Identifying constituents of whey protein concentrates that reduce the pink color defect in cooked ground turkey.

Whey protein concentrate constituents were tested for their ability to reduce naturally occurring pink color defect and pink cooked color induced by sodium nitrite (10ppm) and nicotinamide (1.0%) in ground turkey. ß-lactoglobulin (1.8%), α-lactalbumin (0.8%), bovine serum albumin (0.15-0.3%), lactose (1.0-3.0%), potassium chloride (500-1500ppm), and ferrous iron chloride (0.3-30ppm) had no effects on cooked pink color. Lactoferrin (30-5000ppm) increased or decreased pink color depending on its concentration in samples without added sodium nitrite or nicotinamide. Annatto (0.1-1.0ppm) reduced pink color whereas the higher concentration of magnesium chloride (22-88ppm) and ferric iron chloride (0.3-30ppm) increased pink color in samples with added nicotinamide. Calcium chloride (160-480ppm) was the only tested constituent that consistently reduced pink cooked color in samples with and without added nitrite and nicotinamide. Due to the variability of whey protein concentrates and the number of constituents that do not reduce pink cooked color, the addition of calcium alone or dried milk minerals containing calcium, phosphate, and citrate, represents a better means to regularly prevent the pink color defect in cooked ground turkey.

Investigation of mechanisms by which sodium citrate reduces the pink color defect in cooked ground turkey.

The principal mechanism by which sodium citrate reduces the pink color defect in cooked ground turkey was investigated. Sodium citrate (SC; 0, 0.125, 0.25, 0.5, 1.0, 2.0M), sodium nitrite (0.01, 0.1M), and nicotinamide (0.5, 0.75M) were combined in solutions of bovine hemin to determine SCs ability to bind heme iron and competitively inhibit pink-color-generating ligands from binding. Additionally, the effects of sodium erythorbate (0, 275, 550ppm), ferrous iron chloride (0, 0.3, 3.0, 30ppm), and ferric iron chloride (0, 0.3, 3.0, 30ppm) on SCs ability to reduce pink cooked color was examined. Absorbance curves of hemin+nitrite and hemin+nicotinamide were relatively unaffected by SC, therefore whether or not SC bound heme iron, that did not appear to be a mechanism for inhibiting the pink color defect. Both ferrous and ferric iron chloride had minimal effects on color values, possibly due to sodium tripolyphosphate chelation ability in the meat system and thus their presence did not enhance SCs ability to reduce the pink color defect. However, sodium erythorbate, a reducing agent, inhibited SCs ability to decrease the pink color defect in samples induced pink with sodium nitrite and nicotinamide. Therefore, it appears SC requires the presence of oxygen and may participate in oxidative processes to reduce the pink color defect.

Light microscopy and image analysis of thin filament lengths utilizing dual probes on beef, chicken, and rabbit myofibrils.

Image analysis procedures for immunofluorescence microscopy were developed to measure muscle thin filament lengths of beef, rabbit, and chicken myofibrils. Strips of beef cutaneous trunci, rectus abdominis, psoas, and masseter; chicken pectoralis; and rabbit psoas muscles were excised 5 to 30 min postmortem. Fluorescein phalloidin and rhodamine myosin subfragment-1 (S1) were used to probe the myofibril structure. Digital images were recorded with a cooled charge-coupled device controlled with IPLab Spectrum software (Signal Analytics Corp.) on a Macintosh operating system. The camera was attached to an inverted microscope, using both the phase-contrast and fluorescence illumination modes. Unfixed myofibrils incubated with fluorescein phalloidin showed fluorescence primarily at the Z-line and the tips of the thin filaments in the overlap region. Images were processed using IPLab and the National Institutes of Health's Image software. A region of interest was selected and scaled by a factor of 18.18, which enlarged the image from 11 pixels/microm to approximately 200 pixels/microm. An X-Y plot was exported to Spectrum 1.1 (Academic Software Development Group), where the signal was processed with a second derivative routine, so a cursor function could be used to measure length. Fixation before phalloidin incubation resulted in greatest intensity at the Z lines but a more-uniform staining over the remainder of the thin filament zone. High-resolution image capture and processing showed that thin filament lengths were significantly different (P < 0.01) among beef, rabbit, and chicken, with lengths of 1.28 to 1.32 microm, 1.16 microm, and 1.05 microm, respectively. Measurements using the S1 signal confirmed the phalloidin results. Fluorescent probes may be useful to study sarcomere structure and help explain species and muscle differences in meat texture.

