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Biomedical subjects

R T McKay

Publications and source records attributed to R T McKay.

At least 19 recordsLinked to original sources
PubMed2000

Energetics of the induced structural change in a Ca2+ regulatory protein: Ca2+ and troponin I peptide binding to the E41A mutant of the N-domain of skeletal troponin C.

Structural studies have shown that the regulatory domains of skeletal and cardiac troponin C (sNTnC and cNTnC) undergo different conformational changes upon Ca(2+) binding; sNTnC "opens" with a large exposure of the hydrophobic surface, while cNTnC retains a "closed" conformation similar to that in the apo state. This is mainly due to the fact that there is a defunct Ca(2+)-binding site I in cNTnC. Despite the striking difference, the two proteins bind their respective troponin I (TnI) regions (sTnI(115-131) and cTnI(147-163), respectively) in a similar open fashion. Thus, there must exist a delicate energetic balance between Ca(2+) and TnI binding and the accompanying conformational changes in TnC for each system. To understand the coupling between Ca(2+) and TnI binding and the concomitant structural changes, we have previously engineered an E41A mutant of sNTnC and demonstrated that this mutation drastically reduced the Ca(2+)-binding affinity of site I in sNTnC, and as a result, E41A-sNTnC remains closed in the Ca(2+)-bound state. In the present work, we investigated the interaction of E41A-sNTnC with the sTnI(115-131) peptide and found that the peptide binds to the Ca(2+)-saturated E41A-sNTnC with a 1:1 stoichiometry and a dissociation constant of 300 +/- 100 microM. The peptide-induced chemical shift changes resemble those of Ca(2+) binding to sNTnC, suggesting that sTnI(115-131) induces the "opening" of E41A-sNTnC. In addition, the binding of sTnI(115-131) appears to be accompanied by a conformational change in site I of E41A-sNTnC so that the damaged regulatory site can bind Ca(2+) more tightly. Without Ca(2+), sTnI(115-131) only interacts with E41A-sNTnC nonspecifically. When Ca(2+) is titrated into E41A-sNTnC in the presence of sTnI(115-131), the Ca(2+)-binding affinity of site I was enhanced by approximately 5-fold as compared to when sTnI(115-131) was not present. These observations suggest that the binding of Ca(2+) and TnI is intimately coupled to each other. Together with our previous studies on Ca(2+) and TnI peptide binding to sNTnC and cNTnC, these results allow us to dissect the mechanism and energetics of coupling of ligand binding and structural opening intricately involved in the regulation of skeletal and cardiac muscle contraction.

Alanine↗
PubMed2000

Capability of respirator wearers to detect aerosolized qualitative fit test agents (sweetener and Bitrex) with known fixed leaks.

This study was designed to evaluate and compare the ability of respirator wearers to detect qualitative respirator fit test agents (saccharin and Bitrex) when the respirators were modified to include fixed size leaks. In recent years the number of persons who require fit testing has increased, partly in response to the needs of health care workers with potential exposure to infectious bio-aerosols. Many health care providers have chosen qualitative respirator fit testing using saccharin and/or Bitrex for a variety of reasons, including (but not limited to) low initial equipment cost. Respirators were modified to include a mid-line sampling probe between the nose and mouth for quantitative fit testing with a TSI PortaCount. A second modification included the introduction of a shortened 14-gauge intravenous catheter at the bridge of the nose. The fixed leak was designed to produce fit factors < 100 when unplugged, with an average fit factor of 67 among 26 respirator wearers. A complete fit test was not performed, because one purpose of this study was to determine the ability of respirator wearers to detect a known fixed leak during a single normal breathing exercise, without introducing unknown and potentially variable size leaks. Sensitivity threshold screening included a placebo and requirement to correctly characterize the taste of the agent used. Quantitative fit factors without leaks ranged from 96 to > 20,000 and 22 to 160 with the leak present. Twenty four of 26 subjects had fit factors < 100 (92%) when fixed leaks were induced. All subjects correctly detected Bitrex with fixed leaks (sensitivity = 100%). Nine of 26 subjects (35%) were unable to detect saccharin in the presence of a known fixed leak even though the average fit factor for these subjects was 77. When the two subjects with fit factors > 100 were excluded, only 16 of 24 respirator wearers were able to detect saccharin with fixed leaks (sensitivity = 67%). There were several important aspects of our study design worth noting, including the introduction of a placebo during sensitivity threshold testing, limiting the subject response time to a single maneuver, using a higher concentration of Bitrex than commercially available, and requiring the subjects to correctly characterize the taste of the qualitative test agent. In conclusion, leak detection was correctly identified with Bitrex, but not saccharin.

