High pressure processing (HPP) of pre-rigor longissimus thoracis (strip loin) from prime and bull animals substantially decreased the shear force and improved consumer eating attributes of the final meat product. The improved tenderness in both prime and bull meat was associated with a lower myofibrillar fragmentation index and reduced calpain 1 activity which indicated the mechanism of tenderisation was different from that which occurred in chill aged meat. Light microscopy showed disruption to the fibre packing within the muscle and electron microscopy confirmed significant disruption of the Z discs and M lines and disappearance of the A lines. Thus, HPP is associated with a reduction in the structural integrity and strength of the sarcomeres. These effects were consistent in strip loins sourced from prime and bull stock. HPP also led to the movement of glycogen phosphorylase from the sarcoplasmic fraction to the insoluble myofibrillar fraction in all animals and this was associated with a higher pH at 24 h.

Strip loins from different grades of cattle were subjected to two levels of high pressure processing (HPP) within 1 h of slaughter at a commercial meat processing plant and chilled for 1 day before freezing. The physical and eating quality characteristics of longissimus thoracis (strip loin) steaks from HPP were compared to meat that was chill aged for 1 or 28 days. HPP produced meat after 1 day with 60% lower shear force and higher sensory eating quality scores than 1 day chill aged meat. Extended chill storage for 28 days produced steaks of similar tenderness to HPP meat. HPP also increased the ultimate pH and decreased the cooking loss. Chilled storage of the gluteus medius from prime cattle for 28 days significantly improved the shear force by 18%, whilst HPP improved both the shear force by 43% and the sensory eating quality scores. HPP can produce high eating quality eye of rump medallions within 1 day of slaughter.

PURPOSE:We used sheep with an autosomal dominant gene for cortical cataract as an animal model to evaluate novel macrocyclic calpain inhibitors with potential for the medical treatment of human cataract.METHODS:The macrocyclic aldehyde, CAT811, identified previously as a calpain inhibitor that prevents calcium-induced opacification in cultured sheep lenses, was tested for its ability to protect cytoskeletal proteins from calpain proteolysis. CAT811 and its alcohol analogue, CAT505, were formulated separately into ointments, and each was applied twice daily to the right eye of sheep with early cataracts for five months. Progress of cataracts in the sheep was determined by ophthalmologic examination and comparison with a matched sample of sheep treated similarly with ointment that did not contain the active ingredient.RESULTS:The novel macrocyclic aldehyde, CAT811, was able to inhibit calpain proteolysis of lens cytoskeletal proteins at micromolar concentrations. When applied topically to the eyes of sheep, CAT811 was able to slow cataract development by 27% in the initial three months of treatment (P < 0.05). Its alcohol analogue, CAT505, was not able to slow cataractogenesis significantly.CONCLUSIONS:The inherited sheep cataract provides a reproducible model of cortical cataract over a time scale of several months. The data reported here, using this model, demonstrated the potential of the macrocyclic calpain inhibitor, CAT811, to act as a therapeutic for treatment of cortical cataract.

Two new series of 15-membered macrocyclic peptidomimetics, in which the P1 and P3 residues of the peptide backbone are linked by a bridge containing a 1,4-disubstituted 1H-imidazole, are reported. The structure with an aldehyde at the C-terminus and the imidazole at P3, i.e., 4c, shows significant inhibitory activity against calpain 2, with an IC(50) value of 238 nM. The macrocyclic aldehyde with the imidazole at the alternative P1 position, i.e., 5c, is significantly less active. The relative activities are linked to the ability of the component macrocycles to mimic a β-strand geometry that is known to favor active-site binding. This ability is defined by conformational searches and docking studies with calpain.

Toxoplasma gondii is a parasite that has been extensively studied due to its medical and veterinary importance in terminating pregnancies. Consequently, a satisfactory vaccine is required to control its adverse effects on pregnant animals. The microneme protein, MIC3, is a major adhesion protein that binds to the surface of host cells and parasites, and is therefore a potential vaccine against T. gondii. The viability of MIC3 as a vaccine is investigated in this study. Sheep were injected twice, intramuscularly, with plasmids containing DNA encoding for the mature form of MIC3 protein formulated into liposomes. Control sheep were injected with an empty vector or received no injections. The injection of sheep with DNA plasmids encoding for MIC3 elicited an immune response after the first and second injections as indicated by antibody responses and the production of IFN-gamma. The immune response, as measured by the IgG2 and IgG1 serum levels, was boosted after the injection of the MIC3 DNA vaccine together with high anti-MIC3 antibodies. The results demonstrate that the intramuscular injection of sheep with a plasmid containing DNA coding for MIC3 protein induces a significant and effective immune response against T. gondii.

