Contribution of limb momentum to power transfer in athletic wheelchair pushing.

Pushing capacity is a key parameter in athletic racing wheelchair performance. This study estimated the potential contribution of upper limb momentum to pushing. The question is relevant since it may affect the training strategy adopted by an athlete. A muscle-free Lagrangian dynamic model of the upper limb segments was developed and theoretical predictions of power transfer to the wheelchair were computed during the push phase. Results show that limb momentum capacity for pushing can be in the order of 40J per push cycle at 10m/s, but it varies with the specific pushing range chosen by the athlete. Although use of momentum could certainly help an athlete improve performance, quantifying the actual contribution of limb momentum to pushing is not trivial. A preliminary experimental investigation on an ergometer, along with a simplified model of the upper limb, suggests that momentum is not the sole contributor to power transfer to a wheelchair. Muscles substantially contribute to pushing, even at high speeds. Moreover, an optimal pushing range is challenging to find since it most likely differs if an athlete chooses a limb momentum pushing strategy versus a muscular exertion pushing strategy, or both at the same time. The study emphasizes the importance of controlling pushing range, although one should optimize it while also taking the dynamics of the recovery period into account.

Transgenic overexpression of cardiac actin in the mouse heart suggests coregulation of cardiac, skeletal and vascular actin expression.

Previous studies have shown that depletion of cardiac actin by targeted disruption is associated with increased expression of alternative actins in the mouse heart. Here we have studied the effects of transgenic overexpression of cardiac actin using the alpha-myosin heavy chain promoter. Lines carrying 7 or 8 copies of the transgene showed a 2-fold increase in cardiac actin mRNA and also displayed decreased expression of skeletal and vascular actin in their hearts. In contrast, a line with more than 250 copies of the transgene did not show a similar decrease in the expression of skeletal and vascular actin despite a 3-fold increase in cardiac actin mRNA. While the low copy number transgenic mice displayed hearts that were similar to non-transgenic controls, the high copy number transgenic line showed larger hearts with distinct atrial enlargement and cardiomyocyte hypertrophy. Further, while the low copy number transgenic mouse hearts were mildly hypocontractile when compared with non-transgenic mouse hearts, the high copy number transgenic mouse hearts were significantly so. We conclude that in the presence of a small number of copies of the cardiac actin transgene, homeostatic mechanisms involved in maintaining actin levels are active and negatively regulate skeletal and vascular actin levels in the heart in response to increased expression of cardiac actin. However, these putative mechanisms are either inoperative in the high copy number transgenic line or are countered by the enhanced expression of skeletal and vascular actin during cardiomyocyte hypertrophy.

Differential expression of ras signal transduction mediators in verrucous and squamous cell carcinomas of the upper aerodigestive tract.

CONTEXT: ras gene mutations and expression of its gene product have been described in verrucous and squamous cell carcinomas. Other downstream signal-transduction mediators, extracellular signal-regulated kinases 1 and 2 (ERK-1 and ERK-2) and Raf-1, have not yet been as extensively studied. OBJECTIVE: To determine patterns of expression of ERK-1, ERK-2, and Raf-1 in verrucous and squamous cell carcinomas of the upper aerodigestive tract. DESIGN: Seventeen verrucous carcinomas and 10 squamous cell carcinomas of the upper aerodigestive tract were examined for the immunohistochemical expression of ERK-1, ERK-2, and Raf-1 product. RESULTS: Raf-1 expression was intensely expressed in the most basal portions of the epithelium in verrucous carcinomas, but was minimally expressed in the suprabasalar areas. Anti-Raf-1 staining of the squamous cell carcinomas was diffuse and patchy throughout the tumor cells and was weak in intensity. There was no geographic preference of staining. The cytoplasmic expression of both ERK-1 and ERK-2 was predominantly negative in the most basal layers of the epithelium in the verrucous carcinomas, but was positive in the suprabasalar region of the epithelium. Immunohistochemical expression of ERK-1 and ERK-2 in the squamous carcinomas was diffuse throughout the tumor. CONCLUSION: There is strong correlation of the geographic expression of these mediators of ras signal transduction in verrucous and squamous carcinomas, but the cause of these differences remains unclear at present. The expression of these mediator proteins may have potential for diagnosis, as well as in understanding the biologic behavior of these lesions.