Molecular basis of passive stress relaxation in human soleus fibers: assessment of the role of immunoglobulin-like domain unfolding.

Titin (also known as connectin) is the main determinant of physiological levels of passive muscle force. This force is generated by the extensible I-band region of the molecule, which is constructed of the PEVK domain and tandem-immunoglobulin segments comprising serially linked immunoglobulin (Ig)-like domains. It is unresolved whether under physiological conditions Ig domains remain folded and act as "spacers" that set the sarcomere length at which the PEVK extends or whether they contribute to titin's extensibility by unfolding. Here we focused on whether Ig unfolding plays a prominent role in stress relaxation (decay of force at constant length after stretch) using mechanical and immunolabeling studies on relaxed human soleus muscle fibers and Monte Carlo simulations. Simulation experiments using Ig-domain unfolding parameters obtained in earlier single-molecule atomic force microscopy experiments recover the phenomenology of stress relaxation and predict large-scale unfolding in titin during an extended period (> approximately 20 min) of relaxation. By contrast, immunolabeling experiments failed to demonstrate large-scale unfolding. Thus, under physiological conditions in relaxed human soleus fibers, Ig domains are more stable than predicted by atomic force microscopy experiments. Ig-domain unfolding did not become more pronounced after gelsolin treatment, suggesting that the thin filament is unlikely to significantly contribute to the mechanical stability of the domains. We conclude that in human soleus fibers, Ig unfolding cannot solely explain stress relaxation.

Protein kinase A phosphorylates titin's cardiac-specific N2B domain and reduces passive tension in rat cardiac myocytes.

beta-Adrenergic stimulation of cardiac muscle activates protein kinase A (PKA), which is known to phosphorylate proteins on the thin and thick filaments of the sarcomere. Cardiac muscle sarcomeres contain a third filament system composed of titin, and here we demonstrate that titin is also phosphorylated by the beta-adrenergic pathway. Titin phosphorylation was observed after beta-receptor stimulation of intact cardiac myocytes and incubation of skinned cardiac myocytes with PKA. Mechanical experiments with isolated myocytes revealed that PKA significantly reduces passive tension. In vitro phosphorylation of recombinant titin fragments and immunoelectron microscopy suggest that PKA targets a subdomain of the elastic segment of titin, referred to as the N2B spring element. The N2B spring element is expressed only in cardiac titins, in which it plays an important role in determining the level of passive tension. Because titin-based passive tension is a determinant of diastolic function, these results suggest that titin phosphorylation may modulate cardiac function in vivo.

Titin-actin interaction in mouse myocardium: passive tension modulation and its regulation by calcium/S100A1.

Passive tension in striated muscles derives primarily from the extension of the giant protein titin. However, several studies have suggested that, in cardiac muscle, interactions between titin and actin might also contribute to passive tension. We expressed recombinant fragments representing the subdomains of the extensible region of cardiac N2B titin (tandem-Ig segments, the N2B splice element, and the PEVK domain), and assayed them for binding to F-actin. The PEVK fragment bound F-actin, but no binding was detected for the other fragments. Comparison with a skeletal muscle PEVK fragment revealed that only the cardiac PEVK binds actin at physiological ionic strengths. The significance of PEVK-actin interaction was investigated using in vitro motility and single-myocyte mechanics. As F-actin slid relative to titin in the motility assay, a dynamic interaction between the PEVK domain and F-actin retarded filament sliding. Myocyte results suggest that a similar interaction makes a significant contribution to the passive tension. We also investigated the effect of calcium on PEVK-actin interaction. Although calcium alone had no effect, S100A1, a soluble calcium-binding protein found at high concentrations in the myocardium, inhibited PEVK-actin interaction in a calcium-dependent manner. Gel overlay analysis revealed that S100A1 bound the PEVK region in vitro in a calcium-dependent manner, and S100A1 binding was observed at several sites along titin's extensible region in situ, including the PEVK domain. In vitro motility results indicate that S100A1-PEVK interaction reduces the force that arises as F-actin slides relative to the PEVK domain, and we speculate that S100A1 may provide a mechanism to free the thin filament from titin and reduce titin-based tension before active contraction.