CD154 is a cell surface molecule expressed by activated T cells. CD40 and CD154 interaction is critically important in regulating humoral and cell-mediated immune responses. In this study we have investigated whether a DNA vaccine encoding rhoptry protein 1 (ROP1) of Toxoplasma gondii, and encoding ovine CD154 induces an enhanced ROP1-specific immune response in sheep. Two groups of twelve animals received two intramuscular injections, of a DNA plasmid encoding T. gondii ROP1 antigen (group 1) or an ROP1 antigen fused to ovine CD154 (group 2). There were two control groups of sheep. One was injected with an empty vector (group 3) and the other received no injections at all (group 4). The injection of the plasmid containing ROP1 (group 1) at weeks 0 and 4 induced a significant IgG2 response at week 2 which was amplified at week 4 after the booster injection and persisted to week 8 compared to the control animals in groups 3 and 4. For IgG1, significant differences from the control animals were only observed from week 5 onwards. The fusion of CD154 and ROP1 elicited significant IgG1 and IgG2 responses from week 1 which were amplified from weeks 5 to 8 compared to the control animals in groups 3 and 4. The IgG1 response was significantly higher in group 2 animals receiving pROP1-CD154 compared to group 1 receiving pROP1 only. There was no significant difference in IgG2 responses between groups 1 and 2. Significant differences in IFN-γ levels were only observed in treatment group 1 at week 2 and treatment group 2 at weeks 1 and 2 compared to the control animals. The results demonstrated that an intramuscular injection of pROP1-CD154 gene to sheep significantly enhanced their immune response and induced a mixed Th1/Th2 response while the intramuscular injection of pROP1 only induced a Th1-specific immune response.

Studies of 17 analoges of 3 (SJA6017) in an in silico calpain model are reconciled to measured IC50 values against ovine calpain. The studies validate the potential of the “model” and criteria established for inhibition as a tool to select structures for synthesis to test as calpain inhibitors. Using this screening methodology of virtual libraries led us to synthesize several inhibitors including macrocycle 33, which in vitro sheep eye lens culture experiments showed to substantially slow opacification.

The aim of this study is to compare the immune responses of sheep stimulated by the intramuscular injection of a liposome formulated-DNA plasmid encoding the Toxoplasma gondii MAG1 antigen only or co-expressed with ovine IL-6. Forty-five, 2-year-old sheep were divided into four groups. Group 1 received an empty pVAXIg plasmid, group 2 no treatment, group 3 liposome formulated plasmid pVAXIgMAG1 and group 4, pVAXIgMAG1 plus pVAXovIL-6 plasmids. All the animals were inoculated at weeks 0 and 4. The injection of sheep with a plasmid encoding for MAG1 only or a MAG1 plasmid co-expressed with a plasmid encoding for ovine IL-6 produced humoral immune responses. The plasmids containing MAG1 elevated significantly serum IgG1 and IgG2 levels 2 weeks and onwards after the first injection of the plasmids. Co-expression of IL-6 with MAG1 had no effect on IG1 or IG2 levels illustrating that IL-6 in the formulation used had no modulating effect on any measured immune response.

