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Biomedical subjects

P Manning

Publications and source records attributed to P Manning.

At least 19 recordsLinked to original sources
PubMed1998

Superoxide electrode based on covalently immobilized cytochrome c: modelling studies.

We have recently described an optimised electrode for the detection of enzymatic and cellular superoxide (O2*-) production based on cytochrome c immobilized covalently at a surface-modified gold electrode and applied this system to the study of free radical production by activated human glioblastoma cells. In this paper we report the development of a mathematical model for the O2*- electrode responding to enzymically produced O2*- which should enable the determination of absolute concentrations of O2*- in biological systems when this electrode is employed for direct, real-time monitoring of free radical release and interactions.

Cytochrome c Group↗
PubMed1998

Reduced progression of experimental osteoarthritis in vivo by selective inhibition of inducible nitric oxide synthase.

OBJECTIVE: To evaluate the in vivo therapeutic efficacy of N-iminoethyl-L-lysine (L-NIL), a selective inhibitor of inducible nitric oxide synthase (iNOS), on the progression of lesions in an experimental osteoarthritis (OA) dog model. The effect of L-NIL on metalloprotease activity, levels of interleukin-1beta (IL-1beta), prostaglandin E2 (PGE2), and nitrite/nitrate in synovial fluid was determined. METHODS: The OA model was created by sectioning the anterior cruciate ligament of the right stifle joint of mongrel dogs by a stab wound. Dogs were separated into experimental groups: Group 1 was made up of unoperated dogs that received no treatment, group 2 were operated dogs with no treatment, and group 3 were operated dogs that received oral L-NIL (10 mg/kg/twice daily) starting immediately after surgery. The OA dogs were killed at 10 weeks after surgery. RESULTS: Experiments showed that dog OA cartilage explants in culture produced an increased amount of NO (nitrite). Immunohistochemical study demonstrated that this was due to an increased level of iNOS in chondrocytes. OA dogs treated with L-NIL showed a reduction in the incidence of osteophytes compared with the untreated OA dogs (58% versus 92%) as well as in their size (mean +/- SEM 1.92 +/- 0.58 mm versus 5.08 +/- 0.66 mm). Macroscopically, L-NIL decreased the size of the cartilage lesions by approximately 50% both on condyles and plateaus. The histologic severity of both the cartilage lesions and synovial inflammation was significantly decreased in the L-NIL-treated dogs. Treatment with L-NIL also significantly decreased both collagenase and general metalloprotease activity in the cartilage and the levels of IL-1beta, PGE2, and nitrite/nitrate in synovial fluid. CONCLUSION: This study demonstrated the effectiveness of a selective inhibitor of iNOS, L-NIL, in attenuating the progression of experimental OA. The data suggest that L-NIL may act by reducing the activity of metalloproteases in cartilage and the production of IL-1beta by synovium, both of which are known to play a major role in the pathophysiology of OA structural changes.

Animals↗
PubMed1998

Direct, real-time sensing of free radical production by activated human glioblastoma cells.

Primary brain injury initiates a cascade of events which result in secondary brain damage. Although, at present, the biochemical and molecular mechanisms of nerve cell death are not well understood, sufficient evidence now exists to implicate free radicals in this brain injury response. In the light of the current understanding on the role of free radicals in cell mortality, we report on the use of two specific sensors, which we use to measure the direct, simultaneous and real time electrochemical detection of both superoxide (O2.-) and nitric oxide (NO), produced by activated glioblastoma cells. The development and application of these novel methods has enabled us to show that both the cytokine-mediated induction of the enzymes responsible for the generation of these radical species, and the metabolic requirements of the cell can modulate cell messenger release. Importantly, the data collected provides dynamic information on the time course of free radical production, as well as their interactions and their involvement in the process of cell death. In particular, one of the major advances afforded by this technology is the demonstration that suppression of one of either of the two cellular generated radical species (NO and O2.-) leads directly to a corresponding increase in the species that was not being deliberately inhibited or scavenged. This finding may indicate a mechanism involving inter-enzyme regulation of free radical production in glial cells (a phenomenon which may, in future, also be shown to operate in other relevant cell models).

