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T Braun

Publications and source records attributed to T Braun.

At least 73 records · Page 4Linked to original sources
PubMed1996

Regulation and function of SF/HGF during migration of limb muscle precursor cells in chicken.

Limb muscles of vertebrates are derived from migratory dermomyotomal cells which emanate from a limited number of somites located adjacent to the developing limb buds. We have generated additional limb buds in chicken embryos by implantation of FGF-beads into the interlimb region in order to analyze whether these somites can be programmed to supply ectopic limbs with myogenic precursor cells. We show that migrating myogenic precursor cells are released from somites at the level of the newly formed limb, even when cell migration into the natural limb has been completed. The implantation of FGF beads in the lateral plate mesoderm rapidly induces SF/HGF expression. FGF beads implanted between HH stages 10 and 12 inhibit limb bud formation or shift the normal limb position. When an additional FGF bead was implanted at the original limb position at HH stage 15, SF/HGF expression was transiently induced to low levels without inducing a new limb. This demonstrates that the initial induction of SF/HGF by FGF does not require limb formation. Expression of SF/HGF during early limb bud stages was found in the entire developing bud and the adjacent lateral plate mesoderm with direct contacts to the lateral edge of the dermomyotome. Later, the SF/HGF expression domain retracts to a distal region below the apical ectodermal ridge. To investigate the role of SF/HGF in the migratory process, we implanted beads soaked in SF/HGF-alone or together with FGF into different locations of the developing chick embryo. In the experiments SF/HGF caused delamination of migratory cells from the dermomyotomal epithelium but no chemotactic attraction of migrating cells toward the SF/HGF source.

Animals↗
PubMed1996

Myf-5(m1)/Myf-6(m1) compound heterozygous mouse mutants down-regulate Myf-5 expression and exert rib defects: evidence for long-range cis effects on Myf-5 transcription.

Myf-6 and Myf-5, two members of the family of muscle-specific regulatory genes, are located less than 10 kb apart in the mouse and human genomes. We have shown recently that homozygous mutant mice carrying a pgk-neo-cassette in the first exon of the Myf-6 gene display minor alterations of skeletal musculature but develop a severe rib defect, most likely due to a drastic down-regulation of Myf-5 expression. The mechanism by which the Myf-6 mutation affects the Myf-5 gene is unknown. In order to determine whether Myf-5 transcription is inhibited by the Myf-6 mutation in cis or in trans, we generated compound heterozygous mice carrying inactivated Myf-5 and Myf-6 alleles on different chromosomes. Here, we demonstrate that double-heterozygous mutants exhibit truncated ribs and severe depression of Myf-5 transcription, a phenotype similar to the previously described homozygous Myf-6 mutant mice. These results indicate that the Myf-6 mutation inhibits Myf-5 gene expression by a long-range cis effect.

Animals↗
PubMed1996

Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast.

The endoplasmic reticulum (ER) of the yeast Saccharomyces cerevisiae contains of proteolytic system able to selectively degrade misfolded lumenal secretory proteins. For examination of the components involved in this degradation process, mutants were isolated. They could be divided into four complementation groups. The mutations led to stabilization of two different substrates for this process. The mutant classes were called 'der' for 'degradation in the ER'. DER1 was cloned by complementation of the der1-2 mutation. The DER1 gene codes for a novel, hydrophobic protein, that is localized to the ER. Deletion of DER1 abolished degradation of the substrate proteins. The function of the Der1 protein seems to be specifically required for the degradation process associated with the ER. The depletion of Der1 from cells causes neither detectable growth phenotypes nor a general accumulation of unfolded proteins in the ER. In DER1-deleted cells, a substrate protein for ER degradation is retained in the ER by the same mechanism which also retains lumenal ER residents. This suggests that DER1 acts in a process that directly removes protein from the folding environment of the ER.

Amino Acid Sequence↗
PubMed1996

Myf-5 and myoD genes are activated in distinct mesenchymal stem cells and determine different skeletal muscle cell lineages.

