We investigated the influence of Swedish recommended vitamins A, D3 and E supplementation levels on muscle tenderness and fatty acid (FA) composition under indoor or outdoor finishing programmes. Swedish Red breed steer calves were divided into vitamin supplemented (n = 12) and non-supplemented (n = 15) groups while on pasture prior to the finishing period. This trial began at the beginning of the winter housing period during which the steers were fed a 55 : 45 dry matter barley : grass silage diet indoors. The indoor finished group was comprised of vitamin supplemented (n = 6) and non-supplemented (n = 8) steers slaughtered after about 155 days on feed. Vitamin supplemented steers were provided with 100 g mineral supplement providing 400 000 IU vitamin A, 100 000 IU D3 and 3000 IU E daily as recommended for Swedish production practices. In spring, outdoor finished vitamin supplemented (n = 6) and non-supplemented (n = 7) steers grazed semi-natural grassland for an additional 120 days before slaughter. During pasture, vitamin supplemented steers had free-choice access to a mineral supplement containing vitamins A, D3 and E. The mineral supplement for the non-supplemented steers did not contain vitamins A, D3 and E and was provided at the same amount as the vitamin supplemented steers. Shear force values were similar between vitamin supplemented and non-supplemented steers after ageing 2, 7 and 14 days within indoor and outdoor finishing programmes. The shear force values had decreased by 14 days of ageing within all programmes. The μ- and m-calpain activity did not differ between vitamin supplemented and non-supplemented steers for either the indoor or outdoor finishing programmes. The calpastatin activity was higher for the indoor, vitamin supplemented steers. Indoor finished vitamin supplemented steers had a greater proportion of C18:1c-9 and total monounsaturated fatty acids, whereas the non-supplemented steers had a greater proportion of total saturated fatty acids. We concluded that the meat quality from steers not receiving vitamin supplementation was similar to that of steers receiving vitamins A, D3 and E supplementation at Swedish recommended levels under indoor and outdoor finishing programmes.

In this paper, we aimed to explore the effects of the calpain inhibitor III (MDL28170) and to detect calpain-like molecules (CALPs) in epimastigote forms of Trypanosoma cruzi isolate Dm28c. MDL28170 at 70 μM promoted a powerful reduction in the growth rate after 48 h. The IC50 value was calculated to be 31·7 μM. This inhibitor promoted an increase in the cellular volume, but not cell lysis, resulting in a trypanostatic effect. T. cruzi CALPs presented a strong cross-reactivity with anti-Drosophila melanogaster calpain and anti-cytoskeleton-associated protein from Trypanosoma brucei antibodies, and labelling was found mainly intracellularly. Furthermore, an 80 kDa reactive protein was detected by Western blotting assays. No significant cross-reactivity was found with anti-human brain calpain antibody. The expression of CALPs was decreased in cells kept for long periods in axenic cultures in comparison to a strain recently isolated from mice, as well as in MDL28170-treated cells, the latter being paralleled by an increased expression of cruzipain. Different levels of CALPs expression were also detected in distinct phylogenetic lineages, like Y strain (lineage TCI), Dm28c (TCII) and INPA6147 strain (Z3 zymodeme). These results may contribute for the investigation of the functions of CALPs in trypanosomatids.

