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M T Crow

Publications and source records attributed to M T Crow.

49 records · Page 3Linked to original sources

Myosin expression and specialization among the earliest muscle fibers of the developing avian limb.

Monoclonal antibodies specific to the light- and heavy-chain subunits of chicken skeletal muscle myosin have been used to identify fast and slow myosin-containing fibers in the thigh muscles of embryonic and adult chickens and to determine when in development diversification of muscle fiber types first occurs. Primary generation fibers which expressed different MLC and MHC types were evident within the dorsal and ventral premuscle masses and in the first muscles to form in the limb. These early embryonic muscle fiber types became distributed among and within the individual muscles of the thigh in a characteristic spatial pattern which served as a "blueprint" for guiding future muscle development and predicting the future fiber composition of the muscle. Despite the continuous addition of muscle fibers to the limb throughout development, the pattern remained unchanged. Neither the time of appearance, initial specialization, nor characteristic distribution of these primary fiber types within the limb was altered during the early embryonic period by chronic neuromuscular paralysis induced by D-tubocurarine. In contrast, muscles at later stages of embryonic development were markedly affected by such treatments and underwent atrophy and loss of differential staining characteristics. These results demonstrate that diversification of fibers in terms of myosin content is one of the earliest events in the formation of these muscles and suggest that the development of avian muscles be divided into two phases: an embryonic phase during which fibers of differing myosin content appear independently of innervation to become distributed in a specific topographic pattern within each muscle as it forms, followed by a fetal phase during which innervation becomes essential for maintaining this pattern and modulating the myosin content of its fibers.

Animals↗

Slow and fast myosin heavy chain content defines three types of myotubes in early muscle cell cultures.

We prepared monoclonal antibodies specific for fast or slow classes of myosin heavy chain isoforms in the chicken and used them to probe myosin expression in cultures of myotubes derived from embryonic chicken myoblasts. Myosin heavy chain expression was assayed by gel electrophoresis and immunoblotting of extracted myosin and by immunostaining of cultures of myotubes. Myotubes that formed from embryonic day 5-6 pectoral myoblasts synthesized both a fast and a slow class of myosin heavy chain, which were electrophoretically and immunologically distinct, but only the fast class of myosin heavy chain was synthesized by myotubes that formed in cultures of embryonic day 8 or older myoblasts. Furthermore, three types of myotubes formed in cultures of embryonic day 5-6 myoblasts: one that contained only a fast myosin heavy chain, a second that contained only a slow myosin heavy chain, and a third that contained both a fast and a slow heavy chain. Myotubes that formed in cultures of embryonic day 8 or older myoblasts, however, were of a single type that synthesized only a fast class of myosin heavy chain. Regardless of whether myoblasts from embryonic day 6 pectoral muscle were cultured alone or mixed with an equal number of myoblasts from embryonic day 12 muscle, the number of myotubes that formed and contained a slow class of myosin was the same. These results demonstrate that the slow class of myosin heavy chain can be synthesized by myotubes formed in cell culture, and that three types of myotubes form in culture from pectoral muscle myoblasts that are isolated early in development, but only one type of myotube forms from older myoblasts; and they suggest that muscle fiber formation probably depends upon different populations of myoblasts that co-exist and remain distinct during myogenesis.

Animals↗

Myosin light-chain expression during avian muscle development.

Monoclonal antibodies to adult chicken myosin light chains were generated and used to quantitate the types of myosin light-chain (MLC) isoforms expressed during development of the pectoralis major (PM), anterior latissimus dorsi (ALD), and medial adductor (MA) muscles of the chicken. These are muscles which, in the adult, are composed predominantly of fast, slow, and a mixture of fiber types, respectively. Three distinct phases of MLC expression characterized the development of the PM and MA muscles. The first identifiable pase occurred during the period of 5-7 d of incubation in ovo. Extracts of muscles from the pectoral region (which included the presumptive PM muscle) contained only fast MLC isoforms. This period of exclusive fast light-chain synthesis was followed by a phase (8- 12 d of incubation in ovo) in which coexpression of both fast and slow MLC isoforms was apparent in both PM and MA muscles. During the period, the composition of both fast and slow MLC isoforms in the PM and MA muscles was identical. Beginning at day 12 in ovo, the ALD was also subjected to immunochemical analyses. The proportion of fast and slow MLCs in this muscle at day 12 was similar to that present in the other muscles studied. The third development phase of MLC expression began at approximately 12 d of incubation in ovo and encompassed the transition in MLC composition to the isoform patterns incubation in ovo and encompassed the transition in MLC composition to the isoform patterns typical of adult muscle. During this period, the relative proportion of slow MLC rose in both the MA and ALD and fell in the PM. By day 16, the third fast light chain, LC(3f), was apparent in extracts of both the PM and MA. These results show that there is a developmental progression in the expression of MLC in the two avian muscles studied from day 5 in ovo; first, only fast MLCs are accumulated, then both fast and slow MLC isoforms are expressed. Only during the latter third of development in ovo is the final MLC isoform pattern characteristic of a particular muscle type expressed.

