Farmers without borders-genetic structuring in century old barley (Hordeum vulgare).

The geographic distribution of genetic diversity can reveal the evolutionary history of a species. For crop plants, phylogeographic patterns also indicate how seed has been exchanged and spread in agrarian communities. Such patterns are, however, easily blurred by the intense seed trade, plant improvement and even genebank conservation during the twentieth century, and discerning fine-scale phylogeographic patterns is thus particularly challenging. Using historical crop specimens, these problems are circumvented and we show here how high-throughput genotyping of historical nineteenth century crop specimens can reveal detailed geographic population structure. Thirty-one historical and nine extant accessions of North European landrace barley (Hordeum vulgare L.), in total 231 individuals, were genotyped on a 384 single nucleotide polymorphism assay. The historical material shows constant high levels of within-accession diversity, whereas the extant accessions show more varying levels of diversity and a higher degree of total genotype sharing. Structure, discriminant analysis of principal components and principal component analysis cluster the accessions in latitudinal groups across country borders in Finland, Norway and Sweden. FST statistics indicate strong differentiation between accessions from southern Fennoscandia and accessions from central or northern Fennoscandia, and less differentiation between central and northern accessions. These findings are discussed in the context of contrasting historical records on intense within-country south to north seed movement. Our results suggest that although seeds were traded long distances, long-term cultivation has instead been of locally available, possibly better adapted, genotypes.

Effect of an immunomodulatory feed additive on markers of immunity in pasture-fed dairy cows.

BACKGROUND: Infectious diseases in dairy cows often follow a time of nutritional or physiological stress and the subsequent altered immune system function. This study aimed to determine if the immunomodulatory effects of a feed additive previously observed in experimental animals and housed cattle fed total mixed rations could be reproduced in pasture-fed dairy cattle under Australian conditions. METHODS: The study included 34 pasture-fed dairy cattle given the treatment (n = 17) or placebo (bentonite, n = 17) for an acclimation period of 15 days followed by 60 days of supplementation. Blood tests were taken pre-trial and then 30, 60 and 90 days after acclimation. Blood samples were extracted and preserved in Trizol and analysed for immune markers. RESULTS: Pasture-fed dairy cows in the treatment group had significantly higher levels of the immune markers interleukin-8R and L-selectin in comparison with placebo-fed cows at 60 days after the start of supplementation. CONCLUSION: The immunomodulatory effects of the additive observed in the current study and the associated enhanced neutrophil function demonstrated by other studies suggest a role in decreasing the rates of mastitis and other infectious diseases of dairy cattle, particularly during times of nutritional or physiological stress.

Effect of dietary supplementation on the antimicrobial activity of blood leukocytes isolated from Holstein heifers.

The purpose of this investigation was to evaluate the effect of an immunostimulating feed supplement (OmniGen-AF®) on the antimicrobial properties of blood leukocytes in dairy heifers in an attempt to prevent mastitis. Blood leukocytes from supplemented and unsupplemented controls were used. Phagocytic activity and reactive oxygen species (ROS) production were studied on d 0 (prior to feed supplementation) and on days 30 and 60 after supplementation. L-selectin and IL-8R mRNA expressions on blood leukocytes were evaluated on d 0 (prior to feed supplementation) and monthly thereafter for 15 mo. On d 30 after supplementation, neutrophils from treated heifers exhibited greater binding and internalization of Escherichia coli and greater ROS production compared with unsupplemented controls. L-selectin mRNA expression was increased in supplemented heifers vs. controls; however, IL-8R mRNA expression was not different. Results support the continued study of dietary supplementation as an additional management tool to enhance udder health in dairy heifers.

Organic and inorganic selenium: II. Transfer efficiency from ewes to lambs.