Pulse electrophoresis of muscle myosin heavy chains in sodium dodecyl sulfate-polyacrylamide gels.

We have developed a new method that provides enhanced resolution of myosin heavy chain (MHC) isoforms by sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS-PAGE). The key feature of this protocol involves the application of current to slab SDS gels in a pulsatile, repetitive manner rather than continuously as in standard gel systems. This protocol, designated pulse electrophoresis, was achieved by means of a device that intermittently gates the output of a conventional power supply. When used in long (32 cm) separating gels, pulse electrophoresis not only significantly improves the resolution of MHC isoforms compared to conventional systems, but also reduces common artifacts associated with long running times, such as blurred bands and comingling of closely spaced bands. In addition to the increased resolution of protein bands, pulse electrophoresis also allows detection of bands corresponding to previously unidentified MHC isoforms in mammalian and avian tissue. In rat myocardium, for example, pulse electrophoresis revealed three MHC isoform bands, two of which appeared to correspond to two alpha-MHC subspecies. Alternative splicing of the rat alpha-MHC gene is known to generate two isoform species differing by inclusion (or exclusion) of a single glutamine residue, whose relative levels of expression correspond nicely with the amounts of each band identified in this study. Therefore, we cannot rule out that the system presented here may be sufficiently sensitive to differentiate between high molecular weight proteins differing in a single amino acid.

Identification of new repeating motifs in titin.

Repeating motifs of 26-28 amino acids have been identified in the PEVK region of the giant elastic protein titin. These motifs, termed PPAK for the four amino acids that often constitute the beginning of the motif, occur 60 times in human soleus titin. PPAK motifs occur in groups of 2-12 that are separated by regions rich in glutamic acid (approximately 45%) and termed polyE segments. The fluctuation of the net charge between the PPAK and polyE regions suggests ionic interactions between these segments and their involvement in the elastic function of titin. Proteins 2001;43:145-149.

Kinetic differences in cardiac myosins with identical loop 1 sequences.

The kinetics of nucleotide turnover vary considerably among isoforms of vertebrate type II myosin, possibly due to differences in the rate of ADP release from the nucleotide binding pocket. Current ideas about likely mechanisms by which ADP release is regulated have focused on the hyperflexible surface loops of myosin, i.e. loop 1 (ATPase loop) and loop 2 (actin binding loop). In the present study, we investigated the kinetic properties of rat and pig beta-myosin heavy chains (beta-MHC) in which we have found the sequences of loop 1 (residues 204-216) to be virtually identical, i.e. DQSKKDSQTPKG, with a single conservative substitution (rat E210D pig). Pig myocardium normally expresses 100% beta-MHC, whereas rat myocardium was induced to express 100% beta-MHC by surgical thyroidectomy and subsequent treatment with propylthiouracil. Slack test measurements at 15 degrees C yielded unloaded shortening velocities of 1.1 +/- 0.8 muscle lengths/s in rat skinned ventricular myocytes and 0.35 +/- 0.05 muscle lengths/s in pig skinned myocytes. Similarly, solution measurements at the same temperature showed that actin-activated ATPase activity was 2.9-fold greater for rat beta-myosin than for pig beta-myosin. Stopped-flow methods were then used to assess the rates of acto-myosin dissociation by MgATP both in the presence and absence of MgADP. Although the rates of MgATP-induced dissociation of acto-heavy meromyosin (acto-HMM) were virtually identical for the two myosins, the rate of ADP dissociation was approximately 3.8-fold faster for rat beta-myosin (135 s(-)(1)) than for pig beta-myosin (35 s(-)(1)). ATP cleavage rates were nearly 30% faster for rat beta-myosin. Thus, whereas loop 1 appears from other studies to be involved in nucleotide turnover in the pocket, our results show that loop 1 does not account for large differences in turnover kinetics in these two myosin isoforms. Instead, the differences appear to be due to sequence differences in other parts of the MHC backbone.

Binding of filamin isoforms to myofibrils.

