Displacement of an E-box-binding repressor by basic helix-loop-helix proteins: implications for B-cell specificity of the immunoglobulin heavy-chain enhancer.

The activity of the immunoglobulin heavy-chain (IgH) enhancer is restricted to B cells, although it binds both B-cell-restricted and ubiquitous transcription factors. Activation of the enhancer in non-B cells upon overexpression of the basic helix-loop-helix (bHLH) protein E2A appears to be mediated not only by the binding of E2A to its cognate E box but also by the resulting displacement of a repressor from that same site. We have identified a "two-handed" zinc finger protein, denoted ZEB, the DNA-binding specificity of which mimics that of the cellular repressor. By employing a derivative E box that binds ZEB but not E2A, we have shown that the repressor is active in B cells and the IgH enhancer is silenced in the absence of binding competition by bHLH proteins. Hence, we propose that a necessary prerequisite of enhancer activity is the B-cell-specific displacement of a ZEB-like repressor by bHLH proteins.

Gender differences in long-term beneficial effects of erythropoietin given after neonatal stroke in postnatal day-7 rats.

Recently, we reported that erythropoietin attenuates neonatal brain injury caused by focal cerebral ischemia. The long-term effects of erythropoietin on focal cerebral ischemia-induced injury to the developing brain and the potential gender differences in these long-term effects have not been studied in detail. The current study demonstrated a similarity in the mean infarct volume in both the vehicle-treated male and female rats at 6 and 12 weeks after focal cerebral ischemia. On the other hand, erythropoietin treatment (1000 U/kg x three doses after focal cerebral ischemia) caused a significant reduction in the mean infarct volume in both males and females at 6 weeks after focal cerebral ischemia when compared with the corresponding vehicle-treated animals (males: 141.4+/-48.2 mm3 vs. 194.0+/-59.2 mm3, P<0.05; females: 85.4+/-31.6 mm3 vs. 183.4+/-46.3 mm3, P<0.05). Interestingly, the reduction in the mean infarct volume in the erythropoietin-treated males was significantly less than that in the erythropoietin-treated females at 6 weeks after focal cerebral ischemia (141.4+/-48.2 mm3 vs. 85.4+/-31.6 mm3, P<0.05). At 12 weeks after focal cerebral ischemia, the mean infarct volume in the erythropoietin-treated males significantly increased to 181.0+/-50.4 mm3 (P<0.05). In contrast, the mean infarct volume in the erythropoietin-treated females remained stable (87.0+/-41.7 mm3). Additionally, erythropoietin treatment significantly improved sensorimotor function recovery with a misstep number similar to the sham-operation group at 6 and 12 weeks after focal cerebral ischemia. Moreover, the mean number of missteps in the erythropoietin-treated females was less than that in males at 6 (13.5+/-2.0 vs. 24.5+/-2.5, P<0.05) and 12 (12.5+/-2.0 vs. 20.0+/-2.0, P<0.05) weeks after focal cerebral ischemia. These results indicate that erythropoietin administration after focal cerebral ischemia produces a significant long-term neuroprotective benefit on the developing brain, and that this effect is more beneficial in the female rats.

Cloning of a cDNA encoding a mouse transcriptional repressor displaying striking sequence conservation across vertebrates.

The nucleotide sequence encoding an approx. 120-kDa transcriptional repressor (MEB1) was determined from a cDNA which was cloned from a mouse brain library. An alignment of the deduced amino-acid sequences of the putative functional domains of MEB1 with those from the human, hamster and chicken homologues reveals a dramatic degree of conservation.

Positive and negative elements mediate control of alternative splicing in the AMPD1 gene.

The second exon of the AMP deaminase (AMPD) 1 gene is alternatively spliced in response to stage-specific signals elaborated during myocyte differentiation. Since inheritance of the mutation in exon 2 of the AMPD1 gene has been recently shown to be associated with a better prognosis of congestive heart failure and the alternative splicing of exon 2 modulates the residual activity of AMPD1 in individuals with this mutant allele, the regulatory mechanism of alternative splicing in the AMPD1 gene is clinically intriguing. Retention or exclusion of exon 2 results from the interplay between negative and positive elements in the primary transcript. Exon 2 is intrinsically defective and difficult to recognize. Herein, we show that this property of exon 2 is the consequence of three defects; a suboptimal 3' splice acceptor site, a suboptimal 5' splice donor site and the small size of the exon. An improvement in any one of these defects relieves the masking of this exon. Further, this defective exon can only be identified in the presence of the adjacent downstream intron.