Targeted inactivation of the myogenic determination genes myf-5 and myoD in mice resulted in moderate (Myf-5) or no muscle phenotypes (MyoD) and double knock-out mutants lacking both genes failed to develop any skeletal muscle. In order to determine the mechanism of this apparent genetic redundancy we investigated the basis of functional overlap between the two genes. Here we demonstrate that Myf-5 and MyoD are not expressed within the same muscle precursor cell, but rather determine different muscle cell lineages arising from independently committed stem cell populations. Selective ablation of Myf-5-expressing muscle precursors from differentiating ES cells does not prevent Myo-D-dependent muscle differentiation. The early muscle progenitor cells which normally express Myf-5 do not develop into later appearing MyoD cells, even when the myf-5 gene has been inactivated. Thus skeletal musculature in vertebrates develops from two separate cell lineages and complementation may occur at the cellular level, but not between different myogenic factor genes within one cell.

Alleles↗
PubMed1996

Alterations in somite patterning of Myf-5-deficient mice: a possible role for FGF-4 and FGF-6.

Mice carrying a targeted mutation in the gene for the myogenic factor Myf-5 fail to form major parts of the ribs, which leads to an unstable thorax and perinatal death. Here, we report that somites of Myf-5-deficient mice lack the expression of FGF-4 and FGF-6 while TGF beta-2 is expressed normally. Early sclerotomal markers, such as Pax-1 reveal no substantial reduction of sclerotome size. At E11.5 the condensing mesenchyme of the rib anlagen is considerably reduced in size in Myf-5 mutant mice. This may be caused by the lack of Myf-5-positive, FGF-expressing cells which normally are in close contact with the lateral sclerotome generating the rib progenitors. The potential role of FGFs and TGF beta on sclerotome formation is demonstrated in micromass cultures of early somites. Combinations of FGF-4 or FGF-6 with TGF beta-2 potentiate chondrogenesis suggesting that these growth factors emanating from early myotomal and dermomyotomal cells may have instructive or permissive effects on differentiation or outgrowth of sclerotomal cells.

Animals↗
PubMed1996

Targeted inactivation of myogenic factor genes reveals their role during mouse myogenesis: a review.

The role of the four myogenic regulating genes Myf-5, myogenin, MyoD, and MRF4 (herculin, Myf-6) during mouse embryogenesis has been investigated by targeted gene inactivation. Null mutations for the MyoD gene generate no skeletal muscle phenotype due to a compensatory activation of the Myf-5 gene. Mice carrying a homozygous Myf-5 mutation exert considerably delayed myotome formation with unexpected consequences. While skeletal myogenesis in these mutant mice resumes normally at the onset of MyoD expression, a skeletal defect of the ribs persists. Apparently, Myf-5 and MyoD individually are not absolutely essential for skeletal muscle development, most likely because they have overlapping or redundant functions. In fact, double mutants lacking both, MyoD and Myf-5, fail to develop skeletal musculature and the muscle forming regions seem to be devoid of myoblasts. Homozygous inactivation of the myogenin gene leads to drastically reduced myofiber formation. These mice accumulate apparently normal numbers of myoblasts which are arrested in their terminal differentiation program. Myf-6 null mutant mice exhibit drastically reduced expression of Myf-5 for reasons presently unknown. The phenotype is very similar to Myf-5 mutants with an additional reduction of deep back muscles and minor alterations in sarcomeric protein isoforms. Based on the phenotypes obtained from these various gene "knock-out" mice, we now begin to understand the regulatory network and the homostatic relationship of genes which are critically involved in myogenesis of vertebrates.

Animals↗
PubMed1995

Inactivation of Myf-6 and Myf-5 genes in mice leads to alterations in skeletal muscle development.

