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Biomedical subjects

J M Macdonald

Publications and source records attributed to J M Macdonald.

At least 19 recordsLinked to original sources
PubMed1999

NMR study of the sites of human hemoglobin acetylated by aspirin.

Acetylation of hemoglobin by aspirin and other acetylating agents has been used to generate hemoglobin analogs with altered structural and functional properties, and may prove useful in the treatment of sickle cell disease. We have studied the acetylation of human hemoglobin using [1'-(13)C]acetylsalicylic acid in combination with two-dimensional HMQC and HSQC NMR analysis. The spectra of the acetylated hemoglobin exhibit a number of well resolved resonances. Several spectral assignment strategies were used: blocking the 2, 3-DPG binding site non-covalently with inositol hexaphosphate or covalently with a cross-linking agent, selective carbamylation of the N-terminal valine amino groups with cyanate, spin-labeling the hemoglobin at betaCys93, and analysis of a hemoglobin triple mutant: betaV1MH2DeltaK144R, in which betaLys144 is replaced by an arginine residue. These studies support the conclusion that the most rapidly acetylated residue is betaLys82 rather than betaLys144, as previously reported. Further, it is apparent that acetyl betaLys82 can give rise to several resonances due to additional acetylation of betaLys82' or other nearby residues. An additional assignment strategy involving comparison of the chemical shifts of the acetyl resonances observed for adducts of diamagnetic carbonmonoxyhemoglobin with the shifts observed in paramagnetic cyanomethemoglobin provides information about the location of the acetyl derivatives relative to the heme irons. This approach is limited, however, by the lack of well defined structural information for the lysine residues on the protein surface. Additional tentative assignments have also been made, using the above approaches.

2,3-Diphosphoglycerate↗
PubMed1999

An NMR analysis of the reaction of ubiquitin with [acetyl-1-13C]aspirin.

The acetylation of ubiquitin by [acetyl-1-13C]aspirin has been studied using 2D NMR methods. Studies performed in a 50:50 H2O:D2O medium show doubling of the acetyl carbonyl resonances, indicating that all of the stable adducts formed involved amide linkages. Assignment of the heteronuclear multiple quantum coherence (HMQC) resonances was accomplished based on comparison of resonance intensities with the results of an Edman degradation analysis, pH titration studies of acetylated ubiquitin, and analysis of two ubiquitin mutants, K33R and K63R. The presence of a single tyrosine residue in close proximity to lysine-48 suggested another assignment strategy. Nitration of tyrosine-59 resulted in a small, pH-dependent shift of the resonance assigned to lysine-48, with a pK of 7.0, close to that expected for the nitrotyrosyl hydroxyl group. An additional adduct resonance with very low intensity also was observed and tentatively assigned to the acetylated N-terminal methionine residue. The relative rates of acetylation of the various lysine residues were obtained from time-dependent HMQC studies. Since no sample preparation artifacts were introduced, the levels of modification of the various residues could be determined with relatively high accuracy. Based on the time-dependent intensity data, the relative rate constants for modification of K6, K48, K63, K11, K33, and M1 were 1.0, 0.59, 0.43, 0.26, 0.23, and 0.03, respectively. These results were in much better agreement with amino accessibility predictions based on the crystal structure of the ubiquitin monomer than with predictions based on the ubiquitin structure in the crystallized dimeric and tetrameric forms. This approach provides a useful basis for understanding how local environmental factors can influence protein adduct formation, as well as for comparing the extent and specificity of various acetylation reagents.

Acetylation↗
PubMed1998

NMR spectroscopy and MRI investigation of a potential bioartificial liver.

NMR feasibility was established for a coaxial hydrophobic-membrane bioreactor containing isolated rat hepatocytes with features designed to mimic the human liver. A novel triple-tuned NMR probe and a perfusion system controlling temperature, gas concentrations, flow-rate, and pH were used. We determined the optimum coaxial interfiber distance (i.e. diffusion distance) for maintaining hepatocyte viability in two bioreactor prototypes. Prototype no. 1 and no. 2 had diffusion distances of 500 microns and 200 microns, respectively. Cell viability was established by 31P NMR and trypan blue exclusion. Only prototype no. 2 maintained cell viability for more than 6 h, indicating the importance of diffusion distance. 31P spectra obtained over this 6 h time period were similar to in vivo spectra of rat liver. The 31P spectra were found to be more sensitive to subacute cell viability than trypan blue exclusion. In the 1H and 31P spectra, 1H2O and inorganic phosphate signals were split in two at all flow-rates, probably due to bulk magnetic susceptibility effects originating from the three bioreactor compartments. MRI was useful for quality control and determining flow dynamics, fiber integrity, and cell inoculate distribution. MRI revealed that the inner fibers were not centered in either prototype. Although an increased flow-rate did not influence spectral resolution or chemical shifts, significant degradation of MRI quality occurred above 50 mL/min. NMR spectroscopy and imaging provide valuable, real-time information on cell biochemistry and flow dynamics which can be used in development and monitoring of bioreactors designed as artificial livers.

