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ABSTRACT: Muscle atrophy caused by disuse is accompanied by adverse physiological and functional consequences. Satellite cells are the primary source of skeletal muscle regeneration. Satellite cell dysfunction, as a result of impaired proliferative potential and/or increased apoptosis, is thought to be one of the causes contributing to the decreased muscle regeneration capacity in atrophy. We have previously shown that electrical stimulation improved satellite cell dysfunction. Here we test whether electrical stimulation can also enhance satellite cell proliferative potential as well as suppress apoptotic cell death in disuse-induced muscle atrophy. Eight-week-old male BALB/c mice were subjected to a 14-day hindlimb unloading procedure. During that period, one limb (HU-ES) received electrical stimulation (frequency: 20 Hz; duration: 3 h, twice daily) while the contralateral limb served as control (HU). Immunohistochemistry and western blotting techniques were used to characterize specific proteins in cell proliferation and apoptosis. The HU-ES soleus muscles showed significant improvement in muscle mass, cross-sectional area, and peak tetanic force relative to the HU limb (p<0.05). The satellite cell proliferative activity as detected within the BrdU+/Pax7+ population was significantly higher (p<0.05). The apoptotic myonuclei (detected by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling) and the apoptotic satellite cells (detected by cleaved Poly [ADP-ribose] polymerase co-labeled with Pax7) were reduced (p<0.05) in the HU-ES limb. Furthermore the apoptosis-inducing factor and cleaved caspase-3 were down-regulated while the anti-apoptotic Bcl-2 protein was up-regulated (p<0.05), in the HU-ES limb. These findings suggest that the electrical stimulation paradigm provides an effective stimulus to rescue the loss of myonuclei and satellite cells in disuse muscle atrophy, thus maintaining a viable satellite cell pool for subsequent muscle regeneration. Optimization of stimulation parameters may enhance the outcome of the intervention.
PLoS ONE 01/2012; 7(1):e30348. · 4.09 Impact Factor
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ABSTRACT: TRPC1 and TRPC3 proteins are widely expressed in skeletal muscles in forming calcium-permeable channels. Herein we characterize the expression pattern of TRPC transcripts during skeletal myogenesis in C2C12 myoblasts.
We used polymerase chain reaction and Western blotting to detect expression levels, immunohistochemistry for subcellular localization, and co-immunoprecipitation techniques to assess interaction.
TRPC1 localizes to the cytoplasm and is enriched in the perinuclear region in undifferentiated myoblasts. Expression of TRPC1 increases significantly during myogenesis and resides mainly in differentiated myocytes and myotubes. TRPC3 is absent in undifferentiated myoblasts, is dramatically upregulated in differentiated culture, and is preferentially expressed in myotubes. Physical interaction of TRPC1-TRPC3 was observed, suggesting the possible existence of heteromers.
Expression of TRPC1 and TRPC3 is tightly regulated during myogensis. Evidence of TRPC1-TRPC3 interaction was first demonstrated in a muscle cell line. The functional consequences of this interaction remain to be established.
Muscle & Nerve 09/2011; 44(3):358-65. · 2.37 Impact Factor
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ABSTRACT: Dab2, one of two mammalian orthologs of Drosophila Disabled, has been shown to be involved in cell positioning and formation of visceral endoderm during mouse embryogenesis, but its role in neuronal development is not yet fully understood. In this report, we have examined the localization of the Dab2 protein in the mouse embryonic central nervous system (CNS) at different developmental stages.
Dab2 protein was transiently expressed in rhombomeres 5 and 6 of the developing hindbrain between E8.5 and E11.5, and in the floor plate of the neural tube from E9.5 to E12.5, following which it was no longer detectable within these regions. Dab2 protein was also identified within circumventricular organs including the choroid plexus, subcommissural organ and pineal gland during their early development. While Dab2 was still strongly expressed in the adult choroid plexus, immunoreactivity within the subcommissural organ and pineal gland was lost after birth. In addition, Dab2 was transiently expressed within a subpopulation of Iba1-positive mononuclear phagocytes (including presumed microglial progenitors) within the neural tube from E10.0 and was lost by E14.5. Dab2 was separately localized to Iba1 positive cells from E9.5 and subsequently to F4/80 positive cells (mature macrophage/myeloid-derived dendritic cells) positioned outside the neural tube from E12.5 onwards, implicating Dab2 expression in early cells of the mononuclear phagocyte lineage. Dab2 did not co-localize with the pan-neuronal marker PGP9.5 at any developmental stage, suggesting that Dab2 positive cells in the developing CNS are unlikely to be differentiating neurons.
This is the first study to demonstrate the dynamic spatiotemporal expression of Dab2 protein within the CNS during development.
