Megf10 regulates the progression of the satellite cell myogenic program

We identify here the multiple epidermal growth factor repeat transmembrane protein Megf10 as a quiescent satellite cell marker that is also expressed in skeletal myoblasts but not in differentiated myofibers. Retroviral expression of Megf10 in myoblasts results in enhanced proliferation and inhibited differentiation. Infected myoblasts that fail to differentiate undergo cell cycle arrest and can reenter the cell cycle upon serum restimulation. Moreover, experimental modulations of Megf10 alter the expression levels of Pax7 and the myogenic regulatory factors. In contrast, Megf10 silencing in activated satellite cells on individual fibers or in cultured myoblasts results in a dramatic reduction in the cell number, caused by myogenin activation and precocious differentiation as well as a depletion of the self-renewing Pax7⁺/MyoD⁻ population. Additionally, Megf10 silencing in MyoD^−/− myoblasts results in down-regulation of Notch signaling components. We conclude that Megf10 represents a novel transmembrane protein that impinges on Notch signaling to regulate the satellite cell population balance between proliferation and differentiation.     

Regulation of the Epithelial Adhesion Molecule CEACAM1 Is Important for Palate Formation

Cleft palate results from a mixture of genetic and environmental factors and occurs when the bilateral palatal shelves fail to fuse. The objective of this study was to search for new genes involved in mouse palate formation. Gene expression of murine embryonic palatal tissue was analyzed at various developmental stages before, during, and after palate fusion using GeneChip® microarrays. Ceacam1 was one of the highly up-regulated genes during palate formation, and this was confirmed by quantitative real-time PCR. Immunohistochemical staining showed that CEACAM1 was present in prefusion palatal epithelium and was degraded during fusion. To investigate the developmental role of CEACAM1, function-blocking antibody was added to embryonic mouse palate in organ culture. Palatal fusion was inhibited by this function-blocking antibody. To investigate the subsequent developmental role of CEACAM1, we characterized Ceacam1-deficient (Ceacam1 ^−/−) mice. Epithelial cells persisted abnormally at the midline of the embryonic palate even on day E16.0, and palatal fusion was delayed in Ceacam1 ^−/− mice. TGFβ3 expression, apoptosis, and cell proliferation in palatal epithelium were not affected in the palate of Ceacam1^−/−mice. However, CEACAM1 expression was retained in the remaining MEE of TGFβ-deficient mice. These results suggest that CEACAM1 has roles in the initiation of palatal fusion via epithelial cell adhesion.     

Role of the Vasohibin Family in the Regulation of Fetoplacental Vascularization and Syncytiotrophoblast Formation

Vasohibin-1 (VASH1) and vasohibin-2 (VASH2), the 2 members of the vasohibin family, have been identified as novel regulators of angiogenesis. VASH1 ceases angiogenesis, whereas VASH2 stimulates sprouting. Here we characterized their functional role in the placenta. Immunohistochemical analysis of human placental tissue clarified their distinctive localization; VASH1 in endothelial cells and VASH2 in trophoblasts. We then used a mouse model to explore their function. Wild-type, Vash1^(−/−), and Vash2^(−/−) mice on a C57BL6 background were used in their first pregnancy. As expected, the fetal vascular area was increased in the Vash1^(−/−) mice, whereas it was decreased in the Vash2^(−/−) mice relative to wild-type. In addition, we noticed that the Vash2^(−/−) mice at 18.5dpc displayed thinner villi of the labyrinth and larger maternal lacunae. Careful observation by an electron microscopy revealed that the syncytiotrophoblast formation was defective in the Vash2^(−/−) mice. To test the possible involvement of VASH2 in the syncytiotrophoblast formation, we examined the fusion of BeWo cells, a human trophoblastoid choriocarcinoma cell line. The forskolin treatment induced the fusion of BeWo cells, and the knockdown of VASH2 expression significantly inhibited this cell fusion. Conversely, the overexpression of VASH2 by the infection with adenovirus vector encoding human VASH2 gene significantly increased the fusion of BeWo cells. Glial cell missing-1 and endogenous retrovirus envelope glycoprotein Syncytin 1 and Syncytin 2 are known to be involved in the fusion of trophoblasts. However, VASH2 did not alter their expression in BeWo cells. These results indicate that VASH1 and VASH2 showed distinctive localization and opposing function on the fetoplacental vascularization. Moreover, our study shows for the first time that VASH2 expressed in trophoblasts is involved in the regulation of cell fusion for syncytiotrophoblast formation.     