Animals↗

Correlated reduction of velocity of shortening and the rate of energy utilization in mouse fast-twitch muscle during a continuous tetanus.

Isometric tetani of slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles of the mouse were studied at 20 degrees C. The total energy cost for 3- and 9-s isometric tetani was measured as a function of length above L0 and partitioned into a filament overlap-dependent fraction and a smaller filament overlap-independent fraction. In both muscles, the rate of filament overlap-independent energy cost did not change with tetanic duration. In the EDL, but not in the soleus, the rate of filament overlap-dependent energy utilization was greater in a 3-s tetanus than in a 9-s tetanus. The force-velocity relationships were studied after 3 and 9 s of isometric tetanus. In the soleus, Vmax was 2 fiber lengths/s and was not dependent on the duration of isometric tetanus. In contrast, in the EDL, Vmas decreased from 5.9 fiber lengths/s at 3 s to 3.9 fiber lengths/s at 9 s. The velocity of unloaded shortening (Vus) was examined by the slack test method as a function of the duration of isometric tetanus duration over the range of 1-15 s. In the soleus, Vus did not change, whereas in the EDL, Vus declined progressively from 6.4 to 3.2 fiber lengths/s after an isometric tetanus of increasing duration from 1 to 15 s. These results cannot exclude the hypothesis that in a maintained tetanus there is a decrease in the intrinsic cross-bridge turnover rate in the fast-twitch EDL, but not in the slow-twitch soleus muscle.

Animals↗

Phosphorylation of myosin light chains in mouse fast-twitch muscle associated with reduced actomyosin turnover rate.

Phosphorylation of the 18,000-dalton light chains of the fast-twitch myosin in mouse extensor digitorum longus muscles was correlated with reduction in the rate of the actomyosin adenosinetriphosphatase in vivo, but neither of these changes occurred in the soleus muscle. These results suggest that actomyosin interactions can be down-regulated by a reversible covalent modification of myosin light chains, that a mechanism for thick-filament regulation occurs in vertebrate skeletal muscle, and that the expression of this regulation may be limited to a specific fiber type.

Actomyosin↗

Myosin light chain phosphorylation is associated with a decrease in the energy cost for contraction in fast twitch mouse muscle.

The rate of splitting of energy-rich phosphate compounds and the extent of myosin light chain phosphorylation in contracting mouse extensor digitorum longus (EDL, fast twitch) and soleus (slow twitch) muscles were studied at 20 degrees C. The rate of high energy phosphate-splitting during a maintained isometric tetanus was 1.44 +/- 0.21 mumol . g-1 . s-1 in soleus. In EDL, the splitting rate was higher, 3.71 +/- 0.62 mumol . g-1 . s-1, during the first several seconds and thereafter was reduced to a rate of 1.63 +/- 0.35 mumol . g-1 . s-1 between 12 and 15 s of stimulation. Light chains identified on 2-dimensional gel electrophoretograms from EDL corresponded to the light chain composition of fast twitch muscle (LC1f, LC2f, and LC3f). Soleus is composed of fast twitch and slow twitch fibers because 2 additional light chains were found: LC1s and LC2s. In unstimulated EDL and soleus muscles, 0.1 of the LC1f and LC2s were phosphorylated. Upon stimulation, only LC2f, and only in EDL, increased its extent of phosphorylation. The time course of increase in phosphorylation of LC2f and decrease in rate of high energy phosphate-splitting correspond so that the 2 processes may be mechanistically related. If so, it appears that myosin LC2f phosphorylation represents a thick filament regulatory system capable of downward modulation of actomyosin ATPase in vivo during a maintained contraction.

Animals↗

Chemical energetics of slow- and fast-twitch muscles of the mouse.

The energy utilization associated with contraction was measured in isolated slow- and fast-twitch muscles of the mouse at 20 degrees C. The extent of this utilization was estimated from either the extent of high-energy phosphate splitting occurring during contraction (the initial chemical change, delta approximately P init) or from the extent of recovery resynthesis calculated from the observed oxygen consumption and lactate production occurring during the recovery period (recovery chemical resynthesis, delta approximately P rec). For short tetani, the cost to maintain isometric tension in the fast-twitch extensor digitorum longus (EDL) was approximately threefold greater than that in the slow-twitch soleus. With prolonged stimulation, however, the energy cost in the EDL diminished so that after 12 s of stimulation, the energy cost in the EDL was only 50% greater than that of the soleus. For both the slow-twitch soleus and the fast-twitch EDL and for all tetanus durations (up to 15 s), the extent of the initial chemical change was identical with the amount of recovery chemical resynthesis, showing that a biochemical energy balance existed in these muscles.

