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Biomedical subjects

B O'Connor

Publications and source records attributed to B O'Connor.

At least 37 records · Page 2Linked to original sources
PubMed1998

Inhaled corticosteroids increase interleukin-10 but reduce macrophage inflammatory protein-1alpha, granulocyte-macrophage colony-stimulating factor, and interferon-gamma release from alveolar macrophages in asthma.

We determined the effect of inhaled corticosteroid, budesonide, on the release of the anti-inflammatory cytokine, interleukin-10 (IL-10), and of pro-inflammatory cytokines, macrophage inflammatory protein-1alpha (MIP-1alpha), interferon-gamma (IFN-gamma), and granulocyte-macrophage colony-stimulating factor (GM-CSF), from blood monocytes and alveolar macrophages of mild asthmatic subjects in a double-blind, cross-over, placebo-controlled study. Budesonide reduced bronchial hyperresponsiveness and improved baseline FEV1. Alveolar macrophages were obtained by bronchoalveolar lavage performed at the end of each treatment phase. IL-10 from blood monocytes was not altered, but both IL-10 mRNA and protein expression from alveolar macrophages stimulated by lipopolysaccharide and IL-1beta were increased after corticosteroid therapy. By contrast, alveolar macrophages released significantly less MIP-1alpha, IFN-gamma, and GM-CSF after steroid treatment. In comparison to alveolar macrophages from normal nonasthmatic volunteers, those from asthmatic patients released more MIP-1alpha, IFN-gamma, and GM-CSF but lower amounts of IL-10 particularly at baseline and after IL-1beta stimulation. The ability of steroids to inhibit pro-inflammatory cytokines but to enhance the anti-inflammatory cytokine such as IL-10 may contribute to their beneficial actions in asthma. Asthma is characterized by alveolar macrophages exhibiting both an enhanced capacity to release pro-inflammatory cytokines and a reduced capacity to produce IL-10.

Administration, Inhalation↗
PubMed1997

Proline specific peptidases.

Proline is unique among the 20 amino acids due to its cyclic structure. This specific conformation imposes many restrictions on the structural aspects of peptides and proteins and confers particular biological properties upon a wide range of physiologically important biomolecules. In order to adequately deal with such peptides, nature has developed a group of enzymes that recognise this residue specifically. These peptidases cover practically all situations where a proline residue might occur in a potential substrate. In this paper we endeavour to discuss these enzymes, particularly those responsible for peptide or protein hydrolysis at proline sites. We have detailed their discovery, biochemical attributes and substrate specificities and have provided information as to the methodology used to detect and manipulate their activities. We have also described the roles, or potential roles that these enzymes may play physiologically and the consequences of their dysfunction in varied disease states.

Amino Acid Sequence↗
PubMed1997

The development of two fluorimetric assays for the determination of pyroglutamyl aminopeptidase type-II activity.

Two fluorimetric assays for the determination of pyroglutamyl aminopeptidase type-II activity have been developed. The assays are based on hydrolysis of the quenched-fluorimetric substrate <Glu-His-Pro-7-amino-4-methylcoumarin. Following the removal of the N-terminal <Glu by pyroglutamyl aminopeptidase type-II, liberation of 7-amino-4-methylcoumarin from the metabolite His-Pro-7-amino-4-methylcoumarin is catalyzed by one of two methods: (i) the addition of partially purified bovine serum dipeptidyl aminopeptidase type-IV or (ii) by incubating the reaction mixture for up to 2 h at 80 degrees C, thus promoting the nonenzymatic cyclization of His-Pro-7-amino-4-methylcoumarin to cyclo His-Pro and free 7-amino-4-methylcoumarin. Pyroglutamyl aminopeptidase type-II from bovine brain is used to establish appropriate assay conditions. These fluorimetric assays offer expeditious alternatives to the existing radiolabeled thyrotropin-releasing hormone assays for the determination of PAPII activity.

Aminopeptidases↗
PubMed1997

Intracellular location of thymidylate synthase and its state of phosphorylation.