Two filamin isoforms were purified from bovine tissues and characterized. Muscle filamin and nonmuscle filamin had different SDS gel mobilities, proteolytic digestion patterns, myofibrillar binding distributions and myofibril binding affinities. The muscle specific filamin had an apparent molecular weight of 265 kDa and bound primarily to the Z-lines of myofibrils but also to the I-bands near the Z-lines. The nonmuscle specific filamin had an apparent molecular weight of 275 kDa and bound exclusively to the Z-lines of myofibrils. The filamin myofibril binding was studied quantitatively. Plotting bound fraction (mg filamin/mg myofibril) vs. equilibrium concentration of free filamin yielded a biphasic binding curve. The first hyperbolic binding phase described the binding of filamin to myofibrils but the second phase appeared to be nonspecific due to filamin aggregation. The muscle filamin had a significantly lower (P < 0.05) apparent binding affinity to myofibrils than nonmuscle filamin. However, the muscle filamin showed a significantly higher (P < 0.05) saturation value for myofibrils than nonmuscle filamin. The binding of phosphorylated filamin to myofibrils was significantly lower (P < 0.05) than the corresponding native proteins for both filamin isoforms.

Sequence and mechanical implications of titin's PEVK region.

A widely used titin monoclonal antibody (9D10) was epitope mapped to the PEVK region in the I-band portion of titin. Sequence analysis of the titin PEVK region revealed a large number of 28 amino acid modules (termed "PPAK" repeats) alternating with glutamic acid rich segments. Species differences in cardiac rest tension could not be ascribed to differences in the PEVK length of the N2B titin isoform. The low rest tension generated by dog cardiac muscle also does not appear to be explained by the N2 and PEVK segment lengths in the N2A titin isoform.

From connecting filaments to co-expression of titin isoforms.

The molecular basis of elasticity in insect flight muscle has been analyzed using both the mechanism of extensibility of titin filaments (Trombitás et al., J. Cell Biol. 1998;140:853-859), and the sequence of projectin (Daley et al., J. Mol. Biol. 1998;279:201-210). Since a PEVK-like domain is not found in the projectin sequence, it is suggested that the sarcomere elongation causes the slightly "contracted" projectin extensible region to straighten without requiring Ig/Fn domain unfolding. Thus, the extensible region of the projectin may be viewed as a single entropic spring. The serially linked entropic spring model developed for skeletal muscle titin was applied to titin in the heart. The discovery of unique N2B sequence extension in physiological sarcomere length range (Helmes et al., Circ. Res. 1999;84:1339-1352) suggests that cardiac titin can be characterized as a serially linked three-spring system. Two different cardiac titin isoform (N2BA and N2B) co-exist in the heart. These isoforms can be differentiated by immunoelectron microscopy using antibody against sequences C-terminal of the unique N2B sequence, which is present in both isoforms. Immunolabeling experiments show that the two different isoform are co-expressed within the same sarcomere.

Motor proteins regulate force interactions between microtubules and microfilaments in the axon.

It has long been known that microtubule depletion causes axons to retract in a microfilament-dependent manner, although it was not known whether these effects are the result of motor-generated forces on these cytoskeletal elements. Here we show that inhibition of the motor activity of cytoplasmic dynein causes the axon to retract in the presence of microtubules. This response is obliterated if microfilaments are depleted or if myosin motors are inhibited. We conclude that axonal retraction results from myosin-mediated forces on the microfilament array, and that these forces are counterbalanced or attenuated by dynein-mediated forces between the microfilament and microtubule arrays.

Filamin isogene expression during mouse myogenesis.

The developmental pattern of filamin gene expression has been studied in mouse embryos by using in situ hybridization. The probes used were isoform specific, (35)S-labeled antisense complementary ribonucleic acids (cRNAs) to the 3; untranslated region (3; UTR) of muscle-specific and nonmuscle-specific filamin genes. Northern blot and in situ hybridization results showed that nonmuscle-specific filamin transcripts had a size of 9.5 kb and were expressed in all nonmuscle tissues. Labeling was most intense in tissues containing a substantial proportion of epithelial and smooth muscle cells. Muscle-specific filamin transcripts had a size of 10 kb and were expressed primarily in cardiac and skeletal muscle. The expression of muscle-specific filamin messenger ribonucleicacids (mRNAs) was detected in heart at 8.0 days after coitum, whereas that in the myotomes of somites was not detected until 10.5 days after coitum. The expression of muscle-specific filamin mRNAs in heart and in skeletal muscle continued through the subsequent days of myogenesis. The results showed that muscle-specific filamin gene transcripts are detected before the formation of myotubes in vivo. This is the first study of filamin gene expression at the early stages of skeletal muscle development. Dev Dyn 2000;217:99-108.

