The relationship between animal age and the thermal stability and cross-link content of collagen from five goat muscles.

The thermal stability of intramuscular collagen, as determined using differential scanning calorimetry, was measured in five muscles from 75 goats with known birth dates ranging in age from one day to 13 years. The collagen cross-link pyridinoline, and the collagen-associated, and putative cross-link, Ehrlich Chromogen were also measured. Five different muscles were examined and the effects of age compared to those found in the tendon of the longissimus dorsi muscle. The differences between intramuscular collagen and tendon collagen were found to be much greater than those between the intramuscular collagens of different muscles. Intramuscular collagen is more thermally stable than tendon collagen due to higher levels of heat-stable cross-links. However the increase in thermal stability of intramuscular collagen with age could not be explained simply in terms of the cross-links measured.

Collagen crosslinks and their relationship to the thermal properties of calf tendons.

The hydrothermal isometric tension and thermal transition temperature of collagen were determined in tendons from three different calf muscles. The levels of the nonreducible collagen crosslink, pyridinoline, and the collagen-associated Ehrlich chromogen were also measured in the three tendons. The reducible collagen crosslinks, hydroxylysinonorleucine, dihydroxylysinonorleucine, and histidinohydroxymerodesmosine were measured in two tendons. The thermal properties and levels of crosslinks were found to vary considerably between the different tendons, and also at different sites in two of the tendons. A strong correlation was observed between the thermal transition temperatures and the hydrothermal isometric tensions of the nine tendon sites examined. Both thermal properties correlated with the concentration of both pyridinoline and Ehrlich chromogen. The analogous behavior of the collagen-associated Ehrlich chromogen and the pyridinoline crosslink supports the role of the Ehrlich chromogen as a nonreducible crosslink.

The comparison of electroencephalograms recorded before and after electrical stunning of cattle.

Electroencephalograms (EEGs) were recorded before and after 'head-only' electrical stunning of adult cattle. Epochs of 8·5 s duration derived from the pre-and the post-stun EEG signals were compared for differences in scale and frequency. The frequency structures of two selected epochs from the one animal were evaluated using the periodogram ordinates derived by calculating the Fast Fourier Transform. The comparison of the two pre-stun epochs indicated that, within the one animal, the pre-stun EEG signal had a consistent frequency pattern. Similarly, a comparison of two post-stun epochs indicated that the post-stun EEG signal also had a consistent frequency pattern. The comparison of pre- and post-stun epochs indicated a consistent increases in amplitude after stunning. Additionally, after stunning, there was an increase in the power of frequencies in the range 4-8 Hz and a decrease in the power of certain frequencies in the range 15-25 Hz. Although there was considerable animal-to-animal variation it was demonstrated that electric stunning produced definable changes in the EEG signal.

Thermal transition of collagen in ovine connective tissues.

The thermal transition temperature (T(m)) of collagen in a range of muscle and non-muscle connective tissues from lambs, hoggets and mature sheep was measured by differential scanning calorimetry. The muscles selected were: semimembranosus (SM), semitendinosus (ST), biceps femoris (BF), Longissimus dorsi (LD) and psoas major (PM). Although the T(m) of intramuscular SM collagen from an aged ewe underwent no significant change with time post mortem, that of a 5-months-old lamb had dropped by 0·9°C after 2 days at 19°C. The epimysium of each muscle exhibited a higher T(m) than the corresponding intramuscular connective tissue. The LD and BF tendons each had a lower T(m) than corresponding intramuscular connective tissue but this was not true for the PM. Furthermore, the PM tendon generated an isometric tension more than five times that of the LD, BF or psoas minor tendons. This indicates that the PM tendon is richer in heatstable crosslinks than any of the other tendons investigated. In all tissues, except the liver capsule, there was an increase in T(m) with animal age. However, the rate of change T(m) varied from one tissue to another. For example, the SM intramuscular collagen matured at an earlier age than that of other muscles, the PM being slowest to mature. In keeping with the changes in T(m) values, the Warner-Bratzler peak force of ST muscles increased markedly in older sheep, but there was no significant difference in peak force of SM muscles between lambs, hoggets and mature sheep.

Breakdown of connectin during cooking of meat.

Light-scattering studies on extracts of meat have confirmed the heat-induced breakdown of connectin previously observed by SDS gel electrophoresis. Because of the high subunit MW (∼10(6)) of connectin, the weight-average molecular weight of whole muscle undergoes a relatively large decrease when connectin is broken down during heating of meat. In cold-shortened muscle, breakdown of connectin by proteolysis was as rapid as in control samples, suggesting that connectin exists in an exposed environment rather than as a core to thick filaments. The breakdown of connectin during heating at 60 or 80°C for 40 min was more extensive than during ageing for 3 weeks at 2°C. Hence, the partial proteolysis of connectin during storage at 2°C is unlikely to be responsible for tenderisation induced by ageing.

Species identification of cooked meats by enzyme-staining of isoelectricfocusing gels.

By extracting cooked meat products with 6 m guanidine hydrochloride and then dialysing the extract into 1 % Triton X-100, the activity of adenylate kinase (AK) and creatine kinase (CK) may be at least partly recovered, provided the meat was not heated above ≈120°C (AK) or ≈105°C (CK). A wide range of animal species may be identified in cooked meats by staining isoelectricfocusing gels for these two enzymes. However, if a sample consists of mixed flesh from different species, hybrid creatine kinase dimers form during dialysis and the additional bands complicate interpretation of gel patterns.

Studies on the structure of connectin in muscle.