Adequate Se transfer from ewes to lambs is important to prevent Se-deficiency diseases. To evaluate how different chemical forms of Se administered at comparative dosages to mature ewes affect Se status of their lambs, 240 ewes were divided into 8 treatment groups (n = 30 each) and drenched weekly (at an amount equal to their summed daily intake) with no-Se (controls); at recommended amounts (4.9 mg of Se/wk) with inorganic Na-selenite, inorganic Na-selenate, or organic Se-yeast; or at supranutritional amounts (14.7 and 24.5 mg of Se/wk) with Na-selenite or Se-yeast for 1 yr. Weekly drenching of Se was effective at increasing (P < 0.002) Se concentrations in ewe colostrum and milk at 30 d of lactation and in improving (P < 0.001) the Se status of lambs (whole-blood and serum-Se concentrations at birth, and skeletal-muscle Se concentrations at 14 d of age). Selenium concentrations in lacteal secretions were greater in ewes drenched with Se-yeast (colostrum: 374, 436, and 982 ng/mL at 4.9, 14.7, and 24.5 mg of Se/wk, respectively; milk: 26, 39, 64 ng/mL) compared with ewes drenched with Na-selenite (colostrum: 204, 334, 428 ng/mL; milk: 16, 21, 24 ng/mL), and were also greater (P < 0.001) in their lambs. Selenium concentrations continued to increase (P < 0.001) in lamb whole blood (558 and 695 ng/mL at 14.7 and 24.5 mg of Se/wk, respectively), serum (126, 183 ng/mL), and skeletal muscle (991, 1,696 ng/mL) with supranutritional concentrations of Se-yeast, whereas Se concentrations did not differ in whole blood (304, 332 ng/mL), serum (77, 85 ng/mL), or skeletal muscle (442, 482 ng/mg) of lambs from ewes drenched with 14.7 or 24.5 mg of Se/wk of Na-selenite. We conclude that weekly oral drenching of ewes during gestation and lactation with organic Se-yeast results in a more efficient transfer of Se (over a wide range of supplementation rates) from ewe to lamb than does inorganic Na-selenite.

Organic and inorganic selenium: I. Oral bioavailability in ewes.

Although the essentiality of dietary Se for sheep has been known for decades, the chemical source and Se dosage for optimal health remain unclear. In the United States, the Food and Drug Administration (FDA) regulates Se supplementation, regardless of the source of Se, at 0.3 mg of Se/kg of diet (as fed), which is equivalent to 0.7 mg of Se/d or 4.9 mg of Se/wk per sheep. The objectives of this study were to evaluate the effects of Se source (inorganic vs. organic) and supplementation rate (FDA vs. supranutritional rates of 14.7 and 24.5 mg of Se/wk) on whole-blood (WB) and serum-Se concentrations. Mature ewes (n = 240) were randomly assigned to 8 treatment groups (n = 30 each) based on Se supplementation rate (4.9, 14.7, and 24.5 mg of Se•wk(-1)•sheep(-1)) and source [Na-selenite, Na-selenate (4.9 mg/wk only), and organic Se-yeast] with a no-Se control group (0 mg of Se/wk). Treatment groups were balanced for healthy and footrot-affected ewes. For 1 yr, ewes were individually dosed once weekly with 0, 4.9, 14.7, or 24.5 mg of Se, quantities equivalent to their summed daily supplementation rates. Serum- and WB-Se concentrations were measured every 3 mo in all ewes; additionally, WB-Se concentrations were measured once monthly in one-half of the ewes receiving 0 or 4.9 mg of Se/wk. Ewes receiving no Se showed a 78.8 and 58.8% decrease (P < 0.001) in WB- (250 to 53 ng/mL) and serum- (97 to 40 ng/mL) Se concentrations, respectively, over the duration of the study. Whole-blood Se decreased primarily during pregnancy (-57%; 258 to 111 ng/mL) and again during peak lactation (-44%; 109 to 61 ng/mL; P < 0.001). At 4.9 mg of Se/wk, Se-yeast (364 ng/mL, final Se concentration) was more effective than Na-selenite (269 ng/mL) at increasing WB-Se concentrations (P < 0.001). Supranutritional Se-yeast dosages increased WB-Se concentrations in a dose-dependent manner (563 ng/mL, 14.7 mg of Se/wk; 748 ng/mL, 24.5 mg of Se/wk; P < 0.001), whereas WB-Se concentrations were not different for the Na-selenite groups (350 ng/mL, 14.7 mg of Se/wk; 363 ng/mL, 24.5 mg of Se/wk) or the 4.9 mg of Se/wk Se-yeast group (364 ng/mL). In summary, the dose range whereby Se supplementation increased blood Se concentrations was more limited for inorganic Na-selenite than for organic Se-yeast. The smallest rate (FDA-recommended quantity) of organic Se supplementation was equally effective as supranutritional rates of Na-selenite supplementation in increasing WB-Se concentrations, demonstrating the greater oral bioavailability of organic Se.