Immunization with plasmid DNA, a relatively novel technique, is a promising vaccination technique. To improve the immune response by DNA vaccination various methods have been used, such as chemical adjuvants or immunomodulatory molecules formulated into microparticles or liposomes. The aim of this research is to evaluate the immune responses of sheep immunized with DNA plasmids encoding Toxoplasma gondii dense granule antigen GRA7 formulated into three different adjuvant formulations. Sixty sheep were injected intramuscularly with the DNA plasmids. Twelve received the liposome-formulated plasmid pVAXIgGRA7, 12 Emulsigen P formulated plasmid pVAXIgGRA7 and 12 Emulsigen D formulated plasmid pVAXIgGRA7. Twelve animals were used as a control and received the vector alone. All the animals were inoculated at week 0, and week 4. Immunization of the sheep with plasmids encoding GRA7, with the different adjuvant formulations, effectively primed the immune response. After the first inoculation, moderate to high antibody responses were observed with the three different adjuvant formulations. A significantly elevated specific IgG2 response was observed in the sheep immunized with liposomes and Emulsigen D as adjuvants. In the group immunized with Emulsigen P as an adjuvant, lower IgG1 and IgG2 antibody levels were developed compared to the other treatment groups. In all the immunized groups, DNA immunization stimulated a IFN-γ response. No antibody or IFN-γ responses were detected in the control group immunized with an empty plasmid or not immunized. These results indicate that intramuscular immunization of sheep with a DNA vaccine with the adjuvants liposomes and Emulsigen D induce a significant immune response against T. gondii.

The dense granule proteins of Toxoplasma gondii are investigated as possible vaccine candidates against the parasite. The aim of this research was to evaluate the immune responses of sheep injected twice, intramuscularly, with DNA plasmids encoding T. gondii dense granule antigens GRA1, GRA4, GRA6 and GRA7 formulated into liposomes. Control sheep were injected with an empty vector or received no injections. The injection of sheep with DNA plasmids encoding for GRA1, GRA4, GRA6 or GRA7 elicited an immune response after the first and the second injections as indicated by the moderate to high antibody responses. The injection of pGRA7 induced a significant level of anti-GRA7 IgG2 antibody and IFN-γ responses indicating a Th1-like immune response whereas injection with pGRA1, pGRA4 and pGRA6 stimulated a IgG1 type antibody response with a limited, if any, IFN-γ response. The results demonstrate that the intramuscular injection of sheep with a DNA liposome formulated plasmid coding for GRA proteins is an effective system that induces a significant immune response against T. gondii.

The design and elaboration of a series of macrocyclic templates that exhibit a propensity to adopt a beta-strand-like peptide-backbone conformation led to potent and selective inhibitors of calpain 2. Macrocycle 1 retarded calcium-induced opacification in an ovine-lens culture assay and is a lead compound for the development of a drug for cataract treatment. Cbz=carbobenzyloxy.

OBJECTIVE:To investigate biochemical changes accompanying Ca(2+)-induced lens opacification and the possible role of calpain activation in opacification within an ovine lens culture system.METHODS:Sheep lenses were cultured in minimal media. Lens opacification was induced by exposure to the Ca(2+) ionophore, ionomycin, and graded by digital image analysis. Cell viability was estimated by the release of lactate dehydrogenase into the culture medium. Opaque lenses were fixed and stained for a microscopic view of the lens structural changes. Ionic changes in the lens were measured by atomic absorption spectroscopy. Calpain activation was determined by zymography on casein gels and proteolysis was investigated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis (2DE) and Western blotting. The calpain inhibitor, SJA6017, was used to investigate the involvement of calpains in lens opacification.RESULTS:Treatment of cultured ovine lenses with ionomycin increased total lens Ca(2+) concentration and caused the cortical region of the lens to become opaque. Addition of the Ca(2+) chelator, EGTA, inhibited the ionomycin-induced changes. Progress of opacification correlated with the death of lens cells and lens swelling in differentiating fiber cells. Autolysis of calpain 2, following ionomycin treatment, suggested activation of this protease. 2DE revealed that the ionomycin did not result in substantial proteolysis of the crystallins. However, Western blotting revealed significant breakdown of the cytoskeletal proteins, spectrin and vimentin. The pattern of the breakdown products was consistent with calpain proteolytic activity. SJA6017 retarded the cortical opacity induced by Ca(2+)-overload in the ovine lens.CONCLUSION:The ovine lens with Ca(2+)-induced opacification by ionomycin is associated with calpain activation and the subsequent proteolysis of cytoskeletal proteins. These events could be initial factors contributing to cell death and the loss of lens transparency which occurs in this ovine model of cataractogenesis. The ovine model supports the hypothesis that cytoskeletal proteins and Ca(2+) homeostasis play an important role in maintaining lens transparency.