Adolescent↗
PubMed1999

Defining the region of troponin-I that binds to troponin-C.

The kinetics and energetics of the binding of three troponin-I peptides, corresponding to regions 96-131 (TnI96-131), 96-139 (TnI96-139), and 96-148 (TnI96-148), to skeletal chicken troponin-C were investigated using multinuclear, multidimensional NMR spectroscopy. The kinetic off-rate and dissociation constants for TnI96-131 (400 s-1, 32 microM), TnI96-139 (65 s-1, <1 microM), and TnI96-148 (45 s-1, <1 microM) binding to TnC were determined from simulation and analysis of the behavior of 1H,15N-heteronuclear single quantum correlation NMR spectra taken during titrations of TnC with these peptides. Two-dimensional 15N-edited TOCSY and NOESY spectroscopy were used to identify 11 C-terminal residues from the 15N-labeled TnI96-148 that were unperturbed by TnC binding. TnI96-139 labeled with 13C at four positions (Leu102, Leu111, Met 121, and Met134) was complexed with TnC and revealed single bound species for Leu102 and Leu111 but multiple bound species for Met121 and Met134. These results indicate that residues 97-136 (and 96 or 137) of TnI are involved in binding to the two domains of troponin-C under calcium saturating conditions, and that the interaction with the regulatory domain is complex. Implications of these results in the context of various models of muscle regulation are discussed.

Amino Acid Sequence↗
PubMed1999

Weight change and lung function: implications for workplace surveillance studies.

This study evaluated the relationship between weight change and longitudinal measurement of lung function among 361 men providing at least five pulmonary function tests. The men in this study were participants in a workplace pulmonary surveillance program for subjects with exposure to refractory ceramic fibers (RCFs). Occupational and environmental studies are generally designed to evaluate factors suspected of causing excess decline in lung function. Failure to adequately account for all significant factors may lead to erroneous conclusions regarding change in lung function. This study utilized two different statistical models to evaluate longitudinal changes in a cohort of RCF workers. What was unique to this study was the modeling of longitudinally measured initial weight, weight change, and longitudinal exposure before and during the period of observation. Results showed a strong relationship between weight gain and longitudinal loss in lung function that approximated forced vital capacity declines of 16 mL for every kilogram of weight gain per year in both models. This value is comparable or greater in magnitude and significance to other factors known to be inversely related to lung function, such as age and pack-years smoking to time of initial testing. In conclusion, weight gain was found to have a significant impact on longitudinal change in lung function. Therefore, weight gain becomes a very important variable that requires consideration whenever longitudinal studies of pulmonary function are conducted.

Adult↗
PubMed1998

An industry-wide pulmonary study of men and women manufacturing refractory ceramic fibers.