Inflammation and Gli2 Suppress Gastrin Gene Expression in a Murine Model of Antral Hyperplasia

Chronic inflammation in the stomach can lead to gastric cancer. We previously reported that gastrin-deficient (Gast^−/−) mice develop bacterial overgrowth, inflammatory infiltrate, increased Il-1β expression, antral hyperplasia and eventually antral tumors. Since Hedgehog (Hh) signaling is active in gastric cancers but its role in precursor lesions is poorly understood, we examined the role of inflammation and Hh signaling in antral hyperplasia. LacZ reporter mice for Sonic hedgehog (Shh), Gli1, and Gli2 expression bred onto the Gast^−/− background revealed reduced Shh and Gli1 expression in the antra compared to wild type controls (WT). Gli2 expression in the Gast^−/− corpus was unchanged. However in the hyperplastic Gast^−/− antra, Gli2 expression increased in both the mesenchyme and epithelium, whereas expression in WT mice remained exclusively mesenchymal. These observations suggested that Gli2 is differentially regulated in the hyperplastic Gast^−/− antrum versus the corpus and by a Shh ligand-independent mechanism. Moreover, the proinflammatory cytokines Il-1β and Il-11, which promote gastric epithelial proliferation, were increased in the Gast^−/− stomach along with Infγ. To test if inflammation could account for elevated epithelial Gli2 expression in the Gast^−/− antra, the human gastric cell line AGS was treated with IL-1β and was found to increase GLI2 but decrease GLI1 levels. IL-1β also repressed human GAST gene expression. Indeed, GLI2 but not GLI1 or GLI3 expression repressed gastrin luciferase reporter activity by ∼50 percent. Moreover, chromatin immunoprecipitation of GLI2 in AGS cells confirmed that GLI2 directly binds to the GAST promoter. Using a mouse model of constitutively active epithelial GLI2 expression, we found that activated GLI2 repressed Gast expression but induced Il-1β gene expression and proliferation in the gastric antrum, along with a reduction of the number of G-cells. In summary, epithelial Gli2 expression was sufficient to stimulate Il-1β expression, repress Gast gene expression and increase proliferation, leading to antral hyperplasia.     

The reliability of the ankle-brachial index in the Atherosclerosis Risk in Communities (ARIC) study and the NHLBI Family Heart Study (FHS)

Background

Organ transplantation is presently often the only available option to repair a damaged heart. As heart donors are scarce, engineering of cardiac grafts from autologous skeletal myoblasts is a promising novel therapeutic strategy. The functionality of skeletal muscle cells in the heart milieu is, however, limited because of their inability to integrate electrically and mechanically into the myocardium. Therefore, in pursuit of improved cardiac integration of skeletal muscle grafts we sought to modify primary skeletal myoblasts by overexpression of the main gap-junctional protein connexin 43 and to study electrical coupling of connexin 43 overexpressing myoblasts to cardiac myocytes in vitro.

Methods

To create an efficient means for overexpression of connexin 43 in skeletal myoblasts we constructed a bicistronic retroviral vector MLV-CX43-EGFP expressing the human connexin 43 cDNA and the marker EGFP gene. This vector was employed to transduce primary rat skeletal myoblasts in optimised conditions involving a concomitant use of the retrovirus immobilising protein RetroNectin^® and the polycation transduction enhancer Transfectam^®. The EGFP-positive transduced cells were then enriched by flow cytometry.

Results

More than four-fold overexpression of connexin 43 in the transduced skeletal myoblasts, compared with non-transduced cells, was shown by Western blotting. Functionality of the overexpressed connexin 43 was demonstrated by microinjection of a fluorescent dye showing enhanced gap-junctional intercellular transfer in connexin 43 transduced myoblasts compared with transfer in non-transduced myoblasts. Rat cardiac myocytes were cultured in multielectrode array culture dishes together with connexin 43/EGFP transduced skeletal myoblasts, control non-transduced skeletal myoblasts or alone. Extracellular field action potential activation rates in the co-cultures of connexin 43 transduced skeletal myoblasts with cardiac myocytes were significantly higher than in the co-cultures of non-transduced skeletal myoblasts with cardiac myocytes and similar to the rates in pure cultures of cardiac myocytes.