Organophosphorus pesticide degradation product in vitro metabolic stability and time-course uptake and elimination in rats following oral and intravenous dosing.

Levels of urinary dialkylphosphates (DAPs) are currently used as a biomarker of human exposure to organophosphorus insecticides (OPs). It is known that OPs degrade on food commodities to DAPs at levels that approach or exceed those of the parent OP. However, little has been reported on the extent of DAP absorption, distribution, metabolism and excretion. The metabolic stability of O,O-dimethylphosphate (DMP) was assessed using pooled human and rat hepatic microsomes. Time-course samples were collected over 2 h and analyzed by LC-MS/MS. It was found that DMP was not metabolized by rat or pooled human hepatic microsomes. Male Sprague-Dawley rats were administered DMP at 20 mg kg(-1) via oral gavage and i.v. injection. Time-course plasma and urine samples were collected and analyzed by LC-MS/MS. DMP oral bioavailability was found to be 107 ± 39% and the amount of orally administered dose recovered in the urine was 30 ± 9.9% by 48 h. The in vitro metabolic stability, high bioavailability and extent of DMP urinary excretion following oral exposure in a rat model suggests that measurement of DMP as a biomarker of OP exposure may lead to overestimation of human exposure.

Effects of an immunomodulatory feed additive on the development of mastitis in a mouse infection model using four bovine-origin isolates.

The goal of this study was to examine the ability of a commercially available feed additive (OmniGen-AF) to reduce mammary infections caused by a single strain of mastitic pathogens (Streptococcus uberis, Escherichia coli, Staphylococcus aureus and Klebsiella pneumoniae) and to examine the effects of the additive on markers of mammary immunity. Four experiments were completed using a murine model of bovine mastitis. Infection progression was examined using Sybr-green- and TaqMan-based quantitative PCR assays of 16S ribosomal DNA. Infection of the mammary gland with all pathogens caused rapid (24 to 48 h) appearance of pathogen DNA in mammary tissue. Provision of the feed additive for 2 weeks before infection significantly (P < 0.05) reduced the extent of pathogen DNA accumulation in models of S. uberis, E. coli and S. aureus infection. The additive was ineffective in reducing mammary infections caused by K. pneumoniae. We examined mechanisms of action of the additive through assessment of mammary concentrations of mammary myeloperoxidase (MPO), major histocompatibility complex 2 class II (MHC) and macrophage inflammatory protein-1α (MIP) messenger RNA (mRNA) concentrations and by examining serum complement C3 concentration. Infection of the mammary gland increased concentrations of MPO and MHC mRNAs (P < 0.05). Ability of the pathogen to elicit changes in mammary MPO and MHC gene expression was enhanced by the provision of the additive for 2 weeks before infection. These data imply that the additive increased the mammary inflammatory response and increased antigen presentation during a mammary infection. Value of the additive in preventing mastitis in cattle awaits additional studies using a bovine model and further evaluation of additional strains of the pathogens used in this study.

Use of gene profiling to evaluate the effects of a feed additive on immune function in periparturient dairy cattle.

