Stretch-induced myogenin, MyoD, and MRF4 expression and acute hypertrophy in quail slow-tonic muscle are not dependent upon satellite cell proliferation

 The objectives of these studies were to determine if (1) hypertrophy-stimulated myogenic regulatory factor (MRF) mRNA increases occur in the absense of proliferating satellite cells, and (2) acute hypertrophy occurs without satellite cell proliferation. Adult and aged quails were exposed to 0 or 2500 Rads gamma irradiation, and then wing muscles were stretch-overloaded for 3 or 7 days. MRF mRNA levels in stretch-overloaded and contralateral anterior latissimus dorsi (ALD) muscles were determined after 3 days; hypertrophy was determined after 7 days. The elimination of proliferating cells in irradiated muscles was verified histologically by bromodeoxyuridine incorporation. Relative levels of MRF4, MyoD, and myogenin mRNA were elevated 100%–400% in stretch-overloaded ALD muscles from irradiated adult quails indicating that satellite cell proliferation was not a prerequisite for MRF mRNA increases. Myogenin was the only MRF that exhibited mRNA increases that were lowered by irradiation. This suggests that satellite cells contribute only to myogenin mRNA increases in non-irradiated adult muscles following 3 days of stretch-overload. Stretch-overloaded ALD muscles from aged quails had a relative increase in myogenin mRNA of ∼150%. The myogenin increase was the same in non-irradiated and irradiated aged animals and also the same as that in stretch-overloaded muscles from irradiated adult quails. Together, these data indicate that attenuated increases in MRF expression in muscles from aged animals are attributable to lower satellite cell MRF expression. ALD muscle masses and protein contents in adult irradiated quails approximately doubled after 7 days of stretch-overload demonstrating hypertrophy despite the elimination of satellite cell proliferation. Received: 5 June 1998 / Accepted: 19 November 1998     

Xeroderma pigmentosum variant cells are not defective in the repair of (6-4) photoproducts

Using radioimmunoassays specific for (6-4) photoproducts and cyclobutane dimers, Xeroderma pigmentosum variant cells appear to have a normal capacity for the repair of each of these lesions. However, these assays measure an early stage in the repair pathway and we do not exclude the possibility that repair is not successfully completed following UV irradiation and excision of DNA photoproducts.     

ATR activation and replication fork restart are defective in FANCM‐deficient cells

Fanconi anaemia is a chromosomal instability disorder associated with cancer predisposition and bone marrow failure. Among the 13 identified FA gene products only one, the DNA translocase FANCM, has homologues in lower organisms, suggesting a conserved function in DNA metabolism. However, a precise role for FANCM in DNA repair remains elusive. Here, we show a novel function for FANCM that is distinct from its role in the FA pathway: promoting replication fork restart and simultaneously limiting the accumulation of RPA‐ssDNA. We show that in DT40 cells this process is controlled by ATR and PLK1, and that in the absence of FANCM, stalled replication forks are unable to resume DNA synthesis and genome duplication is ensured by excess origin firing. Unexpectedly, we also uncover an early role for FANCM in ATR‐mediated checkpoint signalling by promoting chromatin retention of TopBP1. Failure to retain TopBP1 on chromatin impacts on the ability of ATR to phosphorylate downstream molecular targets, including Chk1 and SMC1. Our data therefore indicate a fundamental role for FANCM in the maintenance of genome integrity during S phase.     

