Technical note: a rapid method for quantification of calpain and calpastatin activities in muscle

Stepwise and continuous gradient ion-exchange chromatography were compared for yield of calpains and calpastatin from ovine muscle in a study designed to quantify their activities for comparative purposes. In Exp. 1, a continuous (25 to 400 mM NaCl) gradient and a two-step gradient method (200 mM NaCl to coelute mu-calpain and calpastatin together and then 400 mM NaCl to elute mu-calpain) were compared. For the two-step method, mu-calpain activities were determined by subtracting calpastatin activities before and after heat inactivation of mu-calpain. Both the two-step and the continuous gradient method yielded similar results over a broad range of activities. The stepwise gradient method does not require the use of fraction collectors and pumps, and it can be completed in a fraction of the time required for the continuous gradient method. In Exp. 2, the two-step method was compared with a three-step method (100 mM NaCl to elute calpastatin, then 200 mM NaCl to elute mu-calpain, and then 400 mM NaCl to elute m-calpain). Unlike the continuous gradient method, calpastatin and mu-calpain could not be completely separated using the three-step chromatography method. Thus, the three-step gradient method should not be used to quantify the components of the calpain proteolytic system. The present results indicate that the two-step gradient method is a fast and inexpensive method to determine calpain and calpastatin activities in studies designed to quantify the components of the calpain proteolytic system in skeletal muscle.     

Dirofilaria immitis in cats from inner Sydney

Two hundred feral cats from the inner suburbs of Sydney were examined post mortem for adult Dirofilaria immitis and circulating microfilariae, and 101 of these cats were tested for heartworm antigens by an ELISA. Only 2 cats (1%) had adult heartworms, the blood sample from another cat contained a single microfilaria. The blood of a further three cats contained small amounts of D immitis antigen. Although D immitis occurs in cats in Sydney, the prevalence is not high enough to warrant prophylactic treatment.     

Predatory Dinosaurs from the Sahara and Late Cretaceous Faunal Differentiation

Late Cretaceous (Cenomanian) fossils discovered in the Kem Kem region of Morocco include large predatory dinosaurs that inhabited Africa as it drifted into geographic isolation. One, represented by a skull approximately 1.6 meters in length, is an advanced allosauroid referable to the African genus Carcharodontosaurus. Another, represented by a partial skeleton with slender proportions, is a new basal coelurosaur closely resembling the Egyptian genus Bahariasaurus. Comparisons with Cretaceous theropods from other continents reveal a previously unrecognized global radiation of carcharodontosaurid predators. Substantial geographic differentiation of dinosaurian faunas in response to continental drift appears to have arisen abruptly at the beginning of the Late Cretaceous.     

A long-snouted predatory dinosaur from africa and the evolution of spinosaurids

Fossils discovered in Lower Cretaceous (Aptian) rocks in the Tenere Desert of central Niger provide new information about spinosaurids, a peculiar group of piscivorous theropod dinosaurs. The remains, which represent a new genus and species, reveal the extreme elongation and transverse compression of the spinosaurid snout. The postcranial bones include blade-shaped vertebral spines that form a low sail over the hips. Phylogenetic analysis suggests that the enlarged thumb claw and robust forelimb evolved during the Jurassic, before the elongated snout and other fish-eating adaptations in the skull. The close phylogenetic relationship between the new African spinosaurid and Baryonyx from Europe provides evidence of dispersal across the Tethys seaway during the Early Cretaceous.     

Postmortem proteolysis and calpain/calpastatin activity in callipyge and normal lamb biceps femoris during extended postmortem storage.

The present experiment was conducted to determine whether calpastatin inhibits only the rate, or both the rate and extent, of calpain-induced postmortem proteolysis. Biceps femoris from normal (n = 6) and callipyge (n = 6) lamb was stored for 56 d at 4 degrees C. Calpastatin activity was higher (P < .05) in the callipyge muscle at 0 and 14 d postmortem, but not at 56 d postmortem. The activity of mu-calpain did not differ between normal and callipyge biceps femoris at 0 and 56 d postmortem (P > .05), but was higher at 14 d postmortem in the callipyge muscle (P < 0.05). The activity of m-calpain was higher in the callipyge muscle (P < 0.05). Western blot analyses of titin, nebulin, dystrophin, myosin heavy chain, vinculin, alpha-actinin, desmin, and troponin-T indicated that postmortem proteolysis was less extensive in callipyge than in normal biceps femoris at all postmortem times. The results of this experiment indicate that calpastatin inhibits both the rate and extent of postmortem proteolysis.     

Temperament and hypothalamic-pituitary-adrenal axis function are related and combine to affect growth, efficiency, carcass, and meat quality traits in Brahman steers

Associations between temperament, stress physiology, and productivity were studied in yearling Brahman steers (n = 81). Steers differed in calpain system gene marker status; 41 were implanted with a hormonal growth promotant at feedlot entry. Temperament was assessed with repeated measurements of flight speed (FS) and crush score (CS) during 6 mo of backgrounding at pasture and 117 d of grain finishing. Adrenal responsiveness was assessed with ACTH challenge, with plasma samples collected immediately before and 60 min after challenge. Steers with higher FS and CS had higher prechallenge plasma cortisol, glucose, lactate, and nonesterified fatty acid concentrations. The ACTH-induced cortisol response was unrelated to FS or CS, but glucose remained higher after challenge in flightier steers. The hormonal growth promotant reduced adrenal responsiveness; tenderness genotype had no effect. When temperament assessments and cortisol concentrations before and after challenge were combined in a principal components analysis, four vectors accounting for 38%, 25%, 18%, and 9% of the variation were identified. The first vector had significant loadings on temperament and prechallenge cortisol; increasing scores were associated with increased plasma glucose, lactate, and nonesterified fatty acid and with reductions in BW and feedlot growth rates, carcass fatness, and muscle pH. The second vector loaded only on ACTH-induced cortisol response; increased scores related to increased residual feed intake, number of daily feed sessions, and meat marbling score. The third and fourth vectors had different loadings on FS and CS and appeared to identify different aspects of temperament measured by FS or CS. Fewer associations were found between the third or fourth vectors and productivity traits, possibly because of lower variance accounted for by these vectors. In conclusion, temperament was related to prechallenge cortisol but not to ACTH-induced cortisol response. Principal components analysis separated these traits into separate components, which in turn had different relations with productivity traits. The largest component of temperament was described similarly by FS and CS, but there were smaller components that these described differently. There were some temperament-related differences in the metabolic status of the steers which were not related to the variation in cortisol, suggesting involvement of the sympatho-adrenal-medullary axis in these temperament-related effects.