Objectives were to investigate mechanisms by which a nutritional supplement alters immunity in dairy cattle. Our hypothesis was that feeding this product to dairy cattle altered neutrophil gene expression. Eight periparturient Jersey cattle were randomly assigned to one of two treatments: control and treated. Control animals were fed a dry cow ration for 1 month prior to calving. The treated cows were fed the same ration supplemented with OmniGen-AF. Following calving, blood samples were taken and neutrophils were prepared after which RNA was extracted. Gene expression in neutrophils of treated versus control-fed animals was then assessed using bovine-total leukocyte (BOTL-5) arrays. Eighteen genes were differentially regulated in the experimental group and of these, twice as many were up-regulated as down-regulated. Patterns of changes indicated that the additive might alter neutrophil apoptosis, signaling and sensitivity. Two of the regulated genes [interleukin-1beta converting enzyme (ICE) and interleukin-4 receptor (IL-4R)] were investigated in more detail using quantitative reverse transcriptase-polymerase chain reaction (QRT-PCR). Each was found to be elevated by the feeding of experimental product. Increased expression of ICE indicates potential for enhanced neutrophil expression of interleukin-1beta (IL-1beta), a cytokine which plays roles in the inflammatory response and which stimulates adaptive immunity following innate immune activation. Altered expression of IL-4R indicates potential for changes in neutrophil apoptosis. The experiment identified mechanisms by which the additive altered neutrophil gene expression. While many nutrients support the immune system, we have shown that a non-traditional nutritional approach may also have utility in modulating immune function.

Ability of a commercial feed additive to modulate expression of innate immunity in sheep immunosuppressed with dexamethasone.

In the first study, we tested the ability of a commercial feed additive (OmniGen-AF) to affect markers of innate immunity in immunosuppressed sheep and the ability of a pathogen challenge (mould) to affect the immune response to the additive. Treatments consisted of (1) control, (2) immunosuppressed with dexamethasone (DEX), (3) immunosuppressed plus the feed additive, (4) immunosuppressed plus Aspergillus fumigatus and (5) immunosuppressed, A. fumigatus and the additive. Animal health was monitored and indexes of innate immunity (neutrophil L-selectin and interleukin-1ß (IL-1ß)) were collected. DEX caused immunosuppression (i.e. reduced abundance of neutrophil L-selectin and IL-1ß). This immunosuppressive effect was countered by the provision of the additive in the ration. Provision of mould in the ration increased the ability of the additive to regulate markers of innate immune function. A second study was completed to re-assess the properties of the additive and other feed products. The study consisted of seven treatments: (1) immunosuppressed, (2) immunosuppressed with additive, (3) immunosuppressed with additive in pelleted form (low-temperature pellet) and (4) immunosuppressed with additive in a high-temperature pellet. The remaining three treatments assessed abilities of three other additives to regulate markers of innate immune function. In this study, OmniGen-AF increased expression of neutrophil L-selectin abundance in immunosuppressed animals and this was unaffected by the pelleting temperature. None of the other additives affected markers of innate immunity. In these studies we discovered mechanisms by which a feed product may affect the immune function of ruminant livestock. The product countered DEX-dependent down-regulation of markers of innate immune function and its actions were enhanced by the presence of pathogen (mould) in the ration.

The effects of replacing Rhodes grass (Chloris gayana) hay with ghaf (Prosopis cineraria) pods on the performance of Omani native sheep.

A feeding trial using various levels of dry pods of ghaf (Prosopis cineraria) was carried out with 28 Omani native male sheep. Individual feed intake and body weight were determined for 50 days to allow assessment of the effects of the feeds on growth, feed intake, feed conversion and carcase measurements. The ripened ghaf pods contained 91% dry matter (DM), 13.5% crude protein (CP), 14.3% crude fibre (CF), 1.3% ether extract (EE) and 5.2% ash (on DM basis). Four levels of ghaf (0, 15%, 30% and 45%) were used with corresponding decreasing proportions of Rhodes grass (RGH) hay. Animals fed rations containing RGH as the sole source of roughage and no ghaf grew faster (135 g/day) and had better feed conversion than those fed various levels of ghaf. Sheep fed 15% ghaf gained reasonably well (90 g/day) and had carcase attributes similar to those fed no ghaf. Sheep fed 30% ghaf still gained weight but those fed 45% ghaf lost weight. Negative effects on growth appeared to occur after 6 weeks of feeding ghaf.

Internationalization of the animal science undergraduate curriculum: a survey of its current status, barriers to its implementation and its value.

