrexol in serum is quantitative at concentrations greater than 1 micrograms/L. Intra- and interassay coefficients of variation at 50 micrograms/L in serum are 7.1% (n = 9) and 7.5% (n = 33), respectively. The cross-reactivity of naturally occurring folates, folic acid analogs, and the anti-cancer agent methotrexate is minimal. We report the use of the PCFIA during Phase I clinical studies designed to evaluate the pharmacokinetics of lomextrexol after intravenous administration to cancer patients.

Adult↗
PubMed1990

Determination of norepinephrine levels in the adult human prostate.

Tissue levels of norepinephrine were measured in prostate tissue from 24 men ranging in age between 41 and 83 years. Prostatic tissue was obtained from men with subtle palpable prostate nodules undergoing transperineal needle biopsy. None of the patients were shown to have histologic evidence of adenocarcinoma. The severity of the symptoms of prostatism was evaluated prospectively using a standardized micturition symptom score questionnaire. Norepinephrine levels were quantified using a sensitive radioenzymatic assay (REA). Overall, the prostates contained relatively high levels of norepinephrine (1666 +/- 124 ng./gm.). Inverse correlations were observed between tissue norepinephrine levels and severity of symptoms of prostatism (r = -0.58; p = 0.003); age (r = -0.53; p = .008); and prostate size (r = -0.48; p = .02). Norepinephrine levels were also measured in tissue specimens obtained from men undergoing enucleation prostatectomy and transurethral resection of the prostate (TURP). The level of norepinephrine in these prostatectomy specimens (115 ng./gm.) was only 14% the level of the prostate biopsy specimens. The relatively low level of norepinephrine in the specimens obtained from patients with symptoms necessitating prostatectomy provides further evidence that norepinephrine levels are inversely related to the degree of symptomatic bladder outlet obstruction.

Adult↗
PubMed1990

Cross-reactivity of monomeric and dimeric biosynthetic human growth hormone in commercial immunoassays.

Commercial kits give different measurements for concentrations of growth hormone (GH, somatotropin) in serum. Most notably, a two-site monoclonal-antibody-based immunoradiometric assay (IRMA) from Hybritech routinely yields lower values than do conventional RIAs in which polyclonal antibodies are used. We used purified dimeric biosynthetic human GH as a model compound to investigate the specificity of five commercial immunoassays for size variants of GH. In all five assays, biosynthetic monomeric GH was significantly more potent than pituitary-derived standard GH supplied with the kits. Dimeric GH was significantly less potent than monomer in four of the five assays, and cross-reactivities varied more than fivefold, from 15% to 84%. Using three commercial kits selected for their specificity for dimeric GH, we measured GH in serum samples from 18 normal adults. The mean GH concentrations in serum--0.7 (Hybritech, IRMA), 1.8 (Diagnostic Products, RIA), and 3.1 (Cambridge, RIA) micrograms/L--differed significantly, but in the same rank order as that obtained in the experiments on dimer cross-reactivity.

Antibodies, Monoclonal↗
PubMed1989

A sensitive immunoradiometric assay for the quantification of murine monoclonal antibodies in human serum.

The clinical investigation of murine monoclonal antibodies (MoAbs) as potential immunotherapeutic agents necessitates their quantification in human serum. The present paper reports the development of a sensitive, non-competitive "sandwich" immunoradiometric assay specifically optimized for measurement of murine IgG in human serum. Affinity-purified goat anti-mouse IgG antibody covalently bound to acrylic microspheres serves as the solid-phase antibody and 125I-labelled goat anti-mouse IgG antibody functions as tracer. All assay reagents were obtained from commercial sources. The assay is sensitive (capable of detecting 20 ng ml-1 of murine IgG), specific (less than 0.0001% cross-reactivity with human IgG), reproducible (intra-assay % RSD less than 6.3%), and rapid (a 100-tube assay can routinely be processed in 3 h). The assay has a working concentration range of 20-2000 ng ml-1 and is suitable for measuring MoAbs of any antigenic specificity. The assay was validated for use with human serum and cell culture media by comparison studies with flow cytometric and immunonephelometric methods and by high-performance size-exclusion chromatographic studies. Application of this assay should facilitate further investigation of murine MoAbs as potential immunotherapeutic agents.

Animals↗
PubMed1988

Quantification of urinary histamine by a new radioenzymatic assay: documentation of assay specificity and establishment of normal excretion rates.

The goals of this study were to evaluate the specificity of a new radioenzymatic assay for histamine when it is used for urinary-histamine determinations and to establish normal rates of urinary-histamine excretion for male and female subjects. Specificity of the assay was characterized by reacting urine samples at two different incubation temperatures and with varying amounts of highly purified histamine N-methyltransferase and S-adenosyl-L-[methyl-3H]methionine. The radiolabeled products were then separated by thin-layer chromatography and visualized by fluorescence-enhanced autoradiography. Our results indicate that at least one other substrate for histamine N-methyltransferase in addition to histamine is present in human urine, although the identity of this compound could not be determined. Optimization of enzyme-reaction conditions eliminated the methylation of this unidentified substrate while the methylation of histamine was maintained. The average urinary unidentified substrate while the methylation of histamine was maintained. The average urinary excretion of histamine determined for male subjects (N = 37) and female subjects (N = 111) was 18.7 micrograms/24 hr and 27.3 micrograms/24 hr, respectively. More than 12% of the normal female subjects examined had urinary-histamine excretion rates in excess of 50 micrograms/24 hr, which overlaps into the range previously considered to be indicative of abnormal mast cell secretion. In summary, this is the first radioenzymatic assay for histamine with documented specificity for the measurement of histamine in human urine.

