Fatal aluminum phosphide poisoning.

A 39-year-old man committed suicide by ingestion of aluminum phosphide, a potent mole pesticide, which was available at the victim's workplace. The judicial authority ordered an autopsy, which ruled out any other cause of death. The victim was discovered 10 days after the ingestion of the pesticide. When aluminum phosphide comes into contact with humidity, it releases large quantities of hydrogen phosphine (PH3), a very toxic gas. Macroscopic examination during the autopsy revealed a very important asphyxia syndrome with major visceral congestion. Blood, urine, liver, kidney, adrenal, and heart samples were analyzed. Phosphine gas was absent in the blood and urine but present in the brain (94 mL/g), the liver (24 mL/g), and the kidneys (41 mL/g). High levels of phosphorus were found in the blood (76.3 mg/L) and liver (8.22 mg/g). Aluminum concentrations were very high in the blood (1.54 mg/L), brain (36 microg/g), and liver (75 microg/g) compared to the usual published values. Microscopic examination revealed congestion of all the organs studied and obvious asphyxia lesions in the pulmonary parenchyma. All these results confirmed a diagnosis of poisoning by aluminum phosphide. This report points out that this type of poisoning is rare and that hydrogen phosphine is very toxic. The phosphorus and aluminum concentrations observed and their distribution in the different viscera are discussed in relation to data in the literature.

The transcription factor NFAT4 is involved in the generation and survival of T cells.

Nuclear factor of activated T cells (NFAT) is a family of four related transcription factors implicated in cytokine and early response gene expression in activated lymphocytes. Here we report that NFAT4, in contrast to NFATp and NFATc, is preferentially expressed in DP thymocytes. Mice lacking NFAT4 have impaired development of CD4 and CD8 SP thymocytes and peripheral T cells as well as hyperactivation of peripheral T cells. The thymic defect is characterized by increased apoptosis of DP thymocytes. The increased apoptosis and hyperactivation may reflect heightened sensitivity to TcR-mediated signaling. Further, mice lacking NFAT4 have impaired production of Bcl-2 mRNA and protein. NFAT4 thus plays an important role in the successful generation and survival of T cells.

The transcription factor NF-ATc is essential for cardiac valve formation.

Nuclear factor of activated T cells (NF-AT) is the name of a family of four related transcription factors that may be needed for cytokine gene expression in activated lymphocytes. Here we report that mice with a targeted disruption of the NF-ATc gene show an unexpected and dramatic defect in cardiac morphogenesis, with selective absence of the aortic and pulmonary valves, leading to death in utero from congestive heart failure at days 13.5-17.5 of gestation. In contrast, tricuspid and mitral valve morphogenesis is normal. NF-ATc is the first transcription factor known to be expressed only in the endothelial cells of the heart. As in T cells, nuclear translocation of NF-ATc in cardiac endothelial cells is controlled by the calcium-regulated phosphatase calcineurin: NF-ATc remains cytoplasmic in normal embryos cultured with cyclosporin A, an inhibitor of calcineurin. Abnormal development of the cardiac valves and septae is the most frequent form of birth defect, yet few molecular regulators of valve formation are known. Our results indicate that NF-ATc may play a critical role in signal-transduction processes required for normal cardiac valve formation.

The structure of GABPalpha/beta: an ETS domain- ankyrin repeat heterodimer bound to DNA.

GA-binding protein (GABP) is a transcriptional regulator composed of two structurally dissimilar subunits. The alpha subunit contains a DNA-binding domain that is a member of the ETS family, whereas the beta subunit contains a series of ankyrin repeats. The crystal structure of a ternary complex containing a GABPalpha/beta ETS domain-ankyrin repeat heterodimer bound to DNA was determined at 2. 15 angstrom resolution. The structure shows how an ETS domain protein can recruit a partner protein using both the ETS domain and a carboxyl-terminal extension and provides a view of an extensive protein-protein interface formed by a set of ankyrin repeats. The structure also reveals how the GABPalpha ETS domain binds to its core GGA DNA-recognition motif.

Delayed lymphoid repopulation with defects in IL-4-driven responses produced by inactivation of NF-ATc.

The NF-AT family of transcription factors activates early immune response genes such as cytokines. In the adult, NF-ATc is expressed exclusively in the lymphoid system and is induced upon lymphocyte activation. NF-ATc null mutant mice die in utero of cardiac failure, precluding analysis of the role of NF-ATc in lymphocyte activation. By using RAG-2-deficient blastocyst complementation, we now demonstrate that young, highly chimeric mice lacking NF-ATc have impaired repopulation of both thymus and peripheral lymphoid organs. Furthermore, NF-ATc deficiency impaired T lymphocyte activation and secretion of IL-4. B lymphocytes displayed reduced proliferation and a selective loss of IL-4-driven immunoglobulin isotypes both in vivo and in vitro. Our data demonstrate that NF-ATc is essential for the optimal generation and function of mature T and B lineage cells, with an especially profound effect on IL-4-driven responses.