Establishment and characterization of a conditionally immortalized smooth muscle/myometrial-like cell line.

A novel smooth muscle/myometrial-like cell line, SMU1-10, has been generated from the uterus of a H-2Kb-tsA58 transgenic mouse carrying a thermolabile SV40 large T-antigen gene. These cells grow continuously when maintained at the permissive temperature (33 degrees C) for the SV40 large T-antigen but stop dividing when placed at the non-permissive temperature (39 degrees C) and ultimately die within 3 weeks. All of the SMU1-10 cells produce smooth muscle alpha-actin (SMAA) at both 33 degrees C and 39 degrees C. A subset of the cells also contain smooth muscle gamma-actin (SMGA), a hallmark of smooth muscle differentiation, and the fraction of cells staining for this actin increases from about 1% when maintained for three days at 33 degrees C to as much as 30% at 39 degrees C over the same length of time. However, the appearance of SMGA in SMU1-10 cells appears to be regulated mainly at a post-transcriptional level since in situ hybridization indicates that all cells contain SMGA mRNA at both 33 degrees C and 39 degrees C. SMU1-10 cultures also contain smooth muscle myosin heavy chain (SM-MHC) and SM22 alpha, both of which are only found in smooth muscle of the adult mouse. Three additional smooth muscle (myometrium)-related markers, connexin 43, the thromboxane A2 receptor, and the progesterone receptor also are present in these cells. At the nonpermissive temperature for SV40 large T-antigen, the both level of SMGA mRNA and the number of cells staining for this actin are significantly increased in the presence of progesterone, a process that is similar to the upregulation of SMGA in the myometrium late in pregnancy. Overall, SMU1-10 cells provides a potentially useful in vitro model system to study smooth muscle/myometrial differentiation.

Rescue of cardiac alpha-actin-deficient mice by enteric smooth muscle gamma-actin.

The muscle actins in higher vertebrates display highly conserved amino acid sequences, yet they show distinct expression patterns. Thus, cardiac alpha-actin, skeletal alpha-actin, vascular smooth muscle alpha-actin, and enteric smooth muscle gamma-actin comprise the major actins in their respective tissues. To assess the functional and developmental significance of cardiac alpha-actin, the murine (129/SvJ) cardiac alpha-actin gene was disrupted by homologous recombination. The majority ( approximately 56%) of the mice lacking cardiac alpha-actin do not survive to term, and the remainder generally die within 2 weeks of birth. Increased expression of vascular smooth muscle and skeletal alpha-actins is observed in the hearts of newborn homozygous mutants and also heterozygotes but apparently is insufficient to maintain myofibrillar integrity in the homozygous mutants. Mice lacking cardiac alpha-actin can be rescued to adulthood by the ectopic expression of enteric smooth muscle gamma-actin using the cardiac alpha-myosin heavy chain promoter. However, the hearts of such rescued cardiac alpha-actin-deficient mice are extremely hypodynamic, considerably enlarged, and hypertrophied. Furthermore, the transgenically expressed enteric smooth muscle gamma-actin reduces cardiac contractility in wild-type and heterozygous mice. These results demonstrate that alterations in actin composition in the fetal and adult heart are associated with severe structural and functional perturbations.

Muscle isoactin expression during in vitro differentiation of murine embryonic stem cells.

