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N Rosenthal

Publications and source records attributed to N Rosenthal.

At least 19 recordsLinked to original sources
PubMed1997

Developmental regulation of Zbu1, a DNA-binding member of the SWI2/SNF2 family.

The SWI2/SNF2 gene family has been implicated in a wide variety of processes, involving regulation of DNA structure and chromatin configuration, mitotic chromosome segregation, and DNA repair. Here we report the characterization of the Zbu1 gene, also known as HIP116, located on human chromosome band 3q25, which encodes a DNA-binding member of this superfamily. Zbu1 was isolated in this study by its affinity for a site in the myosin light chain 1/3 enhancer. The protein has single-stranded DNA-dependent ATPase activity, includes seven helicase motifs, and a RING finger motif that is shared exclusively by the RAD5, spRAD8, and RAD16 family members. During mouse embryogenesis, Zbu1 transcripts are detected relatively late in fetal development and increase in neonatal stages, whereas the protein accumulates asynchronously in heart, skeletal muscle, and brain. In adult human tissues, alternatively spliced Zbu1 transcripts are ubiquitous with highest expression in these tissues. Gene expression is also dramatically induced in human tumor lines and in Li-Fraumeni fibroblast cultures, suggesting that it is aberrantly regulated in malignant cells. The developmental profile of Zbu1 gene expression and the association of the protein with a tissue-specific transcriptional regulatory element distinguish it from other members of the SWI2/SNF2 family and suggest novel roles for the Zbu1 gene product.

Adenosine Triphosphatases↗
PubMed1996

Modular elements of the MLC 1f/3f locus confer fiber-specific transcription regulation in transgenic mice.

The two proteins encoded by the fast alkali myosin light chain (MLC) 1f/3f locus are developmentally regulated, muscle specific, and expressed exclusively in fast-twitch fibers. Their expression is independently regulated by two separate promoters and a downstream enhancer. Previous studies showed a reporter gene directed by the rat MLC If promoter and MLC enhancer to exhibit correct skeletal muscle-specific expression in transgenic mice during development and to be preferentially expressed in fast-twitch Type IIB fibers [Donoghue et al., (1991) J. Cell B.ol. 115:423-434]. The MLC 3f promoter also directed muscle-specific expression of a CAT reporter gene in adult transgenic mice and showed little dependence upon the enhancer. Here, we show that the MLC 3f promoter also directs transgene expression in the fast-twitch fibers of adult skeletal muscle, but almost exclusively to fiber Types IIA and IIX. MLC 3f transgene expression occurs in only a subset of the fiber types that express the endogenous locus, indicating modular elements included in the transgene confer fiber-specific transcription regulation. MyoD protein was also found to be restricted to fiber Types IIA and IIX, providing evidence for its possible role in mediating fiber-specific gene expression.

Animals↗
PubMed1996

Green fluorescent protein marks skeletal muscle in murine cell lines and zebrafish.

The green fluorescent protein (GFP) acts as a vital dye upon the absorption of blue light. When the gfp gene is expressed in bacteria, flies or nematodes, green fluorescence can be directly observed in the living organism. We inserted the cDNA encoding this 238-amino-acid (aa) jellyfish protein into an expression vector containing the rat myosin light-chain enhancer (MLC-GFP) to evaluate its ability to serve as a muscle-specific marker. Transiently, as well as stably, transfected C2C12 cell lines produced high levels of GFP distributed homogeneously throughout the cytoplasm and was not toxic through several cell passages. Expression of MLC-GFP was strictly muscle-specific, since Cos 7 fibroblasts transfected with MLC-GFP did not fluoresce. When GFP and beta Gal markers were compared, the GFP signal was visible in the cytoplasm of the living cell, whereas visualization of beta Gal required fixation and resulted in deformation of the cells. When the MLC-GFP construct was injected into zebrafish embryos, muscle-specific gfp expression was apparent within 24 h of development. gfp expression was never observed in non-muscle tissues using the MLC-GFP construct. Transgenic fish continued to express high levels of gfp in skeletal muscle at 1.5 months, demonstrating that GFP is an effective marker of muscle cells in vivo.

