Guy Beauregard

Temperature dependence of the radiation inactivation of proteins

The radiation inactivation method allows determination of the relative molecular mass (Mr) of proteins by exposure to high doses of ionizing radiation. The analysis by target theory of biological activity decay curves yields the size of the protein. A correction factor for Mr has been routinely used in the literature when irradiation is conducted at low temperature. Since the radiation inactivation of proteins is affected by temperature, we propose a general equation which relates Mr of a protein to D37,t, the dose in megarads at a given temperature t (in degree C) where 37% of its initial biological activity remains log Mr = 5.89 - log D37,t - 0.0028t. It is concluded that temperature affects the amount of absorbed radiation energy required to inactivate 1 mol of protein.

Radiation inactivation of enzymes at low dose rates: Identical molecular weights of rat liver cytosolic and lysosomal neuraminidases

Abstract A low-dose-rate gamma source ( 60 Co) was calibrated with enzymes of known radiation inactivation behaviors and used for molecular weight determination of rat liver neuraminidase (EC 3.2.1.18). The method allows direct comparison of radiation inactivation of standard and unknown enzymes under identical experimental conditions. The membrane-bound lysosomal neuraminidase had the same molecular weight ( M r = 56,000 ± 8500) as the soluble cytosolic neuraminidase ( M r = 56,000 ± 7000) although they differ in their kinetic properties and substrate specificity.

Studies on the sialidoses. Properties of human leucocyte neuraminidases

At least two components of neuraminidase (acylneuraminyl hydrolase, EC 3.2.1.18) can be distinguished in human leucocytes on the basis of pH optimum, thermolability at 30 degrees C and the effect of the detergent octyl-beta-D-glucoside. With 4-methylumbelliferyl-alpha-D-N-acetylneuraminate as substrate, the A component has a pH optimum of 5.0, is labile at 30 degrees C and is unaffected by 0.2 M octyl-beta-glucoside. The B component has a pH optimum of 4.0-4.2, is stable at 30 degrees C but loses most of its activity in the presence of 0.2 M octyl-beta-glucoside. Both A and B components are membrane-bound but only the A component is solubilized by octyl-beta-glucoside in an active form. Molecular weights of neuraminidases by gamma-ray radiation inactivation (a method that does not require solubilization of the enzyme) were found to be 240 000 +/- 19 000 for the B component, 203 000 +/- 17 000 for the A component and 238 000 +/- 8000 for the octyl-beta-glucoside-solubilized A component. Gel filtration of soluble A component on Sephacryl S-300, in the presence of octyl-beta-glucoside, showed a single peak of activity eluted at or near the void volume suggesting that the enzyme is still in an aggregated form. Profound deficiency of neuraminidase activity was found for both A and B components in leucocytes of patients affected with sialidoses type 1 and 2 (less than 15% normal) and intermediate activity in obligate heterozygotes. These results suggest that the A and B components of leucocyte neuraminidase are closely related from the genetic point of view and that rapid diagnosis of sialidoses can be done by fluorimetric assay of neuraminidase in leucocytes.

The molecular weight of the calcium-transport-ATPase of the human red blood cell determined by radiation inactivation

Radiation inactivation was applied to analyze the molecular weight of the functional unit of (Ca2+ + Mg2+)-ATPase in human erythrocyte membranes. The enzyme activity was stable for at least 7 days at room temperature in membranes lyophilized in the presence of sucrose (150-300 mM). The enzyme activity in the lyophilized membranes and remaining after irradiation from a 60Co source was activated by calmodulin. A Mr of 290,000 +/- 15,000 was determined for (Ca2+ + Mg2+)-ATPase activity. Since the Mr by SDS-polyacrylamide gel electrophoresis is approximately 138,000 (Niggli et al. (1979) J. Biol. Chem. 254, 9955-9958), our results suggest that the Ca2+ pump ATPase functions as a dimer in the native human erythrocyte membrane.

