Abnormal actin binding of aberrant β-tropomyosins is a molecular cause of muscle weakness in TPM2-related nemaline and cap myopathy

NM (nemaline myopathy) is a rare genetic muscle disorder defined on the basis of muscle weakness and the presence of structural abnormalities in the muscle fibres, i.e. nemaline bodies. The related disorder cap myopathy is defined by cap-like structures located peripherally in the muscle fibres. Both disorders may be caused by mutations in the TPM2 gene encoding β-Tm (tropomyosin). Tm controls muscle contraction by inhibiting actin-myosin interaction in a calcium-sensitive manner. In the present study, we have investigated the pathogenetic mechanisms underlying five disease-causing mutations in Tm. We show that four of the mutations cause changes in affinity for actin, which may cause muscle weakness in these patients, whereas two show defective Ca2+ activation of contractility. We have also mapped the amino acids altered by the mutation to regions important for actin binding and note that two of the mutations cause altered protein conformation, which could account for impaired actin affinity.     

Function of the syndecan-4 cytoplasmic domain in oligomerization and association with α-actinin in turkey muscle satellite cells

Syndecan-4 (S4) is a cell membrane heparan sulfate proteoglycan that plays a role in satellite cell mediated myogenesis. S4 modulates the proliferation of myogenic satellite cells, but the mechanism of how S4 functions during myogenesis is not well understood. In other cell systems, S4 has been shown to form oligomers in the cell membrane and interact through its cytoplasmic domain with the cytoskeletal protein α-actinin. This study addressed if S4 forms oligomers and interacts with α-actinin in muscle. The S4 cytoplasmic domain was found to interact with α-actinin in a phosphatidylinositol-4,5-bisphosphate dependent manner, but did not associate with vinculin. Through confocal microscopy, both S4 and syndecan-4 without the cytoplasmic domain were localized to the cell membrane. Although the cytoplasmic domain was necessary for the interaction with α-actinin, S4 oligomer formation occurred in the absence of the cytoplasmic domain. These data indicated that S4 function in skeletal muscle is mediated through the formation of oligomers and interaction with the cytoskeletal protein α-actinin.     

Removal of immunoglobulin-like domains from titin’s spring segment alters titin splicing in mouse skeletal muscle and causes myopathy

Titin is a molecular spring that determines the passive stiffness of muscle cells. Changes in titin’s stiffness occur in various myopathies, but whether these are a cause or an effect of the disease is unknown. We studied a novel mouse model in which titin’s stiffness was slightly increased by deleting nine immunoglobulin (Ig)-like domains from titin’s constitutively expressed proximal tandem Ig segment (IG KO). KO mice displayed mild kyphosis, a phenotype commonly associated with skeletal muscle myopathy. Slow muscles were atrophic with alterations in myosin isoform expression; functional studies in soleus muscle revealed a reduced specific twitch force. Exon expression analysis showed that KO mice underwent additional changes in titin splicing to yield smaller than expected titin isoforms that were much stiffer than expected. Additionally, splicing occurred in the PEVK region of titin, a finding confirmed at the protein level. The titin-binding protein Ankrd1 was highly increased in the IG KO, but this did not play a role in generating small titin isoforms because titin expression was unaltered in IG KO mice crossed with Ankrd1-deficient mice. In contrast, the splicing factor RBM20 (RNA-binding motif 20) was also significantly increased in IG KO mice, and additional differential splicing was reversed in IG KO mice crossed with a mouse with reduced RBM20 activity. Thus, increasing titin’s stiffness triggers pathological changes in skeletal muscle, with an important role played by RBM20.     

Are pioneer axons guided by regulatory gene expression domains in the zebrafish forebrain? High-resolution analysis of the patterning of the zebrafish brain during axon tract formation

Although the principles of axon growth are well understood in vitro the mechanisms guiding axons in vivo are less clear. It has been postulated that growing axons in the vertebrate brain follow borders of neuroepithelial cells expressing specific regulatory genes. In the present study we reexamined this hypothesis by analysing the earliest growing axons in the forebrain of embryonic zebrafish. Confocal laser scanning microscopy was used to determine the spatiotemporal relationship between growing axons and the expression pattern of eight regulatory genes in zebrafish brain. Pioneer axons project either longitudinally or dorsoventrally to establish a scaffold of axon tracts during this developmental period. Each of the regulatory genes was expressed in stereotypical domains and the borders of some were oriented along dorsoventral and longitudinal planes. However, none of these borders clearly defined the trajectories of pioneer axons. In two cases axons coursed in proximity to the borders of shh and pax6, but only for a relatively short portion of their pathway. Only later growing axons were closely apposed to the borders of some gene expression domains. These results suggest that pioneer axons in the embryonic forebrain do not follow continuous pathways defined by the borders of regulatory gene expression domains.     

