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J Bullock

Publications and source records attributed to J Bullock.

At least 19 recordsLinked to original sources
PubMed2001

Gastrin-releasing peptide: a potential growth factor expressed in human neuroblastoma tumors(1).

PURPOSE:Gastrin-releasing peptide (GRP) is a 27-amino acid neuropeptide that has been identified in the cytoplasm of many neuroendocrine tumors. Gastrin releasing peptide has been labeled as an autocrine growth factor in small cell lung carcinomas. Recent work has also shown this to be true in the growth of neuroblastoma cells in vitro. The purpose of this study was to demonstrate GRP and its receptor (GRP-R) in resected human neuroblastomas and to correlate the presence or absence with other known predictors of poor prognosis.To demonstrate the presence of GRP and GRP-R mRNA, total RNA was extracted from human neuroblastoma cells. A reverse transcription-polymerase chain reaction (RT-PCR) was then performed using specific primers. The products of the RT-PCR were then confirmed to be GRP and GRP-R cDNA by Southern blot analysis. The RT-PCR products were then sequenced, and these sequences were compared with the know sequences of GRP and GRP-R DNA.N = 19. GRP and GRP-R mRNA were present in all neuroblastoma specimens. Although no correlation with other known predictors of poor prognosis existed, transcripts of four different sizes (400, 450, 500, and 950 bp) were seen in the GRP-R transcripts. The sequences of the 950 bp-sized transcript reverse transcription PCR products were identical to the known GRP-R.We conclude that gastrin releasing peptide and gastrin releasing peptide receptor mRNA are present in all human neuroblastomas. Although qualitatively it appears to lack prognostic significance, its ubiquitous nature in the tumor suggests it may be a useful target on which to base future treatment modalities.

Journal Article↗
PubMed2001

Mechanisms for cholesterol homeostasis in rat jejunal mucosa: effects of cholesterol, sitosterol, and lovastatin.

The effects of feeding cholesterol, sitosterol, and lovastatin on cholesterol absorption, biosynthesis, esterification, and LDL receptor function were examined in the rat jejunal mucosa. Cholesterol absorption was measured by the dual-isotope plasma ratio method; the rate-limiting enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, was measured as total and expressed enzyme activities (in the absence and presence of a phosphatase inhibitor, NaF, respectively); mucosal total and esterified cholesterol concentrations were determined by gas-liquid chromatography; LDL receptor function was assayed as receptor-mediated binding of (125)I-labeled LDL to mucosal membranes. Feeding 2% sitosterol or 0.04% lovastatin for 1 week significantly (P < 0.01) decreased the amounts of cholesterol absorbed per day (-85% and -63%, respectively). In contrast, feeding 2% cholesterol for 1 week increased the amounts of absorbed cholesterol 27-fold, even though the percent absorption significantly decreased. With all three treatments, there was a coordinate regulation of total HMG-CoA reductase activity and receptor-mediated LDL binding. Cholesterol feeding downregulated both total jejunal HMG-CoA reductase activity (P < 0.05) and receptor-mediated LDL binding (P < 0.01), whereas lovastatin- and sitosterol-supplemented diets significantly upregulated both of these parameters. In the control, cholesterol-fed, and sitosterol-fed animals, about half of the total jejunal HMG-CoA reductase activity was expressed (in functional dephosphorylated form). However, in the lovastatin-treated rats with 4-fold stimulation of HMG-CoA reductase, only 23% of the total enzyme activity was expressed. Changes in total HMG-CoA reductase activity and receptor-mediated LDL binding in all tested groups occurred with no change in total concentrations of mucosal cholesterol, and only cholesterol-fed animals had increased mucosal esterified cholesterol concentrations. Thus, in response to various fluxes of dietary or newly formed cholesterol, HMG-CoA reductase and receptor-mediated LDL binding are coordinately regulated to maintain constant cellular cholesterol concentrations in the jejunum.

Animals↗
PubMed2000

Expression and characterization of novel thrombospondin 1 type I repeat fusion proteins.