Target size analysis by radiation inactivation: A large capacity tube rack for irradiation in a Gammacell 220

Target size analysis by radiation inactivation is now a well-established method to study structure-function relationships in biologically active macromolecules without prior purification or even solubilization. Recently, it was reported that a relatively low-dose-rate but commonly available gamma source such as the Gammacell 220 (Atomic Energy of Canada, Ltd.) can be used to carry out radiation inactivation experiments providing it is appropriately calibrated with enzymes of known radiation sensitivities (G. Beauregard and M. Potier (1982) Anal. Biochem. 122, 379-384). In this report, a tube rack designed to fit into the irradiation chamber of the Gammacell 220 which allows five experiments (at 30 tubes per experiment) to be carried out simultaneously with both standard and unknown samples is described. The dose rates delivered at different positions in the rack were determined by irradiating rat liver cytosolic neuraminidase, an enzyme of known radiation sensitivity. A better than 2.7% agreement was obtained between experimental dose rate and computed values from isodose curves previously published by other authors (O. A. Curzio and H. O. Quaranta (1982) Int. J. Appl. Radiat. Isot. 33, 1-3).

Modulation of erythrocyte acetylcholinesterase by cardiolipin: Effect on subunit coupling revealed by irradiation inactivation

Abstract The functional molecular weights of two kinetically distinct forms of bovine erythrocyte acetylcholinesterase were determined by irradiation inactivation. Whereas both forms have similar molecular weights by hydrodynamic measurements and contain 33 molecules of cardiolipin, the functional molecular weight of form α (140,000) was found to be twice that of form β (73,000). As form β is derived from form α by treatment with high salt concentration in alkaline Ca 2+ -chelating conditions, a procedure which is considered to disrupt the functional association of a Ca 2+ -cardiolipin complex with the enzyme, it is suggested that cardiolipin mediates the energy transfer between enzyme subunits, thereby modulating the kinetic properties of the lipoprotein.

Radiation inactivation of membrane proteins: Molecular weight estimates in situ and after Triton X-100 solubilization

Target size analysis by radiation inactivation is widely used for molecular weight determination of membrane enzymes and receptors in situ without the need for prior solubilization or purification. However, since most molecular weight data available in the literature on membrane proteins involve the use of detergents for solubilization, the target sizes of membrane proteins in situ and after solubilization by detergent treatment have been compared. Using data from the literature and personal results, three different types of behavior of membrane proteins in presence of detergents were found: (i) uncoupling of subunits (electric eel acetylcholinesterase, placental steroid sulfatase, and human nonspecific beta-glucosidase); (ii) coupling of protein molecules (mouse liver neuraminidase, and rat liver insulin receptor regulatory component); and (iii) no major change in quaternary structure (rat liver insulin receptor, kidney gamma-glutamyltransferase, asialoglycoprotein receptor, insulin degrading enzyme, and human leucocyte neuraminidase). For all these proteins, there is a statistically significant increase in target size of about 24% over the value obtained in situ without detergent. A relatively large body of literature data involving a variety of membrane proteins, membrane types, and irradiation conditions (electron accelerators or 60Co sources, and proteins irradiated in lyophilized form or frozen solution) was examined, and it was concluded that target sizes of membrane proteins, irradiated in the presence of Triton X-100, should be diminished by a factor of about 24% to obtain the molecular weight value.

Radiographic abnormalities in cats with feline bronchial disease and intra- and interobserver variability in radiographic interpretation: 40 cases (1999–2006)