tgfβ3 regulation of chondrogenesis and osteogenesis in zebrafish is mediated through formation and survival of a subpopulation of the cranial neural crest

Zebrafish tgfβ3 is strongly expressed in a subpopulation of the migrating neural crest cells, developing pharyngeal arches and neurocranial cartilages. To study the regulatory role of tgfβ3 in head skeletal formation, we knocked down tgfβ3 in zebrafish and found impaired craniofacial chondrogenesis, evident by malformations in selected neurocranial and pharyngeal arch cartilages. Over-expressing tgfβ3 in embryos resulted in smaller craniofacial cartilages without any gross malformations. These defects suggest that tgfβ3 is required for normal chondrogenesis. To address the cellular mechanisms that lead to the observed malformations, we analyzed cranial neural crest development in morphant and tgfβ3 over-expressing fish. We observed reduced pre-migratory and migratory cranial neural crest, the precursors of the neurocranial cartilage and pharyngeal arches, in tgfβ3 knockdown embryos. In contrast, only the migratory neural crest was reduced in embryos over-expressing tgfβ3. This raised the possibility that the reduced number of cranial neural crest cells is a result of increased apoptosis. Consistent with this, markedly elevated TUNEL staining in the midbrain and hindbrain, and developing pharyngeal arch region was observed in morphants, while tgfβ3 over-expressing embryos showed marginally increased apoptosis in the developing pharyngeal arch region. We propose that both Tgfβ3 suppression and over-expression result in reduced chondrocyte and osteocyte formation, but to different degrees and through different mechanisms. In Tgfβ3 suppressed embryos, this is due to impaired formation and survival of a subpopulation of cranial neural crest cells through markedly increased apoptosis in regions containing the cranial neural crest cells, while in Tgfβ3 over-expressing embryos, the milder phenotype is also due to a slightly elevated apoptosis in these regions. Therefore, proper cranial neural crest formation and survival, and ultimately craniofacial chondrogenesis and osteogenesis, are dependent on tight regulation of Tgfβ3 protein levels in zebrafish.     

Regulation of polyribosome formation and protein synthesis in the uterus. Effect of administration of graded doses of oestradiol-17β and actinomycin D on the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of polyribosomes

1. The effects of graded doses of oestradiol-17beta and actinomycin D, administered separately or together, on the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of uterine polyribosomes are described. Preparations of polyribosomes isolated from uteri of ovariectomized adult rats were determined for cytoplasmic concentration in vivo and assayed for [(14)C]leucine-incorporation activity in the cell-free system, exactly as described by Teng & Hamilton (1967b). 2. A minimal dose of 10mug of oestradiol-17beta administered for 10h was found to increase, by about 100%, both the amino acid-incorporation activity in vitro and the cytoplasmic concentration in vivo of the polyribosomes. A minimal dose of 250mug of actinomycin D administered for 10h was found to inhibit, by about 50%, the incorporation activity in vitro of the polyribosomes. All doses of the inhibitor administered for 10h failed to alter the cytoplasmic concentration in vivo of the polyribosomes. 3. A dose of 10mug of oestradiol-17beta restored to the control value the inhibitory effect of a dose of either 50 or 125mug of actinomycin D on the activity in vitro of the polyribosomes, at 10h after treatment with the inhibitor and the hormone. In these experiments, there was an increase of 60-100% in the cytoplasmic concentration in vivo of the polyribosomes. 4. A dose of 125mug of actinomycin D, administered to animals along with 10mug of oestradiol-17beta for 6-36h, abolished the hormone-induced enhancement of the incorporation activity in vitro, but did not prevent an increase of about 200% in the cytoplasmic concentration in vivo of the polyribosomes. However, treatment with 750mug of the inhibitor abolished both stimulatory effects of the hormone. 5. The results reported indicate that the stimulatory effects of oestradiol-17beta in vivo on the number and activity of the cytoplasmic polyribosomes in the uterus of the ovariectomized rat have different sensitivities to actinomycin D, but the primary molecular mechanisms responsible for the results are unknown. The major conclusion drawn is that the formation and appearance in the cytoplasm of newly formed polyribosomes are important features of the early action of oestrogen in the uterus.     