Myf-6, alternatively called MRF4 or herculin, is a member of a group of muscle-specific transcription factors which also comprises Myf-5, myogenin and MyoD. All family members show distinct expression patterns during skeletal muscle development and can convert a variety of cell lines to myocytes. We disrupted the Myf-6 gene in mice to investigate its functional role in the network of regulatory factors controlling myogenesis. Homozygous mice carrying the disrupted Myf-6 gene show pronounced down-regulation of Myf-5 transcription for reasons presently unknown. Consequently, these mice represent a double knock-out model for Myf-6 and Myf-5. The mutants resemble most of the Myf-5 phenotype with aberrant and delayed early myotome formation and lack of distal rib structures. In addition, we find a reduction in the size of axial muscles in the back. Apart from changes in the pattern of some contractile protein isoforms, the existing myofibers appear fairly normal. This suggests that Myf-6 has no major role in the maturation of myotubes, as previously proposed. Our results provide evidence that skeletal myogenesis can proceed in the absence of two myogenic factors, Myf-5 and Myf-6, therefore they must exert largely non-redundant functions in vivo.

Animals↗
PubMed1995

Monoclonal anti-FLAG antibodies react with a new isoform of rat Mg2+ dependent protein phosphatase beta.

The FLAG peptide has been widely used as a multi-purpose tag for the identification and detection of recombinant FLAG fusion proteins. The practicability of this approach depends on specific detection of FLAG fusion proteins with no or very little cross-reactivity to cellular proteins. We have isolated a rat cDNA clone coding for a new splicing isoform of Mg2+ dependent protein phosphatase beta (MPP beta) by screening a rat brain expression library with monoclonal antibody Anti-FLAG M2. MPP beta reacts strongly both as a MPP beta-beta-galactosidase- and as a glutathione S-transferase fusion protein with anti-FLAG M2 antibodies. Sequence analysis of MPP beta revealed a sequence motif with five out of eight amino acid residues identical to the FLAG peptide hitherto believed to be mono-specific.

Amino Acid Sequence↗
PubMed1995

Myogenin's functions do not overlap with those of MyoD or Myf-5 during mouse embryogenesis.

The four myogenic basic helix-loop-helix proteins, MyoD, myogenin, Myf-5, and MRF4, can each activate skeletal muscle differentiation when introduced into nonmuscle cells. During embryogenesis, each of these genes is expressed in a unique but overlapping pattern in skeletal muscle precursors and their descendants. Gene knockout experiments have shown that MyoD and Myf-5 play seemingly redundant roles in the generation of myoblasts. However, the role of either of these genes during differentiation in vivo has not been determined. In contrast, a myogenin-null mutation blocks differentiation and results in a dramatic decrease in muscle fiber formation, yet the role of myogenin in the generation or maintenance of myoblast populations is not known. Because myogenin possesses the same myogenic activity as MyoD and Myf-5 in vitro and the expression patterns of these three genes overlap in vivo, we sought to determine if myogenin shares certain functions with either MyoD or Myf-5 in vivo. We therefore generated mice with double homozygous null mutations in the genes encoding MyoD and myogenin or Myf-5 and myogenin. These mice showed embryonic and perinatal phenotypes characteristic of the combined defects observed in mice mutant for each gene alone. As shown by histological analysis and expression of muscle-specific genes, the numbers of undifferentiated myoblasts and residual myofibers were comparable between myogenin-mutant homozygotes and the double-mutant homozygotes. Myoblasts isolated from neonates of the combined mutant genotypes underwent myogenesis in tissue culture, indicating that no more than two of the four myogenic factors are required to support muscle differentiation. These results demonstrate that the functions of myogenin do not overlap with those of MyoD or Myf-5 and support the view that myogenin acts in a genetic pathway downstream of MyoD and Myf-5.

Animals↗
PubMed1994

A novel NK-related mouse homeobox gene: expression in central and peripheral nervous structures during embryonic development.