Cysteine peptidases of protozoa have been implicated in a variety of biological events, and the expression of these enzymes is modulated in response to distinct stimuli, including environmental changes and differentiation. In the present work, we have examined the expression of cysteine peptidases from Herpetomonas samuelpessoai grown at distinct temperatures and during dimethylsulfoxide (DMSO)-elicited differentiation. We demonstrated that a 45 kDa cysteine peptidase had its activity reduced during the parasite growth at 37°C in comparison to 26°C, and when cultured up to 72 h in the presence of DMSO. The modulation in the 45 kDa cysteine peptidase expression is connected to the differentiation process, since both temperature and DMSO are able to trigger the promastigote to paramastigote transformation in H. samuelpessoai. The possible immunological similarity of H. samuelpessoai proteins with well-known cysteine peptidases produced by trypanosomatid pathogens, including cruzipain (Trypanosoma cruzi) and cysteine peptidase b (cpb) from Leishmania mexicana, was also investigated, as well as with calpain molecules. The protein cellular lysate of H. samuelpessoai reacted with antibodies raised against cpb of L. mexicana and calpain of Drosophila melanogaster; however, no reaction was observed against cruzipain. The 35 kDa cpb-like protein had its expression diminished in DMSO-treated parasites, while the 80 kDa calpain-like molecule was enhanced and an additional 30 kDa calpain-related polypeptide was exclusively observed in these cells. Fluorescence microscopy and flow cytometry analyses corroborated these data. The results described above add H. samuelpessoai to the list of parasites whose differentiation seems to be correlated with cysteine peptidase expression.

Nitric oxide (NO) has been demonstrated to be the principal effector molecule mediating intracellular killing of Leishmania, both in vitro and in vivo. We investigated the type of cell death process induced by NO for the intracellular amastigote stage of the protozoa Leishmania. Specific detection methods revealed a rapid and extensive cell death with morphological features of apoptosis in axenic amastigotes exposed to NO donors, in intracellular amastigotes inside in vitro – activated mouse macrophages and also in activated macrophages of regressive lesions in a leishmaniasis-resistant mouse model. We extended our investigations to the dog, a natural host-reservoir of Leishmania parasites, by demonstrating that co-incubation of infected macrophages with autologous lymphocytes derived from dogs immunised with purified excreted-secreted antigens of Leishmania resulted in a significant NO-mediated apoptotic cell death of intracellular amastigotes. From the biochemical point of view, NO-mediated Leishmania amastigotes apoptosis did not seem to be controlled by caspase activity as indicated by the lack of effect of cell permeable inhibitors of caspases and cysteine proteases, in contrast to specific proteasome inhibitors, such as lactacystin or calpain inhibitor I. Moreover, addition of the products of two NO molecular targets, cis-aconitase and glyceraldehyde-3-phosphate dehydrogenase, also had an inhibitory effect on the cell death induced by NO. Interestingly, activities of these two enzymes plus 6-phosphogluconate dehydrogenase, parasitic enzymes involved in both glycolysis and respiration processes, are overexpressed in amastigotes selected for their NO resistance. This review focuses on cell death of the intracellular stage of the pathogen Leishmania induced by nitrogen oxides and gives particular attention to the biochemical pathways and the molecular targets potentially involved. Questions about the role of Leishmania amastigotes NO-mediated apoptosis in the overall infection process are raised and discussed.

Epidemiological studies have indicated that malnutrition during early life may programme chronic degenerative disease in adulthood. In an animal model of fetal malnutrition, rats received an isoenergetic, low-protein (LP) diet during gestation. This reduced fetal β-cell proliferation and insulin secretion. Supplementation during gestation with taurine prevented these alterations. Since proteases are involved in secretion and proliferation, we investigated which proteases were associated with these alterations and their restoration in fetal LP islets. Insulin secretion and proliferation of fetal control and LP islets exposed to different protease modulators were measured. Lactacystin and calpain inhibitor I, but not isovaleryl-l-carnitine, raised insulin secretion in control islets, indicating that proteasome and cysteinyl cathepsin(s), but not μ-calpain, are involved in fetal insulin secretion. Insulin secretion from LP islets responded normally to lactacystin but was insensitive to calpain inhibitor I, indicating a loss of cysteinyl cathepsin activity. Taurine supplementation prevented this by restoring the response to calpain inhibitor I. Control islet cell proliferation was reduced by calpain inhibitor I and raised by isovaleryl-l-carnitine, indicating an involvement of calpain. Calpain activity appeared to be lost in LP islets and not restored by taurine. Most modifications in the mRNA expression of cysteinyl cathepsins, calpains and calpastatin due to maternal protein restriction were consistent with reduced protease activity and were restored by taurine. Thus, maternal protein restriction affected cysteinyl cathepsins and the calpain–calpastatin system. Taurine normalised fetal LP insulin secretion by protecting cysteinyl cathepsin(s), but the restoration of LP islet cell proliferation by taurine did not implicate calpains.