Embryonic stem (ES) cells are pluripotent cells derived from mouse blastocysts. ES cells can differentiate into complex embryoid bodies (EBs) which exhibit many of the characteristics of 4-10-d embryos, including areas which rhythmically contract. The expression of the four muscle isoactins was examined in EBs by using transcript-specific probes for each of the muscle actin mRNAs and selectively reactive MAbs to muscle actins. Northern blot analyses from undifferentiated ES cells and EBs after 5, 10, 15, and 20 d in suspension culture demonstrated that no muscle actin transcripts could be detected in the undifferentiated cells, whereas during differentiation, the vascular and enteric smooth muscle isoactin mRNAs were easily detected. To further define the pattern of expression polymerase chain reaction analyses were carried out on RNA isolated from individual EBs. The data indicated that all four muscle-specific actin genes are transcribed. We also demonstrated the presence of muscle actins in at least two distinct cell populations within the EBs using selectively reactive MAbs. Fibroblast-like cells exhibit significant levels of the two smooth muscle actins (vascular and enteric) localized to stress fibers. In addition, one or both of the striated muscle actins (cardiac and skeletal) are expressed in cardiomyocyte-like cells. As is the case in embryonic heart, alpha-smooth muscle actin and the striated muscle actin(s) are incorporated into well organized sarcomeres in these cardiomyocyte-like cells. Thus, differentiating EBs provide an in vitro system to study both striated and smooth muscle cell gene expression.

Pregnancy-induced elevation in aortic vascular smooth muscle actin in the Sprague-Dawley rat.

A component of hemodynamic responses during pregnancy may include structural changes in the arterial tree. Smooth muscle alpha-actin is a major structural-contractile protein of muscle cells. We observed a marked increase. In expression of actin during pregnancy in the rat aorta, suggesting a structural alteration of conduit arteries.

Tissue and developmental specific expression of murine smooth muscle gamma-actin fusion genes in transgenic mice.

Smooth muscle gamma-actin (SMGA) is an excellent marker of smooth muscle differentiation because it is essentially restricted to smooth muscle. As a first step toward unraveling the mechanisms underlying smooth muscle development and differentiation, we have examined the tissue-specific and developmental expression patterns of six constructs carrying portions of the murine SMGA gene linked to chloramphenicol acetyltransferase (CAT) in stable lines of transgenic mice. Based on the transgenic studies most, if not all, of the regulatory elements necessary for proper spatial and temporal expression of SMGA are present within a 13.7 kb segment of the SMGA gene containing 4.9 kb of upstream sequence, exon 1, intron 1, and a portion of exon 2 up to the start codon for translation. A second construct (SMGA11.6CAT) that lacks the distal 2.1 kb of upstream sequence but is otherwise identical to SMGA13.7CAT shows a similar level of smooth muscle-specific CAT activity. However, SMGA9.3CAT fusion gene containing only 571 bp of 5' flanking sequence, but otherwise identical to SMGA13.7CAT, and SMGA6.0CAT containing only the 4.9 kb upstream sequence, exon 1, and a miniintron 1 show a more than a 100-fold reduction of CAT activity in most smooth muscle-rich tissues. Furthermore, removal of most or all of intron 1 from a transgene with 571 bp of upstream sequence (SMGA2.0 CAT and SMGA0.6CAT) results in a near-complete or complete loss of activity, respectively, in all tissues. Overall, the studies suggest that upstream elements between -2.7 kb and -571 bp and elements within intron 1 are required for high levels of SMGA gene expression in an appropriate temporal-spatial fashion.

Cloning and sequence analysis of the mouse smooth muscle gamma-enteric actin gene.

Actin represents one of the most highly conserved families of proteins in evolution. The work presented here describes the molecular characterization of the mouse smooth muscle (enteric) gamma-actin gene (SMGA). It represents the largest isoactin gene characterized to date, measuring over 23,000 bp from the transcription start site to the polyadenylation signal. The gene is divided into nine exons and encodes a mature actin protein of 374 amino acids. Putative regulatory elements are noted as well as regions of the gene that have the potential to form non-B DNA conformations that may influence gene expression.

Chicken antibodies to rabbit muscle actin with a restricted repertoire of F-actin recognition.

This report describes a polyclonal antibody against actin with unexpected and unusual properties. The antibody was raised in chicken immunized with a complex of DNase I and rabbit skeletal muscle actin, and purified from egg yolk by affinity chromatography. In Western blots, it reacted with alpha, beta and gamma isoforms of actin. In immunofluorescence and dot blot assays, however, it recognized selectively actin filaments in myofibrils, microvilli of brush border-type epithelium and the "comet tails" of the intracellular parasite Listeria monocytogenes, while it did not react with stress fibers and peripheral belts of fibroblasts and epithelial cells, respectively. This reactivity pattern is reminiscent of that previously described for a monoclonal mouse antibody raised against smooth muscle actin (Sawtell et al., Cell Motil. Cytoskel. 11, 318, 1988). The data presented in this study are consistent with the hypothesis that the chicken antibody recognizes an actin epitope/actin epitopes either accessible in only a subpopulation of microfilaments, or expressed only in a particular conformation of F-actin.

