There is good reason to believe that the known human cysteine protease repertoire is currently incomplete. Recently, two peptide sequences (i.e. CTLA-2alpha and CTLA-2beta) have been identified in the mouse T-lymphocyte. These peptides have been shown to be similar to the propeptide of human cathepsin L. In this project, computer based searches for human homologues to these mouse peptide sequences has been made. One entry from the TIGR express sequence tag database has been identified. As part of this project, the tag was isolated from the human Jurkat cell mRNA, a cell line compatible with the source of RNA used to identify the original sequence tag. / Several novel members of the papain proteases superfamilly have been discovered and characterized in the last couple of years. Of particular note is cathepsin K, which is primarily an osteoclast component that has been shown to be the main mediator of organic matrix degradation during bone resorption. Since many of these proteases demonstrate relevant extracellular matrix degradation in connective tissue, they are of significant interest in the study of joint health and diseases. In this project, qualitative cathepsins mRNA expression analyses in representative human joint-related cells have been assessed by RT-PCR. (Abstract shortened by UMI.)

No description available.

Torsade de pointes (TdP) is a proarrhythmic side effect of antiarrhythmic drugs that prolong the QT interval. The exact electrophysiologic mechanism of TdP is under intense investigation. To study the mechanisms and characteristics of TdP requires an animal model in which the arrhythmia occurs at a high incidence, predictably, reproducibly and resembles the characteristics of the clinical arrhythmia. We have developed such a model in the isolated rabbit heart (chapter II). This model is based on our earlier work showing that in isolated ventricular preparations from the rabbit heart, class III drugs, such as d-sotalol induce a greater increase in action potential duration in Purkinje fibers than in ventricular muscle, resulting in generation of early afterdepolarization-dependent triggered activity in Purkinje fibers and their subsequent propagation to muscle as extra beats (chapter 1). Studies in the whole heart suggested that the initiating beats of d-sotalol induced TdP have a subendocardial origin whereas later beats could be generated by reentry in muscle with disparate monophasic action potential durations (chapter II). The evidence presented in these studies was inconclusive because of the limited number of recording sites. To further clarify the relative contributions of impulse formation in the conducting system and reentry in muscle, we performed epicardial mapping using a sock electrode array to fit the rabbit heart. In addition plunge wires were used to record unipolar electrograms from the endocardial surface. The epicardial and endocardial electrograms were used to measure activation times as well as activation recovery intervals, a measure of local repolarization intervals. Using this technique in our TdP model, we were able to show that the generation of singlet beats and the first beats of couplets, triplets and TdP are due to the same mechanism---ie, a focal mechanism, arising in the ventricular specialized conducting system in a context of incr

The investigation of the interaction of light with tissue has many applications in the field of diagnostic medicine. The objective of the project was to obtain quantitative measurements of constituents in a medium in which the absorption and scattering optical properties vary. Initially the project was divided into two separate experiments, namely multiwavelength steady state and time resolved measurements. Both methods are applicable to complex media, providing information related to absorption and scattering. / The first phase was the study of Near Infrared diffuse reflectance for the determination of pulmonary edema. Non-contact pulmonary edema measurements were made on in vitro isolated perfused animal lungs. Multispectral analysis with Partial Least Squares (PLS) depicted three distinct changes associated with fluid increase: an increase in the O-H absorption, a decrease in the C-H absorption, and a decrease in the spectral baseline or scattering contribution. PLS estimates of relative water content had an R2 of 0.973 with a standard error 10.6 +/- 2.9% relative to the weight of lung. / The behavior of light in a highly scattering medium is a complex phenomenon. A method is described using analytical descriptors for the separate determination of the absorption and scattering properties from time resolved photon distributions. Estimates with the analytical descriptors were tested using simulated data generated from the time dependent diffusion equation. Objective selections of the descriptors were made using a Stepwise Multi-Linear Regression technique to independently estimate the absorption and scattering coefficients. In a medium where scattering and absorption vary simultaneously multiple parameters are required for quantitative estimates. Relative standard errors of less than 0.5% were obtained with the analytical descriptors estimates. / Estimates using the analytical descriptors were tested experimentally with the use of a photon time-of-flight instrument. Different light collection geometries and their relation to the descriptors were investigated in both transmittance and reflectance configuration. Absorption and scattering estimations of less than 9% error were attained for both collection geometries. / Information on both the absorption and scattering properties in highly variable media can assist in quantitative multispectral measurement. A method was developed whereby a multiwavelength scatter correction was made using optical coefficients determined from time resolved distributions. Optical coefficient corrected estimates produced a 70% improvement in the error over non-corrected spectra. The study suggests a practical method to correct and quantitate constituent measurements in variable media.

This study has optimized techniques for the detection and identification of mutations in the genes encoding type I collagen. Two novel polymorphisms were identified which may serve as useful markers in the linkage analysis of inherited disorders resulting from mutations in these genes. The methods were also used to characterize the gene mutations in four patients with osteogenesis imperfecta, a disease associated with bone fragility of heterogeneous severity. The point mutations demonstrated in these patients resulted in four novel glycine substitutions within the triple helical domains of the type I collagen polypeptide chains, and allowed an evaluation of the relationship between disease severity and the nature of the biochemical defects. The description of these mutations has permitted the identification of three regions in the collagen polypeptide chains in which the disease phenotype exhibits a transition between lethal and non-lethal forms, thereby allowing a more accurate prenatal prognosis of phenotypic severity in osteogenesis imperfecta.