Biosensing Techniques↗
PubMed1997

Low vitamin D status is common among elderly Dunedin women.

AIMS: To review vitamin D status and the relationship of serum 25-hydroxyvitamin D levels to hip bone mineral density in a group of healthy elderly women living independently in their own homes in Dunedin. METHODS: Thirty-eight elderly subjects (> 70 years of age) were studied. Serum levels of 25-hydroxyvitamin D (25(OH)D) were measured by radioimmunoassay in summer and winter. Femoral neck bone mineral density was measured by dual x-ray energy absorptiometry. RESULTS: Hip density was correlated with serum 25(OH)D levels at study entry. In summer, 10 of 38 patients (26.3%) had serum 25(OH)D levels below the reference range for healthy adults (40-185 nmol/L). Six patients subsequently withdrew from the study. In winter, 22 of the remaining 32 women (68.8%) had serum 25(OH)D values below the reference range. Subjects with low 25(OH)D values were given halibut oil tablets (400 IU vitamin D3 per day) to improve their serum 25(OH)D levels. CONCLUSIONS: Vitamin D deficiency is common among elderly women with a high risk of fracture who live in southern New Zealand. This is most marked in the winter months. Vitamin D replacement is cheap and effective and should be considered in patients over 70 years of age who have a high risk of fracture and who live in temperate climates.

Absorptiometry, Photon↗
PubMed1997

Gender differences in body fat content are present well before puberty.

To determine whether gender differences in body fat could be detected in prepubertal children using dual energy X-ray absorptiometry (DEXA), body composition was measured in 20 healthy boys aged 3-8 y matched for age, height and weight with 20 healthy girls. Although boys and girls did not differ in age, height, weight, body mass index (BMI) or bone mineral content, the boys had a lower percentage of body fat (13.5 +/- 5.1 vs 20.4 +/- 6.1%, P < 0.01), a lower fat mass (3.2 +/- 2.0 vs 4.9 +/- 3.1 kg, P < 0.01), and a higher bone-free lean tissue mass (18.6 +/- 4.3 vs 17.0 +/- 3.5 kg, P < 0.01) than the girls. Girls had approximately 50% more body fat than the boys. This is the first DEXA study to show that boys aged 3-8 y have less body fat than girls of similar age, height and weight. Thus, this technology demonstrates that significant gender differences in body composition are evident, well before the onset of puberty.

Absorptiometry, Photon↗
PubMed1996

Tyrosinase may protect human melanocytes from the cytotoxic effects of the superoxide anion.

Reactive oxygen species (ROS) are potentially cytotoxic and several mechanisms have evolved to protect against their damaging effects. In melanocytes, tyrosinase may have such a rôle by utilising the superoxide anion (O2-) in the production of melanin. In the present study, we have examined the cytotoxic effects of O2- and hydrogen peroxide (H2O2) in human melanocytes both before and following the activation of tyrosinase. Xanthine oxidase (XO, 5-150 mU.ml-1) and glucose oxidase (GO, 0.1-20 mU.ml-1) were used to generate the O2- and H2O2 respectively, and the cytotoxic effects assessed by measuring cell survival using the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium (MTT) assay. 3 h later, dose-related decreases in melanocyte survival were seen. Similar experiments with keratinocytes and fibroblasts showed that these cells were more resistant to the cytotoxic effects of O2- than were the melanocytes. The effect of increasing tyrosinase activity was examined by growing the melanocytes in the presence of an analogue of melanocyte-stimulating hormone (MSH) Nle4DPhe7 alpha-MSH (10(-8) M), for 48 h. This increased tyrosinase activity, melanin content, the ability to trap O2- and the resistance of the melanocytes to the cytotoxic effects of this ROS, but failed to alter their susceptibility to the damaging effects of H2O2.Nle4DPhe7 alpha-MSH had no effect on the resistance of keratinocytes and fibroblasts to either O2- or H2O2. After 3 h, XO, as opposed to GO, also increased the melanin content of human melanocytes; this effect was not accompanied by an increase in tyrosinase activity. The present results suggest that tyrosinase may utilise O2- to produce melanin and that this process may protect melanocytes from the potentially damaging effects of this ROS.

