The cloning and expression of myogenin, a gene regulating myogenesis

Expression of the differentiated skeletal muscle phenotype is a process that appears to occur in at least two stages. First, pluripotent stem cells become committed to the myogenic lineage. Although undifferentiated and capable of continued proliferation, determined myoblasts are restricted to a single developmental fate. Upon receiving the appropriate environmental signals, these determined myoblasts withdraw from the cell cycle, fuse to form multi-nucleated myotubes, and begin to express a battery of muscle-specific gene products that make up the functional and contractile apparatus of the muscle. This project is aimed at the identification and characterization of factors that control the determination and differentiation of myogenic cells. We have cloned a cDNA, called myogenin, that plays an important role in these processes. Myogenin is expressed exclusively in skeletal muscle in vivo and myogenic cell lines in vitro. Its expression is sharply upregulated during differentiation. When constitutively expressed in fibroblasts, myogenin converts these cells to the myogenic lineage. Transfected cells behave as myogenic tissue culture cells with respect to the genes they express, the way they respond to environmental cues, and are capable of fusing to form multinucleated myotubes. Sequence analysis showed that this cDNA has homology to a family of transcription factors in a region of 72 amino acids known as the basic helix-loop-helix motif. This domain appears to mediate binding to a DNA sequence element known as an E-box (CANNTG) essential for the activity of the enhancers of many muscle-specific genes.^ Analysis of myogenin in tissue culture cells showed that its expression is responsive to many of the environmental cues, such as the presence of growth factors and oncogenes, that modulate myogenesis. In an attempt to identify the cis- and trans-elements that control myogenin expression and thereby understand what factors are responsible for the establishment of the myogenic lineage, we have cloned the myogenin gene. After analysis of the gene structure, we constructed a series of reporter constructs from the 5$\prime$ upstream sequence of the myogenin gene to determine which cis-acting sequences might be important in myogenin regulation. We found that 184 nucleotides of the 5$\prime$ sequence was sufficient to direct high-level muscle-specific expression of the reporter gene. Two sequence elements present in the 184 fragment, an E-box and a MEF-2 site, have been shown previously to be important in muscle-specific transcription. Mutagenesis of these sites revealed that both sites are necessary for full activity of the myogenin promoter, and suggests that a complex hierarchy of transcription factors control myogenic differentiation. ^

Mechanism of dendritic epidermal T cell-mediated tolerance induction and inhibition of proliferation

Dendritic epidermal T cells (DETC) comprise a unique population of T cells that reside in mouse epidermis and whose function remains unclear. Most DETC express a $\gamma\delta$ TCR, although some, including our DETC line, AU16, express an $\alpha\beta$ TCR. Additionally, AU16 cells express CD3, Thy-1, CD45, CD28, B7, and AsGM-1. Previous studies in our laboratory demonstrated that hapten-conjugated AU16 could induce specific immunologic tolerance in vivo and inhibit T cell proliferation in vitro. Both these activities are antigen-specific, and the induction of tolerance is non-MHC-restricted. In addition, AU16 cells are cytotoxic to a number of tumor cell lines in vitro. These studies suggested a role for these cells in immune surveillance. The purpose of my studies was to test the hypothesis that these functions of DETC (tolerance induction, inhibition of T cell proliferation, and tumor cell killing) were mediated by a cytotoxic mechanism. My specific aims were (1) to determine whether AU16 could prevent or delay tumor growth in vivo; and (2) to determine the mechanism whereby AU16 induce tolerance, using an in vitro proliferation assay. I first showed that AU16 cells killed a variety of skin tumor cell lines in vitro. I then demonstrated that they prevented melanoma growth in C3H mice when both cell types were mixed immediately prior to intradermal (i.d.) injection. Studies using the in vitro proliferation assay confirmed that DETC inhibit proliferation of T cells stimulated by hapten-bearing, antigen-presenting cells (FITC-APC). To determine which cell was the target, $\gamma$-irradiated, hapten-conjugated AU16 were added to the proliferation assay on d 4. They profoundly inhibited the proliferation of naive T cells to $\gamma$-irradiated, FITC-APC, as measured by ($\sp3$H) TdR uptake. This result strongly suggested that the T cell was the target of the AU16 activity because no APC were present by d 4 of the in vitro culture. In contrast, the addition of FITC-conjugated splenic T cells (SP-T) or lymph node T cells (LN-T) was less inhibitory. Preincubation of the T cells with FITC-AU16 cells for 24 h, followed by removal of the AU16 cells, completely inhibited the ability of the T cells to proliferate in response to FITC-APC, further supporting the conclusion that the T cell was the target of the AU16. Finally, AU16 cells were capable of killing a variety of activated T cells and T cell lines, arguing that the mechanism of proliferation inhibition, and possibly tolerance induction is one of cytotoxicity. Importantly, $\gamma\delta$ TCR$\sp+$ DETC behaved, both in vivo and in vitro like AU16, whereas other T cells did not. Therefore, these results are consistent with the hypothesis that AU16 cells are true DETC and that they induce tolerance by killing T cells that are antigen-activated in vivo. ^