An industry-wide pulmonary morbidity study was undertaken to evaluate the respiratory health of employees manufacturing refractory ceramic fibers at five US sites between 1987 and 1989. Refractory ceramic fibers are man-made vitreous fibers used for high temperature insulation. Of the 753 eligible current employees, 742 provided occupational histories and also completed the American Thoracic Society respiratory symptom questionnaire; 736 also performed pulmonary function tests. Exposure to refractory ceramic fibers was characterized by classifying workers as production or nonproduction employees and calculating the duration of time spent in production employment. The risk of working in the production of refractory ceramic fibers and having one or more respiratory symptoms was estimated by adjusted odds ratios and found to be 2.9 (95 percent confidence interval 1.4-6.2) for men and 2.4 (95 percent confidence interval 1.1-5.3) for women. The effect of exposure to refractory ceramic fibers on forced vital capacity (FVC), forced expiratory volume in 1 second (FEV1), the ratio of the two (FEV1/FVC), and forced expiratory flow (liters/second) between 25 percent and 75 percent of the FVC curve (FEF(25-75)) was evaluated by multiple regression analysis using transformed values adjusted for height, by dividing by the square of each individual's height. For men, there was a significant decline in FVC for current and past smokers of 165.4 ml (p < 0.01) and 155.5 ml (p = 0.04), respectively, per 10 years of work in the production of refractory ceramic fibers. For FEV1, the decline was significant (p < 0.01) only for current smokers at 134.9 ml. For women, the decline was greater and significant for FVC among nonsmokers, who showed a decrease of 350.3 ml (p = 0.05) per 10 years of employment in the production of refractory ceramic fibers. These findings indicate that there may be important sex differences in response to occupational and/or environmental exposure.

Adult↗
PubMed1998

Structure and interaction site of the regulatory domain of troponin-C when complexed with the 96-148 region of troponin-I.

The structure of the regulatory domain of chicken skeletal troponin-C (residues 1-90) when complexed with the major inhibitory region (residues 96-148) of chicken skeletal troponin-I was determined using multinuclear, multidimensional NMR spectroscopy. This complex represents the first interaction formed between the regulatory domain of troponin-C and troponin-I after calcium binding in the regulation of muscle contraction. The stoichiometry of the complex was determined to be 1:1, with a dissociation constant in the 1-40 microM range. The structure of troponin-C in the complex was calculated from 1039 NMR distance and 111 dihedral angle restraints. When compared to the structure of this domain in the calcium saturated "open" form but in the absence of troponin-I, the bound structure appears to be slightly more "closed". The troponin-I peptide-binding site was found to be in the hydrophobic pocket of calcium saturated troponin-C, using edited/filtered NMR experiments and chemical shift mapping of changes induced in the regulatory domain upon peptide binding. The troponin-I peptide (residues 96-148) was found to bind to the regulatory domain of troponin-C very similarly, but not identically, to a shorter troponin-I peptide (region 115-131) thought to represent the major interaction site of troponin-I for this domain of troponin-C.

Animals↗
PubMed1998

The NMR angle on troponin C.

The calcium-induced structural changes in the skeletal muscle regulatory protein troponin C involve a transition from a closed to an open structure with the concomitant exposure of a large hydrophobic interaction site for target proteins. NMR solution structural studies have served to define this conformational change and elucidate the mechanism of the linkage between calcium binding and the induced structural changes. These structural movements are described in terms of interhelical angles in these largely helical proteins. Oddly, the most recent structure of the cardiac system challenges the central paradigm because the calcium-bound structures are not open. The kinetics, energetics, and dynamics of these proteins have also been investigated using NMR.

Allosteric Regulation↗
PubMed1998

Longitudinal estimates of pulmonary function in refractory ceramic fiber manufacturing workers.

Refractory ceramic fibers (RCF) are man-made vitreous fibers (MMVF) used in high-temperature industrial applications. Between 1987 and 1994, a prospective study evaluated pulmonary function of 361 male workers currently employed in RCF manufacturing and related operations for plausibility of a causal relationship between RCF exposure and pulmonary function changes. Workers included in the analysis provided at least five pulmonary function tests. The exposure-response relationship was modeled with two exposure variables: years in a production job, and cumulative fiber exposure (fiber-mo/cc). Comparison groups were nonproduction workers and workers with up to 15 fiber-mo/cc cumulative exposure. A statistically significant decrease in FVC was demonstrated among workers employed in production jobs more than 7 yr prior to initial test. A similar but nonstatistically significant result was demonstrated for FVC in workers with greater than 60 fiber-mo/cc cumulative exposure prior to initial pulmonary function test. Similar but nonstatistically significant results were obtained for FEV1. These findings, which primarily reflect workers employed before 1980, did not persist with analysis of follow-up production years and accumulated RCF exposure from initial pulmonary function test. Since longitudinal analyses are sensitive to influences that continue to affect annual decline during the study period, lower RCF exposure levels since the 1980s may be responsible for eliminating any further effect on pulmonary function.