Cell Survival↗
PubMed1996

Activation of tyrosinase reduces the cytotoxic effects of the superoxide anion in B16 mouse melanoma cells.

Tyrosinase may protect against oxidative stress by using the superoxide anion (O2-1.) in the production of melanin. We have examined this by comparing its cytotoxic effects in B16/F10 and B16/F10-differential deficient (-DD) mouse melanoma cells that express high and low levels of tyrosinase activity respectively. Xanthine oxidase (XO) was used to generate O2.1 and cytotoxicity assessed by measuring cell survival. XO increased O2.- concentrations and 3 h later dose related decreases in cell survival were seen. F10 cells were more resistant to these cytotoxic effects than the F10-DD cells. [Nle4, DPhe7]MSH increased tyrosinase activity and melanin content, reduced O2.- concentration and increased the resistance of F10 cells to the cytotoxic effects of O2.-. No such effects were seen in F10-DD cells. The effect of [Nle4, DPhe7]MSH on the resistance of the F10 cells was time-dependent and noticeable when tyrosinase activity but not melanin was increased. This suggests that it was the activation of tyrosinase rather than the increase in the melanin that provided the protection against O2.-. In support of this, inhibition of tyrosinase with phenylthiocarbamide reduced the increased resistance induced by [Nle4, DPhe7]MSH. Moreover, although melanin was capable of scavenging O2.- it had little effect at concentrations comparable to those in the activated F10 cells. XO also increased the melanin content of F10 but not F10-DD cells. We conclude that tyrosinase is able to utilise O2.- to produce melanin and this provides pigment cells with a unique anti-oxidant mechanism.

Animals↗
PubMed1996

Parathyroid hormone induces superoxide anion burst in the osteoclast: evidence for the direct instantaneous activation of the osteoclast by the hormone.

We have shown that superoxide anion (O2-) production by the osteoclast can be used as an index of the osteoclast activity since the agents that inhibit and stimulate the osteoclast also diminish and stimulate O2- production respectively. Therefore, we have investigated the mechanism of parathyroid hormone (PTH)-mediated stimulation of osteoclast function in terms of its effect on O2- generation. The determination of O2- generation was carried out by employing cytochrome c immobilised on a surface-modified gold electrode. The basal level of free radical production by the osteoblast-like cells (ROS 17/2.8) was 10(4)-fold lower than by osteoclasts cultured on bone. PTH had no acute effect on free radical production by the osteoblasts. The exposure of the osteoclasts cultured on bone to PTH led to a dramatic and immediate stimulation of O2- generation which was unaffected by the presence of ROS 17/2.8 cells. The osteoclasts co-cultured with ROS 17/2.8 cells and exposed to PTH for 3 h were also found to produce greater stimulation of O2- than the osteoclasts exposed to PTH alone. A competitive leukotriene D4 antagonist REV 5901, which also inhibits 5-lipoxygenase, did not block O2- generation by osteoclasts cultured alone or in the presence of osteoblasts. Therefore, we conclude that PTH directly stimulates osteoclasts to produce O2-; this may be the main mode of activation of the osteoclasts, although an osteoblast-mediated effect of the hormone cannot be ruled out.

Animals↗
PubMed1995

L-N6-(1-iminoethyl)-lysine potently inhibits inducible nitric oxide synthase and is superior to NG-monomethyl-arginine in vitro and in vivo.