Thrombospondin (TSP)1 is a trimeric extracellular matrix protein that is held together by two cysteine residues. It is one of five TSP proteins that have been described to date with almost a universal heparin binding capability (TSP5 being the exception). The existence of two conformationally distinct structures in the TSP family (trimers and pentamers) prompted us to investigate the contribution of TSP1 trimeric structure to its inhibitory role in angiogenesis. We expressed full-length recombinant human TSP1, its type I repeats, and murine TSP3 in a human embryonic kidney cell line and evaluated their effect on human dermal microvascular endothelial cell (HMVEC) proliferation and sprouting into tube-like structures in vitro. Additionally, two chimaeric molecules were constructed so that the type I repeats of TSP1 were expressed as either dimers (TSP1-Ig chimaera) or pentamers (TSP1-TSP3 chimaera). Dimeric and pentameric type I constructs are novel structures. We found that, similarly to full-length TSP1, intact trimeric type I repeats were inhibitory to HMVEC angiogenesis in vitro. However, dimeric and pentameric type I repeats of TSP1 only partially inhibited HMVEC proliferation and sprouting in vitro. TSP3, which is lacking type I repeats, had no inhibitory activity, confirming that type I repeats elicit the anti-angiogenic activity of TSP1.

Amino Acid Sequence↗
PubMed1998

Anterior uveitis associated with latanoprost.

PURPOSE: To report the association of anterior uveitis with the use of latanoprost. METHODS: We studied four patients with complicated open-angle glaucoma who had anterior uveitis associated with the use of latanoprost. The uveitis was unilateral and occurred only in the eye receiving latanoprost in three patients. In one patient, latanoprost was used in both eyes, and the uveitis was bilateral. Four of five eyes had a history of prior inflammation and/or prior incisional surgery. All patients were rechallenged with the drug. RESULTS: The uveitis improved after cessation of latanoprost with or without topical corticosteroids. It recurred after rechallenging with latanoprost in all eyes. CONCLUSION: There is a possible association between latanoprost and anterior uveitis. Topical prostaglandin analogs may be relatively contraindicated in patients with a history of uveitis or prior ocular surgery. This association may also be possible in eyes that have not had previous uveitis or incisional surgery.

Administration, Topical↗
PubMed1998

Emmetropisation, axial length, and corneal topography in teenagers with Down's syndrome.

AIM: To study the refractive status and corneal topography in Down's syndrome. METHOD: A matched cohort subgroup of 50 individuals with Down's syndrome in the Manchester area aged 15-22 years was studied by refraction, corneal topography, A-scan biometry, slit lamp examination, and orthoptic examination. RESULTS: (1) A linear relation was found between axial length and spherical equivalent refraction. There was no statistical relation between keratometry and the axial length. (2) 80% of the group had a hyperopic refraction (mean +2.46 D, range +0.5 to +7.5 D); 18% were myopic (mean -2.75 D, range -0.5 to -8.0 D); and 2% were emmetropic (within plus or minus 0.5 D of zero). The overall mean spherical equivalent refraction was +1.43 (SD 2.86) D. 63% of eyes could see 6/12 or better and 66% of the individuals had a binocular vision of 6/12 or better. (3) Corneal topography was generally of a regular "bow tie" pattern, but there was a high incidence of oblique cylinders. Mean cylinder strength was 1.14 (1.15) D. (4) The prevalence of overt keratoconus was 2%. 6% had corneal topography with inferior steepening which may be a preclinical keratoconic process. CONCLUSIONS: In this cohort of late teenagers with Down's syndrome, emmetropisation has failed to occur in most individuals. In a similar aged group of non-disabled individuals one would expect about 83% emmetropic (plus or minus 0.25 D), 13% myopic, and 4% hyperopic. The wide spread of oblique cylinders and the small proportion of with the rule astigmatism is probably related to this failure of emmetropisation. The prevalence of 2% keratoconus in Down's syndrome compares with that found by other authors of between 5.5 and 15%. The 6% with inferior steepening on topography will be followed up over the next few years to see if there is any development of clinical keratoconus. Hence we will see if corneal topography is useful as a screening tool for preclinical keratoconus in this high risk group.

Adolescent↗
PubMed1997

Comparison of results of various methods used to determine the extent of modification of methoxy polyethylene glycol 5000-modified bovine cupri-zinc superoxide dismutase.

The protein bovine cupri-zinc superoxide dismutase (SOD) was modified by the reaction of lysine residues with an active ester of methoxy polyethylene glycol 5000 (PEG). The extent of modification was determined by capillary zone electrophoresis, matrix-assisted laser desorption/ionization mass spectrometry, Fourier-transform infrared spectroscopy, and Raman spectroscopy and after removing PEG by alkaline hydrolysis of the linkages to SOD followed by quantification of the released PEG using gel-permeation chromatography. There was generally good agreement among the results obtained by these techniques on a typical sample of the PEG-modified protein. The results, extent of protein modification, determined by the preceding methods were compared to that found from the classical trinitrobenzene sulfonic acid (TNBS) procedure. In addition to providing alternatives to the TNBS procedure, results from the described methodologies strongly suggest that the extent of modification determined from the TNBS procedure is overestimated.

