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L Fromm

Publications and source records attributed to L Fromm.

10 recordsLinked to original sources
PubMed2001

Neuregulin-1-stimulated phosphorylation of GABP in skeletal muscle cells.

Localization of acetylcholine receptors (AChRs) to neuromuscular synapses is mediated, in part, through selective transcription of AChR genes in myofiber synaptic nuclei. Neuregulin-1 (NRG-1) is a good candidate for the extracellular signal that induces synapse-specific gene expression, since NRG-1 is concentrated at synaptic sites and activates AChR synthesis in cultured muscle cells. NRG-1-induced transcription requires activation of Erk and Jnk MAP kinases, but the downstream substrates that mediate this transcriptional response are not known. Previous studies have demonstrated that a consensus binding site for Ets proteins is required both for NRG-1-induced transcription and for synapse-specific transcription in transgenic mice. This regulatory element binds GABPalpha, an Ets protein, and GABPbeta, a protein that dimerizes with GABPalpha, raising the possibility that phosphorylation of GABP by MAP kinases induces transcription of AChR genes. To determine whether MAP kinases might directly regulate the activity of GABP, we studied MAP kinase-catalyzed and NRG-1-induced phosphorylation of GABPalpha and GABPbeta. We show that GABPalpha and GABPbeta are phosphorylated in vitro by Erk and by Jnk. Using recombinant proteins containing mutated serine and threonine resides, we show that GABPalpha is phosphorylated predominantly at threonine 280, while serine 170 and threonine 180 are the major phosphorylation sites in GABPbeta. We generated antibodies specific to the major phosphorylation site in GABPalpha and show that NRG-1 stimulates phosphorylation of GABPalpha at threonine 280 in vivo. These results suggest that GABPalpha is a target of MAP kinases in NRG-1-stimulated muscle cells and are consistent with the idea that phosphorylation of GABPalpha contributes to transcriptional activation of AChR genes by NRG-1.

Animals↗
PubMed2000

Induction of cell cycle entry and cell death in postmitotic lens fiber cells by overexpression of E2F1 or E2F2.

PURPOSE: Previous studies have shown that inactivation of the retinoblastoma tumor suppressor protein (pRb) can cause lens fiber cell proliferation and apoptosis. Because pRb is thought to block cell cycle progression by inhibition of E2F transcription factors, experiments were conducted to test whether overexpression of different E2F family members would be sufficient to induce fiber cell proliferation and subsequent apoptosis. The in vivo functions of the transcription factor E2F2 have not previously been analyzed or described in transgenic mice. METHODS: Human E2F1 and E2F2 cDNAs were linked to the alphaA-crystallin promoter. Transgenic mice were generated by microinjection. Changes in cell cycle regulation were assayed by immunohistochemistry for 5-bromo-2'-deoxyuridine (BrdU) incorporation and by in situ hybridization. Cell death was assayed using the TdT-dUTP terminal nick-end labeling (TUNEL) assay. RESULTS: At embryonic day (E)15.5, strong expression of the E2F1 and E2F2 transgenes was detected in lens fiber cells with little or no expression in epithelial cells. BrdU incorporation and TUNEL assays showed that overexpression of either E2F1 or E2F2 in lens fiber cells was sufficient to cause cell cycle entry and subsequent apoptosis. Expression of either E2F1 or E2F2 was sufficient to induce the transcription of cyclins (A2, B1, and E), as well as p53 and Bax in the lens fibercells. CONCLUSIONS: Expression of either E2F1 or E2F2 can induce postmitotic lens fiber cells to re-enter the cell cycle. Inappropriate cell cycle entry is recognized by p53 in each case, and programmed cell death ensues.

Animals↗
PubMed1998

Synapse-specific and neuregulin-induced transcription require an ets site that binds GABPalpha/GABPbeta.