Conclusion

The observed elevated field action potential activation rate in the co-cultures of cardiac myocytes with connexin 43 transduced skeletal myoblasts indicates enhanced cell-to-cell electrical coupling due to overexpression of connexin 43 in skeletal myoblasts. This study suggests that retroviral connexin 43 transduction can be employed to augment engineering of the electrocompetent cardiac grafts from patients' own skeletal myoblasts.     

Identification of a Human Immunodeficiency Virus Type 2 (HIV-2) Encapsidation Determinant and Transduction of Nondividing Human Cells by HIV-2-Based Lentivirus Vectors

Although previous lentivirus vector systems have used human immunodeficiency virus type 1 (HIV-1), HIV-2 is less pathogenic in humans and is amenable to pathogenicity testing in a primate model. In this study, an HIV-2 molecular clone that is infectious but apathogenic in macaques was used to first define cis-acting regions that can be deleted to prevent HIV-2 genomic encapsidation and replication without inhibiting viral gene expression. Lentivirus encapsidation determinants are complex and incompletely defined; for HIV-2, some deletions between the major 5′ splice donor and the gag open reading frame have been shown to minimally affect encapsidation and replication. We find that a larger deletion (61 to 75 nucleotides) abrogates encapsidation and replication but does not diminish mRNA expression. This deletion was incorporated into a replication-defective, envelope-pseudotyped, three-plasmid HIV-2 lentivirus vector system that supplies HIV-2 Gag/Pol and accessory proteins in trans from an HIV-2 packaging plasmid. The HIV-2 vectors efficiently transduced marker genes into human T and monocytoid cell lines and, in contrast to a murine leukemia virus-based vector, into growth-arrested HeLa cells and terminally differentiated human macrophages and NTN2 neurons. Vector DNA could be detected in HIV-2 vector-transduced nondividing CD34⁺ CD38⁻ human hematopoietic progenitor cells but not in those cells transduced with murine vectors. However, stable integration and expression of the reporter gene could not be detected in these hematopoietic progenitors, leaving open the question of the accessibility of these cells to stable lentivirus transduction.     

Enrichment of Denitrifying Methane-Oxidizing Microorganisms Using Up-Flow Continuous Reactors and Batch Cultures

Denitrifying anaerobic methane oxidizing (DAMO) microorganisms were enriched from paddy field soils using continuous-flow and batch cultures fed with nitrate or nitrite as a sole electron acceptor. After several months of cultivation, the continuous-flow cultures using nitrite showed remarkable simultaneous methane oxidation and nitrite reduction and DAMO bacteria belonging to phylum NC10 were enriched. A maximum volumetric nitrite consumption rate of 70.4±3.4 mg-N·L⁻¹·day⁻¹ was achieved with very short hydraulic retention time of 2.1 hour. In the culture, about 68% of total microbial cells were bacteria and no archaeal cells were detected by fluorescence in situ hybridization. In the nitrate-fed continuous-flow cultures, 58% of total microbial cells were bacteria while archaeal cells accounted for 7% of total cell numbers. Phylogenetic analysis of pmoA gene sequence showed that enriched DAMO bacteria in the continuous-flow cultivation had over 98% sequence similarity to DAMO bacteria in the inoculum. In contrast, for batch culture, the enriched pmoA gene sequences had 89–91% sequence similarity to DAMO bacteria in the inoculum. These results indicate that electron acceptor and cultivation method strongly affect the microbial community structures of DAMO consortia.     