L-N6-(1-iminoethyl)-lysine is a novel inhibitor of nitric oxide (NO) synthase, which similar to aminoguanidine but unlike NG-monomethyl-L-arginine is 30-fold more selective for the inducible than for the constitutive isoform of the enzyme. Here, we characterized this inhibitor for the first time in intact cells and during infection of mice with a NO-sensitive parasite (Leishmania major). L-N6-(1-iminoethyl)-lysine potently inhibited the activity of inducible NO-synthase in primary macrophages. After stimulation by interferon-gamma the IC50 of L-N6-(1-iminoethyl)-lysine was 0.4 +/- 0.1 mu M and 10- or 30-fold lower than that of NG-monomethyl-L-arginine or aminoguanidine, respectively. In vivo, L-N6-(1-iminoethyl)-lysine (0.4-9 mM in the drinking water) suppressed inducible NO-synthase activity and caused a dramatic exacerbation of leishmaniasis, despite a counterregulatory increase of inducible NO-synthase protein in the tissue. In contrast, considerably higher concentrations of NG-monomethyl-L-arginine (20-50 mM) were required in order to achieve comparable effects. NG-monomethyl-L-arginine, but not L-N6-(1-imino-ethyl)-lysine led to weight loss, reduced water and food consumption. We conclude that L-N6-(1-iminoethyl)-lysine should be used instead of NG-monomethyl-L-arginine for potent suppression of inducible NO-synthase in vitro and in vivo.

Animals↗
PubMed1995

Effect of calcitonin, elevated calcium and extracellular matrices on superoxide anion production by rat osteoclasts.

The electrochemical determination of superoxide anion generation by isolated mammalian osteoclasts was carried out by employing cytochrome c, immobilized at a surface-modified gold electrode. Osteoclasts cultured on bone generated 15-fold more superoxide anions than cells plated on plastic. Superoxide anion production by osteoclasts cultured on bone was almost completely abolished by salmon calcitonin, and partially abolished following exposure to elevated extracellular calcium. We therefore conclude that osteoclast activity is influenced substantially by the extracellular matrix.

Animals↗
PubMed1994

The prevalence of polycystic ovaries on ultrasound scanning in a population of randomly selected women.

Polycystic ovaries (PCO) diagnosed by ultrasound have been commonly reported amongst healthy women. The study aimed to determine the prevalence of PCO in a population of women from the community, and to relate it to clinical and endocrinological data. Twelve hundred women chosen randomly from electoral rolls were invited to take part in the study. Two hundred and fifty five women (21%) who met eligibility criteria greed to participate and 183 women 916%) finally completed the study. Seventy two women did not attend. An ultrasound scan and blood tests were taken on day 5-9 of their menstrual cycles. Data about their menstrual periods and reproductive history was collected. The prevalence of PCO was 21% (39 of 183). No differences existed between women with PCO and normal ovaries with respect to uterine size, body mass index, luteinizing hormone levels, serum hormone binding globulin levels and fertility status. Hirsutism (Ferriman Gallwey score > 7), elevated testosterone levels and irregular menstrual cycles were significantly more frequent amongst women with PCO. Mean ovarian volume was larger in women with PCO irrespective of the use of hormonal contraception. Fifty nine per cent of women with PCO had irregular menstrual cycles or elevated Ferriman Gallwey scores or both. There was no detectable effect of PCO on parity or miscarriage although only 70% of women with PCO had evidence of an adequate ovulation compared to 95% of women with normal ovaries.

Female↗
PubMed1993

Beclomethasone given after the early asthmatic response inhibits the late response and the increased methacholine responsiveness and cromolyn does not.