Localization of acetylcholine receptors (AChRs) to neuromuscular synapses is mediated by multiple pathways. Agrin, which is the signal for one pathway, stimulates a redistribution of previously unlocalized AChRs to synaptic sites. The signal for a second pathway is not known, but this signal stimulates selective transcription of AChR genes in myofiber nuclei located near the synaptic site. Neuregulin (NRG) is a good candidate for the extracellular signal that induces synapse-specific gene expression, since NRG is concentrated at synaptic sites and activates AChR gene expression in cultured muscle cells. Previous studies have demonstrated that 181 bp of 5' flanking DNA from the AChR delta-subunit gene are sufficient to confer synapse-specific transcription in transgenic mice and NRG responsiveness in cultured muscle cells, but the critical sequences within this cis-acting regulatory region have not been identified. We transfected AChR delta-subunit-hGH gene fusions into a muscle cell line, and we show that a potential binding site for Ets proteins is required for NRG-induced gene expression. Furthermore, we produced transgenic mice carrying AChR delta-subunit-hGH gene fusions with a mutation in this NRG-response element (NRE), and we show that this NRE is necessary for synapse-specific transcription in mice. The NRE binds proteins in myotube nuclear extracts, and nucleotides that are important for NRG responsiveness are likewise critical for formation of the protein-DNA complex. This complex contains GABPalpha, an Ets protein, and GABPbeta, a protein that lacks an Ets domain but dimerizes with GABPalpha, because formation of the protein-DNA complex is inhibited by antibodies to either GABPalpha or GABPbeta. These results demonstrate that synapse-specific and NRG-induced gene expression require an Ets-binding site and suggest that GABPalpha/GABPbeta mediates the transcriptional response of the AChR delta-subunit gene to synaptic signals, including NRG.

Animals↗
PubMed1998

A case series of hospitalized patients with elevated digoxin levels.

PURPOSE: Although there is renewed enthusiasm for the use of digoxin in patients with heart failure, current dosing guidelines are based on a nomogram published in 1974. We studied the incidence of and risk factors for elevated digoxin levels in patients admitted to a community hospital, and compared their dosage regimens to published guidelines. SUBJECTS AND METHODS: We reviewed the charts of all patients who had serum digoxin levels greater than 2.4 ng/mL during a 6-month period. We collected demographic and clinical data, indications for digoxin use, digoxin dosage, concurrent medications, laboratory data, and clinical and electrocardiographic features of digoxin toxicity. RESULTS: Of the 1,433 patients with digoxin assays, 115 (8%) patients had elevated levels. Of the 82 patients with complete records and correctly timed digoxin levels, 59 (72%) had electrocardiographic or clinical features of digoxin toxicity. Patients with serum digoxin levels >2.4 ng/mL were slightly older (78 +/- 8 versus 73 +/- 9 years of age; P = 0.12) and had greater serum creatinine levels (3.1 +/- 7.3 versus 1.4 +/- 0.3 mg/dL; P = 0.01) than those with levels < or =2.4 ng/mL. Forty-seven patients had elevated digoxin levels on admission, including 21 patients admitted for digoxin toxicity. Impaired or worsening renal function contributed to high levels in 37 patients, and a drug interaction was a contributory factor in 10 cases. Twenty (43%) of these patients were taking the recommended maintenance dose based on the scheme employed in the Digitalis Investigation Group study. Thirty-five patients developed high digoxin levels while in hospital. In 26 patients, this followed a loading dose of digoxin for the control of rapid atrial fibrillation. Impaired renal function was implicated in all of these patients. Despite the elevated digoxin level, rate control was achieved in only 11 patients of these patients. CONCLUSIONS: Elevated digoxin levels and clinical toxicity remains a common adverse drug reaction. Elderly patients, particularly those with impaired renal function and low body weights, are at the greatest risk. As published digoxin nomograms often result in toxicity, clinical variables need to be monitored. In patients with congestive heart failure and normal sinus rhythm the potential benefit of digoxin is small; thus, patients should receive a dose that minimizes the risk of toxicity. For patients with new onset atrial fibrillation, other agents may be preferable for rate control.

Age Factors↗
PubMed1997

Inhibition of cell death by lens-specific overexpression of bcl-2 in transgenic mice.

Previous studies on cell cycle regulation in the ocular lens using transgenic mice have shown that inactivation of the retinoblastoma tumor suppressor protein (pRb) can cause postmitotic lens fiber cells to enter the cell cycle. However, when the p53 gene and protein are intact, inactivation of pRb in this terminally differentiated cell type results in cell death, rather than continued proliferation. Since bcl-2 has been shown to act as a cell death repressor, the ability of this gene to block p53-dependent apoptosis in lenses was examined. Transgenic mice were generated that overexpress bcl-2 in a lens-specific fashion. Surprisingly, overexpression of bcl-2 was sufficient to interfere with normal fiber cell differentiation, inducing cataracts, microphakia, vacuolization, fiber cell disorganization, and inhibition of fiber cell denucleation. The bcl-2 mice were mated to mice exhibiting lens-specific expression of the N-terminal region of simian virus 40 large T antigen (termed truncT). The resulting double transgenic mice showed a marked reduction in the truncT-induced fiber cell death. Apoptosis in the truncT mice could also be suppressed by crossing these mice into a p53-deficient background. Either overexpression of bcl-2 or loss of p53 in truncT mice resulted in proliferation of fiber cells around the cortex of the lens. These proliferating fiber cells continue to express beta- and gamma-crystallin proteins, which are normally only expressed following withdrawal from the cell cycle. The p53 protein is known to upregulate expression of certain target genes, including p21, a protein that can block cell cycle progression by inhibition of cyclin-dependent kinases. In order to assess whether bcl-2 interferes with the transcriptional activation activity of p53, transgenic lenses were assayed by in situ hybridization for levels of p21 expression. Lenses that expressed both truncT and bcl-2 showed elevated p21, implying that bcl-2 does not inhibit apoptosis by directly inhibiting p53, but instead may block a later step in the apoptosis pathway. In addition, overexpression of p21 is not sufficient to cause apoptosis. These experiments show that the lenses of transgenic mice represent a valuable in vivo setting for studies of both induction and inhibition of programmed cell death.