Adult↗
PubMed1997

Interaction of the second binding region of troponin I with the regulatory domain of skeletal muscle troponin C as determined by NMR spectroscopy.

Two dimensional 1H,15N-heteronuclear single quantum correlation NMR was used to monitor the resonance frequency changes of the backbone amide groups belonging to the 15N-labeled regulatory domain of calcium saturated troponin C (N-TnC) upon addition of synthetic skeletal N-acetyl-troponin I 115-131-amide peptide (TnI115-131). Utilizing the change in amide chemical shifts, the dissociation constant for 1:1 binding of TnI115-131 to N-TnC in low salt and 100 mM KCl samples was determined to be 28 +/- 4 and 24 +/- 4 microM, respectively. The off rate of TnI115-131 was determined to be 300 s-1 from observed N-TnC backbone amide 1H,15N-heteronuclear single quantum correlation cross-peak line widths, which is on the order of the calcium off rates (Li, M. X., Gagné, S. M., Tsuda, S., Kay, C. M., Smillie, L. B., and Sykes, B. D. (1995) Biochemistry 34, 8330-8340), and agrees with kinetic expectations for biological regulation of muscle contraction. The TnI115-131 binding site on N-TnC was determined by mapping of chemical shift changes onto the N-TnC NMR structure and was demonstrated to be in the "hydrophobic pocket" (Gagné, S. M., Tsuda, S., Li, M. X., Smillie, L. B., and Sykes, B. D. (1995) Nat. Struct. Biol. 2, 784-789).

Animals↗
PubMed1997

Potent pseudosubstrate-based peptide inhibitors for p60(c-src) protein tyrosine kinase.

We recently reported the identification of GIYWHHY as an efficient and specific substrate for p60(c-src) protein tyrosine kinase (PTK) by screening a secondary random peptide library (Q. Lou et al., Bioorg. Med. Chem., 4: 677-682, 1996). Based on the primary structure of GIYWHHY, we designed and synthesized several pseudosubstrate-based peptide inhibitors. Some of these peptide inhibitors are highly potent and specific with IC50 in the low micromolar range. Because both YIYGSFK and GIYWHHY are efficient and specific substrates for p60(c-src) PTK, chimeric branched peptides based on these two sequences were synthesized. These branched peptides inhibit p60(c-src) PTK with high potency, indicating that the enzyme-active site of p60(c-src) PTK can accommodate more than a linear motif. This may explain why seemingly several peptides with very different linear structures can all be phosphorylated by this enzyme.

Amino Acid Sequence↗
PubMed1995

Peptide aldehyde inhibitors of hepatitis A virus 3C proteinase.

Picornaviral 3C proteinases are a group of closely related thiol proteinases responsible for processing of the viral polyprotein into its component proteins. These proteinases adopt a chymotrypsin-like fold [Allaire et al. (1994) Nature 369, 72-77; Matthews et al. (1994) Cell 77, 761-771] and a display an active-site configuration like those of the serine proteinases. Peptide-aldehydes based on the preferred peptide substrates for hepatitis A virus (HAV) 3C proteinase were synthesized by reduction of a thioester precursor. Acetyl-Leu-Ala-Ala-(N,N'-dimethylglutaminal) was found to be a reversible, slow-binding inhibitor for HAV 3C with a Ki* of (4.2 +/- 0.8) x 10(-8) M. This inhibitor showed 50-fold less activity against the highly homologous human rhinovirus (strain 14) 3C proteinase, whose peptide substrate specificity is slightly different, suggesting a high degree of selectivity. NMR spectrometry of the adduct of the 13C-labeled inhibitor with the HAV-3C proteinase indicate that a thiohemiacetal is formed between the enzyme and the aldehyde carbon as previously noted for peptide-aldehyde inhibitors of papain [Lewis & Wolfenden (1977) Biochemistry 16,4890-4894; Gamcsik et al. (1983) J. Am. Chem. Soc. 105, 6324-6325]. The adduct can also be observed by electrospray mass spectrometry.