BMC Developmental Biology 02/2008; 8:76. · 2.79 Impact Factor
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ABSTRACT: Abstract
Background
Dab2, one of two mammalian orthologs of Drosophila Disabled , has been shown to be involved in cell positioning and formation of visceral endoderm during mouse embryogenesis, but its role in neuronal development is not yet fully understood. In this report, we have examined the localization of the Dab2 protein in the mouse embryonic central nervous system (CNS) at different developmental stages.
Results
Dab2 protein was transiently expressed in rhombomeres 5 and 6 of the developing hindbrain between E8.5 and E11.5, and in the floor plate of the neural tube from E9.5 to E12.5, following which it was no longer detectable within these regions. Dab2 protein was also identified within circumventricular organs including the choroid plexus, subcommissural organ and pineal gland during their early development. While Dab2 was still strongly expressed in the adult choroid plexus, immunoreactivity within the subcommissural organ and pineal gland was lost after birth. In addition, Dab2 was transiently expressed within a subpopulation of Iba1-positive mononuclear phagocytes (including presumed microglial progenitors) within the neural tube from E10.0 and was lost by E14.5. Dab2 was separately localized to Iba1 positive cells from E9.5 and subsequently to F4/80 positive cells (mature macrophage/myeloid-derived dendritic cells) positioned outside the neural tube from E12.5 onwards, implicating Dab2 expression in early cells of the mononuclear phagocyte lineage. Dab2 did not co-localize with the pan-neuronal marker PGP9.5 at any developmental stage, suggesting that Dab2 positive cells in the developing CNS are unlikely to be differentiating neurons.
Conclusion
This is the first study to demonstrate the dynamic spatiotemporal expression of Dab2 protein within the CNS during development.
BMC Developmental Biology. 01/2008;
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ABSTRACT: Extracellular adenosine 5'-triphosphate modulates the functions of the adult pancreas via 2 nucleotide receptor families, the P2X and P2Y receptors. Expression of the P2X7 receptor has been demonstrated in islet cells of the pancreas, particularly the mature alpha cells that secrete glucagon. In the streptozotocin-induced diabetic model, a loss of insulin-secreting cells was accompanied by an increase in alpha cells that expressed the P2X7 receptor.
In the present study, we have examined the expression of P2X7 receptors in the developing pancreas from embryonic days 10 (E10) to E18.
We detected P2X7 receptor-immunoreactive cells in pancreatic islet cells as early as E11' before glucagon expression. Subsequently, P2X7 receptors were expressed in glucagon-secreting cells at E12, and complete colocalization was observed at E14. Occasional colocalization of P2X7 receptors and insulin was observed in scattered cells at E12 and E14, but not at E18, when the glucagon- and insulin-secreting cells were almost completely segregated.
It was found that P2X7 receptors were expressed early in a subpopulation of glucagon- and insulin-immunopositive cells in developing islets and subsequently became restricted to glucagon-expressing cells as development proceeded. The possible functional significance of these changes is discussed.
Pancreas 09/2007; 35(2):164-8. · 2.39 Impact Factor
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ABSTRACT: Extracellular nucleotides interact with purinergic receptors, which regulate ion transport in a variety of epithelia. With the use of two different human epithelial carcinoma cell lines (HCT8 and Caco-2), we have shown by RT-PCR that the cells express mRNA for P2X1, P2X3, P2X4, P2X5, P2X6, P2X7, P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, and P2Y12 receptors. Protein expression for P2Y1 and P2Y2 receptors was also demonstrated immunohistochemically, and P2X receptor subtype protein was present in the following decreasing order: P2X4 > P2X7 > P2X1 > P2X3 > P2X6 > P2X5 > P2X2. The functional presence of P2X7, P2Y1, P2Y2, and P2Y4 receptors was shown based on the effect of extracellular nucleotides on apoptosis or cell proliferation, and measurement of nucleotide-dependent calcium fluxes using a fluorometric imaging plate reader in the presence of different selective agonists and antagonists. ATP, at high concentrations, induced apoptosis through ligation of P2X7 and P2Y1 receptors; conversely, ATP, at lower concentrations, and UTP stimulated proliferation, probably acting via P2Y2 receptors. We therefore propose that stimulation or dysfunction of purinergic receptors may contribute at least partially to modulation of epithelial carcinoma cell proliferation and apoptosis.
