Akihito Hattori

Structural weakening of intramuscular connective tissue during conditioning of beef

The structural changes in intramuscular connective tissues endomysium and perimysium during conditioning of beef were investigated using an improved technique of scanning electron microscopy. In beef conditioned for 28 days of 4°C, the endomysium resolved into individual collagen fibrils and the thick sheets of perimysium separated into collagen fibres of 4-8 μm in diameter. These results provide direct evidence for the structural weakening of endomysium and perimysium during conditioning. The structural changes in the intramuscular connective tissue were minimal until 10 days post mortem, but clearly observable after 14 days post mortem. Therefore, it is concluded that intramuscular connective tissue shows the effect on tenderisation of extended conditioning (2-4 weeks) of beef.

A Zn-porphyrin complex contributes to bright red color in Parma ham

The Italian traditional dry-cured ham (Parma ham) shows a stable bright red color that is achieved without the use of nitrite and/or nitrate. In this study we examined the pigment spectroscopically, fluoroscopically and by using HPLC and ESI-HR-MASS analysis. Porphyrin derivative other than acid hematin were contained in the HCl-containing acetone extract from Parma ham. A strong fluorescence peak at 588 nm and a weak fluorescence peak at 641 nm were observed. By HPLC analysis the acetone extract of Parma ham was observed at the single peak, which eluted at the same time as Zn-protoporphyrin IX and emitted fluorescence. The results of ESI-HR-MS analysis showed both agreement with the molecular weight of Zn-protoporphyrin IX and the characteristic isotope pattern caused by Zn isotopes. These results suggest that the bright red color in Parma ham is caused by Zn-protoporphyrin IX.

Decorin enhances the proliferation and differentiation of myogenic cells through suppressing myostatin activity

Decorin, a small leucine‐rich proteoglycan, plays an important role in the regulation of cell growth. Our recent study has shown that immobilized decorin in the collagen matrix sequesters myostatin into the extracellular matrix and prevents its inhibitory action to myoblast proliferation in vitro. However, it still remains unclear whether free decorin could affect the proliferation and differentiation of myogenic cells by regulating myostatin activity. In the present study, we generated stable clonal C2C12 myoblasts that were over‐expressing decorin, and showed that decorin over‐expressing cells had an increased rate of proliferation as compared to control cells. Decorin over‐expressing cells formed multi‐giant hypertrophic myotubes with an elongated morphology and larger size as compared to control cells, although the initiation of differentiation in decorin over‐expressing cells was somewhat delayed as compared to control cells. Western blot analysis demonstrated that MyoD expression in decorin over‐expressing cells was lower than that in control cells until 12 h after induction to differentiate. At 48‐h differentiation, the expressions of MyoD, p21 and myogenin were dramatically increased in cells that over‐expressed decorin. Furthermore, we revealed that over‐expression of decorin suppressed the activity of myostatin endogenously synthesized in C2C12 myoblasts and attenuated the signaling of exogenous myostatin. Consistent with these results, knock‐down of decorin impairs C2C12 myoblast growth by increasing the sensitivity to exogenous myostatin. These results clearly show that decorin enhances the proliferation and differentiation of C2C12 myoblasts through suppressing myostatin activity. J. Cell. Physiol. 215: 856–867, 2008.

Calcium binding to an elastic portion of connectin/titin filaments.

Abstractα-Connectin/titin-1 exists as an elastic filament that links a thick filament with the Z-disk, keeping thick filaments centered within the sarcomere during force generation. We have shown that the connectin filament has an affinity for calcium ions and its binding site(s) is restricted to the β-connectin/titin-2 portion. We now report the localization and the characterization of calcium-binding sites on β-connectin. Purified β-connectin was digested by trypsin into 1700- and 400-kDa fragments, which were then subjected to fluorescence calcium-binding assays. The 400-kDa fragment possesses calcium-binding activity; the binding constant was 1.0 × 107 M−1 and the molar ratio of bound calcium ions to the 400-kDa fragment reached a maximum of 12 at a free calcium ion concentration of approximately 1.0 μM. Antibodies against the 400-kDa fragment formed a sharp dense stripe at the boundary of the A and the I bands, indicating that the calcium-binding domain constitutes the N-terminal region of β-connectin, that is, the elastic portion of connectin filaments. Furthermore, we estimated the N-terminal location of β-connectin of various origins (n = 26). Myofibrils were treated with a solution containing 0.1 mM CaCl2 and 70 μM leupeptin to split connectin filaments into β-connectin and a subfragment, and chain weights of these polypeptides were estimated according to their mobility in 2% polyacrylamide slab gels. The subfragment exhibited a similar chain weight of 1200 ± 33 kDa (mean ± SD), while α- and β-connectins were variable in size according to their origin. These results suggest that the apparent length of the 1200-kDa subfragment portion is almost constant in all instances, about 0.34 μm at the slack condition, therefore that the C-terminus of the 1200-kDa subfragment, that is, the N-terminus of the calcium-binding domain, is at the N2 line region of parent filaments in situ. Because the secondary structure of the 400-kDa fragment was changed by the binding of calcium ions, connectin filaments could be expected to alter their elasticity during the contraction–relaxation cycle of skeletal muscle.

Relationship between degradation of proteoglycans and weakening of the intramuscular connective tissue during post-mortem ageing of beef.