Does one subgenome become dominant in the formation and evolution of a polyploid?

BackgroundPolyploids are common in flowering plants and they tend to have more expanded ranges of distributions than their diploid progenitors. Possible mechanisms underlying polyploid success have been intensively investigated. Previous studies showed that polyploidy generates novel changes and that subgenomes in allopolyploid species often differ in gene number, gene expression levels and levels of epigenetic alteration. It is widely believed that such differences are the results of conflicts among the subgenomes. These differences have been treated by some as subgenome dominance, and it is claimed that the magnitude of subgenome dominance increases in polyploid evolution.ScopeIn addition to changes which occurred during evolution, differences between subgenomes of a polyploid species may also be affected by differences between the diploid donors and changes which occurred during polyploidization. The variable genome components in many plant species are extensive, which would result in exaggerated differences between a subgenome and its progenitor when a single genotype or a small number of genotypes are used to represent a polyploid or its donors. When artificially resynthesized polyploids are used as surrogates for newly formed genotypes which have not been exposed to evolutionary selection, differences between diploid genotypes available today and those involved in the formation of the natural polyploid genotypes must also be considered.ConclusionsContrary to the now widely held views that subgenome biases in polyploids are the results of conflicts among the subgenomes and that one of the parental subgenomes generally retains more genes which are more highly expressed, available results show that subgenome biases mainly reflect legacy from the progenitors and that they can be detected before the completion of polyploidization events. Further, there is no convincing evidence that the magnitudes of subgenome biases have significantly changed during evolution for any of the allopolyploid species assessed.     

Is the Prevalence of Successful Weight Loss and Maintenance Higher in the General Community than the Research Clinic?

BARTLETT, SUSAN J., MYLES S. FAITH, KEVIN R. FONTAINE, LAWRENCE J. CHESKIN, AND DAVID B. ALLISON. Is the prevalence of successful weight loss and maintenance higher in the general community than the research clinic? Obes Res. Objective: The prevalence of successful weight loss remains unclear. In 1982, Schachter concluded that in the general population, the rate of “self-cured” obesity approached 63%–much higher than the rate from clinical trials. Several subsequent studies have addressed this issue. Research Methods and Procedures: Our initial goal was to meta-analyze these studies to evaluate the validity of the original hypotheses and the extent to which additional investigations supported the findings. We began by restating Schachter's hypotheses in precise, testable terms. Results: A systematic review of these studies found many methodological limitations and much heterogeneity among the samples studied, hypotheses addressed, and operational definitions. Some of these limitations appear to stem from the lack of clear, precise statements of the exact hypotheses tested. Differences among studies are delineated, and we outline why meta-analytic pooling of these data appears inappropriate. Conclusions: The current data are inadequate to draw any definite conclusions regarding the cure rate of obesity. Criteria for the adequate study of success rates with “self-cure” in the general population are proposed.     

Dexamethasone-mediated regulation of death and differentiation of muscle cells. Is hydrogen peroxide involved in the process?

The hypothetical involvement of H2O2 in dexamethasone-mediated regulation of muscle cell differentiation and elimination was studied. Rat L6 myoblasts and mouse C2C12 satellite cells were chosen for acute (24 h) and chronic (5 or 10 day) experiments. Mitogenicity and anabolism were both affected by H2O2. Micromolar concentrations of H2O2 inhibited DNA while stimulating protein synthesis. At the millimolar level, H2O2 led to cell death by apoptosis.Synthetic glucocorticoi - dexamethasone (Dex) was shown to effect muscle cell fate similarly to H2O2. Chronic treatment with H2O2 or Dex dose-dependently accelerated either the formation of myotubes or cell elimination. Dex-induced cell death slightly differed from classical apoptosis and was featured by the symptoms of cell senescence such as extensive cytoplasm vacuolisation, accumulation of inclusion-bodies and lack of low molecular weight oligonucleosomal DNA fragmentation but chromatin condensation. Antioxidants (sodium ascorbate, N-acetyl-L-cysteine, catalase) abrogated Dex-dependent cell death. We conclude that H2O2 directly influences myogenesis and muscle cell elimination. Moreover, H2O2 can be considered as the potent mediator of glucocorticoid-dependent effects on muscle cells.     