A comprehensive analysis of the developmental and tissue-specific expression of the isoactin multigene family in the rat.

The present study represents the first comprehensive analysis of isoactin gene expression in the developing rat. Our results clearly demonstrate that the developmental and tissue-specific expression of the actin multigene family is a highly integrated and complex process involving a variety of regulatory paradigms. The distinct temporal patterns of expression reported in this study indicate that there are three key phases in the regulation of expression of the actin multigene family during development. These include early embryonic development, late fetal development, and early postnatal development. The specific spatial patterns of expression observed in this study demonstrate that the expression of the actin multigene family is much more permissive than previously reported. This permissive expression includes a wide range of "ectopic" expression of the striated muscle isoactins as well as an extended expression of the alpha-smooth muscle isoactin. These findings expand our current understanding of the expression of the actin multigene family in development and provide a fundamental basis for future studies directed at investigating these processes.

Chicken cardiac myofibrillogenesis studied with antibodies specific for titin and the muscle and nonmuscle isoforms of actin and tropomyosin.

Myofibrillogenesis was studied in cultured chick cardiomyocytes using indirect immunofluorescence microscopy and antibodies against alpha- and gamma-actin, muscle and nonmuscle tropomyosin, muscle myosin, and titin. Initially, cardiomyocytes, devoid of myofibrils, developed variable numbers of stress fiber-like structures with uniform staining for anti-muscle and nonmuscle actin and tropomyosin, and diffuse, weak staining with anti-titin. Anti-myosin labeled bundles of filaments that exhibited variable degrees of association with the stress fiber-like structures. Myofibrillogenesis occurred with a progressive, and generally simultaneous, longitudinal reorganization of stress fiber-like structures to form primitive sarcomeric units. Titin appeared to attain its mature pattern before the other major contractile proteins. Changes in the staining patterns of actin, tropomyosin, and myosin as myofibrils matured were interpreted as due to longitudinal filament alignment occurring before ordering in the axial direction. Non-muscle actin and tropomyosin were found with sarcomeric periodicity in the initial stages of sarcomere myofibrillogenesis, although their staining patterns were not identical. The localization of the "sarcomeric" proteins alpha-actin and muscle tropomyosin in stress fiber-like structures and the incorporation of non-muscle proteins in the initial stages of sarcomere organization bring into question the meaning of "sarcomeric" proteins in regard to myofibrillogenesis.

Co-distribution of annexin VI and actin in secretory ameloblasts and odontoblasts of rat incisor.

Annexin VI and actin were detected by immunoblot analysis in the enamel- and dentin-related portions of dental tissues. Annexin VI was found mainly in the particulate fraction whereas actin was detected in both the soluble and particulate fractions. By immunoelectron microscopy, annexin VI antibodies conjugated with colloidal gold were seen to label the mitochondria, the cytosol and the nucleus of secretory ameloblasts and odontoblasts of rat incisor. In the processes of these cells, the plasmalemmal undercoat was labeled. Anti-actin antibodies labeled the desmosome-like junctions, the cytosol, and the mitochondria of the cell bodies. Extensive labeling was seen at the periphery of the Tomes' processes and odontoblast processes. These results suggest that annexin VI may play a role in Ca2(+)-regulation in the cell bodies, especially as a calcium receptor protein in the mitochondria. Moreover, annexin VI and actin seem to be co-distributed in secretory processes. Thus, these proteins might be both involved in exocytotic and endocytotic events.

Conserved tissue-restricted expression of HUC 1-1 actin phenotype among eumetazoan organisms.

