We disrupted the FOG-2 gene in mice to define its requirement in vivo. FOG-2(-/-) embryos die at midgestation with a cardiac defect characterized by a thin ventricular myocardium, common atrioventricular canal, and the tetralogy of Fallot malformation. Remarkably, coronary vasculature is absent in FOG-2(-/-) hearts. Despite formation of an intact epicardial layer and expression of epicardium-specific genes, markers of cardiac vessel development (ICAM-2 and FLK-1) are not detected, indicative of failure to activate their expression and/or to initiate the epithelial to mesenchymal transformation of epicardial cells. Transgenic reexpression of FOG-2 in cardiomyocytes rescues the FOG-2(-/-) vascular phenotype, demonstrating that FOG-2 function in myocardium is required and sufficient for coronary vessel development. Our findings provide the molecular inroad into the induction of coronary vasculature by myocardium in the developing heart.

Embryonic stem (ES) cells are immortal and present the ability to self-renew while retaining their ability to differentiate. In contrast, most primary cells possess a limited proliferative potential, and when this is exhausted, undergo an irreversible growth arrest termed senescence. In primary cells, senescence can be also triggered by a variety of stress to which ES cells are highly refractory. Here the authors report that the proliferative capacity of murine ES cells closely correlates with high activity of different glycolytic enzymes, elevated glycolytic flux, and low mitochondrial oxygen consumption. The direct relation between glycolytic flux and the ability of ES cells to proliferate is further remarked in experiments where glycolysis or ES cell self-renewal was specifically inhibited. It was previously reported that the upregulation of glycolysis in primary cells results in life span extension. The authors hypothesize that the naturally high glycolytic flux observed in murine ES cells can be responsible for their unlimited proliferative potential.

We report the isolation and characterization of the cDNAs encoded by the murine and human homeobox genes, Irx4 (Iroquois homeobox gene 4). Mouse and human Irx4 proteins are highly conserved (83%) and their 63-aa homeodomain is more than 93% identical to that of the Drosophila Iroquois patterning genes. Human IRX4 maps to chromosome 5p15.3, which is syntenic to murine chromosome 13. Irx4 transcripts are present in the developing central nervous system, skin, and vibrissae, but are predominantly expressed in the cardiac ventricles. In mice at embryonic day (E) 7.5, Irx4 transcripts are found in the chorion and at low levels in a discrete anterior domain of the cardiac primordia. During the formation of the linear heart tube and its subsequent looping (E8.0-8.5), Irx4 expression is restricted to the ventricular segment and is absent from both the posterior (eventual atrial) and the anterior (eventual outflow tract) segments of the heart. Throughout all subsequent stages in which the chambers of the heart become morphologically distinct (E8.5-11) and into adulthood, cardiac Irx4 expression is found exclusively in the ventricular myocardium. Irx4 gene expression was also assessed in embryos with aberrant cardiac development: mice lacking RXRalpha or MEF2c have normal Irx4 expression, but mice lacking the homeobox transcription factor Nkx2-5 (Csx) have markedly reduced levels of Irx4 transcripts. dHand-null embryos initiate Irx4 expression, but cannot maintain normal levels. These data indicate that the homeobox gene Irx4 is likely to be an important mediator of ventricular differentiation during cardiac development, which is downstream of Nkx2-5 and dHand.

Csx/Nkx2.5 is an evolutionary conserved homeobox gene related to the Drosophila tinman gene, which is essential for the dorsal mesoderm formation. Expression of Csx/Nkx2.5 mRNA is the earliest marker for heart precursor cells in all vertebrates so far examined. Previous studies have demonstrated that Csx/Nkx2.5 mRNA is highly expressed in the heart and at lower levels in the spleen, tongue, stomach, and thyroid in the murine embryo. Since some developmental genes are regulated by posttranscriptional mechanisms, we analyzed the developmental pattern of Csx protein expression at the single-cell level using Csx-specific antibodies. Immunohistochemical analysis of murine embryos at 7.8 days post coitum revealed that Csx protein is strongly expressed in the nucleus of endodermal and mesodermal cells in the cardiogenic plate. Subsequently, in the heart, Csx protein was detected only in the nucleus of myocytes of the atrium and the ventricle through the adult stage. During the fetal period, Csx protein expression in the nucleus was also noted in the spleen, stomach, liver, tongue, and anterior larynx. Unexpectedly, confocal microscopy revealed that Csx immunoreactivity was detected only in the cytoplasm of a subset of cranial skeletal muscles. Csx protein was not detected in the thyroid glands. The expression of Csx protein in all organs was markedly downregulated after birth except in the heart. These results raise the possibility that Csx/Nkx2.5 may play a role in the early developmental process of multiple tissues in addition to its role in early heart development.