Adenosine Triphosphate↗

Altered transcriptional regulation of human interstitial collagenase in cultured skin fibroblasts from older donors.

Primary human dermal fibroblasts isolated from the medial aspect of the proximal forearm of young and old donors were compared for the expression of interstitial collagenase, 72 kDa type IV collagenase, the tissue inhibitor of metalloproteinase type 1, and pro-alpha 2 (I) collagen mRNA at basal levels and after stimulation with the tumor promotor 12-O-tetradecanoyl-phorbol-13-acetate. Higher basal and induced steady-state mRNA levels of interstitial collagenase were found in the cells from older donors. Ratios of basal and induced steady-state mRNA levels of interstitial collagenase to pro-alpha 2 (I) collagen, and interstitial collagenase to the tissue inhibitor of metalloproteinases type 1 were also higher in the cells from older donors. Seventy-two kiloDalton type IV collagenase and pro-alpha 2 (I) collagen mRNA showed similar levels of expression in the cells from young and old donors and were not altered by treatment with 12-O-tetradecanoyl-phorbol-13-acetate. Transient transfection assays with the interstitial collagenase promoter linked to a reporter gene showed increased activity of the reporter in cell strains with high interstitial collagenase mRNA levels. Mobility shift assays demonstrated increased binding activity to the specific 12-O-tetradecanoyl-phorbol-13-acetate response element in nuclear extracts from the cell strains with higher induced collagenase mRNA levels and higher reporter gene activity. These findings are consistent with the observed phenotype of interstitial collagenase and its specific tissue inhibitor in the senescent fibroblast aging model.

Adolescent↗

Identification of specific mRNAs affected by treatments producing long-term facilitation in Aplysia.

Neural correlates of long-term sensitization of defensive withdrawal reflexes in Aplysia occur in sensory neurons in the pleural ganglia and can be mimicked by exposure of these neurons to serotonin (5-HT). Studies using inhibitors indicate that transcription is necessary for production of long-term facilitation by 5-HT. Several mRNAs that change in response to 5-HT have been identified, but the molecular events responsible for long-term facilitation have not yet been fully described. To detect additional changes in mRNAs, we investigated the effects of 5-HT (1.5 hr) on levels of mRNA in pleural-pedal ganglia using in vitro translation. Four mRNAs were affected by 5-HT, three of which were identified as calmodulin (CaM), phosphoglycerate kinase (PGK), and a novel gene product (protein 3). Using RNase protection assays, we found that 5-HT increased all three mRNAs in the pleural sensory neurons. CaM and protein 3 mRNAs were also increased in the sensory neurons by sensitization training. Furthermore, stimulation of peripheral nerves of pleural-pedal ganglia, an in vitro analog of sensitization training, increased the incorporation of labeled amino acids into CaM, PGK, and protein 3. These results indicate that increases in CaM, PGK, and protein 3 are part of the early response of sensory neurons to stimuli that produce long-term facilitation, and that CaM and protein 3 could have a role in the generation of long-term sensitization.

Amino Acid Sequence↗

Increased expression of membrane-type matrix metalloproteinase and preferential localization of matrix metalloproteinase-2 to the neointima of balloon-injured rat carotid arteries.

BACKGROUND: Remodeling of the injured vascular wall is dependent on the action of several extracellular proteases. Previous studies have shown that expression of matrix metalloproteinases (MMP-2 and MMP-9) is upregulated after vascular injury and that MMP-2 is required for the migration of cultured vascular smooth muscle cells across complex extracellular matrix barriers. The present study examined changes in the expression of membrane-type metalloproteinase (MT-MMP-1), a putative regulator of MMP-2, in the tissue localization of MMP-2, and in the expression of activated and latent forms of MMP-2 and the tissue inhibitor of metalloproteinases, TIMP-2, in rat carotid arteries subjected to balloon catheter injury. METHODS AND RESULTS: MT-MMP-1 mRNA levels increased sixfold after 3 days of injury, coinciding with an increase in MMP-2 activation assessed by gelatin zymography. Western blotting and gelatin zymography showed an increase in MMP-2 protein levels beginning 5 to 7 days after injury; immunocytochemistry and Western blotting showed that the increase occurred preferentially in the developing neointima. CONCLUSIONS: These results show that increased expression of MT-MMP-1 and activation of MMP-2 occurs early after injury to the rat carotid artery and that at later times MMP-2 is preferentially localized to the developing neointima.

Angioplasty, Balloon↗