Hypoxia affects positively the proliferation of bovine satellite cells and their myogenic differentiation through up-regulation of MyoD

Hypoxia alters the biological functions of skeletal muscle cells to proliferate and differentiate into myotubes. However, the cellular responses of myoblasts to hypoxia differ according to the levels of oxygen and the types of cells studied. This study examined the effect of hypoxia (1% oxygen) on bovine satellite cells. Hypoxia significantly increased the proliferation of satellite cells cultured in a growth medium. In addition, the levels of PCNA, cyclin D1, cyclin-dependent kinase-1 (CDK1) and CDK2 expression were increased. Hypoxia facilitated the formation of myotubes as well as the stimulation of MyoD, myogenin, and myosin heavy chain (MHC) expression in differentiating medium (DM) cultures. In particular, satellite cells cultured under hypoxic/DM conditions showed increased p21 expression but not p27. The transfection of satellite cells with antisense MyoD oligonucleotides resulted in a decrease in the MHC, myogenin, MRF4 RNA and protein levels with the concomitant decrease in fused cells to levels similar to those observed under normoxia/DM conditions. This indicates that MyoD up-regulation is closely associated with hypoxia-stimulated myogenic differentiation. In conclusion, hypoxia stimulates the proliferation of satellite cells and promotes their myogenic differentiation with MyoD playing an important role.     

In vitro culture and induced differentiation of sheep skeletal muscle satellite cells

Skeletal muscle satellite cells are adult muscle-derived stem cells receiving increasing attention. Sheep satellite cells have a greater similarity to human satellite cells with regard to metabolism, life span, proliferation and differentiation, than satellite cells of the rat and mouse. We have used 2-step enzymatic digestion and differential adhesion methods to isolate and purify sheep skeletal muscle satellite cells, identified the cells and induced differentiation to examine their pluripotency. The most efficient method for the isolation of sheep skeletal muscle satellite cells was the type I collagenase and trypsin 2-step digestion method, with the best conditions for in vitro culture being in medium containing 20% FBS+10% horse serum. Immunofluorescence staining showed that satellite cells expressed Desmin, α-Sarcomeric Actinin, MyoD1, Myf5 and PAX7. After myogenic induction, multinucleated myotubes formed, as indicated by the expression of MyoG and fast muscle myosin. After osteogenic induction, cells expressed Osteocalcin, with Alizarin Red and ALP (alkaline phosphatase) staining results both being positive. After adipogenic induction, cells expressed PPARγ2 (peroxisome-proliferator-activated receptor γ2) and clear lipid droplets were present around the cells, with Oil Red-O staining giving a positive result. In summary, a successful system has been established for the isolation, purification and identification of sheep skeletal muscle satellite cells.     

Mutants of satellite bacteriophage P4 that are defective in the suppression of transcriptional polarity

The satellite bacteriophage P4 relies on a helper such as P2 to supply the gene products necessary for virion assembly and cell lysis (Six, 1975). P4 has the unique capacity to activate the late genes of P2 by a mechanism that differs from the one normally used by P2 itself. This process has been termed transactivation (Calendar et al., 1977). In addition, P4 is able to suppress the strong polarity associated with certain P2 amber mutations. The isolation of P4 mutants solely defective in polarity suppression (psu^?) demonstrates that the ability of P4 to suppress polarity is non-essential for P4 growth. In particular, polarity suppression plays no essential role in either transactivation or head size determination. The product of the P4 psu gene has been identified as a 19,900 M_r P4 late protein.     

BioBreeding/Worcester (BB/Wor) rats are deficient in the generation of functional cytotoxic T cells