Thymidylate synthase (TS), an enzyme that is essential for DNA synthesis, was found to be associated mainly with the nucleolar region of H35 rat hepatoma cells, as determined both by immunogold electron microscopy and by autoradiography. In the latter case, the location of TS was established through the use of [6-3H]5-fluorodeoxyuridine, which forms a tight ternary complex of TS with 5-fluorodeoxyuridylate (FdUMP) and 5, 10-methylenetetrahydrofolylpolyglutamate within the cell. However, with H35 cells containing 50-100-fold greater amounts of TS than unmodified H35 cells, the enzyme, although still in the nucleus, was located primarily in the cytoplasm as shown by autoradiography and immunohistochemistry. In addition, TS was also present in mitochondrial extracts of both cell lines, as determined by enzyme activity measurements and by ternary complex formation with [32P]FdUMP and 5,10-methylenetetrahydrofolate. Another unique observation is that the enzyme appears to be a phosphoprotein, similar to that found for other proteins associated with cell division and signal transduction. The significance of these findings relative to the role of TS in cell division remains to be determined, but suggest that this enzyme's contribution to the cell cycle may be more complex than believed previously.

Animals↗
PubMed1997

Identification and initial characterisation of a N-benzyloxycarbonyl-prolyl-prolinal (Z-Pro-prolinal)-insensitive 7-(N-benzyloxycarbonyl-glycyl-prolyl-amido)-4-methylcoumarin (Z-Gly-Pro-NH-Mec)-hydrolysing peptidase in bovine serum.

A group of enzymes exists that specifically recognises proline within proteins and peptides. Prolyl endopeptidase is one such enzyme, which cleaves on the carboxyl side of proline within peptide substrates. Its broad specificity towards bioactive peptides has led to its implication in various disease states including neurodegenerative and psychiatric disorders. This association has been based primarily on the abnormal levels of activity observed following the enzymes detection with the reportedly specific fluorimetric substrate, 7-(N-benzyloxycarbonyl-glycyl-prolyl-amido)-4-methylcoumarin (Z-Gly-Pro-NH-Mec). In this study, we report the discovery and preliminary characterisation of a Z-Gly-Pro-NH-Mec-hydrolysing activity that is distinct from prolyl oligopeptidase (prolyl endopeptidase). Following the production of serum from bovine whole blood, Z-Gly-Pro-NH-Mec hydrolysis in serum was determined to be 7.2 U/mg protein. In the presence of 350 nM Z-Pro-prolinal, a specific inhibitor of prolyl endopeptidase, residual Z-Gly-Pro-NH-Mec hydrolysis of 2.6 U/mg protein was observed. This residual activity was resistant to inhibition by Z-Pro-prolinal at concentrations in excess of 200 times its reported Ki value for prolyl endopeptidase and could not be inhibited under conditions of prolonged incubation with the inhibitor. Following cation-exchange chromatography, Z-Gly-Pro-NH-Mec-hydrolysing activity was resolved into two distinct entities. The first of these activities was inhibited by Z-Pro-prolinal, demonstrated activity towards the thyroliberin analogue, 7-(pyroglutamyl-histidyl-prolyl)-4-methylcoumarin (Glp-His-Pro-NH-Mec), and was catalytically enhanced under reduced assay conditions. This activity was subsequently designated prolyl endopeptidase. The second activity was totally resistant to Z-Pro-prolinal inhibition, demonstrated no activity towards Glp-His-Pro-NH-Mec, and was unaffected when assayed under reduced conditions. It was subsequently designated Z-Pro-prolinal-insensitive Z-Gly-Pro-NH-Mec-hydrolysing peptidase (ZIP).

Animals↗
PubMed1997

Periosteal new bone formation in a canine neuropathic model of osteoarthritis.

OBJECTIVE: To characterize, for the first time, periosteal new bone formation in a well-established canine model of accelerated osteoarthritis (OA) with features of neuropathic arthropathy. METHODS: Seven dogs underwent left L4-S1 dorsal root ganglionectomy (DRG), followed 3 weeks later by transection of the anterior cruciate ligament of the ipsilateral knee (ACLT). Eight weeks thereafter, a postmortem examination was performed to assess the severity of cartilage changes of OA and the formation of new bone on the distal femur and proximal tibia in the cruciate-deficient limb. RESULTS: As described previously, extensive full-thickness ulceration of the articular cartilage was present in the unstable knee of every dog. The femoral shaft immediately proximal to the condyles in the unstable limb was consistently wider (mean +/- SD diameter 22.4 +/- 2.2 mm) than that in the contralateral limb (19.9 +/- 1.3 mm; P = 0.01). Xeroradiography and histologic examination of the distal femur revealed extensive formation of woven bone on the periosteal surfaces of the medial, lateral, and anterior aspects of the femoral shaft in the OA limb of every dog. These bony changes were not seen in radiographs of dogs that underwent DRG with the cruciate ligament left intact (n = 8) or of neurologically intact dogs that underwent ACLT (n = 7) and were examined 24 weeks after surgery. CONCLUSION: Formation of new periosteal bone on the distal femur and tibia is a feature of this model of accelerated OA that is not seen in the conventional ACLT model of OA in the neurologically intact dog. This observation suggests that interruption of sensory input from the limb may affect the regulation of osteogenesis in the mechanically unstable joint.