Regulation of angiogenesis by interferon-beta

The purpose of these studies was to investigate the role of interferon-beta (IFN-$\beta$) in angiogenesis. IFN-$\alpha/\beta$ have been implicated in inhibiting a number of steps in the angiogenic pathway. We examined the balance of angiogenesis-regulating molecules in several systems including human infantile hemangiomas, UV-B irradiated mice, and dorsal incisional wound healing in mice. In each system, epidermal hyperplasia and cutaneous angiogenesis were directly related to the expression of positive angiogenic factors (bFGF and VEGF) and inversely related to the expression of endogenous IFN-$\beta.$ The re-expression of IFN-$\beta$ correlated with tumor regression and/or resolution of wound healing. In contrast to control mice, UV-B-induced cutaneous angiogenesis and hyperplasia persisted in IFN-$\alpha/\beta$ receptor knock-out mice. In normal mice, endogenous IFN-$\beta$ was expressed by all differentiated epithelial cells exposed to environmental stimuli. The expression of endogenous IFN-$\beta$ was necessary but insufficient for complete differentiation of epidermal keratinocytes.^ The tumor organ microenvironment can regulate angiogenesis. Human bladder carcinoma cells growing in the bladder wall of nude mice express high levels of bFGF, VEGF, and MMP-9, have higher vascular densities, and produce metastases to lymph nodes and lungs, whereas the same cells growing subcutaneously express less bFGF, VEGF, and MMP-9, have lower vascular densities, and do not metastasize. IFN-$\alpha/\beta$ was found to inhibit bFGF and MMP-9 expression both in vitro and in vivo in human bladder carcinoma cells. Systemic therapy with human IFN-$\alpha$ of human bladder cancer cells growing orthotopically in nude mice, resulted in decreased vascularity, tumorigenicity, and metastasis as compared to saline treated mice. Human bladder cancer cells resistant to the antiproliferative effects of IFN were transfected with the human IFN-$\beta$ gene. Hu-IFN-$\beta$ transfected cells expressed significantly less bFGF protein and gelatinase activity than parental or control-transfected cells and did not grow at ectopic or orthotopic sites. Collectively the data provide direct evidence that IFN-$\alpha/\beta$ can inhibit angiogenesis via down-regulation of angiogenesis-stimulating cytokines. ^