Animals↗
PubMed1996

Proliferation precedes differentiation in IGF-I-stimulated myogenesis.

The insulin-like growth factors (IGFs) have dramatic and complex effects on the growth of many tissues and have been implicated in both the proliferation and differentiation of skeletal muscle cells. A detailed analysis of gene expression was performed in L6E9 myoblast cultures treated with IGF-I to dissect the early events leading to the stimulation of myogenic differentiation by this growth factor. A time course of transcript accumulation in confluent L6E9 myoblasts treated with defined media containing IGF-I revealed an initial transient decrease in myogenic factors, accompanied by an increase in cell cycle markers and cell proliferation. This pattern was reversed at later time points, when the subsequent activation of myogenic factors resulted in a net increase in structural gene expression and larger myotubes. The data presented here support the hypothesis that IGF-I activates proliferation first, and subsequently stimulates events leading to the expression of muscle-specific genes in myogenic cell cultures.

Animals↗
PubMed1996

Distinct gene expression patterns in skeletal and cardiac muscle are dependent on common regulatory sequences in the MLC1/3 locus.

The myosin light-chain 1/3 locus (MLC1/3) is regulated by two promoters and a downstream enhancer element which produce two protein isoforms in fast skeletal muscle at distinct stages of mouse embryogenesis. We have analyzed the expression of transcripts from the internal MLC3 promoter and determined that it is also expressed in the atria of the heart. Expression from the MLC3 promoter in these striated muscle lineages is differentially regulated during development. In transgenic mice, the MLC3 promoter is responsible for cardiac-specific reporter gene expression while the downstream enhancer augments expression in skeletal muscle. Examination of the methylation status of endogenous and transgenic promoter and enhancer elements indicates that the internal promoter is not regulated in a manner similar to that of the MLC1 promoter or the downstream enhancer. A GATA protein consensus sequence in the proximal MLC3 promoter but not the MLC1 promoter binds with high affinity to GATA-4, a cardiac muscle- and gut-specific transcription factor. Mutation of either the MEF2 or GATA motifs in the MLC3 promoter attenuates its activity in both heart and skeletal muscles, demonstrating that MLC3 expression in these two diverse muscle types is dependent on common regulatory elements.

Animals↗
PubMed1995

Activation of a muscle-specific enhancer by the Ski proto-oncogene.

In transgenic mice, muscle-specific expression of the c-ski oncogene induces hypertrophy exclusively in a subset of fast muscle fibers. Here we report that regulatory elements from two genes expressed in fast fibers, myosin light chain 1/3 (MLC) and muscle creatine kinase (MCK), were activated when co-transfected with c-ski expression vectors in myoblasts. The expression from the MLC enhancer was reduced when the c-ski oncogene was cotransfected with MyoD into NIH3T3 fibroblasts. Activation of the MLC enhancer by Ski also occurred in vivo, since bigenic progeny generated by mating MLC-CAT and MSV-skitransgenic mice displayed higher CAT activity in their muscles than did the MLC-CAT parental line. Identification of gene targets for the fiber-specific action of the c-ski gene product provides a molecular model that could be used for the further dissection of Ski-induced hypertrophy, both in tissue culture and in vivo.

3T3 Cells↗
PubMed1995

Regulation of a muscle-specific transgene by retinoic acid.

Retinoic acid (RA) has been shown to have variable effects on myogenic differentiation in cell culture. The application of RA on primary cultures of embryonic somites, limb buds, and neonatal limbs inhibited myogenic differentiation in a dose-dependent way as indicated by the repression of: (a) myotube formation, (b) myosin heavy chain protein accumulation, (c) myosin light chain (MLC) 1/3, alpha sk-actin and myogenic factor transcript expression. Expression of retinoic acid receptors (RAR) was also affected by RA treatment, specifically RAR gamma transcripts were induced. To further understand the pleiotropic action of RA on myogenesis, we took advantage of two muscle-specific transgene markers which consisted of CAT reporter genes driven by regulatory elements either from the myosin light chain 1/3 locus (MLC-CAT) or the alpha-skeletal actin gene (alpha sk actin-CAT). RA inhibited MLC-CAT transgene but not alpha sk actin-CAT transgene expression in primary cultures from these mice. Analysis of MLC-CAT expression in transgenic mouse primary cultures and in stably transfected C2C12 cells demonstrated that repression of MLC-CAT activity by RA was dependent upon diffusible factors in chick embryo extract. We hypothesize that during development, the pleiotropic effects of RA on myogenesis do not depend solely on the distribution and concentration of RA itself, but are also influenced by extracellular signals in the embryonic environment.

