Fresh vein allograft survival in dogs after cyclosporine treatment.

Synthetic grafts are widely used for peripheral arterial reconstructions when autologous veins are not available, but their results have not been satisfactory. Venous allograft may be used as an alternative to synthetic prostheses. The aim of the study was to explore the immunosuppressive efficacy of Cyclosporine A (CyA) as a means of preventing venous allograft failures and rejection. We utilized 56 mongrel dogs. Immunological incompatibility was checked with the skin graft method. Donor inferior vena cava was transplanted into the infrarenal abdominal aorta of recipient animals. One group (group 1, 10 dogs) served as a control and three groups received CyA treatment regimens. Group 2 (10 dogs) received postoperative oral CyA treatment for 30 days. Group 3 (12 dogs) received a vein graft pretreated with a CyA solution without postoperative immunosuppressive therapy. Group 4 (9 dogs) received a vein graft pretreated with a CyA solution and postoperative CyA treatment for 30 days. Allografts were examined at 30 days for patency, aneurysmal dilatation, gross structural changes, inflammatory response, and lymphocytic infiltration. Sex chromatine assessment determined the origin (donor or recipient) of the endothelial cells. The allografts from groups 1 and 3 showed significant aneurysmal dilatation and perivenous inflammation when compared to dogs treated with oral CyA therapy (P < 0.0002). Moreover allografts treated with CyA therapy had a better-developed venous neointima (P < 0.009) with less fibrin (P < 0.02) and thinner medial (P < 0.0009) with less fibrin (P < 0.02), and thinner medial (P < 0.0009) and adventitial layers (P < 0.02). No significant differences were observed in neointimal thickness among the four groups. Lymphocytic infiltration was greater in the group of animals who did not receive oral CyA therapy (P < 0.0004). Barr bodies status showed significant differences between oral CyA treated groups and nontreated groups (P < 0.0003). Oral CyA therapy reduced aneurysmal dilatation and immunological response, promoted the development of a neoendothelium, and preserved the structure of the venous layers. Graft pretreatment with CyA flushing did not have a significant immunosuppressive effect.

Esophagitis in Sprague-Dawley rats is mediated by free radicals.

Free radical-mediated esophagitis was studied during duodenogastroesophageal reflux (mixed reflux) or acid reflux in rats. The influence of reflux on esophageal glutathione levels was also examined. Mixed reflux caused more gross mucosal injury than acid reflux. Gross mucosal injury occurred in the mid-esophagus. Total glutathione (GSH) in the esophageal mucosa of control rats was highest in the distal esophagus. The time course of esophageal GSH in rats treated by mixed reflux showed a significant decrease 4 hr after initiation of reflux, followed by a significant increase from the 12th hour on. Mucosal GSH was increased in both reflux groups after 24 hr but significantly more so in the mixed than in the acid reflux group. The free radical scavenger superoxide dismutase (SOD) prevented esophagitis and was associated with decreased GSH levels. GSH depletion by buthionine sulfoximine (BSO) prevented esophagitis and stimulated SOD production in the esophageal mucosa. It is concluded that gastroesophageal reflux is associated with oxidative stress in the esophageal mucosa. The lower GSH levels in the mid-esophagus may predispose to damage in this area. Duodenogastroesophageal reflux causes more damage than pure acid reflux. Oxidative stress leads to GSH depletion of the esophageal mucosa in the first few hours following damage but then stimulates GSH production. GSH depletion by BSO does not worsen esophagitis since it increases the esophageal SOD concentration.

Comparison of proteolytic variables in a lean and obese strain of pig at the ages of 2.5 and 7 months.

The mode(s) of skeletal muscle protein turnover as well as muscle and animal growth may be studied by using lean and obese animals as models. The objectives of this study were to look at proteolytic variables implicated in these processes. A lean and obese strain of swine from similar genetic lineage (Duroc x Yorkshire, 50:50) have been well established and may prove ideal for this purpose. This study was done in two phases. Phase I included eight lean and eight obese pigs at 2.5 months of age, and phase II was identical, but the pigs were 7 months old. Longissimus muscle samples were processed immediately after euthanasia for activity measurements of mu-calpain, m-calpain, calpastatin, and lysosomal cathepsins B and B + L. Additional samples were taken for DNA, RNA, and total protein determinations. In phase I, total calpastatin activity, total and specific cathepsin B+L activity, and total protein/g muscle were greater in the obese pigs than in the lean pigs. In contrast, DNA and RNA/g muscle were greater in the lean pigs. No other differences were observed in phase I. In phase II, total calpastatin activity and total cathepsin B activity were greater in the obese pigs than in the lean pigs. No other differences were observed in phase II. From phase I to phase II, mu-calpain total activity increased in the lean pigs but not in the obese pigs and calpastatin activity decreased in both lean and obese pigs; however, the phase-II-obese and phase-I-lean total calpastatin concentrations were not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)

Postmortem proteolysis in longissimus muscle from beef, lamb and pork carcasses.