Animals↗
PubMed1996

Regulation of cyclin and cyclin-dependent kinase gene expression during lens differentiation requires the retinoblastoma protein.

The retinoblastoma protein (pRb) functions as a negative regulator of the cell cycle and is essential to maintain certain cell types in a post-mitotic state during terminal differentiation. In the ocular lens, inactivation of this protein is sufficient to cause lens fiber cells, which are normally post-mitotic, to enter the cell cycle. The current studies address whether regulation of the cell cycle during lens fiber differentiation in normal lenses or in lenses in which pRB has been inactivated is accompanied by changes in expression of cyclin and cyclin-dependent kinase genes. In the normal lens, our experiments using in-situ hybridization reveal that the expression of cyclin A, cyclin B1, cdc2 and cdk2 is restricted to the proliferative epithelial cells, with no expression in the differentiating fiber cells. Cyclins D1 and D2 and cdk4 show a less restrictive pattern and are expressed in some of the post-mitotic cells. Lenses from RB-deficient embryos, in contrast, show inappropriate expression in the fiber cells of cyclins A, B1 and E, as well as cdc2 and cdk2. The lens fiber cells in these embryos express protein markers for differentiation, such as beta- and gamma-crystallins, even though the cells do not withdraw from the cell cycle. These results indicate that the regulated expression of multiple cell cycle regulatory genes during lens fiber cell differentiation requires the presence of pRb.

Animals↗
PubMed1994

The retinoblastoma protein-binding region of simian virus 40 large T antigen alters cell cycle regulation in lenses of transgenic mice.

Regulation of the cell cycle is a critical aspect of cellular proliferation, differentiation, and transformation. In many cell types, the differentiation process is accompanied by a loss of proliferative capability, so that terminally differentiated cells become postmitotic and no longer progress through the cell cycle. In the experiments described here, the ocular lens has been used as a system to examine the role of the retinoblastoma protein (pRb) family in regulation of the cell cycle during differentiation. The ocular lens is an ideal system for such studies, since it is composed of just two cell types: epithelial cells, which are capable of proliferation, and fiber cells, which are postmitotic. In order to inactivate pRb in viable mice, genes encoding either a truncated version of simian virus 40 large T antigen or the E7 protein of human papillomavirus were expressed in a lens-specific fashion in transgenic mice. Lens fiber cells in the transgenic mice were found to incorporate bromodeoxyuridine, implying inappropriate entry into the cell cycle. Surprisingly, the lens fiber cells did not proliferate as tumor cells but instead underwent programmed cell death, resulting in lens ablation and microphthalmia. Analogous lens alterations did not occur in mice expressing a modified version of the truncated T antigen that was mutated in the binding domain for the pRb family. These experimental results indicate that the retinoblastoma protein family plays a crucial role in blocking cell cycle progression and maintaining terminal differentiation in lens fiber cells. Apoptotic cell death ensues when fiber cells are induced to remain in or reenter the cell cycle.

Animals↗
PubMedDate not supplied

Transcriptional pathways for synapse-specific, neuregulin-induced and electrical activity-dependent transcription.

Innervation-dependent expression of acetylcholine receptor (AChR) genes in skeletal muscle is mediated by multiple transcriptional pathways. One pathway leads to activation of AChR genes selectively in synaptic nuclei and requires an Ets binding site that binds GABP. A second pathway leads to repression of AChR transcription in nuclei throughout the myofiber and requires inactivation of E-box-binding proteins, including myogenic bHLH proteins. Taken together, these studies indicate that separate pathways regulate innervation-dependent transcription.

Animals↗