Calpain and calpastatin in myoblast differentiation and fusion: Effects of inhibitors

Myoblast differentiation and fusion to multinucleated muscle cells can be studied in myoblasts grown in culture. Calpain (Ca²⁺-activated thiol protease) induced proteolysis has been suggested to play a role in myoblast fusion. We previously showed that calpastatin (the endogenous inhibitor of calpain) plays a role in cell membrane fusion. Using the red cell as a model, we found that red cell fusion required calpain activation and that fusibility depended on the ratio of cell calpain to calpastatin. We found recently that calpastatin diminishes markedly in myoblasts during myoblast differentiation just prior to the start of fusion, allowing calpain activation at that stage; calpastatin reappears at a later stage (myotube formation). In the present study, the myoblast fusion inhibitors TGF-β, EGTA and calpeptin (an inhibitor of cysteine proteases) were used to probe the relation of calpastatin to myoblast fusion. Rat L8 myoblasts were induced to differentiate and fuse in serum-poor medium containing insulin. TGF-β and EGTA prevented the diminution of calpastatin. Calpeptin inhibited fusion without preventing diminution of calpastatin, by inhibiting calpain activity directly. Protein levels of μ-calpain and m-calpain did not change significantly in fusing myoblasts, nor in the inhibited, non-fusing myoblasts. The results indicate that calpastatin level is modulated by certain growth and differentiation factors and that its continuous presence results in the inhibition of myoblast fusion.     

Strain-Dependent Myeloid Hyperplasia, Growth Deficiency, and Accelerated Cell Cycle in Mice Lacking the Rb-Related p107 Gene

To investigate the function of the Rb-related p107 gene, a null mutation in p107 was introduced into the germ line of mice and bred into a BALB/cJ genetic background. Mice lacking p107 were viable and fertile but displayed impaired growth, reaching about 50% of normal weight by 21 days of age. Mutant mice exhibited a diathetic myeloproliferative disorder characterized by ectopic myeloid hyperplasia in the spleen and liver. Embryonic p107^−/− fibroblasts and primary myoblasts isolated from adult p107^−/− mice displayed a striking twofold acceleration in doubling time. However, cell sort analysis indicated that the fraction of cells in G₁, S, and G₂ was unaltered, suggesting that the different phases of the cell cycle in p107^−/− cells was uniformly reduced by a factor of 2. Western analysis of cyclin expression in synchronized p107^−/− fibroblasts revealed that expression of cyclins E and A preceded that of D1. Mutant embryos expressed approximately twice the normal level of Rb, whereas p130 levels were unaltered. Lastly, mutant mice reverted to a wild-type phenotype following a single backcross with C57BL/6J mice, suggesting the existence of modifier genes that have potentially epistatic relationships with p107. Therefore, we conclude that p107 is an important player in negatively regulating the rate of progression of the cell cycle, but in a strain-dependent manner.     

Isolation and characterization of terminally differentiated chicken and rat skeletal muscle myoblasts

The ability of skeletal muscle myoblasts to differentiate in the absence of spontaneous fusion was studied in cultures derived from chicken embryo leg muscle, rat myoblast lines L6 and L8, and the mouse myoblast line G8. Following 48–96 hr of culture in a low-Ca²⁺ (25 μm), Mg²⁺-depleted medium, chicken myoblasts exhibited only 3–5% fusion whereas up to 64% of the cells fused in control cultures. Depletion of Mg²⁺ led to preferential elimination of fibroblasts, with the result that 97% of the mononucleated cells remaining at 120 hr exhibited a bipolar morphology and stained with antibodies directed against M-creatine kinase, skeletal muscle myosin, and desmin. Mononucleated myoblasts rarely showed visible cross-striations or M-line staining with anti-myomesin unless the medium was supplemented with 0.81 mM Mg²⁺, suggesting that Mg²⁺ plays a role in sarcomere assembly. Conditions of Ca²⁺ and Mg²⁺ depletion inhibited myoblast fusion in the rodent cell lines as well, but mononucleated myoblasts failed to differentiate under these conditions. Differentiated individual myoblasts from rat cell lines and from chicken cell cultures were obtained when fusion was inhibited by growth in cytochalasin B (CB). CB-treated rat myoblast cultures accumulated MM-CK to nearly twice the specific activity found in extensively fused control cultures of comparable age. Spherical cells which accumulated during CB treatment were isolated and shown to contain nearly eight times the CK specific activity present in nonspherical cells from the same cultures. Approximately 90% of these cells exhibited immunofluorescent staining with antibodies to skeletal muscle myosin, failed to incorporate [³H]thymidine or to form colonies in clonal subculture, and thus represent terminally differentiated rat myoblasts. Quantitative microfluorometric DNA measurements on individual nuclei demonstrated that the terminally differentiated myoblasts obtained in these experiments from both chicken and rat contain 2cDNA levels, suggesting arrest in the G₀ stage of the cell cycle.     