The BioBreeding/Worcester (BB/Wor) rat provides a good model of spontaneous autoimmune diabetes. There are several sublines of the BB/Wor rat. The diabetes prone (DP) sublines develop diabetes at a frequency of 50 to 80% from 60 to 120 days of age. The DP rats are lymphopenic, have a severe deficit in phenotypic OX 19+ OX 8+ cytotoxic T cells (Tc), and lack RT 6.1 T cells. These rats have a relative increase in OX 19- OX 8+ natural killer (NK) cells and in NK activity as compared with the diabetes resistant (DR) sublines. The DR sublines have a normal complement of phenotypic Tc and RT 6.1 T cells, fewer NK cells, and lower NK activity than the DP rat. The ability to elicit functional Tc in the BB/Wor rat has not been well studied. In these experiments, by using a model of lymphocytic choriomeningitis virus (LCMV) infection in DP and DR rats, we have studied the functional activity of Tc in these lines. Seven days after infection with LCMV, DR rats develop lymphocytes which are cytotoxic for LCMV-infected syngeneic fibroblasts. These cytotoxic lymphocytes are phenotypic Tc (OX 19+ OX 8+), and do not kill Pichinde virus-infected syngeneic fibroblasts or LCMV-infected allogeneic fibroblasts. This cytotoxic activity is accompanied by an increase in phenotypic Tc from 17 to 33%. DP rats produced neither functional nor phenotypic Tc. These studies confirm that NK cells are the predominant cytotoxic lymphocyte in the BB/Wor rat and suggest that these rats may not utilize a Tc mechanism in islet destruction or another immunologic process such as graft rejection.     

Selection of a dominant negative retinoblastoma protein (RB) inhibiting satellite myoblast differentiation implies an indirect interaction between MyoD and RB

Satellite myoblasts serve as stem cells in postnatal skeletal muscle, but the genes responsible for choosing between growth versus differentiation are largely undefined. We have used a novel genetic approach to identify genes encoding proteins whose dominant negative inhibition is capable of interrupting the in vitro differentiation of C2C12 murine satellite myoblasts. The screen is based on fusion of a library of cDNA fragments with the lysosomal protease cathepsin B (CB), such that the fusion protein intracellularly diverts interacting factors to the lysosome. Among other gene fragments selected in this screen, including those of known and novel sequence, is the retinoblastoma protein (RB) pocket domain. This unique dominant negative form of RB allows us to genetically determine if MyoD and RB associate in vivo. The dominant negative CB-RB fusion produces a cellular phenotype indistinguishable from recessive loss of function RB mutations. The fact that the dominant negative RB inhibits myogenic differentiation in the presence of nonlimiting concentrations of either RB or MyoD suggests that these two proteins do not directly interact. We further show that the dominant negative RB inhibits E2F1 but cannot inhibit a forced E2F1-RB dimer. Therefore, E2F1 is a potential mediator of the dominant negative inhibition of MyoD by CB-RB during satellite cell differentiation. We propose this approach to be generally suited to the investigation of gene function, even when little is known about the pathway being studied.     

Ataxia-telangiectasia cells are not uniformly deficient in poly(ADP-ribose) synthesis following X-irradiation

Inducibility of 6-thioguanine-resistant (6TGr) mutants and single-strand scission of DNA by cadmium chloride (CdCl2) was investigated in cultured Chinese hamster V79 cells. Frequency of 6TGr mutants increased concentration dependently by 24-h treatment with CdCl2 up to 3 X 10(-6) M but decreased beyond 3 X 10(-6) M. Mutagenic potency of cadmium in the absence of S9 was about half that of benzo[a]pyrene in the presence of S9 at equitoxic concentrations. Treatment of the cultured cells with cadmium after benzo[a]pyrene treatment was not synergistic but additive to the mutagenicity of benzo[a]pyrene. Single-strand scission of DNA by alkaline elution techniques was observed in the cells treated with CdCl2 for 2 h in a concentration-dependent manner. The single-strand scission by cadmium was detected only in combination with proteinase K digestion of the cell lysates, indicating formation of DNA--protein cross-linking by the metal. These biological and biochemical findings indicate that cadmium is mutagenic in mammalian cells, and its mutagenic effect seems to be accompanied by single-strand scission of DNA.     