Clinical Enzyme Tests↗
PubMed1987

Dopamine in rat adrenal glomerulosa.

There is increasing evidence that dopamine (DA) inhibits aldosterone production, but the source of DA for this dopaminergic influence is not known. In the present study we examined the adrenal's zona glomerulosa for the presence of DA. Rats maintained on an intake of regular food were killed by decapitation and the adrenal capsule (containing zona glomerulosa) and the remainder of the gland (containing both cortex and medulla) were examined for their content of DA and also for norepinephrine (NE) and epinephrine (E). DA was found in adrenal glomerulosa in substantial quantity, 1.92 +/- 0.17 (SEM) ng/mg wet weight, representing an approximate concentration of DA of 1-100 microM. DA in adrenal capsule represented 12.2% of the total adrenal content of DA. NE and E were also present in glomerulosa, 3.46 +/- 0.32 and 18.7 +/- 2.1 ng/mg respectively, but, unlike DA, about 98% of the total adrenal content of NE and E was contained in adrenal medulla. The NE/E ratio in capsule and medulla were similar, although slightly higher in adrenal medulla, suggesting that the medulla is the source of the NE and E found in glomerulosa. On the other hand, the DA/E ratio was several-fold higher in glomerulosa than medulla--suggesting that glomerulosa DA was derived at least partially from a source other than adrenal medulla. We also found that short-term culturing of the adrenal reduced DA levels to 1/3 that observed in fresh tissue. This could explain in part why cultured glomerulosa has been shown to be more responsive to administered stimuli. In summary, the findings indicate a significant concentration of DA in adrenal glomerulosa, and suggest that the effects of DA on aldosterone production are mediated locally within the adrenal.

Adrenal Glands↗
PubMed1987

Chemical, physical, and biological characterization of a dimeric form of biosynthetic human growth hormone.

A dimer of biosynthetic human growth hormone (HGH) has been isolated and characterized. This entity, which is the predominant dimeric species in biosynthetic HGH, is chemically identical to monomeric HGH and exists in a noncovalent dimeric form which is dissociated to monomeric HGH on polyacrylamide electrophoresis gels or in aqueous solutions containing 30% acetonitrile. This substance, found in all production lots of pituitary HGH, biosynthetic HGH, and biosynthetic methionyl HGH examined, is much less biopotent than monomeric HGH and can be distinguished from monomeric HGH by a monoclonal antibody. These data demonstrate that polyacrylamide gel electrophoresis is not a valid method for measuring this dimer and that size-exclusion chromatography under aqueous conditions is required.

Amino Acids↗
PubMed1986

An improved radioenzymatic assay for plasma norepinephrine using purified phenylethanolamine N-methyltransferase.

Radioenzymatic assays have been developed for norepinephrine (NE) using either catechol O-methyltransferase (COMT) or phenylethanolamine N-methyltransferase (PNMT). Assays using PNMT are specific for NE but have been considered less sensitive than the more complex assay procedures employing COMT. An improved purification procedure for bovine PNMT has permitted development of a NE assay with substantially improved sensitivity (less than 0.5 pg), reproducibility, and decreased manipulative effort. PNMT was purified by sequential pH 5.0 treatment and dialysis and by column chromatographic procedures using DEAE-Sephacel, Sephacryl S-200 and Phenyl Boronate-agarose. Recovery of PNMT activity through the purification scheme was 50% while blank recovery was less than 0.001%. Norepinephrine can be directly quantified in 25 microliters of human plasma and a seventy-tube assay can be routinely completed within 4 h. The capillary to venous plasma NE gradient was examined in eight normotensive male subjects. Capillary plasma NE (211 +/- 21.7 pg/ml) was significantly lower than venous plasma NE (367 +/- 32.7 pg/ml) in all subjects (p less than 0.005). This difference suggests the concentration of NE in capillary blood may be a unique indicator of sympathetic nervous system activity in vivo.

Adrenal Medulla↗
PubMed1986

The effect of hypertonic saline administration or stalk transection on histamine and histamine N-methyltransferase in the rat posterior pituitary.

There is evidence to suggest that histamine is a neurotransmitter in the CNS and functions in the regulation of arg-vasopressin (AVP) secretion. The posterior pituitary contains high levels of histamine and histamine N-methyltransferase (HNMT). Therefore, posterior pituitary histamine could also modulate the release of AVP. Paralleling the effect on AVP levels, the concentration of histamine in the rat posterior pituitary decreased from 18.8 +/- 2.7 ng/mg protein (x +/- SEM) to 12.9 +/- 1.9 ng/mg protein following 2 days of 2% (w/v) hypertonic saline administration and to 11.5 +/- 0.9 ng/mg protein with 7 days of treatment. Conversely, posterior pituitary HNMT activity was significantly elevated after hypertonic saline administration. Pituitary stalk transection did not reduce the concentration of histamine in the rat posterior pituitary although HNMT activity was reduced from 18.8 +/- 0.82 munits/gland to 9.22 +/- 1.56 munits/gland (x +/- SEM). These results indicate that histamine released from posterior pituitary mast cells could facilitate AVP release as part of the overall mechanism for osmotic stimulation of AVP secretion and support the concept that most posterior pituitary histamine is not neuronally derived from the brain. HNMT, on the other hand, may be contained in neurons disrupted by stalk section.

Animals↗