AJP Gastrointestinal and Liver Physiology 06/2005; 288(5):G1024-35. · 3.43 Impact Factor
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ABSTRACT: Extracellular ATP mediates diverse biological effects by activating two families of receptors, the P2X and P2Y receptors. There is growing evidence to show that activation of G protein-coupled P2Y receptors can produce trophic effects in many cell types. Yet the expression and function of the P2Y receptors in development has rarely been studied and has never been investigated in mammalian development. This study used the reverse transcription-polymerase chain reaction and immunohistochemistry to demonstrate the abundant and dynamic expression of P2Y receptors in rat development. These receptors were expressed in a wide range of embryonic structures, notably somites, skeletal muscle, the central and peripheral nervous system, the heart, lung, and liver. All the P2Y receptors studied were expressed as early as embryonic day 11, when most embryonic organs were far from being functional and still in the process of being formed. P2Y receptor proteins were strongly expressed in temporary, developmental structures that do not have a correlate in the adult animal, including the somites (P2Y1, P2Y2, and P2Y4) and the floor plate of the neural tube (P2Y1). P2Y receptors were also dynamically expressed, with receptor mRNA and protein being both up- and down-regulated at different developmental stages. The down-regulation of the P2Y1, 2, and 4 receptor proteins in skeletal muscle and heart, and the disappearance of the P2Y4 receptor from the brainstem and ventral white matter of the spinal cord postnatally, demonstrated that many P2Y receptors were likely to be involved in functions specific to embryonic life. Thus, these findings strongly suggest that P2Y receptors play an important role in the development of many tissues, and pioneer further studies into the role of purinergic signalling in development.
Developmental Dynamics 11/2003; 228(2):254-66. · 2.54 Impact Factor
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ABSTRACT: It is well known that extracellular ATP mediates rapid excitatory signaling by means of the ionotropic P2X receptors. One of its subunits, the P2X(3) receptor, is well documented to be associated with sensory innervation in adult animals. It is speculated that the P2X(3) receptor may have already been present in the early sensory system. The aim of this study was to investigate the distribution of the P2X(3) receptor during neurogenesis by using immunohistochemistry on rat embryos from embryonic day (E)9.5-18.5. The P2X(3) receptor was first identified in the hindbrain neural tube and the sensory ganglia in E11-11.5 embryos. At E14.5, the optic tract and retina, nucleus tractus solitarius, mesencephalic trigeminal nucleus, and sensory nerves in both respiratory and digestive tract showed positive staining. The facial nucleus, the prepositus hypoglossal nucleus, and the sympathetic ganglia also showed P2X(3) immunoreactivity, even though these are not sensory associated. P2X(3) immunoreactivity was detected in the vestibular nucleus, the nerves in mesentery, bladder, and kidney in E16.5 and in nerves in vibrissae in E18.5. P2X(3) immunoreactivity in the facial nucleus, spinal trigeminal tract, the mesencephalic trigeminal nucleus, and the vestibular nucleus were undetectable in postnatal day 16 rat brainstem. The P2X(3) receptor was coexpressed with the P2X(2) receptor in nucleus tractus solitarius, dorsal root ganglion, nodose ganglion, and the taste bud in E16.5 embryo, which was 5 days later than the first appearance of the native P2X(3) receptor. In summary, we present a detailed expression pattern of the P2X(3) receptor during neurogenesis and report that P2X(3) immunoreactivity is down-regulated in early postnatal brainstems.
The Journal of Comparative Neurology 03/2002; 443(4):368-82. · 3.81 Impact Factor
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ABSTRACT: Background: Dab2, 1 of the 2 mammalian orthologs of the Disabled protein in Drosophila, is a mitogen-responsive phosphoprotein and a cytoplasmic adaptor involved in several signal transduction pathways. Deficiency in Dab2 affects endodermal cell positioning and is embryonically lethal. Aims and Results: In the present study, the Dab2 expression pattern in mouse embryos from embryonic day (E)7.5 to E9.5 was first determined by specific immunohistochemical staining. Dab2 protein was expressed exclusively in the extraembryonic endoderm at E7.5 and was detected within the cranial mesenchyme, heart and foregut epithelium at E8.5. By E9.5, the protein was also localized in the roof plate of the hindbrain neural tube and within the branchial arch mesenchyme and epithelium. Next, to help define the role of Dab2 protein in early embryonic development, antisense oligodeoxynucleotides (ODNs) were microinjected into mouse embryos to inhibit Dab2 expression. The protein expression within the neural tube and cranial mesenchyme was greatly reduced following microinjection at E8.5 and culturing embryos intact with a whole-embryo culture system for 8 h in vitro. Moreover, 26.7% of the injected embryos exhibited a twisted neural tube or an unturned body axis. Twenty-four hours following microinjection of antisense ODNs, 70% of the injected embryos showed structural abnormalities mainly affecting the body axis, otic placode, forelimb buds, branchial arches and neural tube. Excessive cell death was detected histologically in the ventral part of the neural tube, although the expression of Dab2 protein had resumed by this stage (24 h following microinjection). Conclusion: We conclude that Dab2 plays an important role in mouse embryonic development between E8.5 and E9.5, as a reduction in Dab2 expression during this period was found to disturb normal development.
Neuroembryology and Aging 08/1970; 5(3):89-99.
