Biomedical subjects

M L Greaser

Publications and source records attributed to M L Greaser.

At least 19 recordsLinked to original sources

PubMed2004

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.

Actin Cytoskeleton↗

PubMed2000

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.

Animals↗

PubMed2000

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.

Amino Acid Sequence↗

PubMed2000

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.

Animals↗

PubMed1999

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.

Animals↗

PubMed1999

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.

Animals↗

PubMed1999

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.

Animals↗

PubMed1998

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.

Adrenergic beta-Antagonists↗

PubMed1998

Can pale, soft, exudative pork be prevented by postmortem sodium bicarbonate injection?

Previous attempts at eliminating the problem of PSE pork by genetic selection or rapid postmortem cooling have been only partially successful. A new approach, namely, postmortem injection of sodium bicarbonate (SBC), was tested on halothane-positive gilts. Sixteen pigs were used to establish a suitable SBC concentration. At approximately 15 min after death, the longissimus of one side of the carcass was injected with 10% (by weight) of .2 to .4 M SBC solutions containing .7% NaCl (wt/vol). All concentrations resulted in a higher ultimate pH, improved muscle color, and reduced drip loss. In a second experiment, with 23 pigs, .3 M SBC was injected into the longissimus and the biceps femoris at either 15 min or 24 h after death and with or without inclusion of .7% NaCl (wt/vol). Compared with controls, the 15-min SBC + NaCl injected samples had darker color (L* of 47 vs 53 in controls), higher ultimate pH (5.6 vs 5.3), lower drip loss (5% vs 10%), and increased protein solubility (140 vs 115 mg/g). Injection at 24 h reduced drip loss (from 10% to 5.7%) but did not correct the color defect. The SBC alone and SBC + NaCl treatments had essentially the same effects in reducing drip loss, increasing ultimate pH, and improving color; but the SBC-NaCl injected samples had improved juiciness and flavor compared with SBC. Early postmortem sodium bicarbonate injection seems to prevent the development of PSE pork when injected into carcasses of halothane-sensitive pigs.

Animals↗

PubMed1998

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.

Animals↗

PubMed1997

Characteristics of troponin C binding to the myofibrillar thin filament: extraction of troponin C is not random along the length of the thin filament.

Troponin C (TnC) is the Ca(2+)-sensing subunit of troponin responsible for initiating the cascade of events resulting in contraction of striated muscle. This protein can be readily extracted from myofibrils with low-ionic-strength EDTA-containing buffers. The properties of TnC extraction have not been characterized at the structural level, nor have the interactions of TnC with the native myofibrillar thin filament been studied. To address these issues, fluorescein-labeled TnC, in conjunction with high-resolution digital fluorescence microscopy, was used to characterize TnC binding to myofibrils and to determine the randomness of TnC extraction. Fluorescein-5-maleimide TnC (F5M TnC) retained biological activity, as evidenced by reconstitution of Ca(2+)-dependent ATPase activity in extracted myofibrils and binding to TnI in a Ca(2+)-sensitive manner. The binding of F5M TnC to highly extracted myofibrils at low Ca2+ was restricted to the overlap region under rigor conditions, and the location of binding was not influenced by F5M TnC concentration. The addition of myosin subfragment 1 to occupy all actin sites resulted in F5M TnC being bound in both the overlap and nonoverlap regions. However, very little F5M TnC was bound to myofibrils under relaxing conditions. These results suggest that strong binding of myosin heads enhances TnC binding. At high Ca2+, the pattern of F5M TnC binding was concentration dependent: binding was restricted to the overlap region at low F5M TnC concentration, whereas the binding propagated into the nonoverlap region at higher levels. Analysis of fluorescence intensity showed the greatest binding of F5M TnC at high Ca2+ with S1, and these conditions were used to characterize partially TnC-extracted myofibrils. Comparison of partially extracted myofibrils showed that low levels of extraction were associated with greater F5M TnC being bound in the nonoverlap region than in the overlap region relative to higher levels of extraction. These results show that TnC extraction is not random along the length of the thin filament, but occurs more readily in the nonoverlap region. This observation, in conjunction with the influence of rigor heads on the pattern of F5M TnC binding, suggests that strong myosin binding to actin stabilizes TnC binding at low Ca2+.

Animals↗

PubMed1997

Interaction between titin and thin filaments in intact cardiac muscle.