The goal of this project was to identify the current level at which internationalization has been adopted as a theme in the North American animal science curriculum and to identify its value and the barriers to its implementation. We surveyed animal, dairy, and poultry science departments across Canada and the United States. One hundred twenty-four surveys were mailed and 60% were returned. Associations between aspects of internationalization and student outcomes (admission to veterinary and graduate schools and starting salaries) were examined. Although administrators strongly believed internationalization had value, implementation was limited. The most common practices included international content in core animal science classes, advising, international internships, and participation of faculty in international scholarly activities. Few departments have incorporated internationalization into their mission statements or developed a specific international-themed class, scholarships devoted to international activities, or roles for international students. Few departments reported participation of students in international programs. Barriers included finances and limited commitment from higher administration. Student outcomes were positively associated with faculty size, percentage of international faculty, the ratio of international students to the total student population, international content in core animal science classes, a specific international-themed class, availability of international internships, and exchange of class material internationally via the Internet. Departments that did not offer international opportunities had a negative association (r = -0.79) with starting salary, but these relationships may not be causal. Alternatively, progressive departments may attract and retain exceptional students. The analysis indicated an awareness of the value of international programs, positive impacts in student outcomes, and financial barriers to implementation.

Assessment of molasses-urea blocks for goat and sheep production in the Sultanate of Oman: intake and growth studies.

The goal of this study was to develop a molasses-urea block (MUB) for purposes of supplementing trace minerals to domestic ruminant livestock in Oman. To accomplish this, the utility of molasses and date syrup as fermentable energy sources, of straw, date flakes and wheat bran as fibre sources, and of cement and lime as binders were evaluated. The proportion of cement needed for adequate hardening of the block was also studied. Molasses- and date syrup-based blocks hardened equally well. However, the higher cost of date syrup precluded its use. Wheat straw yielded a low-density block that hardened slowly. Date fibre retained moisture and hardened extremely slowly. Wheat bran-based blocks hardened quickly and yielded dense blocks. Hence, wheat bran was judged to be the superior source of fibre. Lime did not effectively bind the blocks. A cement content of 15% allowed hardening of the blocks within 2-3 weeks. A level of 10% cement in the block reduced the hardening rate by about 50%. Sheep and goats consumed both the straw- and wheat bran-based blocks but at different rates. Consumption of the straw-based block by sheep ranged from 50 to 179 g/head per day, whereas the denser wheat bran-based block was consumed at a rate of 8-20 g/head per day. Consumption of the straw-based block by goats was low (8 g/head per day) compared to that of wheat bran-based blocks (16-24 g/head per day). On the basis of the intake of the bran-based block by sheep, a block was designed that would provide approximately 50% of an animal's trace mineral requirements per day. This block consisted of 45% molasses, 10% urea, 5% trace minerals, 2.5% NaCl, 22.5% wheat bran and 15% cement. Sheep consuming this block gained more weight than sheep fed a conventional mineral block or sheep receiving no mineral supplementation. MUBs are inexpensive (9.5 US cents/kg). We conclude that MUBs have utility for providing trace elements in ruminant diets.

Identification of glycogen phosphorylase and creatine kinase as calpain substrates in skeletal muscle.

The goal of this study was to identify calpain substrates in muscle cells. Our hypothesis was that the yeast two-hybrid method could be used to identify novel calpain substrates. To accomplish this, native mu- and m-calpains, as well as a variety of calpain DNA fragments, were expressed in yeast cells and used to screen for binding proteins in a human skeletal muscle cDNA library. Calpain constructs that were used in the screening process included native mu- and m-calpains, a dominant negative (DN) m-calpain (i.e. active site modified), N-terminal truncated DN m-calpain (i.e. autolyzed DN-m-calpain) and, finally, an N- and C-terminal truncated m-calpain (i.e. autolyzed DN-m-calpain lacking a calcium-binding domain). Yeast cells were transformed using yeast two-hybrid expression vectors containing the different calpain constructs as "baits". Beta-galactosidase activity was assayed as an index of interaction between calpain and its potential target proteins. From this analysis, four clones (Ca2+-ATPase, novel nebulin-related protein (N-RAP), creatine kinase and glycogen phosphorylase) were recovered. Two of these, creatine kinase and glycogen phosphorylase, were selected for further study. In in-vitro assays, calpain was able to partially digest both proteins, suggesting that both creatine kinase and glycogen phosphorylase are natural calpain substrates.

