Submitted to: American Society of Animal Science
Publication Type: Abstract Only
Publication Acceptance Date: 3/12/2003
Publication Date: 6/22/2003
Citation: Elsasser, T.H., Kahl, S., Sartin, J.L., Fayer, R., Martinez, A., Cuttitta, F., Hinson, J. 2003. Muscle protein tyrosine nitration patterns during chronic subclinical intramuscular parasitism: Co-localization to fiber type and ubiquitin [abstract]. Journal of Animal Science. v 8(1):84.
Technical Abstract: The present study was conducted to determine whether the inflammatory oxidative response to chronic intramuscular parasitism, as modeled with the protozoan parasite Sarcocystis cruzi, results in protein nitration damage and whether a pattern to it localization can be characterized. Holstein steer calves (n=10; av.wt.= 124 kg) were assigned equally to control (C) and infected (I, 25,000 Sarco sporocysts) groups. Calves were slaughtered on d56 postinfection and samples of rectus femoris (RF) and psoas major (PM) harvested. Xanthine oxidase (XO) was measured in muscle homogenates by fluorescence (resorufin, 587 nm). Frozen sections (9 microns) were immunostained (IHC; horseradish peroxidase/DAB) for nitrotyrosine (NT) or ubiquitin (UBI) or co-localization of NT with fibertype (staining v nonstaining with mouse anti-myosin fast twitch), or NT with UBI via confocal immunofluorescence. Extracted muscle proteins were extracted, separated on 4-20% SDS PAGE gels, transferred to nitrocellulose, and probed for nitrated proteins using a anti-NT or antibovine-UBI. XO activity, a source of superoxide, was 2.3 times greater in I than C (P<0.01). Western blot demonstrated that >80% of the increase in NT was associated with an increased number of protein bands (P<0.04, I v C) >75 kD. IHC demonstrated very low levels of both NT and UBI staining in RF and PM of C but increased NT (42% more NT+ fibers, P<0.05) in both RF and PM of I. NT immunostaining could be categorized into three distinct forms: a) peripheral fiber (I and C), b) dispersed intrafiber (I), and c) cyst-specific (I). Both fast and slow fibers displayed the peripheral localization of NT and UBI. Only slow twitch oxidative fibers displayed extensive co-localized intrafiber NT staining regardless of muscle source. The sarcocyst itself was highly nitrated and muscle proteins in the immediate vicinity of the cyst displayed increased NT co-localized with UBI. The data suggest that the oxidative inflammatory response to chronic low-level muscle-resident parasitism generates nitrated muscle proteins. The nitration appears to be more pronounced in slow oxidative fibers and supports prior observations of more severe impact of this parasitism on muscles with higher percentages of slow twitch fibers.