A 'freeze break' technique and immunoelectron microscopy were used to study the elastic properties of cardiac titin filaments. Small bundles consisting of a few fibres from freshly prepared dog papillary muscle were quickly frozen and broken under liquid nitrogen to fracture sarcomeres in planes perpendicular to the filament axes. Breaks occurred at each of several regions along the sarcomeres. The still-frozen specimens were thawed during fixation to allow elastic filaments to retract. The broken muscle segments were then treated with monoclonal titin antibody 9D10 which labelled a unique epitope in the I-band. In sarcomeres broken at the A-I junction, the titin filaments reacted toward the Z-line, independently of the thin filaments. The retracted epitopes did not reach the Z-line; retraction stopped at the N1-line level. In sarcomeres broken near the Z-line, the titin filaments retracted in the opposite direction, to the tip of the thick filaments. When the break occurred in the A-band, by contrast, the titin-epitope position was unaffected. On the basis of these results, and despite the reported interaction of titin and actin in vitro, it appears that cardiac titin molecules form elastic filaments that are functionally independent of the thin filaments. Near the Z-line, however, the titin filaments seem to associate firmly with the thin filaments.

Actins↗

PubMed1997

Arachidonic acid stimulates protein kinase C-epsilon redistribution in heart cells.

Arachidonic acid is elevated in a variety of cell types in response to extracellular stimuli, and has been hypothesized to exert at least some of its intracellular actions via activation of protein kinase C. Here we show that arachidonic acid stimulates a unique pattern of translocation of the epsilon-isoform of protein kinase C in isolated adult rat cardiac myocytes. Using western blot analysis, the majority of epsilon-protein kinase C was found in a cytosolic fraction in unstimulated cells. Treatment with 50 microM arachidonic acid caused a transient increase of epsilon-protein kinase C in a membrane fraction within 1 minute, then after 5-20 minutes most was found in a filament/nuclear fraction. Immunofluorescence and confocal microscopy of the filament fraction revealed a striated staining pattern with epsilon-protein kinase C localized near the Z-line where actin filaments are anchored and where transverse tubules are closely apposed to the myofilaments. delta-Protein kinase C, another isoform highly expressed in these cells, did not redistribute significantly in response to arachidonic acid, but in response to phorbol ester displayed a predominantly nuclear localization. Arachidonic acid also stimulated phosphorylation of the thin filament protein, troponin I, consistent with a filament localization for activated PKC. The physiological relevance of these findings was supported by the observation that 50 microM arachidonic acid promoted a 2.3-fold enhancement of myocyte twitch amplitude, an effect that was significantly blocked by the protein kinase C antagonist chelerythrine. Moreover, the onset of this physiological response correlated in time with translocation of epsilon-protein kinase C to the filaments. The results suggest that arachidonic acid initiates a redistribution of epsilon-protein kinase C to myofilament structures at or near the Z-line where this isozyme would be strategically located to regulate myofilament function and excitation-contraction coupling.

Animals↗

PubMed1997

Effect of rapid rigor mortis processes on protein functionality in pectoralis major muscle of domestic turkeys.

The pale, soft, exudative (PSE) phenomenon in turkey pectoralis major (breast) muscle was studied using a combination of biochemical, meat quality, microscopic, and gel electrophoresis techniques. Breast muscle samples were collected from turkeys characterized by slow vs fast postmortem glycolysis assessed by muscle pH at 20 min after death. The PSE group was characterized by lower muscle ATP (P < .05) and higher lactate levels (P < .05) compared with the normal group. Excess water-holding capacity and cooking yield were significantly lower (P < .05) in the PSE group than in normal turkeys. Breast muscle of the PSE group was also lighter (P < .05) than that in the normal group as determined by Minolta L* values. The SDS-PAGE, Western blotting, and immunofluorescence microscopy revealed that phosphorylase, a soluble enzyme, became tightly associated with the myofibrils in muscle from the PSE group. Also, less myosin could be solubilized from PSE vs normal myofibril samples. The results indicate that irreversible myosin insolubility due to low pH and high-temperature conditions is decisive in the development of PSE turkey breast muscle.

Adenosine Triphosphate↗

PubMed1996

X-ray structures of Mn, Cd and Tb metal complexes of troponin C.

The crystal structures of three metal complexes of troponin C (TnC) have been determined and refined where the two occupied structural Ca(2+) sites in the C domain have been substituted by Mn(2+), Cd(2+) and Tb(3+). The X-ray intensity data were collected to 2.1, 1.8 and 1.8 A resolution, respectively, on the three metal complexes, which are isomorphous with Ca-TnC. The three complexes have r.m.s. deviations of 0.27, 0.25 and 0.35 A, respectively, for all protein atoms, from Ca-TnC. Irrespective of the charge on the metal (+2 or +3), the occupied sites 3 and 4 exhibit a distorted pentagonal bipyramidal coordination, like Ca-TnC, with seven ligands, six from the 12-residue binding loop and the seventh from a water molecule. Mn(2+) at site 4 seems to display a longer distance to one of the carboxyl bidentate ligands representing an intermediate coordination simulating the six-coordinate Mg(2+). The carboxyl O atoms of the bidentate Glu12 are displaced on the side of the equatorial plane passing through the remaining three ligands with one O atom closer to the plane (Delta of 0.11 to 0.76 A) than the other (Delta of 0.93 to 1.38 A). The two axial ligands are an aspartic carboxyl O atom and a water molecule. The metal is displaced (0.18 to 0.56 A) towards the water facing the water channel.