What are the roles of substrate-assisted catalysis and proximity effects in peptide bond formation by the ribosome?

The action of the peptidyl transferase center of the large ribosomal unit presents a fundamental step in the evolution from the RNA world to the protein world. Thus, it is important to understand the origin of the catalytic power of this ancient enzyme. Earlier studies suggested that the ribosome catalyzes peptide bond formation by using one of its groups as a general base, while more recent works have proposed that the catalysis is due to proximity effects or to substrate-assisted catalysis. However, the actual nature of the catalytic mechanism remains controversial. This work addresses the origin of the catalytic power of the ribosome by using computer simulation approaches and comparing the energetics of the peptide bond formation in the ribosome and in water. It is found that a significant part of the observed activation entropy of the reference solution reaction is due to solvation entropy, and that the proximity effect is smaller than previously thought. It is also found that the 2'-OH of the A76 ribose, which is associated with a large rate acceleration in the ribosome reaction, does not catalyze peptide bond formation in water. Thus, the catalytic effect cannot be attributed to substrate-assisted catalysis but rather to the effect of the ribosome on the reacting system. Overall, our calculations indicate that the reduction of the activation free energy is mainly due to electrostatic effects. The nature of these effects and their relationship to catalytic factors in modern enzymes is analyzed and discussed.     

Are cross-bridging structures involved in the bundle formation of intermediate filaments and the decrease in locomotion that accompany cell aging?

Five different fibroblast strains derived from donors of a wide range of ages were used for investigation of senescence-associated changes in the organization of intermediate filaments (IFs) and the activity of cell locomotion. Results of immunofluorescence microscopy demonstrate that, in large and flat in vitro aged fibroblasts, vimentin-containing IFs are distributed as unusually organized large bundles. Electron microscopic examination shows that these large bundles are indeed composed of filaments of 8-10 nm. Such a profile of large bundles is rarely seen in young fibroblasts whose IFs are usually interdispersed among microtubules. Within the large filament bundles of senescent fibroblasts, cross-bridge-like extensions are frequently observed along the individual IFs. Immunogold labeling with antibody to one of the cross-bridging proteins, p50, further illustrates the abundance of interfilament links within the IF bundles. The senescence-related increase in interfilament association was also supported by the results of co-precipitation between vimentin and an associated protein of 50,000 D. Time-lapse cinematographic studies of cell locomotion reveal that accompanying aging, fibroblasts have a significantly reduced ability to translocate across a solid substratum. These results led me to suggest that the increased interfilament links via cross-bridges may in part contribute to the mechanism that orchestrates the formation of large filament bundles. The presence of enormous bundles in the cytoplasm may physically impede the efficiency of locomotion for these nondividing cells.     

What causes the differences in cavitation resistance of two shrubs? Wood anatomical explanations and reliability testing of vulnerability curves

Relationships between xylem anatomical traits and cavitation resistance have always been a major content of plant hydraulics. To know how plants cope with drought, it is extremely important to acquire detailed knowledge about xylem anatomical traits and assess the cavitation resistance accurately. This study aims to increase our knowledge in the methods determining cavitation resistance and xylem anatomical traits. We selected a semi-ring-porous species, Hippophae rhamnoides L., and a diffuse-porous species, Corylus heterophylla F., to clarify the reasons for the difference in cavitation resistance based on detailed xylem anatomical traits and reliable vulnerability curves (VCs). Both Cavitron and bench dehydration (BD) were used to construct VCs. Xylem anatomical traits, including pit membrane ultrastructure of these two species, were determined. The VCs obtained by the two different techniques were of different types for H. rhamnoides, its Cavitron VCs might be unreliable because of open-vessel artifacts. On the basis of BD VCs, H. rhamnoides showed higher cavitation resistance than C. heterophylla, and this is attributed to its low vessel connectivity as well as non-porous and thicker pit membranes.     

Do independent processes control the activation and inactivation of potassium contracture tension in rat skeletal muscle?

Potassium (K⁺) contracture tension, measured in small bundles of rat soleus muscle fibers during maintained depolarization, increases to a peak value and then decays either to the baseline or to a pedestal level. We have tested the hypothesis that the rise and fall of tension are determined by independent activation and inactivation processes. If the “Independence” hypothesis is correct, tension during the decay of K⁺ contractures should equal tension predicted from the product of the activation and inactivation parameters determined from the same K⁺ contractures. Both the measured and predicted tensions decayed to a pedestal level that was increased in amplitude in the presence of perchlorate ions. However, the measured tensions in normal solutions and in the presence of perchlorate were three to five times smaller than the predicted tensions. This result indicates that the activation and inactivation of processes controlling the rise and decay of K⁺ contracture tension are not independent.     