AbstractPurpose: The aim of this study is to evaluate the therapeutic potential of a newly synthesized calpain inhibitor, CAT0059, using a naturally occurring in vivo sheep cataract model.Methods: The selectivity of CAT0059 was investigated by an in vitro protease assay. The efficacy of CAT0059 in preventing proteolysis of lens cytoskeletal proteins by calpain 2 was investigated using a lens‐based cell‐free method. The cytotoxicity and stability of CAT0059 in physiological conditions were examined using cultured sheep lenses. Protein binding of CAT0059 by ocular proteins was assessed and quantified by a modified high‐performance liquid chromatography assay. CAT0059 was formulated in an eye drop solution and as an eye ointment. These were applied in vivo daily to one eye of the cataract lambs, over a 67‐ and 97‐day trial period, respectively. The progression of cataracts in the treated and untreated eyes was assessed by an independent veterinary ophthalmologist using a slit‐lamp microscope.Results: In vitro assays revealed that CAT0059 was selective for cysteine proteases and also protected lens cytoskeletal proteins from degradation. CAT0059 was stable in physiological conditions and non‐toxic to the lens. Only 15% of CAT0059 is bound to proteins in the aqueous humour but >90% bound to lens homogenate. The 67‐day CAT0059 eye drop treatment was not effective in slowing the rate of cataract development. However, application of CAT0059 in an eye ointment initially slowed cataract development compared with the untreated eye. This effect was temporary.Conclusions: In vitro assays confirmed CAT0059 to be a potent calpain inhibitor. The two in vivo trials addressed the ability of CAT0059 to reach the lens and established its limitations as a therapeutic molecule for cataract treatment.

A series of N-heterocyclic dipeptide aldehydes 4-13 have been synthesised and evaluated as inhibitors of ovine calpain 1 (o-CAPN1) and ovine calpain 2 (o-CAPN2). 5-Formyl-pyrrole 9 (IC(50) values of 290 and 25nM against o-CAPN1 and o-CAPN2, respectively) was the most potent and selective o-CAPN2 inhibitor, displaying >11-fold selectivity. The amino acid sequences of o-CAPN1 and o-CAPN2 have been determined. Because of the lack of available structural information on the ovine calpains, in silico homology models of the active site cleft of o-CAPN1 and o-CAPN2 were developed based on human calpain 1 (h-CAPN1) X-ray crystal structure (PDB code 1ZCM). These models were used to rationalise the observed SAR for compounds 4-13 and the selectivity observed for 9. The o-CAPN2 selective inhibitor 9 (CAT0059) was assayed in an in vitro ovine lens culture system and shown to successfully protect the lens from calcium-induced opacification.

Calpains, particularly u-calpain, have been implicated in myofibrillar protein degradation. Calpastatin is a natural occurring inhibitor of calpains and consequently the balance of calpain -calpastatin activity in muscles is believed to dictate the rate of tenderization in post-mortem meat. Research in beef cattle has shown that there is a link between the presence of specific calpastatin variants and post-mortem meat tenderization, whilst other researchers have found there are no linkages. This research investigates whether polymorphic variants in ovine calpastatin in lambs (n = 152) are associated with any post-mortem differences in the tenderness of the LD or in the yield of retail meat cuts. There were no associations between the presence of any one of the eight allelic variations of calpastatin in the lambs with the tenderness of lamb loins. There was a significant association (p=0.05) between the presence of three calpastatin variants and the yield of short-loins. One calpastatin variant resulted in an 15% increase in the weight of the short-loins. The research illustrates that in lambs there is a lack of association between the measured allelic variants of calpastatin and meat tenderness, but the yield of short-loins is influenced by specific calpastatin allelic variants. The relationship between the allelic variants and muscle calpastatin activity has yet to be determined.

Figure S1 Frequency histogram of tenderness data collected from the 120 lamb LD samples. Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Medical practitioners practising in the field of pharmaceutical medicine, whether in industry, regulatory bodies or an academic environment, are bound by the same ethical standards which apply to all doctors. Their work, however, leads to some very specific ethical considerations which may not be fully explored in ethical codes based in clinical medicine. This document aims to establish some guiding principles which should underpin a working ethical framework for pharmaceutical physicians. It clearly places the protection of patients (and research subjects) and the doctor's duties to wider society ahead of responsibilities to an individual employer while emphasising the importance of adherence to high standards of research, including dissemination of findings. These principles form the basis of a fuller report which offers more specific practical advice on possible ethical conflicts or dilemmas.