Actin has evolved from a single protein into a family of more than six distinct isoforms in mammals. Based on amino acid sequence data, actins segregate into two major classes, the "cytoplasmic" or nonmuscle actins, present in all animals, and the "a-" or muscle actins, a group restricted to vertebrate muscle. We have recently identified two unique actin isoforms in rat intestinal brush border which combine features of these two classes. The amino terminal regions of these actins indicate that they are of a cytoplasmic type and yet the carboxy terminal regions contain an epitope (defined by Mab HUC 1-1) which, among mammalian actins, is restricted to the muscle isoforms. We report here that in addition to the rat, all species thus far examined which have an intestinal "brush border" express actins containing the HUC 1-1 epitope in this region. Furthermore, we show that the actins present in the muscle tissue of nonvertebrate eumetazoans, which are all of the cytoplasmic type, also contain this epitope. Thus these findings suggest that the HUC 1-1 epitope appeared early on a subset of cytoplasmic-type actins and was retained among actins expressed in muscle tissue throughout the evolutionary divergence of these cytoplasmic-type actins to the "a-" muscle actins.

Smooth muscle actin expression during P19 embryonal carcinoma differentiation in cell culture.

P19 embryonal carcinoma (EC) cells can be induced in vitro to differentiate into cells resembling those normally formed in the embryo. Among these cell types is one whose morphology is fibroblast-like. Using indirect immunofluorescence and Western blot analysis with antibodies directed against various isoforms of actin, many of these fibroblast-like cells were found to express smooth muscle actin isoforms. Northern blot analysis of RNA indicated the presence of a smooth muscle-specific isoform of myosin heavy-chain mRNA in immortal lines of these fibroblast-like cells. These results suggest that these fibroblast-like cells resemble fetal myofibroblastic or myoepithelial cells, which have a wide distribution during embryonic development.

Cellular distribution of smooth muscle actins during mammalian embryogenesis: expression of the alpha-vascular but not the gamma-enteric isoform in differentiating striated myocytes.

The cellular distribution of the alpha-vascular and gamma-enteric smooth muscle actin isoforms was analyzed in rat embryos from gestational day (gd) 8 through the first neonatal week by in situ antigen localization using isoactin specific monoclonal antibodies. The alpha-vascular actin isoform was first detected on gd 10 in discrete cells lining the embryonic vasculature. By gd 14, this isoform was also present in the inner layers of mesenchymal cells condensing around the developing airways and gut. The gamma-enteric actin, however, was not detected until gd 15 when cells surrounding the developing aorta, airways, and gut labeled with the gamma-enteric-specific probe. There was continued expression of these two actin isoforms in regions of developing smooth muscle through the remainder of gestation and first neonatal week at which time their distribution coincided with that found in the adult. In addition to developing smooth muscle, the alpha-vascular actin isoform was expressed in differentiating striated muscle cells. On gd 10, there was intense labeling with the alpha-vascular specific probe in developing myocardiocytes and, within 24 h, in somitic myotomal cells. Although significant levels of this smooth muscle actin were present in striated myocytes through gd 17, by the end of the first postnatal week, alpha-vascular actin was no longer detectable in either cardiac or skeletal muscle. Thus, the normal developmental sequence of striated muscle cells includes the transient expression of the alpha-vascular smooth muscle actin isoform. In contrast, the gamma-enteric smooth muscle actin was not detected at any time in embryonic striated muscle. The differential timing of appearance and distribution of these two smooth muscle isoforms indicates that their expression is independently regulated during development.

Expression of actin isoforms in developing rat intestinal epithelium.

A minimum of six very similar but distinct actin isoforms are encoded by the mammalian genome. Developmental regulation of these genes results in a tissue-specific distribution of the isoforms in the adult. Using a panel of actin specific monoclonal antibodies (MAb), we recently reported the expression of two unique actin isoforms in adult rat intestinal brush border. In this report, we examine the developmental expression of these and other actin isoforms in rat intestinal epithelial cells. Isoforms containing the HUC 1-1 and/or C4 epitopes are present by day 15 of gestation and are continuously expressed throughout adult life. Unexpectedly, the gamma-enteric smooth muscle isoactin, defined by the B4 epitope, is transiently expressed in these non-muscle cells late in gestation. The alpha-vascular smooth muscle isoform, however, is not expressed in intestinal epithelial cells during development and, as previously reported, both smooth muscle isoforms are absent in epithelial cells of adult intestine. In addition, we demonstrate that although multiple isoforms are expressed simultaneously in these cells, they are not uniformly distributed at the subcellular level, suggesting that the cell recognizes the actin isoforms as functionally distinct entities.