The regulatory role of NF-κB in autophagy-like cell death after focal cerebral ischemia in mice.

Autophagy may contribute to ischemia-induced cell death in the brain, but the regulation of autophagic cell death is largely unknown. Nuclear factor kappa B (NF-κB) is a regulator of apoptosis in cerebral ischemia. We examined the hypothesis that autophagy-like cell death could contribute to ischemia-induced brain damage and the process was regulated by NF-κB. In adult wild-type (WT) and NF-κB p50 knockout (p50(-/-)) mice, focal ischemia in the barrel cortex was induced by ligation of distal branches of the middle cerebral artery. Twelve to 24h later, autophagic activity increased as indicated by enhanced expression of Beclin-1 and LC3 in the ischemic core and/or penumbra regions. This increased autophagy contributed to cell injury, evidenced by terminal deoxynucleotidyltransferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) co-staining and a protective effect achieved by the autophagy inhibitor 3-methyladenine. The number of Beclin-1/TUNEL-positive cells was significantly more in p50(-/-) mice than in WT mice. Neuronal and vascular cell death, as determined by TUNEL-positive cells co-staining with NeuN or Collagen IV, was more abundant in p50(-/-) mice. Immunostaining of the endothelial cell tight junction marker occludin revealed more damage to the blood-brain barrier in p50(-/-) mice. Western blotting of the peri-infarct tissue showed a reduction of Akt-the mammalian target of rapamycin (mTOR) signaling in p50(-/-) mice after ischemia. These findings provide the first evidence that cerebral ischemia induced autophagy-like injury is regulated by the NF-κB pathway, which may suggest potential treatments for ischemic stroke.

Tissue-specific gene activation by MyoD: determination of specificity by cis-acting repression elements.

MyoD is a muscle-specific transcriptional activator; E12 is a B-cell activator. An IgH enhancer is activated almost 100-fold by E12 but not at all by Myo; an MCK enhancer is activated almost 1000-fold by MyoD and not at all by E12. MyoD and E12 are both basic helix-loop-helix proteins that bind to similar E-box sequences (CANNTG); the IgH enhancer contains the same E boxes as the MCK enhancer, yet each retains exclusive specificity for either E12 or MyoD, respectively. We show that the IgH enhancer contains a cis-acting negative element that is directed at MyoD, but not at E12. This repression requires the mu E5 E box within the IgH enhancer; however, the specificity for repression, as opposed to activation, is associated with 2 bp flanking each side of the mu E5 E box. The target for repression of MyoD in the IgH enhancer is the bHLH region of MyoD. Our results suggest that MyoD only activates myogenic genes because nonmuscle enhancers that contain E boxes also contain negative elements that prevent MyoD activity.

ABCIXIMAB: a new antiaggregant used in angioplasty.

OBJECTIVE: To review the scientific literature on the pharmacology and clinical uses of abciximab. DATA SOURCES: MEDLINE, Index Medicus, and bibliographic literature searches of English-language articles pertaining to abciximab, 7E3, m7E3, and c7E3 were performed. Eli Lilly also provided unpublished results of the Evaluation of 7E3 for the Prevention of Ischemic Complications trial. DATA SELECTION: The selection of data presented focused on controlled trials using abciximab at doses currently approved by the Food and Drug Administration. DATA SYNTHESIS: Abciximab is a humanized chimeric Fab fragment of 7E3. 7E3 is a murine antibody directed against the integrin glycoprotein IIb)/IIIa receptor (GPIIb/IIIa) located on platelets. These receptors play an integral part in platelet aggregation by allowing fibrinogen to bind to them and interconnect platelets. When administered intravenously, abciximab binds to GPIIb/IIIa and hinders platelet aggregation. Bleeding times and activated clotting times are increased and the platelets' response to adenosine diphosphate is reduced with the use of abciximab. Clinical trials have indicated that abciximab can reduce the incidence of abrupt closure and restenosis associated with percutaneous transluminal coronary angioplasty (PTCA) performed in high-risk patients. Clinical trials also suggest that abciximab may have a role in the treatment of unstable angina and the acute therapy of myocardial infarctions. Complications associated with abciximab include bleeding and thrombocytopenia. The thrombocytopenia is likely related to immunologic mechanisms. In addition, the production of antimurine antibodies has been demonstrated with abciximab use. Abciximab is currently approved for the prevention of abrupt coronary closure associated with PTCA in patients at high risk for this event. CONCLUSIONS: Abciximab is effective in preventing platelet aggregation and has been proven to be of clinical benefit in selected high-risk patients receiving PTCA.