Actins↗
PubMed1995

Role of methylation in maintenance of positionally restricted transgene expression in developing muscle.

In transgenic mouse embryos, expression of a muscle-specific reporter, consisting of a chloramphenicol acetyltransferase gene linked to regulatory sequences from the rat myosin light chain 1/3 locus (MLC-CAT), is graded in developing axial muscles along the rostrocaudal axis and in cell cultures derived from these muscles. Here we demonstrate that maintenance of positional differences in MLC-CAT transgene expression cannot be attributed to differences in the transcriptional competence of corresponding muscles. Rather, patterns of transgene expression are reflected in the extent of CpG demethylation of both MLC1 promoter and MLC enhancer sequences. Variations in reporter gene expression can be reconstituted by in vitro methylation of specific CpGs in transfected MLC-CAT DNA. As the MLC-CAT transgene is activated during embryogenesis, demethylation of the MLC1 promoter lags behind that of the downstream MLC enhancer, which appears to be the initial target for epigenetic modification. In developing somites, demethylation of the transgenic MLC enhancer is not graded and therefore does not reflect early regional differences in MLC-CAT transgene expression patterns. These studies implicate selective methylation in the maintenance rather than in the establishment of transcriptional differences in developing muscles.

Animals↗
PubMed1993

Alveolar macrophage surface carbohydrate expression is altered in interstitial lung disease as determined by lectin-binding profiles.

Cell-surface-associated glycoconjugates play important roles in cellular functions such as antigen presentation and cell adhesion, functions that may be modulated in patients with interstitial lung disease. Because carbohydrate residues can be recognized by specific lectins, we designed our study to establish baseline data for bronchoalveolar-lavage-derived cells from normal volunteers and to compare the lectin-binding properties of these cells with cells recovered from patients presenting with interstitial lung disease. Cells were obtained from patients with idiopathic pulmonary fibrosis (n = 10), patients with sarcoidosis (n = 20), and patients receiving amiodarone without evidence of clinical lung disease (n = 10) as well as from normal volunteers (n = 8). To determine the pattern of cell-surface glycoconjugate expression on alveolar macrophages (AM), we used a panel of 21 fluorochrome-coupled plant lectins and employed flow cytometry to determine their binding to AM. The labeling profiles of AM were found to be highly reproducible for normal subjects. At the lectin concentrations used for this study, some lectins showed very little binding to AM and some displayed intermediate binding, but the majority of the lectins labeled nearly all AM in samples. Fluorescence intensity varied characteristically for cells labeled with different lectins, providing further refinement and permitting discrimination beyond that provided by data restricted to percent of labeling. AM from patients with interstitial lung disease showed increased binding for the plant-derived lectins PNA, UEA-I, BSL-I, VVL, and SJA compared with AM from normal subjects, being most augmented for AM from patients with idiopathic pulmonary fibrosis. Because peripheral blood monocytes from normal subjects show a higher percentage of labeling with PNA, UEA-I, SJA, and BSL-I than did AM, the increased expression of binding sites for these four lectins by AM from patients with interstitial lung disease may reflect the influx of immature blood monocytes and/or the emergence of a proinflammatory macrophage phenotype. This study demonstrated heterogeneous expression of surface carbohydrate residues by AM and blood monocytes from normal subjects and alterations in carbohydrate receptor expression in interstitial lung disease. Lectin-binding properties may prove useful, therefore, in the evaluation of mononuclear phagocyte populations in interstitial lung disease, especially by the identification of functional subsets and/or changed activation states.

Adolescent↗