Adaptation of a super-sensitive epitope detection technique for the immunoelectron microscopy of titin filaments in vertebrate striated muscle.

A super-sensitive epitope-detection technique based on gold-silver intensification was adapted for pre-embedding immunolabelling of titin filaments in vertebrate striated muscle. Indirect immunoelectron microscopy of titin filaments was performed with monoclonal titin antibodies as primary antibodies and Fab anti-mouse IgG conjugated with 1.4 nm gold particles as secondary antibodies. The secondary antibodies penetrated easily into the tissue owing to their reduced size and the very small gold particles. After the labelling procedure, the tissue was fixed in glutaraldehyde. Since the gold particles were not visible by conventional transmission electron microscopy, they were intensified with a silver developing system. Although the particle size varied nonlinearly with the developing time, very fine grain size was achievable. The technique provided super-sensitive detection with excellent contrast and demonstrated epitopes with both strong and weak affinities.

Using pork to teach students quality variations and how they are measured. 1998 UW-AS-305 Class.

Using a total of eight pork loins representing DFD (dark, firm, and dry) and PSE (pale, soft, and exudative) conditions, 35 students conducted a series of objective and subjective measurements to demonstrate extremes in meat quality in a single 2-h laboratory. Students learned to objectively assess appearance, water-holding and water-binding capacity, ultimate pH (pHu), and shear force (cooked samples) by operating seven commonly known laboratory instruments. They also learned how to prepare and present samples for organoleptic analysis using hedonic and triangle tests. Finally, the students learned the factors related to meat quality and how extremely they can vary. Within one laboratory, they observed that DFD, when compared with PSE, averaged 1.5 units higher in pHu, 4.7% (absolute) less drip loss, bound 136% (absolute) more water, was darker in color (26 units lower L* value), was firmer with a more attractive structure requiring 1 kg/cm less force to shear, and was superior in organoleptic properties (overall 21% more desirable). Having 35 replicates to use for the data set, the results illustrated statistically significant variations in meat-quality traits and how they could be objectively measured. Nine months later, 12 of the students were surveyed. It was their perception that the laboratory was not any more effective than other laboratories in the same class, but they were able to remember 85% of the methods used to measure quality; about twice that of other methods taught in other laboratory sessions.

Myogenin, MyoD, and myosin heavy chain isoform expression following hindlimb suspension.

BACKGROUND: Myogenin expression is associated with a slow myofiber phenotype, and MyoD expression is associated with a fast myofiber phenotype. Hindlimb suspension (HS) will induce muscular atrophy, and a transition from a slow to fast myofiber phenotype in the rat soleus. HYPOTHESIS: Hindlimb suspension will induce myofiber atrophy, and a slow to fast myofiber type transition with corresponding changes in myogenin and MyoD expression. METHODS: Myofiber phenotype was evaluated by electrophoretically separating the myosin heavy chain isoforms. Myogenin expression was evaluated by Northern analysis, while MyoD expression was evaluated by Northern analysis and semiquantitative RT-PCR. RESULT: After 28 d of hindlimb suspension, there was significantly (p < 0.05) less myosin heavy chain Type IIA, and more (p < 0.05) myosin heavy chain Type IIX in the soleus muscles of hindlimb suspended rats compared with soleus muscles from weight-bearing (WB) rats. Although there was a shift to a faster myosin heavy chain phenotype in soleus muscles from hindlimb suspended rats, there was no change in myogenin expression, and MyoD expression was undetectable by Northern analysis. Semi-quantitative RT-PCR revealed an up-regulation of MyoD expression following 14 d of hindlimb suspension. CONCLUSIONS: Myogenin expression levels do not change during the slow to fast myofiber phenotypic transition that occurs during hindlimb suspension; MyoD expression appears to increase at the same time as the phenotypic transition. Thus, MyoD expression or the Myogenin: MyoD mRNA ratio may be important in the phenotypic transition. Neither myogenin nor MyoD appear to play a critical role in the muscular atrophy that occurs during weightlessness.