The present study was conducted to determine the effect of growth rate of calves on the activity of the calpain system post mortem, post mortem desmin degradation, myofibrillar fragmentation index and meat tenderness of m. longissimus lumborum (LL) and m. supraspinatus (SS). Twenty-four Friesian heifer calves were allocated to two treatment groups: MM and HH. The MM calves were given food to achieve a moderate growth rate (678 g/day from 5 days of age to 90 kg body weight (BW) (period I) and 770 g/day from 90 kg BW to slaughter at 250 kg BW (period II)) and the HH calves were given food to achieve a high growth rate (period I: 895 g/day and period II: 1204 g/day). The myofibril fragmentation index (MFI), desmin degradation and the activity of µ-calpain, m-calpain and calpastatin were measured in LL and SS at slaughter, and 1 and 7 days post mortem. Shear force was measured in LL and SS after 1 and 7 days of ageing and a sensory panel evaluated the eating quality of the loin aged 7 days. MFI (P < 0·01) and tenderness (P < 0·01) were higher and shear force was lower (P < 0·001) in meat from HH calves compared with meat from MM calves. However, growth rate did not affect the activity of µ-calpain, m-calpain or calpastatin or the degradation pattern of desmin at any time post mortem. Besides growth rate and MFI at day 7 post mortem, which both correlated positively with meat tenderness (P < 0·001 and P < 0·01, respectively), fibre type traits and DNA or RNA concentrations were not generally related to final tenderness. Thus, it was concluded that increased growth rate of calves before slaughter increased the ease of fragmentation (MFI) of the meat and improved the tenderness but these findings could not be related to any changes in the activity of the calpain system.

The objective of this study was to determine the effect of growth rate on muscle fibre characteristics, concentration of nucleic acids (RNA and DNA) as indicators of muscle protein synthesis capacity and activity of the calpain system at time of slaughter in m. longissimus lumborum (LL) and m. supraspinatus (SS) from calves, in order to elucidate the effect of growth rate on muscle protein turn-over at time of slaughter. Twenty-four Friesian heifer calves were allocated to two different feeding regimens that allowed for a moderate/moderate (MM) or high/high (HH) growth rate from 5 days of age to 90 kg body weight (BW) (period I) and from 90 kg BW to slaughter at 250 kg BW (period II), respectively. The growth rates in the two periods and the weight of LL and SS at slaughter were recorded. Within 30 min after exsanguination, samples were removed from LL and SS, snap-frozen, and later analysed for muscle fibre type frequency and cross-sectional area, DNA and RNA concentration and the activity of the calpain system. High growth rate (i.e. 895 g/day and 1204 g/day in periods I and II, respectively), compared with moderate growth rate (678 g/day and 770 g/day in periods I and II, respectively) had a marked effect on muscle weight and muscle characteristics. High compared with moderate growth rate resulted in hypertrophic growth of type I, IIA and IIB fibres in LL and of type IIA and type IIB fibres in SS, but had no effect on the muscle fibre type frequency in either of the muscles. High growth rate increased total DNA and RNA content and the RNA: DNA ratio in LL, indicating a greater potential for protein synthesis in this muscle, whereas the effect of growth rate was smaller in SS. The activity of µ-calpain, m-calpain and calpastatin was higher in the red SS muscle compared with the whiter LL muscle. However, these enzyme activities were not affected by growth rate, and thus, did not indicate a higher myofibrillar proteolysis in vivo in calves exhibiting high growth rate compared with moderate growth rate. Overall the results showed that different types of muscles react differently to high versus moderate growth rate. High growth rate induced muscle hypertrophy and increased protein synthesis capacity especially in LL and less in SS, but the activities of the enzymes in the calpain system did not show any concomitant increase in muscle protein degradation that would be in favour of improved meat tenderness.