Animals↗
PubMed1997

Variant cDNA sequences of human ATP:citrate lyase: cloning, expression, and purification from baculovirus-infected insect cells.

ATP:citrate lyase (ACL) is a major generator of cytosolic acetyl-coenzymeA, which is required for both fatty acid and cholesterol biosynthesis. The human ACL (hACL) cDNA was cloned by RT-PCR, and our results indicate the existence of previously unknown sequence variations in hACL. Expression of the hACL cDNA in Spodoptera frugiperda 9 insect cells resulted in the production of high levels of soluble, active enzyme. The recombinant protein (re-hACL) was purified to homogeneity from the soluble lysate of infected cells and was observed to exist as a tetramer by gel filtration chromatography. Kinetic analyses indicated that the re-hACL and rat ACL have very similar enzymological properties. The facile preparation of milligram quantities of purified, active re-hACL affords the opportunity to characterize the enzyme for structure-based design of hypolipidemic drugs, and to further examine the functional significance of the sequence variations.

ATP Citrate (pro-S)-Lyase↗
PubMed1997

Nasal nitric oxide is increased in patients with asthma and allergic rhinitis and may be modulated by nasal glucocorticoids.

Nitric oxide (NO) is produced in large amounts in the noses of normal individuals. We have measured NO by chemiluminescence in the noses and exhaled air of subjects with symptomatic allergic rhinitis, some of whom had concomitant asthma, during the pollen season and compared this with values measured in normal subjects and in patients treated with nasal and/or inhaled glucocorticoids. We found that nasal levels of NO were significantly (p < 0.001) elevated in patients with untreated rhinitis (1527 +/- 87 ppb, n = 12) compared with normal individuals (996 +/- 39 ppb, n = 46) or subjects treated with nasal steroids (681 +/- 34 ppb, n = 10), whereas exhaled NO in patients with untreated rhinitis was similar to that in normal subjects (10 +/- 2 ppb vs 7 +/- 0.6 ppb, respectively). In five subjects who were nasally challenged with allergen, there was a significant decrease in nasal NO 1 hour after challenge, and this was significantly correlated with increased rhinitis symptoms. In patients with rhinitis and concomitant asthma, nasal NO was also significantly elevated (1441 +/- 76 ppb, n = 16) but not when they were treated with nasal or inhaled steroids; whereas exhaled NO was elevated in untreated patients and in patients treated with nasal, but not inhaled, steroids. Our data suggest that the increase in exhaled NO in patients with allergic rhinitis is likely to be due to increased local production, caused by long-term exposure to allergen, which is suppressed by locally administered steroids. Measurement of nasal NO may be useful to study the inflammatory response in rhinitis and its response to antiinflammatory treatments.

Administration, Inhalation↗
PubMed1997

Effects of diacerhein in an accelerated canine model of osteoarthritis.

OBJECT: To determine whether diacerhein has a disease-modifying effect in an accelerated canine model of osteoarthritis. DESIGN: Fourteen adult mongrel dogs underwent unilateral L4-S1 dorsal root ganglionectomy (DRG), followed 3 weeks later by ipsilateral anterior cruciate ligament transection. Seven dogs received diacerhein (15-20 mg/kg) daily throughout the interval between DRG and sacrifice, eight weeks after ligament transection. The other seven dogs served as OA controls. RESULTS: The mean volume of synovial fluid obtained from the OA knee of the diacerhein-treated dogs was approximately 40% less than that from the OA knee of the controls. In addition, diacerhein appeared to reduce the severity of fibrillation (femoral condyle) and full-thickness ulceration (trochlear ridge) of the articular cartilage and the level of collagenase activity in extracts of the OA cartilage, and to increase net PG synthesis in the OA cartilage, although none of the above changes were statistically significant. CONCLUSION: The differences between the diacerhein group and untreated OA controls, even though not statistically significant, suggest that diacerhein was active in this rapidly progressive model of OA. Because changes associated with initiation of OA may be different than those associated with progression, whether diacerhein has a disease-modifying effect should be examined in a less rapidly progressive model.