Postmortem proteolysis in skeletal muscle and factors affecting this process were examined in pork, lamb and beef longissimus muscles (LM) to determine the cause of differences in meat tenderness among these species. Fat thickness differed among species in the following order: pork greater than beef greater than lamb. The following patterns were observed for rate of temperature and pH decline: lamb greater than pork greater than beef and pork greater than beef greater than lamb, respectively. At 1 d postmortem, pork was the most tender, followed by beef and lamb, respectively. Between 1 and 14 d of postmortem storage, lamb LM was the most improved in tenderness, followed by beef and pork, respectively. Species did not differ (P greater than .05) in LM collagen solubility. Pork LM from fed pigs had the highest (P less than .05) level of cathepsins B + L and cystatin(s) activities, whereas no differences (P greater than .05) were observed among the species for cathepsin B activity. The lowest (P less than .01) Ca2(+)-dependent protease (CDP)-II and CDP inhibitor activities were observed in pork LM. Beef LM had the highest CDP inhibitor activity (P less than .05) but was intermediate in CDP-II activity. No differences were observed among species for CDP-I activity. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of myofibrils isolated at 0, 1 and 14 d postmortem indicated that by d 1, desmin hydrolysis was most extensive in pork muscle, followed by lamb and beef.(ABSTRACT TRUNCATED AT 250 WORDS)

In vitro muscle cell proliferation and protein turnover as affected by serum from pigs fed antimicrobials.

The effect of antimicrobial supplementation of pigs on the capacity of their sera to influence proliferation and protein turnover in cultured muscle cells was evaluated. Mitogenic activity of sera increased when pigs were fed ASP250 (P less than .005) or carbadox (P less than .001), whereas the mitogenic activity of serum from pigs receiving the basal diet remained unchanged (P = .5). Additionally, sera from ASP250-fed pigs significantly decreased (P less than .001) total cellular protein degradation compared with sera obtained from the same pigs prior to supplementation. Neither ASP250 nor carbadox stimulated proliferation of myogenic cells when added to the culture media. Inclusion of ASP250 in swine diets altered the composition of their sera in a way that stimulated muscle cell proliferation and reduced the rate of protein degradation in cultured myogenic cells. Likewise, the inclusion of carbadox in swine diets increased the ability of their sera to stimulate cultured muscle cell proliferation.

Comparisons of four methods for quantification of lysosomal cysteine proteinase activities.

Four methods were compared to optimize the measurement of the activities of cathepsin B and cathepsin L in porcine skeletal muscle. These methods were: Method A (lysosomal enriched fraction obtained by differential centrifugation), Method B (muscle extract in the absence of detergent), Method C (muscle extract in the presence of detergent) and Method D (the same as method C, but passed through a S-carboxymethylated-papain-Sepharose affinity column). Results indicated that, of the methods tested, Method D yielded greater cathepsin B and consistently greater cathepsin B + L activities per gram of muscle. Hence, Method D is the method of choice for quantification of these enzyme activities. Studies indicated that for cathepsin B with Z-Arg-Arg-NMec as substrate, Km and Vmax values were .416 mM and 4,405 pmol.min-1.mg protein-1, respectively. The Km and Vmax for cathepsins B + L were .132 mM and 9,346 pmol.min-1.mg protein-1, respectively. The relationship between enzyme activity and incubation time was linear for the incubation times studied (up to 60 min). Also, the relationship between enzyme activities and amount of protein in the assay was linear at the concentrations studied (up to 20 micrograms protein). The same preparations were assayed by conditions commonly used by many investigators (.005 mM substrate, approximately 75 micrograms protein, 30 min at 37 degrees C) and by conditions established in this study (1.0 mM substrate, 10 micrograms protein and 15 min at 37 degrees C).(ABSTRACT TRUNCATED AT 250 WORDS)

Alterations in postmortem degradation of myofibrillar proteins in muscle of lambs fed a beta-adrenergic agonist.