BACKGROUND: Single doses of inhaled beclomethasone or inhaled cromolyn, given before allergen inhalation, inhibit allergen-induced late asthmatic responses (LARs) and increased airway responsiveness (delta log methacholine PC20). We hypothesized that when given 2 hours after allergen, beclomethasone might work better than cromolyn. METHODS: In 10 patients with mild, stable, atopic asthma with LARs or delta log PC20 or both, we performed a double-blind, double-dummy, random-order trial comparing a single dose of inhaled beclomethasone (500 micrograms), cromolyn (20 mg), and placebo, administered 2 hours after allergen challenge on LAR and delta log PC20. RESULTS: The treatment effect on LAR was significant (p < 0.001). The LAR after beclomethasone (7.3% +/- 6.1%) was significantly less than after cromolyn (20.4% +/- 15.2%) or placebo (26.4% +/- 8.2%); cromolyn was not different from placebo. There was a borderline treatment effect on delta log PC20 (p = 0.056) with beclomethasone (0.12 +/- 0.31) less than placebo (0.37 +/- 0.39) but not less than cromolyn (0.34 +/- 0.18). CONCLUSION: Beclomethasone (500 micrograms) administered 2 hours after allergen challenge markedly inhibited the LAR and had a small effect on allergen-induced airway responsiveness. Cromolyn (20 mg) was not effective on maximal LAR; a small effect on the early part of the LAR was suggested.

Adolescent↗
PubMed1993

Acceptance of direct physician access to a computer-based patient record in a managed care setting.

Kaiser Permanente Mid-Atlantic States has developed a fully integrated outpatient information system which currently runs on an IBM ES9000 on a VM platform written in MUMPS. The applications include Lab, Radiology, Transcription, Appointments. Pharmacy, Encounter tracking, Hospitalizations, Referrals, Phone Advice, Pap tracking, Problem list, Immunization tracking, and Patient demographics. They are department specific and require input and output from a dumb terminal. We have developed a physician's work station to access this information using PC compatible computers running Microsoft Windows and a custom Microsoft Visual Basic 2.0 environment which draws from these 14 applications giving the physician a comprehensive view of all electronic medical records. Through rapid prototyping, voluntary participation, formal training and gradual implementation we have created an enthusiastic response. 95% of our physician PC users access the system each month. The use ranges from 0.2 to 3.0 screens of data viewed per patient visit. This response continues to drive the process toward still greater user acceptance and further practice enhancement.

Ambulatory Care Information Systems↗
PubMed1991

Hypertrophic osteoarthropathy in association with pulmonary tuberculosis.

Three white male patients with advanced cavitary pulmonary tuberculosis presented with HOA. No other pathology to explain the osteoarthropathy was detected. The osteoarthropathy responded symptomatically to NSAI drugs and treatment of tuberculosis but resolved radiologically in only one patient.

Adult↗
PubMed1989

Giardia cyst wall-specific carbohydrate: evidence for the presence of galactosamine.

Gas chromatographic (GC), mass spectrometric (MS), lectin binding and enzymatic analyses of the carbohydrates from Giardia cyst walls, intact cysts and trophozoites were performed to investigate the carbohydrate composition of Giardia cyst walls and to test the hypothesis that the Giardia cyst wall is composed largely of chitin. Galactosamine, verified by MS, was present in Giardia cyst walls and intact cysts (ca. 47 nmol 10(-6) cysts). Since not even trace amounts of it were detected in trophozoites by either GC or lectin binding, galactosamine is hypothesized to be a cyst wall-specific amino hexose. Based on the putative binding affinity of Phaseolus limensis lectin, galactosamine may be present in cyst walls as N-acetylgalactosamine. Neither glucosamine nor sialic acid were detected in as much as 11 mg dry weight of cysts, cyst walls, or trophozoites. Glucose, the most abundant carbohydrate, and ribose were detected in Giardia cysts and trophozoites. Galactose (ca. 10 nmol 10(-6) cysts) was detected in cysts but not in trophozoites. The lack of detectable levels of (1) glucosamine in cyst wall hydrolysates, (2) cyst staining by Calcofluor M2R, (3) endogenous chitinase activity and (4) N-acetylglucosamine when cysts served as a substrate for exogenous chitinase suggests that the Giardia cyst wall is not composed largely of chitin as previously reported. beta-N-Acetylgalactosaminidase, EC 3.2.1.32, activity was detected in cysts and trophozoites and represents the first carbohydrate splitting hydrolase detected in Giardia.

Animals↗