Animals↗
PubMed1996

Capillary electrophoretic purity method for the novel metal chelator TMT-NCS.

A capillary electrophoretic method (CE) was developed for determining the purity of the novel metal chelator TMT-NCS. The separation of TMT-NCS from its degradation products, synthetic intermediates and by-products was accomplished using free solution CE in an aqueous-organic solvent system. This compound exhibits a complex impurity profile with the potential for over 30 degradants/impurities. The CE separation was optimized with respect to buffer type and concentration, pH, organic solvent and competitive chelator additive, allowing the resolution of all impurities in under 20 min. The specificity was established by examining stressed samples and evaluating peak purity using a diode-array detector. The sensitivity for low level impurities was optimized using sample stacking. Preliminary validation data were accumulated to support the utility of this method for estimating the purity of this drug intermediate.

2,2'-Dipyridyl↗
PubMed1995

Assay for the dianhydride of diethylenetriaminepentaacetic acid and its major degradation products by capillary electrophoresis.

DTPA-DA, the dianhydride of diethylenetriaminepentaacetic acid (DTPA), is a drug intermediate used for preparing functional derivatives of the DTPA metal chelator as well as for coupling DTPA to polypeptides, antibodies and other macromolecules. The DTPA functions as a complexing agent for various types of metal ions or radionuclides for diagnostic as well as therapeutic applications. A capillary electrophoretic (CE) method has been developed to assess the purity of DTPA-DA. The method involves derivatization of the DTPA-DA to form a diamide and subsequent complexation of the diamide with Zn2+ followed by free solution CE with UV absorbance detection at 200 nm. The procedure is capable of separating and quantitating the two major degradation products of DTPA-DA, the monoanhydride (DTPA-MA) and the free pentaacetic acid (DTPA). The method was validated with respect to linearity, precision/reproducibility, limit of detection and accuracy. This procedure was shown to be a fast and reliable method to assay this highly reactive drug intermediate.

Anhydrides↗
PubMed1995

Screening for glaucoma in a Brisbane general practice--the role of tonometry.

OBJECTIVES: (1) To pilot, and evaluate, a tonometry training intervention for general practitioners; (2) to evaluate the efficacy of three types of tonometer (Perkins, Schiotz and Tonopen) in the hands of these general practitioners; (3) to evaluate the predictive value of tonometry in screening for glaucoma in a general practice population; and (4) to evaluate the acceptability of tonometry to general practice patients. DESIGN: After being trained, three general practitioners recruited 73 of their patients over 50 years of age to attend for measurement of intraocular pressure by tonometry. Intraocular pressure was initially measured by an ophthalmologist using the Goldmann applanation tonometer, and then recorded in random order by GPs using three types of tonometer--the Perkins, Schiotz and Tonopen. SETTING: A group general practice in a middle-class suburb in southern Brisbane. OUTCOME MEASURES: (1) Comparison of measurements on the Perkins, Schiotz and Tonopen tonometers with that of the 'gold standard', the Goldmann Applanation tonometer; (2) prevalence of disc and perimetric abnormality suggestive of glaucoma among those patients with increased intraocular pressure; and (3) the acceptability of contact tonometry to general practice patients. RESULTS: There was considerable variability between intraocular values obtained across doctors and across instruments (6% to 95% of values within 4 mm of the 'gold standard'). The Schiotz tonometer provided the most uniform assessment of intraocular pressure across the groups. Nineteen of the 73 patients (26%) had intraocular pressures > or = 21 mmHg using the 'gold standard'. Of these, 18 were followed up with funduscopy, gonioscopy, repeat measurement of intraocular pressure and visual field assessment. Five had persistent elevations of intraocular pressure > or = 21 mmHg on subsequent assessment, and two had mild abnormalities of cup-disc ratio with normal fields on testing with Humphrey computerised perimetry. CONCLUSION: No one hand-held tonometer proved highly accurate in the hands of all three doctors. Even measured optimally, increased intraocular pressure alone was a poor predictor of glaucoma. Of the population screened, two patients (3%) showed evidence of mild cup-disc abnormality requiring follow-up.

Australia↗
PubMed1994

Deficient ileal 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in sitosterolemia: sitosterol is not a feedback inhibitor of intestinal cholesterol biosynthesis.