3C Viral Proteases↗
PubMed1994

Visual field loss while wearing full-face respiratory protection.

The loss of visual field was quantified for 21 test subjects while they wore three full-face respirators. Changes in visual field were quantified for each type of respirator using a modified Goldmann projection perimeter. The loss of visual field was determined by calculating the area under the curve while wearing a respirator to that while wearing no respirator. Distinct patterns of visual field loss were apparent for the different style respirators. Analysis of the patterns could lead to the design of full-face respirators with improved visual qualities, which could improve worker safety for certain occupations. This technique also could be of help when selecting models of respiratory protection when certain visual fields must be maintained.

Adolescent↗
PubMed1988

Toluene diisocyanate-induced airway hyperreactivity in guinea pigs depleted of granulocytes.

The influence of cyclophosphamide-induced granulocyte depletion on toluene diisocyanate (TDI)-related changes in airway reactivity and pathology was assessed in guinea pigs. Twelve cyclophosphamide-treated and 12 control animals comprising each group were studied physiologically before and 2 h after a single 10-min exposure to 3 ppm of TDI. Reactivity was determined in intact unanesthetized animals by measuring specific airway conductance before and during intravenous acetylcholine infusion. After testing, tracheal tissue for light microscopic examination was obtained from three hyperreactive guinea pigs in each exposed group and compared with tissue from treated and control animals (n = 3 each) that had not been TDI exposed. Cyclophosphamide treatment caused substantial decreases in both circulating and airway granulocyte counts. However, the incidence and degree of bronchial hyperreactivity that occurred 2 h post-TDI was similar in the untreated and treated groups. Our results indicate that TDI-induced bronchial hyperreactivity 1) occurs shortly after a brief high concentration exposure and 2) appears independent of circulating or airway granulocyte counts.

Acetylcholine↗
PubMed1986

Toluene diisocyanate-induced airway hyperreactivity and pathology in the guinea pig.

We assessed the nature and progression of airway mucosal disease and histaminic reactivity in English short-haired guinea pigs at 2, 24, 72, 168, and 504 hours after toluene diisocyanate (TDI) exposure (4 hours of 3 ppm of TDI for 5 consecutive days). To also determine whether TDI-specific, IgE-like antibodies developed in TDI-exposed animals, passive cutaneous anaphylaxis testing was done 28 days after TDI. Bronchial reactivity was determined serially by measuring specific airway conductance as a function of increasing doses of aerosolized histamine in six exposed and three control animals studied intact and unanesthetized. The remaining 10 exposed and 10 control guinea pigs were sacrificed in groups of two at each time point to obtain airway tissue for light microscopic examination. We found that airway hyperreactivity to histamine occurred after TDI in all animals tested. It was maximal 2 hours after the 5-day exposure and remitted by 72 hours. In addition, marked airway obstruction occurred after TDI that persisted for at least 168 hours. There were dramatic signs of airway mucosal damage associated with the bronchial hyperreactivity that included substantial decreases in epithelial cilia, mucin content, and mast cells, as well as squamous metaplasia, numerous mitotic figures, and a prominent polymorphonuclear leukocytic infiltrate. Passive cutaneous anaphylaxis tests in exposed animals were negative. Our results suggest that TDI-induced bronchial hyperreactivity may be related to airway mucosal injury and inflammation.

Airway Resistance↗