PEVK extension of human soleus muscle titin revealed by immunolabeling with the anti-titin antibody 9D10.

Titin is a giant protein that spans half of the striated muscle sarcomere. The I-band portion of titin extends as the sarcomere is stretched, developing what is known as passive force. This portion of the molecule is composed mainly of tandem immunoglobulin (Ig) segments, consisting of serially linked Ig-like domains, and a recently discovered unique sequence termed the PEVK segment. The tandem Ig and PEVK segments have been suggested to extend sequentially when sarcomeres are stretched, with PEVK extension dominating at moderate to high degrees of sarcomere stretch (M. Gautel and D. Goulding, 1996, FEBS Lett. 385, 11-14; W. Linke et al., 1996, J. Mol. Biol. 261, 62-71; K. Trombitás et al., 1998). Previously we observed that the anti-titin antibody 9D10 labels a region in the I-band that increases in width as sarcomeres are stretched. Here we tested whether 9D10 labels the PEVK segment. The 9D10-labeled region of human soleus fibers was followed by immunoelectron microscopy as sarcomeres were stretched. It was found that 9D10 labeled a region in the I-band that was approximately 100 nm wide at a sarcomere length of 2.4 micrometer and approximately 550 nm wide at a sarcomere length of 4.0 micrometer. Results were compared with those obtained with sequence-specific antibodies that were used to mark the boundaries of the PEVK segment. Findings indicate that 9D10 labels the PEVK segment from close to its N-terminal end to its C-terminal end. Consistent with this conclusion are the results on cardiac myocytes that express a much shorter PEVK segment than skeletal muscle and where 9D10 labels a region that is much less wide than in skeletal muscle. The anti-titin antibody 9D10 is a useful tool for investigating the extensible behavior of the PEVK segment in both skeletal and cardiac muscles.

Myogenin, MyoD, and myosin expression after pharmacologically and surgically induced hypertrophy.

The relationship between myogenin or MyoD expression and hypertrophy of the rat soleus produced either by clenbuterol and 3,3', 5-triiodo-L-thyronine (CT) treatment or by surgical overload was examined. Mature female rats were subjected to surgical overload of the right soleus with the left soleus serving as a control. Another group received the same surgical treatment but were administered CT. Soleus muscles were harvested 4 wk after surgical overload and weighed. Myosin heavy chain isoforms were separated by using polyacrylamide gel electrophoresis while myogenin and MyoD expression were evaluated by Northern analysis. CT and functional overload increased soleus muscle weight. CT treatment induced the appearance of the fast type IIX myosin heavy chain isoform, depressed myogenin expression, and induced MyoD expression. However, functional overload did not alter myogenin or MyoD expression in CT-treated or non-CT-treated rats. Thus pharmacologically and surgically induced hypertrophy have differing effects on myogenin and MyoD expression, because their levels were associated with changes in myosin heavy chain composition (especially type IIX) rather than changes in muscle mass.

Effect of postmortem storage on the Z-line region of titin in bovine muscle.

Myofibrils were prepared from bovine muscles (cutaneous trunci, rectus abdominis, psoas major, and masseter) and compared between different aging periods at 4 degrees C (0, 1, 2, 4, 8, and 16 d). Myofibrils were stained with an antibody directed against a 56-kDa fragment (FE-RE) of titin located in the Z-line region. Unaged myofibrils from all four muscles showed a single stained band at the Z-line with similar intensities. Postmortem time did not significantly affect the total amount of fluorescence in the sarcomere, suggesting the titin FE-RE epitope was not degraded nor were titin fragments containing this epitope released during storage. However, the fluorescence patterns were altered. The relative fluorescence intensity at the Z-line decreased but that in the I-band increased gradually, showing the translocation of some titin FE-RE epitopes during the aging period. This suggested that a cleavage occurred in a region of titin very close to the Z-line during postmortem storage. Usually the position of maximum fluorescence remained at the Z-line, although about 1/3 of the myofibrils from rectus abdominis showed a two-band pattern around the Z-line after 16 d of aging. The titin changes observed may be related to the increased fragility of the myofibril and the improvement of meat tenderness during postmortem storage.