A novel bipartite intronic splicing enhancer promotes the inclusion of a mini-exon in the AMP deaminase 1 gene.

Alternative splicing of the 12-base exon 2 of the adenosine monophosphate deaminase (AMPD) gene is subject to regulation by both cis- and trans-regulatory signals. The extent of exon 2 inclusion is stage- and cell type-specific and is subject to the physiological state of the cell. In adult skeletal muscle, a cell type that regulates the activity of this allosteric enzyme at several levels, the exon 2-plus form of AMPD, predominates. We have performed a systematic analysis of the cis-acting regulatory sequences that reside in the intron immediately downstream of this mini-exon. A complex element comprising sequences that enhance exon 2 inclusion and sequences that counteract this effect resides in the middle of this intron. We demonstrate that the enhancing component is bipartite, with more than a kilobase of sequence separating the two functional sites. The presence of even minimal levels the mini-exon in the fully processed AMPD mRNA requires both of these sites, neither of which appears in any other published splicing enhancer. An RNA binding activity derived from a muscle cell line requires both of the enhancing sites. Mutations in either of the sites that eliminate exon 2 inclusion abrogate this binding activity.

Plasma protein and liver mRNA levels of two closely related murine alpha 1-protease inhibitors during the acute phase reaction.

Plasma levels of alpha 1-PI(T) and alpha 1-PI(E), two closely related murine alpha 1-protease inhibitors, having affinities for trypsin and elastase, respectively, were compared to changes in specific liver mRNA levels after induction of the acute-phase reaction by subcutaneous injection of turpentine. In earlier, qualitative experiments an increase in plasma levels of alpha 1-PI(E), but not alpha 1-PI(T), during the acute-phase reaction had been shown. It is now shown that stimulation of plasma alpha 1-PI(E) levels reaches a maximum of 35-50% above baseline 12 h after induction of the acute-phase response using either a functional or immunological assay to measure protease inhibitor activity. Consistent with earlier observations, little or no change in plasma levels of alpha 1-PI(T) is seen. Determination of mRNA levels in the mouse liver specific for alpha 1-PI(E) and alpha 1-PI(T) was accomplished using a cell-free translation system followed by immunoprecipitation of the 35S-labeled protease inhibitors. The apparent Mr's of alpha 1-PI(E) and alpha 1-PI(T) synthesized in vitro are 42K and 46K, respectively. Apparent Mr's of the native proteins in plasma are 55K and 65K. Unexpectedly, mRNA levels for both alpha 1-PI(E) and alpha 1-PI(T) were found to increase after induction of the acute-phase reaction. Maximal stimulation for both mRNAs was approximately 300% and occurred 9 h after turpentine administration. Under these conditions, levels of translatable albumin mRNA in the mouse liver decreased to 40% of baseline in 6-9 h.

cDNA cloning of esterase 1, the major esterase activity in mouse plasma.

We report here the cloning of a partial cDNA for Esterase 1, the major esterase activity in mouse plasma. A 470 base pair insert was isolated from a lambda gt11 cDNA library constructed from mouse liver poly A+ RNA, and identified by hybrid selected translation. We show that the sexual dimorphism displayed in the plasma levels of this protein is caused by a difference at the level of transcription. In addition, RFLP data using mouse recombinant inbred strains mapped this clone at the Es-1 locus on mouse chromosome 8.

Transcriptional repression of the IL-2 gene in Th cells by ZEB.

Th1- and Th2-type cells mediate distinct effector functions via cytokine secretion in response to immunologic challenge. Precursor Th cells transcribe IFN-gamma, IL-2, and IL-4 upon activation. Repeated stimulation of Th precursor cells in the presence of IL-4 leads to terminally differentiated Th2 cells that have lost the ability to transcribe the IL-2 gene. We provide evidence that repression of IL-2 gene expression in Th2 cells and partial repression in Th1 cells are mediated by ZEB, a zinc finger, E box-binding transcription factor. This factor binds to a negative regulatory element, NRE-A, in the IL-2 promoter, thereby acting as a potent repressor of IL-2 transcription.