Effects of ciliary neurotrophic factor (CNTF) on protein turnover in cultured muscle cells.

The goal of the study was to evaluate the mechanism by which ciliary neurotrophic factor (CNTF) regulated protein metabolism in skeletal muscle. L8 myotubes were cultured and effects of various times and doses of CNTF on protein synthesis and degradation were evaluated. Effects of CNTF on turnover of specific pools of proteins (myofibrillar and non-myofibrillar) were also evaluated. Protein synthesis was assayed by incorporation of radioactive tyrosine into muscle proteins. Degradation was assessed by release of labelled tyrosine from pre-labelled myotubes. Effects of CNTF on protein turnover were found to be time- and dose-dependent. CNTF (1 and 10 ng/ml) increased myofibrillar protein synthesis after 12 h of exposure but had no effect on non-myofibrillar protein synthesis. Longer exposures of CNTF (24 h) reduced non-myofibrillar protein synthesis and had no effect on myofibrillar protein synthesis. High concentrations of CNTF (10 and 20 ng/ml) reduced myofibrillar protein degradation but had no effect on degradation of non-myofibrillar proteins. To evaluate the mechanism by which CNTF exerts control of protein turnover, we completed a Northern blot for CNTF receptor alpha-subunit (CNTFRalpha). This was non-detectable via conventional northern analysis. Use of RT-PCR, however, confirmed expression of CNTFRalpha, albeit at a low level compared to rat skeletal muscle. This low expression of the receptor in L8 myotubes may explain the limited effect of CNTF in vitro compared to the larger effects typically detected in vivo. CNTF regulated protein turnover through control of protein synthesis and degradation. Effects were dose and timedependent. These observations may explain ability of CNTF to exert both anabolic and catabolic actions in vivo.

The effects of selenium deficiency on differentiation, degradation, and cell lysis of L8 rat skeletal muscle cells.

We investigated the effects of selenium (Se) deficiency on differentiation, protein degradation, and cell lysis in cultured skeletal muscle cells, using L8 rat skeletal muscle cells cultured in serum-free (SF) medium to induce differentiation and to maintain myotubes. Creatine kinase activity was reduced (p < 0.05) by approximately 15% without Se supplementation for 96 h. Confluent myoblasts were treated with SF media with four different levels of vitamin E (0, 10, 35, and 100 microM) in the absence and presence of Se (0 and 0.25 microM, respectively). After 96 h, vitamin E at a high dose (100 microM) was effective in the prevention of the decrease of differentiation caused by Se deficiency (p < 0.05). Following differentiation, the effects of three Se concentrations (0, 0.25, and 2.5 microM) on degradation of proteins as assessed by release of 3H-labeled free amino acids secreted into the media were studied. Selenium supplementation did not affect (p > 0.05) total protein degradation. However, Se deficiency increased (p < 0.05) lactate dehydrogenase released from lyzed dead cells. The results indicate that Se is required to maintain an optimal rate of muscle cell differentiation and health of myotube cultures.

Selenoprotein W, Selenium and Glutathione Peroxidase in Rat and Sheep Brains and in Brain Cell Cultures.