Animals↗
PubMed1998

Fructose-1,6-bisphosphate preserves adenosine triphosphate but not intracellular pH during hypoxia in respiring neonatal rat brain slices.

BACKGROUND: Fructose-1,6-bisphosphate (FBP) sometimes provides substantial cerebral protection during hypoxia or ischemia. 31P/1H nuclear magnetic resonance spectroscopy of cerebrocortical slices was used to study the effects of FBP on hypoxia-induced metabolic changes. In addition, 13C-labeled glucose was administered and 13C nuclear magnetic resonance spectroscopy was used to search for FBP-induced modulations in glycolysis and the pentose-phosphate pathway. METHODS: In each experiment, 80 slices (350 microm) obtained from ten 7-day-old Sprague-Dawley rat litter mates were placed together in a 20-mm nuclear magnetic resonance tube, perfused, and subjected to 30 min of hypoxia (PO2 < 3 mmHg). Nine experiments were performed, with n = 3 in each of three groups: (1) no treatment with FBP; (2) 60 min of prehypoxia treatment with FBP (2 mM); and (3) 60 min of posthypoxia treatment with FBP (2 mM). 31P/1H Interleaved nuclear magnetic resonance spectra at 4.7 T provided average adenosine triphosphate, intracellular pH, and lactate. Cresyl violet stains of random slices taken at predetermined time points were studied histologically. Some experiments had [2-13C]glucose in the perfusate. Slices from these studies were frozen for perchloric acid extraction of intracellular metabolites and studied with high-resolution 13C nuclear magnetic resonance spectroscopy at 11.75 T. RESULTS: With no pretreatment with FBP, hypoxia caused an approximately 50% loss of adenosine triphosphate, an approximately 700% increase in lactate, and a decrease in intracellular pH to approximately 6.4. Pretreatment with FBP resulted in no detectable loss of adenosine triphosphate, no increase in lactate, and minimal morphologic changes but did not alter decreases in intracellular pH. 13C Nuclear magnetic resonance spectra of extracted metabolites showed that pretreatment caused accumulation of [1-13C]fructose-6-phosphate, an early pentose-phosphate pathway metabolite. Posthypoxic treatment with FBP had no effects compared with no treatment. CONCLUSIONS: During severe hypoxia, pretreatment with FBP completely preserves adenosine triphosphate and almost completely preserves cell morphology but does not alter hypoxia-induced decreases in intracellular pH. Pretreatment also substantially augments the flux of glucose into the pentose-phosphate pathway.

Adenosine Triphosphate↗
PubMed1996

An unusual congenital dorsal midline thoracic mass.

OBJECTIVE: To describe the presentation and investigation of an unusual form of congenital dorsal midline thoracic mass. RESULTS: An infant born by emergency Caesarean section at 34 weeks gestation was found to have a large dorsal midline thoracic mass. The infant had normal neurological function in all limbs. Radiological investigation showed no abnormality in the vertebrae. Ultrasonographic investigation suggested the mass to consist of subcutaneous oedema. The mass resolved completely within the first 2 weeks. CONCLUSIONS: The lesion observed in this infant represents a very unusual location for a benign condition caused by cervical pressure on the presenting fetal part. The use of ultrasonography enabled rapid exclusion of the more common, potentially serious, causes of a congenital midline dorsal thoracic mass.

Back↗
PubMed1994

13-cis-retinoic acid-mediated growth inhibition of DU-145 human prostate cancer cells.

The purpose of this study was to examine the effects of 13-cis-retinoic acid (13-cis-RA) on the growth regulation of DU-145 human prostatic cancer cells. The results of these experiments demonstrate that cell growth and metastatic potential of DU-145 cells were significantly inhibited by 13-cis-RA (10 microM). In order to elucidate the possible molecular mechanisms of 13-cis-RA action on prostate cancer cells, we examined the expression of nuclear receptor genes (hRXR alpha) and found that 13-cis-RA treated cells showed higher mRNA expression for hRXR alpha nuclear receptors compared to untreated cells. To elucidate further the possible biochemical mechanisms associated with these alterations, we analyzed the phosphorous metabolites by MR spectroscopy and found that the major metabolites were PME, (PC, PE) and DPDE (UDP-GalNAc, UDP-GLcNAc). The DU-145 cells and xenografts, which were both treated with 13-cis-RA, showed a two-fold decrease in DPDE's, compared to their controls. The higher resolution spectra of perfused cells revealed that phosphocholine levels were twice as high in 13-cis-RA-treated DU-145 cells as compared to untreated cells. These investigations demonstrate for the first time that 13-cis-RA inhibits the growth of human prostatic cancer cells, and this inhibition is associated with an increase in hRXR alpha nuclear receptor gene expression and alterations in phosphorous metabolites detected by 31P MR spectroscopy.