Animals↗
PubMed1997

A prospective study of the role of lipoprotein(a) in the pathogenesis of unstable angina.

AIM: Lipoprotein(a) is considered to be a risk factor for atherothrombotic diseases. The aim of the study was to examine the potential role of lipoprotein(a) concentrations in patients admitted with unstable angina with particular reference to their cardiac Troponin T concentrations, which identifies a subgroup at high risk of subsequent cardiac events. METHODS AND RESULTS: Consecutive patients admitted with chest pain to a single coronary care unit were studied. One hundred and sixty-seven patients had a final diagnosis of unstable angina, of whom 56 were cardiac Troponin T-positive. Admission lipoprotein(a) concentrations were significantly higher in the cardiac Troponin T-positive unstable angina group as compared to the cardiac Troponin T-negative unstable angina group: median and interquartile ranges 22.25 mg.dl-1 (6.25, 32.0) vs 6.0 mg.dl-1 (2.22, 14.8) respectively) P = 0.0004. A highly significant correlation was also found between the level of cardiac Troponin T at diagnosis and the lipoprotein(a) concentration: rs = 0.2798, P = 0.0001. CONCLUSION: This study provides the first evidence in man of a significant role for lipoprotein(a) in unstable angina. The correlation between lipoprotein(a) concentration and cardiac Troponin T concentration suggests that lipoprotein(a) may be significantly involved in the early failure of plaque rupture stabilization.

Adult↗
PubMed1997

Bone scintigraphy in the canine cruciate deficiency model of osteoarthritis. Comparison of the unstable and contralateral knee.

OBJECTIVE: To examine the effects on 99mTc medronate (MDP) bone scintigraphy of unilateral anterior cruciate ligament (ACL) transection in the unstable and contralateral knee of dogs. METHODS: Bone scintigraphy was performed in 5 dogs at baseline and 6 and 12 weeks after ligament transection, with images of both knees obtained 3-4 h after intravenous injection of the radionuclide. An image of the T4-T5 vertebral region was also acquired as an internal standard. In 3 additional dogs, scintigraphy was performed at baseline and 5 1/2 weeks after sham ACL transection. RESULTS: A marked increase in uptake of 99mTc MDP was seen in the unstable knee. In contrast, in all but one case uptake in the contralateral knee did not increase significantly. Sham ligament transection had no effect on uptake of the radionuclide. CONCLUSION: Changes occur in subchondral bone as well as in articular cartilage in the unstable knee in this model. The bony changes, which could be either primary or secondary to early changes in the biomechanical properties of the cartilage, may contribute to the pathogenesis of cartilage damage. No appreciable effects on bone turnover in the contralateral knee could be proved in this study.

Animals↗
PubMed1996

Ileal pouch-anal anastomosis: pregnancy, delivery and pouch function.

OBJECTIVE: To evaluate the pregnancies, deliveries and functional results of patients who have undergone and ileal pouch-anal anastomosis. DESIGN: A retrospective survey by questionnaire. SETTING: The study was conducted at a university hospital. SUBJECTS: Twelve women who had undergone an ileal pouch-anal anastomosis at the Inflammatory Bowel Disease Centre, Mount Sinai Hospital and who had subsequently become pregnant, were identified from the hospital records. A follow up questionnaire was completed by all subjects. RESULTS: Twelve patients had 16 deliveries. There were 10 vaginal deliveries and 6 caesarian sections. There were no pouch-related complication during the 16 pregnancies and there were two postpartum complications. CONCLUSIONS: Pregnancy is safe in women with an ileal pouch-anal anastomosis. Functional results are altered minimally. Vaginal delivery is safe and obstetric reasons should determine whether a caesarian section is performed.

Adolescent↗
PubMed1996

Purification and characterization of a novel membrane-bound form of prolyl endopeptidase from bovine brain.