The pain experience of elderly hospice patients with cancer

Purpose/objectives. A grounded theory design was used to identify, describe, and generate a theoretical analysis of the pain experience of elderly hospice patients with cancer. ^ Sample. Eleven participants over the age of 65, receiving services from a for-profit hospice were interviewed in their homes. ^ Methods. Broad unstructured face to face audio-taped interviews were transcribed verbatim and analyzed using constant-comparative method of analysis. ^ Findings. Pain was described as a hierarchy of chronic, acute, and psychological pain with psychological pain as the worst. Suffering was the basic social problem of pain. Participants dealt with suffering by the basic social process of enduring. Enduring had two sub-processes, maintaining hope and adjusting. Trusting in a higher being and finding meaning were mechanisms of maintaining hope. Mechanisms of adjusting were dealing with uncertainty, accepting, and minimizing pain. ^ Implications for nursing practice. Nurses need to recognize and value the hard work of enduring to deal with suffering. Assisting elderly hospice patients with cancer to address the sub-processes of enduring and their mechanisms can foster enduring. ^

AN EPIDEMIOLOGIC CASE-CONTROL ANALYSIS OF CHILDHOOD INTRACRANIAL AND SPINAL CORD TUMORS IN RELATION TO PATERNAL OCCUPATION AT BIRTH (BRAIN, CANCER)

Epidemiologic case-control studies of small groups of childhood nervous system tumor patients have suggested that parental employment in occupations with exposure to hydrocarbons is a risk factor for disease. The main focus of this case-control study was to assess the paternal occupation at the time of birth of offspring who later developed childhood intracranial and spinal tumors. All children under 15 years of age dying of such tumors in Texas, during the period 1964-1980, were selected as cases. Disease and demographic data were abstracted from death certificates. The birth certificate for each child of the final group of 499 cases was located and parental occupation information, as well as demographic and obstetric data, were collected. The comparison group consisted of a random sample from all Texas live births with the same birth year, race and sex distribution as the cases.^ The paternal occupations were categorized into broad classifications of those involving hydrocarbon exposure versus those that did not, based on the occupation criteria used in the previous studies. Odds ratios did not indicate any increased risk associated with general paternal hydrocarbon exposure in the workplace. In prior studies, increased risk estimates were detected with narrower groups of occupations involving exposure to hydrocarbon materials. The data from this study were classified according to these groups, and again, no increased risks were indicated except for a statistically insignificant but elevated odds ratio for fathers who were paper and pulp mill workers.^ Odds ratios were calculated for specific occupations and industries previously implicated as risk factors. Significantly associated odds ratios (OR) were detected for electricians (OR = 3.5), especially those working for construction companies (OR = 10.0), for employment in the printing occupations (OR = 4.5), particularly graphic arts workers (OR = 21.9), and in the electronics and electronic machinery industries (OR = 3.5). Analysis of the petroleum refining and chemical industries, which were not found in previous study populations, revealed significantly elevated odds ratios of 3.0 for occupations with probable heavy exposure to chemicals and petroleum compounds and 10.0 for salesmen of chemical products. ^

THE TRANSLATION PRODUCTS OF MOLONEY MURINE SARCOMA VIRUS 124 GENOMIC RNA

Murine sarcoma viruses constitute a class of replication-defective retroviruses. Cellular transformation may be induced by these viruses in vitro; whereas, fibrosarcomas may result in animals infected with them in vivo (Tooze, 1973; Bishop, 1978). Hybridization studies suggest that murine sarcoma viruses arose by recombination between nondefective murine leukemia virus sequences and certain cellular sequences present in uninfected mouse cells (Hu et al., 1977). A specific gene product, however, has not been implicated in murine sarcoma virus transformation.^ One line of murine sarcoma virus-producing cells, Mo-MuSV-clone 124, (Ball et al., 1973), was studied biochemically because it mainly produces the sarcoma virus as a pseudotype packaged with helper murine leukemia virus proteins. The sarcoma viral RNA was translated in a sophisticated cell-free protein synthesizing system (Murphy and Arlinghaus, 1978). The translation products were analyzed by a number of techniques, including electrophoresis in denaturing gels of SDS polyacrylamide, immunoprecipitation, and peptide mapping. The major products of the total RNA purified from the virus preparation were shown to have molecular weights of about 63,000 (P63('gag)), 42,000 (P42), 40,000 (P40), 38,000 (P38), and 23,000 (P23). The size class of mRNA coding for each of the cell-free products was estimated using a poly(A) selection technique and sucrose gradient fractionation. These analyses were used to localize the coding information related to each of the in vitro synthesized cell-free products within the sarcoma virus genome.^ The major findings of these studies were: (1) the 5' half of the sarcoma viral RNA codes for the 63,000 dalton polypeptide and 42,000 - 38,000 dalton polypeptides derived from the "gag" gene; and (2) the 3' half of the sarcoma viral RNA codes for a 38,000 dalton polypeptide and possibly derived from the cellular acquired sequences. ^