Skeletal muscle myofibrillogenesis as revealed with a monoclonal antibody to titin in combination with detection of the alpha- and gamma-isoforms of actin.

The distribution of titin during myofibrillogenesis was examined using rat skeletal muscle myogenic cultures and fluorescent-antibody staining. Efforts were made to compare the distribution and temporal sequence of incorporation of titin relative to that of the alpha- and gamma-isoforms of actin. The present observations suggested the following sequence of titin assembly: (1) newly synthesized titin molecules are distributed in a diffuse pattern throughout the sarcoplasm, (2) the titin molecules gradually associate with alpha- and gamma-actin-positive stress fiber-like structures (SFLS), (3) groups of titin molecules begin to segregate on the SFLS, and (4) titin molecules align in a mature doublet configuration in the sarcomeres of nascent myofibrils. Titin assembly on the SFLS often appeared prior to the onset of either alpha- or gamma-actin periodicity on nascent myofibrils; the latter result suggested a role for titin in sarcomeric organization. Actin distribution on SFLS and its periodicity on nascent myofibrils was usually identical between the alpha- and gamma-isoforms. This suggested that gamma-actin participated in myofibrillogenesis in a manner indistinguishable from that of alpha-actin. The transition seen from continuous actin staining of SFLS to the I-band staining pattern of mature myofibrils is discussed in relation to the corresponding reorganization of actin filaments and the molecular associations that this would entail.

The development expression of the rat alpha-vascular and gamma-enteric smooth muscle isoactins: isolation and characterization of a rat gamma-enteric actin cDNA.

We have isolated and characterized two cDNA clones from whole rat stomach, pRV alpha A-19 and pRE gamma A-11, which are specific for the alpha-vascular and gamma-enteric smooth muscle isoactins, respectively. The rat gamma-enteric smooth muscle actin contains a single amino acid substitution of a proline for a glutamine at position 359 of the mature peptide when compared with the chicken gizzard gamma-actin sequence (J. Vandekerckhove and K. Weber, FEBS Lett. 102:219, 1979). Sequence comparisons of the 5' and 3' untranslated (UT) regions of the two smooth muscle actin cDNAs demonstrate that these regions contain no apparent sequence similarities. Additional comparisons of the 5' UT regions of the two smooth muscle actin cDNAs to all other known actin sequences reveal no apparent sequence similarities for the rat gamma-enteric isoactin within the 15 base pairs of sequence currently available, while the rat alpha-vascular isoactin contains two separate sequences which are similar to sequences within the 5' UT regions of the human and chicken alpha-vascular actin genes. A similar comparison of the 3' UT regions of the two smooth muscle actins demonstrates that the alpha-vascular isoactins do not contain the high degree of cross-species sequence conservation observed for the other isoactins and that the gamma-enteric isoactin contains an inverted sequence of 52 nucleotides which is similar to a sequence found within the 3' UT regions of the human, chicken, and rat beta-cytoplasmic isoactins. These observations complicate the apparent cross-species conservation of isotype specificity of these domains previously observed for the other actin isoforms. Northern blot analysis of day 15 rat embryos and newborn, day 19 postbirth, and adult rats demonstrates that the day 15 rat embryo displays low to undetectable levels of smooth muscle isoactin mRNA expression. By birth, the stomach and small intestine show dramatic increases in alpha-vascular and gamma-enteric actin expression. These initially high levels of expression decrease through day 19 to adulthood. In the adult rat, the uterus and aorta differ in their content of smooth muscle isoactin mRNA. These results demonstrate that the gamma-enteric and alpha-vascular isoactin mRNAs are coexpressed to various degrees in tissues which contain smooth muscle.