Changes in proteoglycans (PGs) during post-mortem ageing of bovine m. semitendinosus were studied. Electron microscopic observations made it clear that there were two types of PGs in bovine m. semitendinosus immediately post-mortem: PGs were arranged regularly in the basement membrane and PGs associated with collagen fibrils in the perimysium. After 28 days ageing at 4 °C, no PG was observed in the basement membrane, and the greater part of PGs in the perimysium had disappeared. The total amount of PGs decreased with time post-mortem. SDS-polyacrylamide gel electrophoresis indicated that PGs with a high molecular weight disappeared within 7 days post mortem. These results suggest that PGs are degraded during post-mortem ageing of beef. The degradation of PGs seems likely to be the main factor in the weakening of intramuscular connective tissues, i.e., separation of collagen fibrils and fibres from the endomysium and the perimysium, which results in the partial tenderization of beef during post-mortem ageing.

Developmental expression of extracellular matrix components in intramuscular connective tissue of bovine semitendinosus muscle

Abstract We have investigated the expression patterns of extracellular matrix components in intramuscular connective tissue during the development of bovine semitendinosus muscle by means of indirect immunofluorescence techniques. Types I, III, V, and VI collagen and fibronectin were located in the endomysium and the perimysium. Type IV collagen, laminin, and heparan sulfate proteoglycans (PGs) were exclusively located in the endomysium, and dermatan sulfate PGs existed only in the perimysium. The localization of these components in the intramuscular connective tissue of semitendinosus muscle remained unchanged throughout prenatal and postnatal growth of cattle, suggesting that they are essential for forming and maintaining structures of the endomysium and perimysium in bovine semitendinosus muscle. On the other hand, decorin was undetectable in the endomysium of neonates, although other matrix components were already expressed. It was expressed slightly in the endomysium of 2-month-old calves, and clearly detectable in the endomysium of cattle more than 6 months old. Chondroitin sulfate PGs were barely detectable in the perimysium of fetuses and neonatal calves, and progressively appeared during postnatal development of the muscle. It seems likely that these PGs are closely related to the postnatal development of the endomysium and perimysium.

Establishment of a model experiment system to elucidate the mechanism by which Zn–protoporphyrin IX is formed in nitrite-free dry-cured ham

The aim of this study was to establish a model experiment system to elucidate the mechanism by which Zn-protoporphyrin IX (ZPP) is formed in Parma ham. The established model consisted of myoglobin, meat and antibiotics, and incubation under anaerobic conditions resulted in a greater yield of ZPP. Formation of ZPP was observed even in the presence of various antiseptics. The amount of ZPP formed increased as the period of incubation increased. ZPP formation was inhibited by heating meat homogenate depending on the heating temperature. Our results show that anaerobic conditions are suitable for the formation of ZPP in meat products without nitrate or nitrite and that endogenous enzymes as well as microorganisms may be involved in ZPP formation.

Ultrastructure of the Intramuscular Connective Tissue in Bovine Skeletal Muscle

The three-dimensional arrangement of intramuscular connective tissues in bovine semitendinosus muscle was investigated using the cell-maceration method for scanning electron microscopy, by which cellular elements were eliminated and collagen fibrils and fibers were exposed. The 60-100 microns diameter honeycomb structures of the endomysium housing individual muscle fibers were clearly observed. The sheath of endomysium was membranous and consisted of tightly arranged collagen fibrils 30-70 nm in diameter. The perimysium was composed of several layers of 100-200 microns-thick sheets surrounding the sheaths of the endomysium. The sheets of the perimysium were wavy and consisted of collagen fibers made up of tightly bundled fibrils. The epimysium was composed of two distinct layers of thick sheets. The inner layer consisted of thousands of collagen fibers lying in an extremely regular wavy pattern parallel to the axis of the muscle fiber. The outer layer of epimysium was composed of several wavy sheets of collagen fibers which ran transversely to the axis of the muscle fiber. In a region of the epimysium, there was a thick wall of about 1 mm thickness, where fiber bundles of 200-300 microns in diameter ran longitudinally parallel to the axis of muscle fiber.

Myosin is solubilized in a neutral and low ionic strength solution containing L-histidine.

Myosin, one of the major myofibrillar proteins, is insoluble at low and physiological ionic strength and soluble at high ionic strength. In this study, the behavior and morphology of myosin solubilized in a low ionic strength solution containing l-histidine (l-His) was investigated. More than 80% of myosin was solubilized in a low ionic strength solution with dialysis against a solution containing 1mM KCl and 5mM l-His. Transmission electron microscopy with rotary shadowing demonstrated that the rod of myosin in a low ionic strength solution containing l-His is longer than that of myosin in a high ionic strength solution. The elongation of the myosin rod in a low ionic strength solution containing l-His would inhibit the formation of a filament, resulting in the solubilization of myosin.

Heat-induced gelation of myosin in a low ionic strength solution containing L-histidine

Binding properties are important for meat products and are substantially derived from the heat-induced gelation of myosin. We have shown that myosin is solubilized in a low ionic strength solution containing L-histidine. To clarify its processing characteristics, we investigated properties and structures of heat-induced gels of myosin solubilized in a low ionic strength solution containing L-histidine. Myosin in a low ionic strength solution formed transparent gels at 40-50°C, while myosin in a high ionic strength solution formed opaque gels at 60-70°C. The gel of myosin in a low ionic strength solution with L-histidine showed a fine network consisting of thin strands and its viscosity was lower than that of myosin in a high ionic strength solution at 40-50°C. The rheological properties of heat-induced gels of myosin at low ionic strength are different from those at high ionic strength. This difference might be caused by structural changes in the rod region of myosin in a low ionic strength solution containing L-histidine.