Sphingolipid-free Leishmania are defective in membrane trafficking, differentiation and infectivity

Sphingolipids are structural components of the eukaryotic plasma membrane that are involved, together with cholesterol, in the formation of lipid microdomains (rafts). Additionally, sphingolipid metabolites have been shown to modulate a wide variety of cellular events, including differentiation and apoptosis. To investigate the role of de novo sphingolipid biosynthesis in Leishmania, we have focused on serine palmitoyltransferase (SPT), which catalyses the first, rate-limiting step in the synthetic pathway. Genetic ablation of one SPT subunit, LmLCB2, yields viable null parasites that can no longer synthesize ceramide and sphingolipids de novo. Unexpectedly, LmLCB2 expression (and sphingolipid biosynthesis) is stage regulated in Leishmania, being undetectable in intramacrophage parasites. As expected from this observation, the LmLCB2 null mutants maintain infectivity in vivo. However, they are compromised in their ability to form infective extracellular parasites, correlating with a defect in association of the virulence factor, leishmanolysin or GP63, with lipid rafts during exocytosis and an observed relocalization of a second virulence factor, lipophosphogycan, during differentiation. Thus, de novo sphingolipid biosynthesis is critical for membrane trafficking events in extracellular Leishmania but has at best a minor role in intracellular pathogenesis.     

Mitochondria from leaf mesophyll cells of C(4) plants are deficient in the H protein of glycine decarboxylase complex

Mesophyll mitochondria from green leaves of the C(4) plants Zea mays (NADP-ME-type), Panicum miliaceum (NAD-ME-type) and Panicum maximum (PEP-CK-type) oxidized NADH, malate and succinate at relatively high rates with respiratory control, but glycine was not oxidized. Among the mitochondrial proteins involved in glycine oxidation, the L, P and T proteins of glycine decarboxylase complex (GDC) and serine hydroxymethyltransferase (SHMT) were present, while the H protein of GDC was undetectable. In contrast, mesophyll mitochondria from etiolated leaves of Z. mays oxidized glycine at a slow rate and with no respiratory control, and contained the H protein as well as the other GDC proteins and SHMT. The T and P proteins and SHMT were present in the mitochondria from etiolated leaves at significantly higher levels than in those from green leaves of Z. mays. The content of the L protein was almost identical in all three C(4) plants examined and close to the value obtained for mesophyll mitochondria from the C(3) plant Pisum sativum, whereas the other GDC proteins and SHMT were less abundant than the L protein. We discuss possible reasons for the H protein's absence in mesophyll mitochondria of C(4) plants, as well as the role(s) the other GDC components could play in its absence.     

Retinoic acid stimulates the differentiation of PC12 cells that are deficient in cAMP-dependent protein kinase

Retinoic acid (RA) induced neuronal differentiation in A126-1B2 cells and 123.7 cells, two mutant lines of PC12 that are deficient in cAMP-dependent protein kinase, but not in the parental PC12 cell line. A single exposure to RA was sufficient to cause neurite formation and inhibit cell division for a period of greater than 3 wk, suggesting that RA may cause a long-term, stable change in the state of these cells. In A126-1B2 cells, RA also induced the expression of other markers of differentiation including acetylcholinesterase and the mRNAs for neurofilament (NF-M) and GAP-43 as effectively as nerve growth factor (NGF). Neither NGF nor RA stimulated an increase in the expression of smg-25A in A126-1B2 cells, suggesting that the cAMP-dependent protein kinases may be required for an increase in the expression of this marker. RA also caused a rapid increase in the expression of the early response gene, c-fos, but did not effect the expression of egr-1. RA equivalently inhibited the division of A126-1B2 cells, 123.7 cells and parental PC12 cells, so RA induced differentiation is not an indirect response to growth arrest. In contrast, the levels of retinoic acid receptors (RAR alpha and RAR beta), and retinoic acid binding protein (CRABP) mRNA were strikingly higher in both A126-1B2 cells and 123.7 cells than in the parental PC12 cells. The deficiencies in cAMP-dependent protein kinase may increase the expression of CRABP and the RARs; and, thus, cAMP may indirectly regulate the ability of RA to control neurite formation and neural differentiation. Thus, RA appears to regulate division and differentiation of PC12 cells by a biochemical mechanism that is quite distinct from those used by peptide growth factors.