Most new cases of spinal-cord-injured (SCI) persons in Canada have incomplete loss of sensory and/or motor function, but only a minority are able to walk. The study of animal models of spinal cord injury, especially the chronic spinal cat, has shown that monoaminergic drugs can modulate locomotion and spinal reflexes. Clonidine, a noradrenergic agonist, and cyproheptadine, a serotonergic antagonist, have each been associated with improved walking in SCI subjects. Baclofen, a GABA agonist, is frequently prescribed for spasticity in SCI patients, but its effects on walking have not been quantified. The objective of this doctoral project was to compare the effects of clonidine, cyproheptadine and baclofen on walking in incomplete SCI subjects. Subjects were evaluated on a motorized treadmill. Severely disabled subjects required harness support for their evaluations. The treadmill and harness system are described in detail, and their potential uses in the evaluation and rehabilitation of gait are discussed. A repeated single-subject design was employed for the twelve subjects. The greatest effects were found in the the subjects with greater severity of disability. Cyproheptadine was associated with greatly reduced need for assistance, increases in maximum treadmill speed (MTS) and reduced clonus, among other improvements in walking patterns. Clonidine was associated with increases in MTS, and a generally more upright posture, among other improvements in walking patterns. Baclofen was not associated with changes in walking, although two subjects showed small improvements following washout of baclofen. Among subjects with less severe motor disability, drug effects were less marked. Following washout of cyproheptadine or clonidine, subjects frequently retained walking improvements such as increases in MTS and reduced need for assistance that had first been evident in the drug periods. The significance and implications of the drug effects and the retention of effects dur

Cell transplantation to the brain and spinal cord is a well-established tool for studies on cellular and molecular mechanisms involved in development and plasticity. Experimental data from animals has suggested that restoration or preservation of function through cell transplantation has potential as a useful therapeutic approach in neurodegenerative disorders. Experimental evidence of graft-induced behavioral ameliorations has raised the question of the applicability of this technique to the cognitive neurodegenerative disorder, Alzheimer's disease (AD). / This present study is based on previous work from our lab that has indicated a potential for cortical grafts of chromaffin cells of the adrenal medula to alleviate nucleus basalis magnocellularis (nbm) lesion-induced cognitive and neurochemical deficits; such lesions produce deficits which mimic certain of the deficits seen in AD. This study has found that chromaffin cell grafts ameliorate behavioral deficits and that a neurochemical recovery occurs in the cortex consequential to it. This particular behavioral improvement was evidenced 6 weeks but not 5 days post-graft, indicating a gradual effect by the graft. Additionally, in this behavioral test, control grafts of kidney cells were found to have no ameliorating effects. A final objective to establish immunocytochemical techniques for detection of graft constituents was also actualized.

In order to determine the role of nutritional status as a predictor of mortality in patients with chronic obstructive pulmonary disease (COPD), 348 patients with severe airway obstruction were followed for 1 to 3 years. Baseline measurements were done on patients recruited for a clinical trial of negative pressure ventilation in severe COPD. When adjusted for the severity of disease, as assessed by the forced expiratory volume in one second (FEV$ sb1$) and the use of oxygen therapy, body mass index (BMI) was a significant predictor of mortality in the recruited group. The relative risk (RR) for BMI comparing the 25$ sp{ rm th}$ to the 75$ sp{ rm th}$ percentile was 1.86 (1.76, 1.97). Both BMI and triceps skinfold thickness (TSF) were the nutritional parameters which significantly predicted mortality in the subgroup of randomized patients when adjusted for the partial pressure of carbon dioxide in arterial blood (PaCO$ sb2$) and use of oxygen therapy. The RR's comparing the 25$ sp{ rm th}$ to the 75$ sp{ rm th}$ percentile for BMI and TSF respectively, were 2.09 (1.92, 2.27) and 1.80 (1.66, 1.96).

Controversy exists among clinicians and caregivers as to whether reduced fluid intake contributes to the suffering of those dying of advanced cancer. This study explored the distribution of proposed "dehydration state" symptoms among inpatient palliative care cancer patients. Fifty-two subjects responded to a seven item self-report questionnaire using visual analogue scales. Associations were determined between the symptom self-reports and the possible predictor variables fluid intake, serum sodium, urea and osmolality. Confounding variables considered were age, oral disease and mouth care regime. Mean symptom ratings (range 0-100 mm) were: thirst 53.8, dry mouth 60.0, bad taste 46.6, nausea 24.0, pleasure to drink 61.6, fatigue 61.8, and pain 33.5. No significant association was determined between symptom ratings and the predictor or confounding variables. Although the symptoms appear to be rated moderately severe, there was no demonstrable association between severity and fluid intake, the key concern of clinicians and families.

The objective of this study was to determine how alterations in the insulin-receptor trafficking pathway affect ligand-dependent cellular action in Chinese hamster lung fibroblasts. Cell lines altered in their trafficking pathway were selected using a toxic insulin-diphtheria A-chain molecule (DTaI) which specifically interacts with the insulin receptor. One cell variant (IV-Al-j) which exhibited decreased insulin binding and enhanced insulin degradation did not demonstrate insulin or IGF-1 dependent cell proliferation. IV-Al-j cells also failed to produce an insulin-stimulated tyrosine phosphorylated protein (pp175). IGF-1 binding, insulin-stimulated hexose uptake, epidermal growth factor and thrombin stimulated cell proliferation were unaltered in IV-Al-j cells. These results suggest that the insulin and IGF-1 receptor may share a common pathway post-ligand binding for cell proliferation which does not involve EGF or thrombin, and that enhanced insulin degradation and loss of pp175 may be responsible for the loss of insulin-stimulated cell proliferation.