OBJECTIVE To determine prevalence of various radiographic signs in cats with feline bronchial disease (FBD) and evaluate intra- and interobserver variability in radiographic interpretation for examiners with variable degrees of experience in radiographic interpretation. DESIGN Retrospective case series. ANIMALS 40 cats with FBD and 40 control cats without thoracic disease. PROCEDURES Radiographic abnormalities in cats with FBD were scored by consensus of 2 radiologists. Radiographs of control cats and cats with FBD were examined twice by 5 other individuals, and diagnostic accuracy and observer agreement were assessed. RESULTS In cats with FBD, the most common radiographic signs were bronchial (n=37) and unstructured interstitial (30) lung patterns, lung hyperinflation (31) and hyperlucency (21), aerophagia (19), and lung soft tissue opacities (11). Ratios of lung inflation on ventrodorsal views were significantly higher in cats with FBD. For the 5 examiners, sensitivity ranged from 71% to 89% and specificity ranged from 43% to 74%. Intraobserver agreement was good (N=0.47 to 0.60), but the agreement between examiners was only poor to good (N=0.22 to 0.70). For most examiners, significant associations were found between examiner diagnosis (correct vs incorrect), level of examiner certainty, and bronchial pattern severity. CONCLUSIONS AND CLINICAL RELEVANCE Findings suggested that several radiographic abnormalities can commonly be seen in cats with FBD but highlighted the limitations of thoracic radiography. Examiner diagnosis and level of confidence were significantly associated with severity of a bronchial pattern.

Palpation and Dorsal Acetabular Rim Radiographic Projection for Early Detection of Canine Hip Dysplasia: A Prospective Study

OBJECTIVE To determine the value of 2 diagnostic methods: (1) the reduction angle (RA) using the Ortolani maneuver and (2) the dorsal acetabular slope (DAS) from the dorsal acetabular rim (DAR) radiographic projection, to predict osteoarthritis (OA) in dogs with hip dysplasia. STUDY DESIGN Prospective study. SAMPLE POPULATION Dogs (n = 73). METHODS Hip-extended ventrodorsal (VD) radiographic projections, RA, and DAS were evaluated when dogs were 6, 12, and 24 months of age. VD projections were qualitatively scored for OA. RA was determined using the Ortolani maneuver in dorsal recumbency and DAS using the DAR projection. Distraction index (DI) measurements from the compression-distraction radiographic projections (PennHIP method) were also performed at 6 months of age. Statistical analyses were used to establish the range of values of normal and abnormal RA and DAS, to document the temporal variation in RA and DAS, to compare the ability of the different methods to predict coxofemoral OA, to determine the influence of pure passive laxity and of the DAS on the occurrence of an Ortolani sign and on the magnitude of the RA, to establish the relationship between the DAS and subsequent development of passive laxity and coxofemoral OA, and to evaluate the influence of the DAS and RA on the occurrence of coxofemoral OA with severe, moderate, and minimal coxofemoral passive joint laxity, respectively. RESULTS VD, RA, DAS, and DI methods of coxofemoral joint evaluation correlated significantly with the status of the coxofemoral joints at 2 years of age. The risk of occurrence of coxofemoral OA increased, as the RA, DAS, or DI increased. There was a significant positive linear relationship between RA and DI (P = .015, r(2) = 0.32), RA and DAS (P = .0078, r(2) = 0.38), and DAS and DI (P = .015, r(2) = 0.33). A negative Ortolani sign was at all times significantly predictive of absence of coxofemoral OA at 2 years of age. DAS best predicted coxofemoral OA for DI ≥ 0.7, whereas RA best predicted coxofemoral OA for 0.3 < DI < 0.7; however, RA proved to be the best overall predictor of coxofemoral OA. CONCLUSION RA measured at 6 months of age in dorsal recumbency was the best predictor of coxofemoral OA at 2 years of age.

Sialidase Deficiency in the SM/J Mouse: A Physically Altered Mutant Enzyme in the Liver

The glycosidase sialidase (acylneuraminyl glycohydrolase, EC 3.2.1.18) hydrolyzes terminal sialic acid residues of glycoproteins, oligosaccharides and glycolipids, and is widely distributed in viruses, bacteria and mammals.1 Recessively inherited syndromes characterized by sialidase deficiency, the sialidoses, have been described in man2–4 and genetic sialidase deficiency has been reported in the mouse strain SM/J5,6 and the rat strain KGH.7 In SM/J mice, the deficiency involves a labile component (named A) which denatures rapidly at 0°C.5