The effects of various selenium (Se) levels on seleno-protein W (SeW) content, Se concentration and glutathione peroxidase (GPX) activity in various parts of the brain from sheep and rats, and in glial and neuronal cells from rats were determined. Se concentration and GPX activity were significantly lower in whole brains from Se deficient lambs than from those receiving Se, but the SeW levels were not different. This is in contrast to the heart and muscle where SeW levels were significantly lower in deficient lambs. As the Se status of lambs increased, GPX and SeW increased in the brain cortex, cerebellum and thalamus. Different patterns from the lambs were noted for the increase of GPX and SeW levels in the cortex and cerebellum from rats fedvarious levels of Se. SeW increased at a faster rate in glial cells than either in L8 muscle or neuronal cells but a different pattern was found for GPX activity. Although GPX activity increased with Se content, the differences between cells were not as great as with SeW. The rate of decline was different for SeW levels and GPX activity upon removal of Se from the media with the three types of cells. SeW and GPX activity decreased at about the same rate in L8 muscle cells, GPX activity decreased faster than SeW in neuronal cells, but SeW decreased faster than GPX activity in glial cells when Se was removed from the medium. Thus, Se appears to be metabolized differently by various parts of the brain and by different brain cells.

Role of calpain in skeletal-muscle protein degradation.

Although protein degradation is enhanced in muscle-wasting conditions and limits the rate of muscle growth in domestic animals, the proteolytic system responsible for degrading myofibrillar proteins in skeletal muscle is not well defined. The goals of this study were to evaluate the roles of the calpains (calcium-activated cysteine proteases) in mediating muscle protein degradation and the extent to which these proteases participate in protein turnover in muscle. Two strategies to regulate intracellular calpain activities were developed: overexpression of dominant-negative m-calpain and overexpression of calpastatin inhibitory domain. To express these constructs, L8 myoblast cell lines were transfected with LacSwitch plasmids, which allowed for isopropyl beta-D-thiogalactoside-dependent expression of the gene of interest. Inhibition of calpain stabilized fodrin, a well characterized calpain substrate. Under conditions of accelerated degradation (serum withdrawal), inhibition of m-calpain reduced protein degradation by 30%, whereas calpastatin inhibitory domain expression reduced degradation by 63%. Inhibition of calpain also stabilized nebulin. These observations indicate that calpains play key roles in the disassembly of sarcomeric proteins. Inhibition of calpain activity may have therapeutic value in treatment of muscle-wasting conditions and may enhance muscle growth in domestic animals.

Evidence for the participation of the proteasome and calpain in early phases of muscle cell differentiation.

Objectives were to investigate the role of the proteasome and m-calpain to muscle cell differentiation. Accordingly, we investigated the effects of lactacystin, a proteasome inhibitor, and calpain inhibitor-II (CI-II) on L8 muscle cell differentiation and assessed concentrations of proteasomal and calpain subunit mRNAs during differentiation. L8 myoblasts were induced to differentiate by culturing in mitogen-depleted medium. To assess the importance of the proteasome and calpain to differentiation, we examined effects of lactacystin and CI-II on creatine kinase (CK) activity. In the absence of inhibitor, CK activity was detectable within 48 h of mitogen depletion and myotubes were formed. Addition of lactacystin or CI-II to cultures drastically reduced CK activity and prevented formation of myotubes. Hence, proteasome and calpain are both necessary for differentiation. In order to identify which proteasomal subunits were regulated during differentiation, we examined the concentrations of two 20S core subunits (C8 and C9) and three 22S ATPases (MSS1, S4 and TBP1) during differentiation. Concentrations of m-calpain and beta-tubulin mRNAs were also assessed. Differentiation was associated with slight increases (ca. 30%) in concentrations of mRNAs encoding the proteasomal 20S core subunits (C8 and C9) and with large increases (approximately 2-fold) in mRNAs encoding the regulatory subunit ATPases. m-calpain mRNA concentration also increased two-fold following mitogen depletion. beta-Tubulin mRNA concentration remained unchanged early in the differentiation process and thereafter declined. Of interest, changes in proteasomal and m-calpain mRNAs occurred within 6-24 h of mitogen depletion (i.e., at least 24-36 h prior to detectable changes in creatine kinase activity). These results indicate that changes in expression of proteasome and calpains subunits occur early in the differentiation process. These changes may be required for the normal course of differentiation to proceed. Differentiation is associated with larger changes in proteasomal ATPase mRNAs than in 20S core particle mRNAs indicating that either turnover rates of the 22S ATPase subunits are more rapid in differentiating cells than of the 20S core particles or that functions of the regulatory subunits become more important during muscle cell differentiation.