Dietary administration of 4 ppm of the beta-agonist L-644,969 (Merck Sharpe and Dohme Research Laboratories) to finishing lambs induced a decrease (10 to 14%, P less than .05) in extractable calpain I activity in the longissimus muscle (LD) at death (d 0). At 4 d postmortem (d 4), extractable calpain I levels in the LD of both control and treated lambs were reduced (P less than .001) from those present at d 0, but the extractable activity in the LD was reduced to a greater extent in control than in treated lambs. Calpain II activity was increased 42% (P less than .005) in LD of treated lambs; however, no significant differences were observed between d 0 and d 4 calpain II activity within treated or control LD samples (P greater than .1). Calpastatin activity was higher in the LD of treated lambs (74% on d 0, P less than .001 and 430% on d 4, P less than .001) than in the LD of control lambs. Measurable cathepsin B activity was decreased (29% on d 0, P less than .05) and measurable cathepsin H activity was increased (10% on d 0, P less than .05 and 10% on d 4, P less than .05) in the LD of treated lambs compared with controls. On d 2, 4 and 6 postmortem, degradation in myofibrils isolated from the LD was lower for treated than for control lambs. Warner-Bratzler shear values for loin chops from treated lambs were higher on both d 3 (111%) and 6 (108%) postmortem than for chops from control lambs (P less than .001). L-644,969-induced decreases in muscle proteolytic capacity may limit postmortem myofibril degradation and contribute to the reduced tenderness observed. This decreased proteolytic capacity may contribute to increased muscularity of L-644,969-treated lambs.

Partial purification of a serum fraction from fasted pigs that inhibits proliferation of cultured myogenic cells.

Sera from pigs fasted as little as 24 h appears to contain a factor(s) that inhibits proliferation of myogenic cells in culture. An inhibitor of myogenic cell proliferation has been partially purified from this sera by using a combination of gel filtration and immunoaffinity chromatography. The inhibitory activity elutes from a Sephacryl S-300 column at a Kav (elution minus void volume divided by total minus void column volume) between .41 and .59. Proteins banding at 76 and 67 kilodaltons appear to predominate on sodium dodecyl sulfate polyacrylamide gels of this fraction. Small quantities of each of these proteins were electrophoretically purified and used to elicit production of anti-76 and anti-67 immunoglobulin G in rabbits. These antibodies were used to prepare anti-76 and anti-67 column was particularly useful in isolating the inhibitor because it removed mitogens that made detection of the inhibitory activity difficult. The partially purified inhibitor inhibits proliferation of L6 myogenic cells in a concentration-dependent manner. On sodium dodecyl sulfate polyacrylamide gels, the predominant proteins in the inhibitor fraction band at approximately 63 and 61 kilodaltons. Inhibitors of myogenic cell proliferation may play an important role in balancing the effects of positive growth factors.

In vivo effect of a beta-adrenergic agonist on activity of calcium-dependent proteinases, their specific inhibitor, and cathepsins B and H in skeletal muscle.

DEAE-Sephacel and phenyl-Sepharose chromatography were compared as methods for separating and quantitatively isolating calpain I, calpain II, and calpastatin from lamb muscle extracts. DEAE-Sephacel chromatography gave greater than 90% recovery of all three proteins, while phenyl-Sepharose gave only 70, 66, and 48% of the DEAE recovery of calpain I, calpain II, and calpastatin, respectively. Additionally, DEAE-Sephacel chromatography was shown to effectively separate calpastatin and calpain I. Consequently DEAE-Sephacel appears to be superior to phenyl-Sepharose for quantitative isolation of the components of the calcium-dependent proteinase system from muscle extracts. Dietary administration of beta-agonist (L-644, 969; Merck Sharpe & Dohme Research Laboratories) decreases extractable calpain I activity in lamb longissimus dorsi (LD) muscle by 10-14% (P less than 0.05), increases calpain II activity by 34-42% (P less than 0.001), and increases calpastatin activity by 59-75% (P less than 0.001). Additionally, net cathepsin B activity is reduced by 30% (P less than 0.05) in the LD of beta-agonist-treated lambs. Reduced activity of the calcium-dependent or catheptic proteinase systems may contribute to the increased protein accretion in muscles of beta-agonist-treated lambs.