Biomedical subjects
Publications and source records attributed to R A Harrison.
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The fourth component of complement (C4) is inactivated by treatment with methylamine. This property is shared wit the third component (C3) and with alpha 2-macroglobulin. In each instance, the reaction with methylamine is stoichiometric, covalent, and accompanied by the appearance of a thiol group. These data are consistent with the presence of an internal thiolester bond. Incubation of C4 with [14C] methylamine in the presence of activated thiol-Sepharose resulted in immobilization of the protein via its active-site thiol. Analysis of bound C4 indicated incorporation of 1.12 mol of [14C]methylamine per mol of protein. Digestion of the immobilized protein with porcine elastase resulted in the release of C4 beta- and gamma-chains and lower molecular weight fragments. The 14C label, however, was retained on the Sepharose beads. Subsequent release of bound material with L-cysteine indicated that the radiolabel was associated with two polypeptides of Mr 25,000 [C4d(ela25)]. The released material was dialyzed and the active-site thiol was radioalkylated with iodo[2-3H]acetic acid. C4d(ela25) was further purified by chromatography on Sephadex G-100 and, after reduction and alkylation, on Sepharose CL-6B in 0.2% NaDodSO4. The C4d(ela25) pool, containing 0.83 mol of [14C]methylamine per mol of iodo[2-3H]acetic acid, was subjected to automated sequence analysis. S-carboxy-[3H]methylcysteine was released at step 21 and gamma-glutamyl-[14C]methylamide was released at step 24. The recovery of radiolabel at positions 21 and 24 confirmed the originally calculated 14C/3H incorporation ratio and further indicated that the radiolabels were present at single sites in the C4 molecule. Comparison of the derived primary structure for the thiolester site in C4 with those for the corresponding regions in C3 and alpha 2-macroglobulin has shown sequence identity. Further comparisons among these three proteins have indicated additional homologies on both the NH2- and COOH-terminal sides of the thiolester site.
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A brother and sister with classic, biopsy proved pulmonary alveolar proteinosis are described. Both had low serum and low normal secretory immunoglobulin A (IgA) levels. A tendency for familial occurrence is possible and it is recommended that patients with pulmonary alveolar proteinosis, and their families, be evaluated for immunologic deficiencies.
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Samples of ram, bull and boar semen were subjected to cold shock and then stained using an indirect immunocytochemical method for detecting acrosin. It was found that the shock treatment abolished staining of the acrosomal region in all but a very few cells. This finding was interpreted as a great loss of proacrosin or acrosin from damaged acrosomes. Using a fluorescent label in the system, sperm cells in both control and treated samples could be scored easily and reproducibly as "stained" or "unstained". Results using a peroxidase label were less satisfactory because staining was not as consistent. Biochemical assessment of sperm acrosin content before and after cold shock confirmed that such treatment caused acrosin loss from the cells into the seminal plasma, but as a quantitative measure of cellular integrity the biochemical method exhibited a number of deficiencies compared with the immunofluorescent cytochemical method. Preliminary data are presented for semen evaluation comparing this latter method with a "live-dead" stain.
Treatment of the third component of human complement (C3) with methylamine results in a loss of hemolytic function and the appearance of a thiol group. Studies with [14C]methylamine have indicatd a stoichiometric and covalent reaction with the native protein. Hydrazine-inactivated C3 and C3b prepared with bovine trypsin were unreactive with [14C]-methylamine. Alkylation experiments with [1-14C]iodoacetamide have further established a 1:1 correspondence between methylamine incorporation and expression of the reactive thiol. Autoradiographic analyses of [14C]methylamine-treated C3 and methylamine-inactivated [1-14C]carboxyamidomethylated C3 after NaDodSO4/polyacrylamide gel electrophoresis have shown a specific incorporation of each radiolabel into the alpha polypeptide chain. [14C]Methylamine-treated C3 was immobilized on Sepharose 4B by reaction of the protein thiol with a mixed disulfide. Digestion with bovine trypsin in 4 M urea released 96% of the bound absorbance (at 280 nm) units; the radiolabel remained associated with the Sepharose beads. Peptide material labeled with 14C was eluted with dithiothreitol, carboxymethylated with [3H]iodoacetic acid, and chromatographed on Sephadex G-75. On Edman degradation S-[3H]carboxymethylcysteine was released at step 9 and gamma-glutamyl[14C]-methylamide was released at step 12. We interpret these data to indicate the presence of an internal thiolester bond in native C3. In addition, evidence is presented for an identical reactive site in alpha 2-macroglobulin.
The spectral absorbance of Intralipid, a phospholipid emulsion, was investigated to discern its effect, when parenterally administered, on the spectrophotometric measurement of hemoglobin (Hb), oxyhemoglobin (HbO2), and carboxyhemoglobin (HbCO). While accounting for dilutional factors, various concentrations of Intralipid in both water and hemoglobin solutions were analyzed at six wavelengths commonly used to measure Hb, HbO2 and HbCO. Absorbance increased linearly with Intralipid concentration at all wavelengths, and ranged from 0.034 at 505 nm to 0.019 at 626.6 nm per mg of Intralipid. Therefore, in patients receiving Intralipid, significant errors in Hb, HbO2 and HbCO measurements can be introduced if these measurements are made by oximetry, and the authors suggest that such measurements should be accomplished by methods other than spectrophotometry.
In 100 critically ill patients, intrapulmonary shunts (Qsp/Qt) calculated by assuming a carboxyhemoglobin (HbCO) of zero and a hemoglobin saturation (HbO2) derived from the Severinghaus nomogram were compared to shunts calculated utilizing measured values of HbCO and HbO2. The differences were statistically significant (p less than 0.001). These 100 patients had a mean Hb of 11.9 g/dl and a mean HbCO of 1.7%. Measured shunt calculations in 30 critically patients were prospectively compared with shunt calculations utilizing the mean assumed values derived from the 100 patients initially studies. No statistically significant differences occurred between these two shunts. It is, therefore, concluded that errors attributable to the common practice of assuming zero HbCO and deriving HbO2 from nomograms will produce falsely high calculated intrapulmonary shunt values in critically ill patients. It is, therefore, important to measure accurately HbCO and HbO2 concentrations when monitoring intrapulmonary shunting in critically ill patients. However, if such measurements cannot be obtained, utilization of an assumed value of 1.5% for HbCO and HbO2 nomogram values will minimize errors in the shunt calculation.