Animals↗
PubMed1994

Tobacco counselling among Alberta dentists.

Evidence suggests that dentists, with their existing commitment to prevention and their opportunity for regular interaction with patients, have the potential to significantly decrease tobacco-related morbidity and mortality. In 1992, dentists belonging to the Alberta Dental Association were surveyed to determine their attitudes, behavior and perceived obstacles to tobacco counselling. The 755 respondents represent 55 per cent of the province's practising dentists. Their attitudes toward involvement in tobacco counselling varied. Over 90 per cent agreed that dentists should show leadership and set a good example. However, only 60-70 per cent indicated that they should try to convince or actively help patients quit, and 25 per cent indicated that intervention was not appropriate. Little difference was found in the preferred counselling approach, with most dentists limiting their counselling efforts to discussing the hazards of smoking and benefits of quitting. Very few provided patients with specific strategies to change their smoking behavior. Perceived obstacles to tobacco counselling included a lack of coordination and exchange of information between dentistry and cessation services, pessimism about the patients' ability to change their tobacco habit, the need for further training, and the fact that counselling was not a high priority. This survey suggests that dentists could expand their role as tobacco counsellors. Professional associations and educational institutions have the potential to greatly assist the dental community in this regard, and attention should be given to the development and dissemination of informational campaigns and educational workshops.

Adult↗
PubMed1993

Citrate alterations in primary and metastatic human prostatic adenocarcinomas: 1H magnetic resonance spectroscopy and biochemical study.

The objectives of this study were to quantitatively verify the low levels of citrate previously observed in primary human prostatic adenocarcinomas and to determine whether citrate was further reduced in metastatic prostatic cancer. This was accomplished by comparison of citrate concentrations of DU 145 xenografts (a poorly differentiated human prostatic adenocarcinoma cell line grown in nude mice) with concentrations in primary human adenocarcinomas. Following in vivo 1H NMR studies of DU 145 xenografts, citrate concentrations of DU 145 xenografts and surgically removed primary prostatic adenocarcinoma tissue were determined by quantitative high resolution 1H NMR and enzymatic assay. The most significant findings of this study were that citrate concentrations in primary human adenocarcinomas (3.74 +/- 0.19 mumol/g wet weight) were significantly lower than those observed for normal and benign hyperplastic (BPH) prostatic tissues. Furthermore there was a further ten-fold reduction of citrate associated with DU 145 xenografts (0.31 +/- 0.028 mumole/g wet weight) compared with primary prostatic cancer. DU 145 xenografts also exhibited higher levels of uridine diphosphosugars and choline containing metabolites relative to primary prostatic adenocarcinomas. These findings support the hypothesis that citrate is low in primary prostatic cancer and further reduced in metastatic disease.

Adenocarcinoma↗
PubMed1993

Effect of glucose and confluency on phosphorus metabolites of perfused human prostatic adenocarcinoma cells as determined by 31P MRS.

A series of perfused cell 31P MRS studies were conducted using a well established human prostate adenocarcinoma cell line (DU 145) at different phases of growth, and exposed to varying glucose concentrations during growth. The spectral characteristics of perfused DU 145 cells were compared with the same cells grown in nude mice (xenografts). Perfused DU 145 cells had lower levels of inorganic phosphate and phosphocreative relative to in vivo nude mice xenografts. 31P MR spectra obtained from perfused cells at different phases of growth and exposed to varying glucose concentrations during grown suggest that increases in diphosphodiester levels are associated with high glucose concentrations and confluency. Perfused DU 145 cells grown in 5.5 mM glucose and harvested at log phase of growth best reflected the phosphorus MR spectra of the same cell line grown in nude mice.

Adenocarcinoma↗
PubMed1991

Emergence and recovery response of phosphate metabolites and intracellular pH in intact Mytilus edulis as examined in situ by in vivo 31P-NMR.

We employed surface probe-localized 31P-NMR spectroscopy to examine in situ the impact of short-term emergence (hypoxia) and resubmergence on phosphate metabolites and intracellular pH (pHi) in intact mussels. The use of intact organisms ensured that all intrinsic responses remained active while monitoring of individuals minimized uncertainties resulting from stochastic behavior and other individual differences. The use of a photoetched, balanced-match foil probe combined with 1H-NMR images allowed 31P-NMR spectra to be acquired from the posterior adductor muscle with good signal-to-noise. Upon emergence, all mussels exhibited an increase in [Pi], a decline in [phosphoarginine] and pHi, and very little changes in [ATP] with time. The complementary behavior of [phosphoarginine] and [Pi] indicated a precursor-product relationship involved in the maintenance of [ATP] but the similarity between [phosphoarginine] and pHi time-courses cannot be so readily explained. Irregularity in the time-courses of some parameters could have reflected stochastic gaping activity. Resubmergence responses exhibited a reversal of the emergence responses, except that the pHi eventually became supraalkaline with irregular fluctuations. This might be related to the 'oxygen debt' phenomenon and increased oxidative phosphorylation.