Heterozygous mutations of the cardiac transcription factor Nkx2-5 cause atrioventricular conduction defects in humans by unknown mechanisms. We show in KO mice that the number of cells in the cardiac conduction system is directly related to Nkx2-5 gene dosage. Null mutant embryos appear to lack the primordium of the atrioventricular node. In Nkx2-5 haploinsufficiency, the conduction system has half the normal number of cells. In addition, an entire population of connexin40 -/connexin45 + cells is missing in the atrioventricular node of Nkx2-5 heterozygous KO mice. Specific functional defects associated with Nkx2-5 loss of function can be attributed to hypoplastic development of the relevant structures in the conduction system. Surprisingly, the cellular expression of connexin40, the major gap junction isoform of Purkinje fibers and a putative Nkx2-5 target, is unaffected, consistent with normal conduction times through the His-Purkinje system measured in vivo. Postnatal conduction defects in Nkx2-5 mutation may result at least in part from a defect in the genetic program that governs the recruitment or retention of embryonic cardiac myocytes in the conduction system. 1130The Nonstandard abbreviations used: AV nodal effective refractory period (AVERP); connexin40 (Cx40); embryonic day (E); interventricular septum (IVS); intracardiac electrogram (IEGM); stimulus (S).

HIV‐1 efficiently infects susceptible cells and causes AIDS in humans. Although HIV can also enter the cells of Old World monkeys, it encounters a block before reverse transcription. Data have shown that this species‐specific restriction is mediated by tripartite motif (TRIM)5α, whose molecular function is still undefined. Here, we show that TRIM5α functions as a RING‐finger‐type E3 ubiquitin ligase both in vitro and in vivo and ubiquitinates itself in cooperation with the E2 ubiquitin‐conjugating enzyme UbcH5B. In addition to the self‐ubiquitination, we show that TRIM5α is ubiquitinated by another E3 ubiquitin ligase, Ro52, and deubiquitinated by YopJ, one of the pathogenic proteins derived from Yersinia species. Thus, the ubiquitination of TRIM5α is catalyzed by itself and Ro52 and downregulated by YopJ. Unexpectedly, although TRIM5α is ubiquitinated, our results have revealed that the proteasome inhibitors MG115 and MG132 do not stabilize it in HeLa cells, suggesting that the ubiquitination of TRIM5α does not lead to proteasomal degradation. Importantly, TRIM5α is clearly conjugated by a single ubiquitin molecule (monoubiquitination). Our monoubiquitin‐fusion assay suggests that monoubiquitination is a signal for TRIM5α to translocate from cytoplasmic bodies to the cytoplasm.

A total of 239 patients undergoing serial coronary angiography with a concomitant ergonovine provocation test were studied between July 1974 and June 1987. The progression of coronary artery disease was evaluated in relation to risk factors, especially coronary artery spasm. Patients were classified into three groups: 1) new myocardial infarction group (39 patients); 2) progression without infarction group (90 patients); and 3) nonprogression group (110 patients). To assess how risk factors and coronary spasm are related to the occurrence of new myocardial infarction and progression without infarction, 11 variables in the three groups were examined: age, gender, the time interval between the studies, fasting blood sugar, systolic blood pressure, diastolic blood pressure, smoking, serum cholesterol, triglyceride, uric acid and a positive response to the ergonovine provocation test. Multiple regression analysis selected three independent predictors of progression without infarction: cholesterol (p less than 0.01), systolic blood pressure (p less than 0.05) and a positive response to the ergonovine provocation test (p less than 0.001). Multiple regression analysis also selected three independent predictors of the occurrence of new myocardial infarction: fasting blood sugar (p less than 0.01), systolic blood pressure (p less than 0.05) and a positive response to the ergonovine provocation test (p less than 0.001). A positive response to the ergonovine provocation test was the strongest factor for occurrence of both new myocardial infarction and progression without infarction. To evaluate segmental arterial changes, 3,275 coronary artery segments were analyzed in the 239 patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Upon activation, NF-κB translocates into the nucleus and initiates many biological events. This NF-κB signaling is mainly induced by the protein kinase IKKβ. Early in this signaling pathway, IKKβ is phosphorylated for activation by several factors, such as pro-inflammatory cytokines and the Tax oncoprotein of human T-cell leukemia virus type 1 (HTLV-1). In cells expressing Tax protein, IKKβ is persistently phosphorylated, which chronically activates NF-κB signaling. But the active IKKβ is conjugated with a monoubiquitin by the E3 ubiquitin ligase Ro52, and the IKKβ-induced NF-κB signaling is downregulated. However, the mechanism of the downregulation has been unknown. Here, we show that Ro52-mediated monoubiquitination is involved in the subcellular translocation of active IKKβ to autophagosomes. Furthermore, using reporter assays, we show that Ro52 suppresses IKKβ-induced NF-κB signaling and that this suppression is blocked by an autophagy inhibitor. These results suggest that Ro52-mediated monoubiquitination plays a critical role in the downregulation of active IKKβ through autophagy.