Enzymic hydrolysis, followed by amino acid analysis, provided no evidence for the presence of epsilon-(gamma-glutamyl) lysine or other isopeptide crosslinks in connectin. Gel elecrrophoresis in the presence of sodium dodecyl sulphate did not reveal any difference in connectin between normal and lathyritic muscle, indicating that lysyl oxidase does not initiate cross-link formation in connectin. Although connectin may be covalently crosslinked by some unknown mechanism, the available evidence suggests that the subunit of MW approximately to 900 000 is synthesised as a single polypeptide chain. In developing fetal muscle, myosin heavy chains are apparent some weeks earlier than connectin. This, together with the known susceptibility of connectin to hydrolysis, suggests that connectin exists in an exposed environment rather than as a core to the thick filament.

Heat-induced tenderisation of meat by endogenous carboxyl proteases.

The rôle of carboxyl proteases in tenderising meat was investigated by injecting the inhibitors, pepstatin and EPNP, into pre-rigor muscle. The increase in shear force values induced by these inhibitors provided a minimum estimate of the extent to which endogenous carboxyl proteases normally tenderise meat at 60°C. Gel electrophoresis showed that connectin was hydrolysed to a greater extent than other muscle proteins at this temperature and that breakdown of connectin was inhibited by pepstatin and EPNP. Thus it is likely that, when intact, connectin may contribute to the strength of cooked meat.

Proteolytic degradation of connectin, a high molecular weight myofibrillar protein, during heating of meat.

When homogenised muscle (pH 5·5) was heated at 55°C, connectin was extensively degraded, whereas actin and myosin heavy chains were apparently unaffected. It was concluded that carboxyl proteases (e.g. cathepsin D) were largely responsible, because the breakdown of connectin was inhibited by the addition of pepstatin. When whole muscle samples were heated at 50-70°C, degradation of connectin was inversely related to the ultimate pH of the source muscle, again indicating the role of carboxyl proteases. The greater activity of carboxyl proteases in tissues from older animals was apparently responsible for the more extensive degradation of connectin in muscle from older sheep. Because connectin is extensively degraded in cooked meat, it is unlikely to contribute to meat toughness.

Tensile strength of heat-coagulated myosin fibres.

Myosin fibres, formed by heat-coagulation under conditions of pH and ionic strength similar to those prevailing in post-rigor meat, exhibited tensile strengths which decreased with fibre diameter. Tensile strengths of the smaller diameter fibres (10-100 µm) ranged from 30 to 3 kg/cm(2). These values resemble breaking strengths reported for cooked meat (e.g. Davey & Gilbert, 1977) but are an order of magnitude higher than strengths reported for heat-set myosin gels (Nakayama & Sato, 1971) and myosin-bonded meat pieces (Macfarlane et al., 1977).

Isoelectric variants of actin in raw and cooked meat.

Isoelectric focusing of myofibrillar proteins from raw meat gave one major and several minor actin bands. Extending the time, or raising the temperature, of a heat treatment progressively increased the proportion of variants of isoelectric point lower than the primary component. Because of the concomitant release of ammonia on heating, it was concluded that these variants arose from the hydrolysis pf amide groups of asparagine and/or glutamine. Determination of the relative proportion of deamidated actin components may provide a measure of the severity of heat treatment given to a sample of cooked meat.

Deficiency of serum bactericidal activity against Salmonella typhimurium in sickle cell anaemia.

Systemic salmonellosis is a recognized complication of sickle cell anemia (SCA). In our initial study of SCA host defences against salmonella, we evaluated the bactericidal activity of serum against Salmonella typhimurium. When compared to controls, sera from eight out of nineteen SCA patients were deficient in bactericidal function. Levels of factor B, haemolytic complement and agglutinating antibody were similar in SCA and control sera. However, abnormalities that might theoretically account for the decreased antibacterial activity were observed in many SCA sera. These abnormal findings included: (a) defective function of the alternative complement pathway (decreased bacterial killing in the presence of Mg EGTA); (b) low serum C3 concentration; and (c) decreased total iron-binding capacity (TIBC), with a resultant increase in per cent saturation of iron-binding capacity. Of these deficiencies only the abnormal alternative pathway function was significantly associated with decreased serum bactericidal activity. A suggested function of serum bactericidal activity is prevention of bacteraemia by susceptible organisms. Thus diminished serum bactericidal capacity may increase the risk of Salmonella bacteraemia in some individuals with sickle cell disease.

Activation of alveolar macrophages after lower respiratory tract infection.

Alveolar macrophage function has been studied in relation to bacterial infection of the lower respiratory tract. First, LRT macrophages were examined after exposure of rabbits to Listeria monocytogenes aerosols. Macrophages obtained from the LRT of animals 10 to 48 days after infection were activated, as evidenced by greater adherence to culture dishes and increased ability to ingest and kill both the original infecting organism and unrelated organisms, when compared to normal alveolar macrophages. Next, the in vitro effects on normal alveolar macrophages of incubation supernatants of control and antigen-stimulated lymphocytes (LRT and lymph node) from animals infected with L. monocytogenes or Streptococcus pneumoniae were evaluated. As manifested by increased adherence and phagocytosis, and an enhanced nonspecific bactericidal activity, alveolar macrophages were activated by the antigen-stimulated supernatants. These stimulated lymphocyte supernatants contain lymphokines (MIF), but the exact nature of the alveolar macrophage activating factor(s) remains to be determined. These observations, together with recent evidence that alveolar macrophages respond to lymphokines (MIF), suggest that the effector mechanism for cell-mediated immunity in the LRT is intact.