An die hohe Stände-Versammlung des Königreichs Bayern : gehorsamste Beschwerde des Distrikt-Rabbiners

[Samson Wolf] Rosenfeld

Mémoire an die hohe Stände-Versammlung des Königreichs Baiern : über verschied'ne gegen die Juden gemachte Anträge ; nebst einigen Wünschen u. Bitten

von Samson Wolf Rosenfeld

Feierlicher Act der Religionsprüfung der zu entlassenden Schulkinder : entworfen und abgehalten in der Synagoge zu Carlsruhe am Sabbath den 23. April 1836 (6. Jjar 5596)

von M. Rosenfeld

Der jüdische Religionsunterricht : Reflexionen ; mit bes. Berücks. d. Wiener Schulverhältnisse

von M. Rosenfeld

Der Wiener jüdische Jugend-Gottesdienst und seine Reorganisation : vorangeht ein kurzer Abriß seiner Geschichte ; Referat erstattet d. Konferenz d. isr. Religionslehrer an d. Wiener Mittelschulen am 17. Mai 1917

von M. Rosenfeld

Lieder des Ghetto

von Morris Rosenfeld. Autor. Übertr. aus d. Jüd. von Berthold Feiwel mit Zeichnungen von E. M. Lilien

An ARHGEF10 deletion is highly associated with a juvenile-onset inherited polyneuropathy in Leonberger and Saint Bernard dogs

An inherited polyneuropathy (PN) observed in Leonberger dogs has clinical similarities to a genetically heterogeneous group of peripheral neuropathies termed Charcot-Marie-Tooth (CMT) disease in humans. The Leonberger disorder is a severe, juvenile-onset, chronic, progressive, and mixed PN, characterized by exercise intolerance, gait abnormalities and muscle atrophy of the pelvic limbs, as well as inspiratory stridor and dyspnea. We mapped a PN locus in Leonbergers to a 250 kb region on canine chromosome 16 (Praw = 1.16×10-10, Pgenome, corrected = 0.006) utilizing a high-density SNP array. Within this interval is the ARHGEF10 gene, a member of the rho family of GTPases known to be involved in neuronal growth and axonal migration, and implicated in human hypomyelination. ARHGEF10 sequencing identified a 10 bp deletion in affected dogs that removes four nucleotides from the 3'-end of exon 17 and six nucleotides from the 5'-end of intron 17 (c.1955_1958+6delCACGGTGAGC). This eliminates the 3'-splice junction of exon 17, creates an alternate splice site immediately downstream in which the processed mRNA contains a frame shift, and generates a premature stop codon predicted to truncate approximately 50% of the protein. Homozygosity for the deletion was highly associated with the severe juvenile-onset PN phenotype in both Leonberger and Saint Bernard dogs. The overall clinical picture of PN in these breeds, and the effects of sex and heterozygosity of the ARHGEF10 deletion, are less clear due to the likely presence of other forms of PN with variable ages of onset and severity of clinical signs. This is the first documented severe polyneuropathy associated with a mutation in ARHGEF10 in any species.

Deutschtum und Judentum

Hans Rosenfeld