How do the migratory and adhesive properties of the neural crest govern ganglia formation in the avian peripheral nervous system?

The peripheral nervous system derives mainly from the neural crest both in the head and trunk. Using markers such as fibronectin (FN), neural cell-adhesion molecule (NCAM), the nucleolar marker for quail cells in chimaeric embryos, and NC-1, a monoclonal antibody specific to crest cells and their neural derivatives, we have attempted to reconstruct the processes that lead to the formation of peripheral ganglia. Our observations allow us to propose a model of the formation of ganglia based on morphogenetic movements and on variations of crest cell adhesiveness. In most cases, crest cells migrate in morphologically defined and transient pathways that lead them to their final site of arrest; these pathways are always associated with FN, which appears necessary for crest cell attachment and movement in vitro. The directionality of crest cell migration is probably dictated by the cells' motile properties and population pressure in restricted areas suitable for cell movement. The disappearance of the pathways and of the substrate necessary for migration while the population is rapidly dividing may be responsible for the aggregation of crest cells in the case of the sensory ganglia. To the contrary, the aggregation of crest cells into autonomic ganglia (sympathetic, enteric, and ciliary ganglia) does not seem to obey the same rules, no disappearance of the substratum or of the pathways being obvious; rather, their formation seems correlated with the de novo synthesis of adhesive molecules such as NCAM.     

Molecular interplays involved in the cellular uptake of octaarginine on cell surfaces and the importance of syndecan-4 cytoplasmic V domain for the activation of protein kinase Cα

Arginine-rich cell-penetrating peptides (CPPs) are promising carriers for the intracellular delivery of various bioactive molecules. However, many ambiguities remain about the molecular interplays on cell surfaces that ultimately lead to endocytic uptake of CPPs. By treatment of cells with octaarginine (R8), enhanced clustering of syndecan-4 on plasma membranes and binding of protein kinase Cα (PKCα) to the cytoplasmic domain of syndecan-4 were observed; these events potentially lead to the macropinocytic uptake of R8. The cytoplasmic V domain of syndecan-4 made a significant contribution to the cellular uptake of R8, whereas the cytoplasmic C1 and C2 domains were not involved in the process.     

Distribution of the Galβ1-4Gal Epitope among Birds: Species-Specific Loss of the Glycan Structure in Chicken and Its Relatives

The Galβ1-4Gal epitope is rarely found in mammals, and the natural antibody against Galβ1-4Gal is rich in human. In contrast, we have previously demonstrated the presence of Galβ1-4Gal in pigeon and ostrich, and the absence of this epitope in chicken. Here, to further investigate the expression of this glycan among birds, egg white glycoproteins and egg yolk IgG from nine species of birds, namely, chicken, duck, emu, guineafowl, ostrich, peafowl, pigeon, quail, and turkey, were analyzed by western blot using an anti-(Galβ1-4Gal) antibody. The results indicated that some egg white glycoproteins from emu, ostrich, and quail, and heavy chains of IgG from all of the birds, except chicken and quail, were stained with the antibody. The presence of Galβ1-4Gal on N-glycans of IgGs from guineafowl, peafowl, and turkey were confirmed by mass spectrometry (MS), MS/MS, and MSⁿ analyses. In quail, the presence of Galβ1-4Gal was confirmed by detecting the activities of UDP-galactose: β-galactoside β1,4-galactosyltransferase (β4GalT(Gal)) in various tissues, and by detecting Galβ1-4Gal by western blotting. In contrast, bamboo partridge, which is a close relative of chicken, did not show any detectable activities of β4GalT(Gal) or Galβ1-4Gal on glycoproteins. Because quail, peafowl, turkey, chicken, and bamboo partridge belong to the same family, i.e., Phasianidae, expression of Galβ1-4Gal was most likely differentiated within this family. Considering that Galβ1-4Gal is also expressed in ostrich, emu, and pigeon, which are phylogenetically distant relatives within modern birds, Galβ1-4Gal expression appears to be widely distributed among birds, but might have been abolished in the ancestors of chicken and bamboo partridge.