Herpes Simplex Virus 2 ICP0? Mutant Viruses Are Avirulent and Immunogenic: Implications for a Genital Herpes Vaccine

Herpes simplex virus 1 (HSV-1) ICP0 ⁻ mutants are interferon-sensitive, avirulent, and elicit protective immunity against HSV-1 (Virol J, 2006, 3:44). If an ICP0 ⁻ mutant of herpes simplex virus 2 (HSV-2) exhibited similar properties, such a virus might be used to vaccinate against genital herpes. The current study was initiated to explore this possibility. Several HSV-2 ICP0 ⁻ mutant viruses were constructed and evaluated in terms of three parameters: i. interferon-sensitivity; ii. virulence in mice; and iii. capacity to elicit protective immunity against HSV-2. One ICP0 ⁻ mutant virus in particular, HSV-2 0ΔNLS, achieved an optimal balance between avirulence and immunogenicity. HSV-2 0ΔNLS was interferon-sensitive in cultured cells. HSV-2 0ΔNLS replicated to low levels in the eyes of inoculated mice, but was rapidly repressed by an innate, Stat 1-dependent host immune response. HSV-2 0ΔNLS failed to spread from sites of inoculation, and hence produced only inapparent infections. Mice inoculated with HSV-2 0ΔNLS consistently mounted an HSV-specific IgG antibody response, and were consistently protected against lethal challenge with wild-type HSV-2. Based on their avirulence and immunogenicity, we propose that HSV-2 ICP0 ⁻ mutant viruses merit consideration for their potential to prevent the spread of HSV-2 and genital herpes.     

Experimental Colitis Is Associated with Transcriptional Inhibition of Na+/Ca2+ Exchanger Isoform 1 (NCX1) Expression by Interferon γ in the Renal Distal Convoluted Tubules

NCX1 is a Na⁺/Ca²⁺ exchanger, which is believed to provide a key route for basolateral Ca²⁺ efflux in the renal epithelia, thus contributing to renal Ca²⁺ reabsorption. Altered mineral homeostasis, including intestinal and renal Ca²⁺ transport may represent a significant component of the pathophysiology of the bone mineral density loss associated with Inflammatory Bowel Diseases (IBD). The objective of our research was to investigate the effects of TNBS and DSS colitis and related inflammatory mediators on renal Ncx1 expression. Colitis was associated with decreased renal Ncx1 expression, as examined by real-time RT-PCR, Western blotting, and immunofluorescence. In mIMCD3 cells, IFNγ significantly reduced Ncx1 mRNA and protein expression. Similar effects were observed in cells transiently transfected with a reporter construct bearing the promoter region of the kidney-specific Ncx1 gene. This inhibitory effect of IFNγ is mediated by STAT1 recruitment to the proximal promoter region of Ncx1. Further in vivo study with Stat1^−/− mice confirmed that STAT1 is indeed required for the IFNγ mediated Ncx1 gene regulation. These results strongly support the hypothesis that impaired renal Ca²⁺ handling occurs in experimental colitis. Negative regulation of NCX1- mediated renal Ca²⁺ absorption by IFNγ may significantly contribute to the altered Ca²⁺ homeostasis in IBD patients and to IBD-associated loss of bone mineral density.     

Targeting Photoreceptors via Intravitreal Delivery Using Novel,Capsid-Mutated AAV Vectors