Journal Article↗

PubMed1996

Myofibrillar calcium sensitivity of isometric tension is increased in human dilated cardiomyopathies: role of altered beta-adrenergically mediated protein phosphorylation.

To examine the role of alterations in myofibrillar function in human dilated cardiomyopathies, we determined isometric tension-calcium relations in permeabilized myocytesized myofibrillar preparations (n = 16) obtained from left ventricular biopsies from nine patients with dilated cardiomyopathy (DCM) during cardiac transplantation or left ventricular assist device implantation. Similar preparations (n = 10) were obtained from six normal hearts used for cardiac transplantation. Passive and maximal Ca2+-activated tensions were similar for the two groups. However, the calcium sensitivity of isometric tension was increased in DCM compared to nonfailing preparations ([Ca2+]50=2.46+/-0.49 microM vs 3.24+/-0.51 microM, P < 0.001). In vitro treatment with the catalytic subunit of protein kinase A (PKA) decreased calcium sensitivity of tension to a greater degree in failing than in normal preparations. Further, isometric tension-calcium relations in failing and normal myofibrillar preparations were similar after PKA treatment. These findings suggest that the increased calcium sensitivity of isometric tension in DCM may be due at least in part to a reduction of the beta-adrenergically mediated (PKA-dependent) phosphorylation of myofibrillar regulatory proteins such as troponin I and/or C-protein.

Adolescent↗

PubMed1996

Contractile properties and protein isoforms of single fibres from the chicken pectoralis red strip muscle.

1. The contractile properties of single muscle fibres of the red strip region of adult chicken pectoralis major (PM) muscle, some of which are known to express an embryonic isoform of myosin heavy chain (MHC), were determined and compared with the properties of the fast white fibres of the PM and the slow tonic fibres of the anterior latissimus dorsi (ALD) muscle. 2. The red strip fibres could be classified into two groups, fast and slow. The mean velocity of unloaded shortening (Vmax) in fast red strip fibres was approximately half the Vmax of fast white fibres. Vmax of slow red strip fibres was less than 20% of the value for fast red strip fibres and was not different from Vmax of ALD fibres. 3. The tension-generating ability, i.e. the maximal isometric tension/fibre cross-sectional area (P0/CSA), was the same in fast red strip fibres and fast white fibres. P0/CSA was approximately 30% lower in slow red strip fibres compared with fast red strip fibres but was 70% greater in slow red strip fibres compared with ALD fibres. 4. The tension-pCa relation of fast red strip fibres was shifted to lower pCa values, indicating a lower calcium sensitivity compared with fast white fibres, and this difference was associated with a difference in troponin T isoform composition. The tension-pCa relation of slow red strip fibres was not different from that in ALD fibres. 5. The difference in Vmax between fast red strip fibres and fast white fibres was associated with different MHC compositions of these fibres. 6. The myofibrillar protein isoform composition of slow red strip fibres was identical to that of the slow tonic fibres of ALD muscle and these two groups of fibres had very similar contractile properties.

Animals↗

PubMed1996

Primary structure of the kinase domain region of rabbit skeletal and cardiac muscle titin.

A 2.3 kb region of rabbit cardiac and skeletal muscle titin has been cloned. The cDNA sequences of the two tissues are identical and show 91% identity on the nucleotide level with the corresponding region of human cardiac muscle titin. On the amino acid level the identity is 96% and similarity is 98%. Alignment of predicted amino acid sequences of several homologous kinase domains reveals that the rabbit titin kinase has all the necessary elements of an active catalytic domain and carries a potential regulatory region on its C-terminal end. The distance of the 2.3 kb contig from the 3' end of the message was determined to be 5.7 kb in both tissues using oligonucleotide directed RNase H cleavage of titin mRNAs. This is essentially identical with the length of the fully sequenced human cardiac titin C-terminal end. It therefore appears unlikely that there are major tissue specific differences in this 8 kb cDNA region which encodes the C-terminus of rabbit skeletal and cardiac titin.

Amino Acid Sequence↗