We correlated the activity of the rate-limiting enzyme of cholesterol biosynthesis, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, with the sterol content and composition of mucosal cells from the ileum of three homozygous sitosterolemic subjects and one control subject. In this inherited disease, whole-body cholesterol biosynthesis is decreased and increased amounts of sitosterol are absorbed from the intestine and deposited in tissues. For comparison, similar measurements were obtained in the ileal mucosa of sitosterol-fed rats where sitosterol accounted for 11% of enterocyte sterols. In the three sitosterolemic homozygotes, sitosterol represented 9% to 11% of the total microsomal sterols in the intestinal mucosa, although normal architecture for both crypts and villi is observed. The mean ileal microsomal HMG-CoA reductase activity in the three homozygotes was less than half of control values. In the ileum of sitosterol-fed rats with increased mucosal sitosterol concentrations, microsomal HMG-CoA reductase activity was not inhibited. These results show that in three sitosterolemic homozygotes, abnormally low HMG-CoA reductase activity was detected in the ileum, as previously demonstrated in mononuclear leukocytes and liver. The failure of the increased tissue sitosterol pool to inhibit HMG-CoA reductase in rat ileum suggests that deficient cholesterol biosynthesis in homozygous sitosterolemia is inherited and is not due to feedback inhibition by tissue sitosterol.

Adult↗
PubMed1994

The effect of increased hepatic sitosterol on the regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase and cholesterol 7 alpha-hydroxylase in the rat and sitosterolemic homozygotes.

We investigated hepatic cholesterol homeostasis in four homozygous sitosterolemic subjects from two unrelated families who showed enhanced absorption, diminished removal and increased tissue and plasma concentrations of sitosterol (24-ethyl cholesterol). Measurements of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activities were correlated with steady state messenger RNA levels and related to cholesterol 7 alpha-hydroxylase activities in the sitosterolemic homozygotes and nine controls. Similar determinations were made in rats infused intravenously with sitosterol so that hepatic and plasma sitosterol concentrations increased to about 10% of total sterols to resemble the human disease sitosterolemia. In the four sitosterolemic homozygotes, hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activities were markedly reduced (12% of normal), and steady state 3-hydroxy-3-methylglutaryl coenzyme A reductase messenger RNA levels barely detected. In contrast, hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activities and messenger RNA levels were not decreased in rats with similarly elevated hepatic sitosterol concentrations. However, hepatic cholesterol 7 alpha-hydroxylase activity was inhibited 30% in both the sitosterolemic homozygotes and rats with high liver sitosterol concentrations. Plasma cholesterol concentrations increased 120% in the sitosterol-infused rats and 29% in the untreated human homozygotes. These results demonstrate that high-tissue sitosterol concentrations do not inhibit hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase activityor steady state messenger RNA levels and that they competitively block cholesterol 7 alpha-hydroxylase activity and raise plasma cholesterol levels. Thus the deficiency of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the liver of sitosterolemic homozygotes is inherited and not due to the hepatic accumulation of sitosterol.(ABSTRACT TRUNCATED AT 250 WORDS)

Adolescent↗
PubMed1993

Capillary zone electrophoresis and packed capillary column liquid chromatographic analysis of recombinant human interleukin-4.

Capillary zone electrophoresis (CZE) and packed capillary column liquid chromatography (micro-LC) have been applied to the analysis of the recombinant human protein interleukin-4 (rhIL-4). Separations for both the parent protein and its enzymatic digest were developed for the purpose of characterizing protein purity and identity. CZE separations of the intact protein were investigated over the pH range of 4.5 to 8.0 using uncoated fused silica capillaries. Gradient reversed-phase micro-LC was performed using 0.32 mm packed capillary columns at flow-rates of 5-6 microliters/min. Emphasis was placed on the ability of these methods to separate close structural variants and degradation products of the protein. Peptide mapping of the tryptic digest of rhIL-4 using a combination of CZE and micro-LC provided complimentary high resolution methods for establishing protein identity. Reproducible separations were achieved using sub-picomol amounts of sample. The advantages and problems encountered with these two techniques for characterizing rhIL-4 were assessed.

Capillary Action↗
PubMed1993

Microvascular clearance of macromolecules in skeletal muscle of spontaneously diabetic rats.