Development of viral vectors capable of transducing photoreceptors by less invasive methods than subretinal injection would provide a major advancement in retinal gene therapy. We sought to develop novel AAV vectors optimized for photoreceptor transduction following intravitreal delivery and to develop methodology for quantifying this transduction in vivo. Surface exposed tyrosine (Y) and threonine (T) residues on the capsids of AAV2, AAV5 and AAV8 were changed to phenylalanine (F) and valine (V), respectively. Transduction efficiencies of self-complimentary, capsid-mutant and unmodified AAV vectors containing the smCBA promoter and mCherry cDNA were initially scored in vitro using a cone photoreceptor cell line. Capsid mutants exhibiting the highest transduction efficiencies relative to unmodified vectors were then injected intravitreally into transgenic mice constitutively expressing a Rhodopsin-GFP fusion protein in rod photoreceptors (Rho-GFP mice). Photoreceptor transduction was quantified by fluorescent activated cell sorting (FACS) by counting cells positive for both GFP and mCherry. To explore the utility of the capsid mutants, standard, (non-self-complementary) AAV vectors containing the human rhodopsin kinase promoter (hGRK1) were made. Vectors were intravitreally injected in wildtype mice to assess whether efficient expression exclusive to photoreceptors was achievable. To restrict off-target expression in cells of the inner and middle retina, subsequent vectors incorporated multiple target sequences for miR181, an miRNA endogenously expressed in the inner and middle retina. Results showed that AAV2 containing four Y to F mutations combined with a single T to V mutation (quadY−F+T−V) transduced photoreceptors most efficiently. Robust photoreceptor expression was mediated by AAV2(quadY−F+T−V) −hGRK1−GFP. Observed off-target expression was reduced by incorporating target sequence for a miRNA highly expressed in inner/middle retina, miR181c. Thus we have identified a novel AAV vector capable of transducing photoreceptors following intravitreal delivery to mouse. Furthermore, we describe a robust methodology for quantifying photoreceptor transduction from intravitreally delivered AAV vectors.     

The Gene Expression Profile of CD11c+CD8α? Dendritic Cells in the Pre-Diabetic Pancreas of the NOD Mouse

Two major dendritic cell (DC) subsets have been described in the pancreas of mice: The CD11c⁺CD8α⁻ DCs (strong CD4+ T cell proliferation inducers) and the CD8α⁺CD103⁺ DCs (T cell apoptosis inducers). Here we analyzed the larger subset of CD11c⁺CD8α⁻ DCs isolated from the pancreas of pre-diabetic NOD mice for genome-wide gene expression (validated by Q-PCR) to elucidate abnormalities in underlying gene expression networks. CD11c⁺CD8α⁻ DCs were isolated from 5 week old NOD and control C57BL/6 pancreas. The steady state pancreatic NOD CD11c⁺CD8α⁻ DCs showed a reduced expression of several gene networks important for the prime functions of these cells, i.e. for cell renewal, immune tolerance induction, migration and for the provision of growth factors including those for beta cell regeneration. A functional in vivo BrdU incorporation test showed the reduced proliferation of steady state pancreatic DC. The reduced expression of tolerance induction genes (CD200R, CCR5 and CD24) was supported on the protein level by flow cytometry. Also previously published functional tests on maturation, immune stimulation and migration confirm the molecular deficits of NOD steady state DC. Despite these deficiencies NOD pancreas CD11c⁺CD8α⁻ DCs showed a hyperreactivity to LPS, which resulted in an enhanced pro-inflammatory state characterized by a gene profile of an enhanced expression of a number of classical inflammatory cytokines. The enhanced up-regulation of inflammatory genes was supported by the in vitro cytokine production profile of the DCs. In conclusion, our data show that NOD pancreatic CD11c⁺CD8α⁻ DCs show various deficiencies in steady state, while hyperreactive when encountering a danger signal such as LPS.     

Ultrastructural plasma membrane asymmetries in tension and curvature promote yeast cell fusion

Cell–cell fusion is central for sexual reproduction, and generally involves gametes of different shapes and sizes. In walled fission yeast Schizosaccharomyces pombe, the fusion of h+ and h− isogametes requires the fusion focus, an actin structure that concentrates glucanase-containing vesicles for cell wall digestion. Here, we present a quantitative correlative light and electron microscopy (CLEM) tomographic dataset of the fusion site, which reveals the fusion focus ultrastructure. Unexpectedly, gametes show marked asymmetries: a taut, convex plasma membrane of h− cells progressively protrudes into a more slack, wavy plasma membrane of h+ cells. Asymmetries are relaxed upon fusion, with observations of ramified fusion pores. h+ cells have a higher exo-/endocytosis ratio than h− cells, and local reduction in exocytosis strongly diminishes membrane waviness. Reciprocally, turgor pressure reduction specifically in h− cells impedes their protrusions into h+ cells and delays cell fusion. We hypothesize that asymmetric membrane conformations, due to differential turgor pressure and exocytosis/endocytosis ratios between mating types, favor cell–cell fusion.