Prolyl endopeptidase has been predominantly described as a cytosolic activity capable of cleaving a number of important neuropeptides (including TRH, LHRH, Bradykinin, Angiotensin, Substance P, Neurotensin, Oxytocin and Vasopressin) on the carboxy side of proline. In this paper, we report, for the first time, on the complete purification and characterization of a membrane-bound form of prolyl endopeptidase. This novel activity has been isolated from the synaptosomal (plasma membranes) membranes of bovine brain. Following gel filtration, hydroxylapatite and hydrophobic interaction chromatographies, the prolyl endopeptidase activity was purified 1400-fold with a 23% recovery of activity. The enzyme was shown to have a relative molecular mass of 87 kDa and a Km of 60 microM for its specific fluorimetric substrate, Z-GlyProMCA. The purified enzyme demonstrated a relatively broad substrate specificity and a relatively high affinity for proline-containing neuropeptides. It was shown to be inhibited by certain thiol-protease inhibitors and by the metal chelator, 1,10-phenanthroline, thus possibly classifying it as a 'thimet' activity. The purified particular form of proyl endopeptidase displayed a similar substrate specificity to the previously reported cytosolic forms of the enzyme. However, there were differences between the two forms in term of their sensitivity to inhibitors, their affinities for the peptide substrates and their relative molecular masses. The different subcellular location (i.e. the synaptosomal membrane) of the particulate prolyl endopeptidase is also of potential physiological significance given that here it is more likely to come in contact with the vesicle-bound neuropeptides than is its cytosolic counterpart.

Animals↗
PubMed1996

Bovine brain pyroglutamyl aminopeptidase (type-1): purification and characterisation of a neuropeptide-inactivating peptidase.

Pyroglutamyl aminopeptidase type-1 (PAP-I) is reported to be a soluble, broad specificity aminopeptidase, capable of removing the pyroglutamic acid (pGlu) residue from the amino terminus of pGlu-peptides (e.g. TRH, LHRH, neurotensin and bombesin). The central aim of this study was to undertake, for the first time, the complete purification and characterisation of a PAP activity observed within the cytosolic fraction of bovine whole brain and to compare the properties of the enzyme with previous findings. A series of chromatographic steps (DEAE-Sepharose, Sephacryl S-200 and Activated Thiol Sepharose 4B) generated a soluble PAP activity purified to near homogeneity with a total active yield of 6.6% The enzyme displayed a native molecular mass of approximately 23,700 Da, which compares well with that value obtained under denaturing conditions via SDS-PAGE (24,000 Da), suggesting that the enzyme exists as a monomer. The expression of PAP activity displayed an absolute requirement for the presence of a disulphide bond-reducing agent such as DTT, whilst optimum activity was observed at pH 8.5. strong inhibition of PAP activity was observed with a number of different agents, including transition metal ions, sulphydryl-blocking agents and 2-pyrrolidone (a pGlu analog). A broad pyroglutamyl substrate specificity, which excludes substrates commencing with the pGlu-Pro bond, was also demonstrated for the bovine brain enzyme. Based on a comparison of these findings with those reported for PAP-I in other mammalian tissues, the soluble PAP activity observed in bovine whole brain can tentatively be classified as a pyroglutamyl aminopeptidase type-1 (EC 3.4.19.3).

Animals↗
PubMed1996

Nutritional status and function in patients following Whipple procedure compared with controls.

OBJECTIVE: Despite the potential for nutritional deficits in patients undergoing pancreaticoduodenectomy or Whipple procedure, long-term assessment of nutritional status has largely been ignored. This study assessed nutritional status of 24 Whipple patients compared with matched post-cholecystectomy controls. METHODS: Clinical assessment was by subjective global assessment, body composition was assessed by bioelectric impedance analysis and functional assessment was by respiratory muscle strength and skeletal muscle function performed by electrical stimulation of the ulnar nerve of the wrist and hand-grip dynamometry. RESULTS: Whipple patients studied 4.6+/-0.7 years since surgery and controls (4.8+/-0.7 years since surgery) were all judged clinically to be in a good nutritional state. Compared with controls, Whipple patients had significantly lower body weight (Whipple: 72.5+/-2.8 kg, control: 83.9+/-3.3 kg, p<0.05) however, the mean body weight of both Whipple and controls was above ideal weight (Whipple: 113.3+/-4.3%, control: 122.3+/-3.7% p = NS). No significant differences in functional performance were observed between groups. Energy intake of Whipple and controls was also comparable. In the Whipple group, neither the extent of gastric resection or the pathological diagnosis had an effect on the nutritional parameters studied. CONCLUSIONS: Long-term follow-up of patients having undergone Whipple procedure failed to reveal the presence of any nutritional or functional deficits suggesting that a full nutritional recovery is possible after this surgery.

Aged↗