Microvascular clearance of FITC-Dextran 150 (fluorescein isothiocyanate dextran, MW 150,000) was studied in cremaster muscles of control (BB/W-R) and diabetic (BB/W-DM) rats following daily injections of a full (FD) or a 1/2 (reduced; RD) dose of insulin. The cremaster muscle was placed in an intravital chamber and superfused with bicarbonate buffer (pH 7.4, equilibrated with 95% N2-5% CO2). A 1-hour period of stabilization was followed by the i.v. injection of FITC-dextran 150 and an equilibration period of 45 min. Suffusate samples were collected for 1 h for control measurements. Following this period, bradykinin was applied topically at a concentration of 10(-7) M. Samples were collected for a final hour for the assessment of clearance. The mean +/- SEM FITC-dextran 150 clearance values (microliter/60 min/g) for BB/W-R, BB/W-DM FD, and BB/W-DM RD were 11.5 +/- 1.8, 14.8 +/- 3.4, and 90.5 +/- 12.0, respectively. The corresponding values after topical application of 10(-7) M bradykinin were 23.7 +/- 5.9, 24.2 +/- 4.4, and 98.7 +/- 25.0. Our results indicate that bradykinin evokes a twofold increase in FITC-dextran 150 clearance in the BB/W-R (control) animals and in the BB/W-DM rats receiving full-dose insulin. In contrast, bradykinin does not further enhance the eightfold increase in FITC-Dextran 150 clearance observed in the reduced-insulin dose-treated BB/W-DM group. Thus, our data show that insulin, administered at a full dose, protects the functional integrity of microvascular perm-selectivity in diabetes mellitus.

Administration, Topical↗
PubMed1992

Characterization of the heterogeneity of polyethylene glycol-modified superoxide dismutase by chromatographic and electrophoretic techniques.

Covalent attachment of polyethylene glycol (PEG) chains to the enzyme Cu,Zn-superoxide dismutase (SOD) produces a heterogeneous mixture of modified protein species. The heterogeneity of the product (PEG-SOD) derives from a variable stoichiometric combination of PEG with individual SOD molecules in addition to the polydispersity of the PEG reagent. Characterization of PEG-SOD presents significant challenges due in part to this heterogeneity in addition to the hybrid nature of the modified enzyme. The application of classical methods of protein characterization is not always successful for these PEG-proteins requiring the development of alternative or modified procedures. A series of chromatographic techniques including reversed-phase, ion-exchange, size-exclusion, and hydrophobic interaction high-performance liquid chromatography along with electrophoretic techniques including isoelectric focusing, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and capillary zone electrophoresis have been developed for assessing the degree of heterogeneity of PEG-SOD samples which encompass a range of different stoichiometries. Examples will be given demonstrating the application of these techniques to characterize PEG-SOD samples of different composition produced during the course of the reaction between SOD and an activated PEG reagent.

Animals↗
PubMed1992

Infra red coagulation for bleeding mucosal telangiectasia.

The technique of infra red coagulation is well suited to the destruction of superficial blood vessels in the skin and/or mucosal surfaces. A method is described here for the destruction of resistant bleeding telangiectasia of the palate and lip in Osler-Weber-Rendu syndrome.

Diathermy↗
PubMed1991

The SEC receptor recognizes a pentapeptide neodomain of alpha 1-antitrypsin-protease complexes.

Formation of the covalently stabilized alpha 1-antitrypsin (alpha 1-AT)-neutrophil elastase complex, the archetype of serpin-enzyme complexes, results in a structurally rearranged alpha 1-AT molecule that possesses chemo-attractant activities, mediates an increase in synthesis of alpha 1-AT by mononuclear phagocytes and hepatocytes, and is more rapidly cleared from the circulation than is the native alpha 1-AT molecule. We have recently identified an abundant, high affinity cell surface receptor on human hepatoma HepG2 cells and human monocytes that binds alpha 1-AT-elastase complexes, mediates endocytosis and lysosomal degradation of alpha 1-AT-elastase complexes, and induces an increase in synthesis of alpha 1-AT. We have referred to this receptor as the serpin-enzyme complex, or SEC, receptor because it also recognizes complexes of serpins antithrombin III, alpha 1-antichymotrypsin, and C1 inhibitor with their cognate enzymes. In the current study, we show that a pentapeptide domain in the carboxyl terminal fragment of alpha 1-AT (amino acids 370-374, FVFLM) is sufficient for binding to the SEC receptor. A synthetic analog of this pentapeptide (peptide 105C, FVYLI) blocks binding and internalization of alpha 1-AT-125I-trypsin complexes by HepG2 cells. 125I-Peptide 105C binds specifically and saturably to HepG2 cells, and its binding is blocked by alpha 1-AT-trypsin or alpha 1-AT-elastase complexes. Alterations of this sequence introduced into synthetic peptides (mutations, deletions, or scrambling) demonstrate that binding of the pentapeptide domain is sequence-specific. Comparisons with the sequences of other serpins in the corresponding region indicate that this pentapeptide neodomain is highly conserved.

Amino Acid Sequence↗