Adaptation, Physiological↗
PubMed1989

Monitoring of metabolic responses of intact Haliotis (abalones) under salinity stress by 31P surface probe localized NMR.

Surface probe localized 31P NMR spectroscopy was employed to record the metabolic responses of the foot of intact Haliotis cracherodii and H. rufescens (black and red abalones) under hyper- and hypoosmotic stresses. Use of the surface probe allowed spectral localization on the foot of intact abalones, facilitated monitoring of different sizes of animals, and minimized constraints on aquatic chamber design normally imposed by homogeneous-field probes. Generally, hyperosmotic stress (51%) elicited more rapid changes of phosphate metabolites than hypoosmotic stress (17%). As with the well-studied hypoxic stress in intact mammalian and excised molluscan tissue, both salinity treatments caused drops in the phosphagen and increases in inorganic phosphate levels. However, osmotic stress was distinct from hypoxic stress in that intracellular pH did not change and nucleotide triphosphate (NTP) concentrations dropped immediately. Although these findings are preliminary, they demonstrate the utility of the surface probe approach for studies of environmental stress in intact marine invertebrates.

Animals↗
PubMed1988

Effect of diameter and depth on the response to solid polysulfone intracorneal lenses in cats.

Impermeable, solid polysulfone intracorneal lenses (ICLs) can change corneal refractive power but will not allow diffusion of nutrients from the aqueous to the anterior stroma. Lenses of 4, 5, 6, and 7 mm in diameter were implanted in 50 cat eyes at depths ranging from 45% to 95% of corneal thickness to determine the effect of depth and diameter. Two types of stromal opacities occurred during follow-up: peripheral amorphous opacity (100%) and crystalline-granular lipid central opacity (79%). No eyes implanted with a 4-mm ICL ulcerated, but ulceration occurred in 28% of the eyes overall. Because opacification occurred with all lens diameters and implantation depths, metabolic demands of the cornea are not met. No safe limit exists at which these ICLs can be predictably used in cat eyes.

Animals↗
PubMed1987

Effect of inflammation on the corneal endothelial pump and barrier.

Corneal thickness is a reflection of endothelial barrier and pump functions. The corneal edema that occurs during intraocular inflammation is a consequence of the breakdown of one or both of these parameters. Results of this study demonstrate that, during intraocular inflammation induced by an intravitreal injection of bovine serum albumin (BSA), the permeability of rabbit corneal endothelia to inulin was increased. By comparison, treatment with oral aspirin and/or subconjunctival triamcinolone acetonide prevented the endothelial barrier breakdown induced by the BSA. Concomitant with the loss of the barrier function, endothelial ouabain binding decreased in the BSA injected eye, indicating a reduction in endothelial Na/K ATPase pump site density. A subconjunctival injection of triamcinolone prevented this decrease in pump sites. The increase in endothelial permeability and the decrease in pump site density correlated with an increase in corneal thickness. It can be concluded that the intraocular inflammation induced by BSA effects corneal edema by both an increase in endothelial permeability and a decrease in Na/K ATPase pump site density. Subconjunctival triamcinolone is effective in preventing this response.

Animals↗
PubMed1975

L-Ascorbic acid and D-isoascorbic acid in a common cold survey.

In a survey of double-bind design, 3 groups of approximately 70 student volunteers took 1 g L-ascorbic acid, 1 g D-isoascorbic acid, or placebo tablets every day for 15 weeks and the incidence and duration of colds were recorded. The group taking D-isoascrobic acid suffered 34% fewer colds than the other two groups; the results were also analyzed in relation to the sex and smoking habits of the volunteers. The rationale for investigating D-isoascorbic acid, an isomer of L-ascorbic acid which has limited antiscorbutic activity, is discussed.

Ascorbic Acid↗
PubMed1975

Survey of a neonatal population for the prevalence of cytomegalovirus.

A neonatal population of 542 was tested for cytomegaloviruria. 293 were normally grown and full term, 172 normally grown but premature, and 77 were undergrown. Two normally grown term infants and one premature were excreting the virus (o.55%). Only one, a normally grown term infant, had signs of classical cytomegalic inclusion disease. None of the undergrown infants was infected.

Birth Weight↗