We have identified three novel mouse homeobox genes that are related to the Drosophila NK gene family. Two genes without direct homologues in Drosophila were designated Nkx-5.1 and Nkx-5.2; the third gene Nkx-1.1 constitutes the mouse homologue to NK1.Nkx-5.1 and Nkx-5.2 are closely linked on mouse chromosome 7, whereas Nkx-1.1 is located on a different chromosome. Here, we report the spatiotemporal expression pattern of Nkx-5.1 during prenatal mouse development. Nkx-5.1 gene activity begins at Embryonic Day 10.5 in the developing ear, the neural tube, and dorsal root ganglia. It continues to be active throughout prenatal life in discrete regions of the brain with an anterior border in the ventral diencephalon at the optic chiasma and expression domains in mesencephalon, metencephalon, and myelencephalon. At midgestation, Nkx-5.1 is also expressed in mesenchyme of the head and branchial arches, and in some cranial ganglia, as well as in derivatives of neural crest, such as the truncus sympathicus and myenteric ganglia. The time pattern of Nkx-5.1 expression and its confinement to primarily postmitotic cells of the central and peripheral nervous system suggest that Nkx-5.1 may play a role in the specification of neuronal cell types.

Amino Acid Sequence↗
PubMed1994

ES-cells carrying two inactivated myf-5 alleles form skeletal muscle cells: activation of an alternative myf-5-independent differentiation pathway.

Both alleles of the myogenic regulatory gene myf-5 have been inactivated in mouse embryonic stem cells by different strategies involving either consecutive gene targeting with neomycin and hygromycin replacement vectors or spontaneous loss of heterozygosity in cells targeted with the neomycin replacement vector alone. Both selection schemes provided homozygous myf-5 mutant ES-cells with normal developmental potential in vitro. Embryoid bodies differentiated into skeletal muscle cells as assessed by their typical morphology and skeletal muscle markers. The extent of differentiation in homozygous mutant myf-5 embryonic stem cells was virtually indistinguishable from control cultures, suggesting that myf-5 is not required for the early steps of myogenic development in vitro. While myocyte populations derived from wild-type and heterozygous myf-5 mutant ES-cells contained myoD-positive and myoD-negative cells, no myoD-negative muscle cells were found among myf-5 homozygous mutants. Differentiated myf-5 double-knockout cells also showed a premature expression of myoD. These results indicate a compensatory role of myoD and myf-5 during early myocyte development and suggest a possible down-regulation of myoD by myf-5 during early myogenesis.

Alleles↗
PubMed1994

MyoD expression marks the onset of skeletal myogenesis in Myf-5 mutant mice.

The expression pattern of myogenic regulatory factors and myotome-specific contractile proteins was studied during embryonic development of Myf-5 mutant mice by in situ hybridization and immunohistochemistry. In contrast to somites in wild-type embryos, no expression of myogenin and Myf-6 (MRF4), or any other myotomal markers was detected in mutant animals at E9.0 and E10.0 indicating that Myf-5 plays a crucial role during this developmental period. Significantly, the onset of MyoD expression in rostral somites of E10.5 embryos was unaffected by the Myf-5 mutation suggesting that the activation of the MyoD gene occurs independently of Myf-5 at the correct developmental time. Immediately after the activation of MyoD myogenin transcripts and protein accumulated within the myotome. The first contractile proteins of the sarcomeric apparatus appeared slightly later. By E11.5 the expression of muscle markers were indistinguishable between wild-type and Myf-5 mutant mice. The migration of muscle precursor cells that leave the somites to form limb musculature was monitored in Myf-5-mutant mice by Pax-3 expression. Pax-3-positive cells were equally found in somites and limbs of E10.0 wild-type and mutant mice indicating that myogenic factor expression at the level of somites is not a prerequisite for determination and subsequent migration of limb precursor cells.

Animals↗
PubMed1993

MyoD or Myf-5 is required for the formation of skeletal muscle.

Mice carrying null mutations in the myogenic regulatory factors Myf-5 or MyoD have apparently normal skeletal muscle. To address whether these two factors functionally substitute for one another in myogenesis, mice carrying mutant Myf-5 and MyoD genes were interbred. While mice lacking both MyoD and Myf-5 were born alive, they were immobile and died soon after birth. Northern blot and S1 nuclease analyses indicated that Myf-5(-1-);MyoD(-1-) mice expressed no detectable skeletal muscle-specific mRNAs. Histological examination of these mice revealed a complete absence of skeletal muscle. Immunohistochemical analysis indicated an absence of desmin-expressing myoblast-like cells. These observations suggest that either Myf-5 or MyoD is required for the determination of skeletal myoblasts, their propagation, or both during embryonic development and indicate that these factors play, at least in part, functionally redundant roles in myogenesis.

Animals↗
PubMed1993

cAMP-dependent protein kinase represses myogenic differentiation and the activity of the muscle-specific helix-loop-helix transcription factors Myf-5 and MyoD.

Myf-5 and MyoD are members of a family of muscle-specific basic helix-loop-helix (bHLH) proteins that are fundamental for myogenic cell differentiation and transcriptional activation of muscle-specific genes. Here we report that elevated levels of the intracellular signaling molecule cAMP and overexpression of cAMP-dependent protein kinase (PKA) inhibit myogenic differentiation. PKA represses the transcriptional activation of muscle-specific genes by the myogenic regulators Myf-5 and MyoD. The repression is directed at the basic HLH domain and is mediated through the E-box DNA consensus motif to which these proteins bind. However, phosphorylation of Myf-5 and MyoD by PKA in vitro does not affect their ability to bind to DNA. PKA specifically inhibits the activity of myogenic bHLH proteins, but not of other HLH proteins, such as the ubiquitously expressed E2A gene products E12 and E47 (E2-5). Our results demonstrate that PKA mediates the cAMP-induced inhibition of muscle cell differentiation by repressing the activity of Myf-5 and MyoD. The inhibition by PKA occurs post-translationally and presumably affects the transactivation process at a step following DNA-binding. The regulation of Myf-5 and MyoD function by a cAMP-dependent pathway may partly explain how external signals generated by serum and certain peptide growth factors can be transduced to the nucleus and inhibit dominant-acting factors that are responsible for myoblast differentiation.

Animals↗
PubMed1993

Extended administration of oral etoposide and oral cyclophosphamide for the treatment of advanced non-small-cell lung cancer: a Southwest Oncology Group study.

PURPOSE: We designed an all-oral regimen of etoposide and cyclophosphamide for use in advanced non-small-cell lung cancer. PATIENTS AND METHODS: Eligible patients were chemotherapy-naive and had histologically confirmed assessable or measurable stage IV non-small-cell lung cancer. Patients received etoposide 50 mg/m2/d orally days 1 through 14 and cyclophosphamide 50 mg/m2/d orally days 1 through 14 every 28 days. Doses on later cycles were adjusted for myelosuppression. RESULTS: Sixty-six patients (64 eligible patients) received 192 cycles of oral extended etoposide/cyclophosphamide therapy (median, two cycles; range, zero to 15). Therapy was well tolerated with the mean dose per cycle being 104% of the originally scheduled dose. Two patients (3%) achieved a complete response and six (9%) achieved a partial response. Leukopenia, anemia, nausea/vomiting, and alopecia were the most common toxicities. Median survival was 6 months, and the 1-year survival rate was 25.6%, comparable to more intensive treatments. CONCLUSION: Oral extended etoposide/cyclophosphamide is a well-tolerated alternative for the treatment of stage IV non-small-cell lung cancer and can be used as a basis for the design of further outpatient regimens.

Administration, Oral↗
PubMed1992

Targeted inactivation of the muscle regulatory gene Myf-5 results in abnormal rib development and perinatal death.

The Myf-5 gene, a member of the myogenic basic HLH factor family, has been inactivated in mice after homologous recombination in ES cells. Mice lacking Myf-5 were unable to breathe and died immediately after birth, owing to the absence of the major distal part of the ribs. Other skeletal abnormalities, except for complete ossification of the sternum, were not apparent. Histological examination of skeletal muscle from newborn mice revealed no morphological abnormalities. Northern blot analysis demonstrated normal levels of muscle-specific mRNAs including MyoD, myogenin, and Myf-6. However, the appearance of myotomal cells in early somites was delayed by several days. These results suggest that while Myf-5 plays a crucial role in the formation of lateral sclerotome derivatives, Myf-5 is dispensable for the development of skeletal muscle, perhaps because other members of the myogenic HLH family substitute for Myf-5 activity.

Animals↗