Hyperproliferation and dysregulation of IL-4 expression in NF-ATp-deficient mice.

NF-ATp is a member of a family of genes that encodes the cytoplasmic component of the nuclear factor of activated T cells (NF-AT). In this study, we show that mice with a null mutation in the NF-ATp gene have splenomegaly with hyperproliferation of both B and T cells. They also display early defects in the transcription of multiple genes encoding cytokines and cell surface receptors, including CD40L and FasL. A striking defect in early IL-4 production was observed after ligation of the TCR complex by treatment with anti-CD3 in vivo. The transcription of other cytokines including IL-13, GM-CSF, and TNF alpha was also affected, though to a lesser degree. Interestingly, the cytokines IL-2 and IFN gamma were minimally affected. Despite this early defect in IL-4 transcription, Th2 development was actually enhanced at later timepoints as evidenced by increased IL-4 production and IgE levels in situations that favor the formation of Th2 cells both in vitro and in vivo. These data suggest that NF-ATp may be involved in cell growth, and that it is important for the balanced transcription of the IL-4 gene during the course of an immune response.

Identification of the promoter of the mouse obese gene.

Primer extension and RACE (rapid amplification of cDNA ends) assays were used to identify and sequence the 5' terminus of mouse ob mRNA. This sequence was used to obtain a recombinant bacteriophage containing the first exon of the encoding gene. DNA sequence analysis of the region immediately upstream of the first exon of the mouse ob gene revealed DNA sequences corresponding to presumptive cis-regulatory elements. A canonical TATA box was observed 30-34 base pairs upstream from the start site of transcription and a putative binding site for members of the C/EBP family of transcription factors was identified immediately upstream from the TATA box. Nuclear extracts prepared from primary adipocytes contained a DNA binding activity capable of avid and specific interaction with the putative C/EBP response element; antibodies to C/EBP alpha neutralized the DNA binding activity present in adipocyte nuclear extracts. When linked to a firefly luciferase reporter and transfected into primary adipocytes, the presumptive promoter of the mouse ob gene facilitated luciferase expression. When transfected into HepG2 cells, which lack C/EBP alpha, the mouse ob promoter was only weakly active. Supplementation of C/EBP alpha by cotransfection with a C/EBP alpha expression vector markedly stimulated luciferase expression. Finally, an ob promoter variant mutated at the C/EBP response element was inactive in both primary adipocytes and HepG2 cells. These observations provide evidence for identification of a functional promoter capable of directing expression of the mouse ob gene.

Quail myoD is regulated by a complex array of cis-acting control sequences.

Quail myoD (QmyoD) is the earliest myoD family member expressed in quail somites and its transcription is initiated in response to early developmental signals. We have investigated the transcriptional regulation of QmyoD to define the cis-acting sequences required for tissue-specific and correct developmental expression. The QmyoD gene locus was isolated and sequenced and its regulatory properties were characterized. We identified three distinct regions of cis-acting regulatory sequences that control the expression of reporter gene constructs following DNA transfection into cell lines and cultured primary quail cells. The first, a complex distal control region (DCR), 11.5 kb upstream of the gene, contains three separable enhancer activities. Two of these DCR enhancer activities are tissue specific and can be autoactivated. In addition, these same two enhancers and the entire DCR direct somite- and muscle-specific expression of a reporter gene in transgenic mice. Sequence analysis of the DCR enhancers reveals clusters of E-boxes, MEF2 binding motifs, and the stretches of sequence identity with the human myoD enhancer. Second, the promoter region has sequences which act positively to direct expression in both muscle and nonmuscle cells as well as sequences that repress expression specifically in nonmuscle cells. The third control region, the PR, is located -3.3 to -5 kb from the transcription start site and directs muscle-specific expression in cultured cells. This analysis demonstrates that QmyoD has multiple control regions and that some features of myoD regulation are conserved between mammals and birds.

Molecular and genetic characterization of GABP beta.

This report outlines three observations relating to GABP beta, a polypeptide constituent of the heterotetrameric transcription factor GABP. Evidence is presented showing that the mouse genome encodes two highly related GABP beta polypeptides, designated GABP beta 1-1 and GABP beta 2-1. Genomic and cDNA copies of the newly defined Gabpb2 gene were cloned and characterized, providing the conceptually translated amino acid sequence of GABP beta 2-1. The genes encoding these two proteins, as well as GABP alpha, were mapped to three unlinked chromosomal loci. Although physically unlinked, the patterns of expression of the three genes were strikingly concordant. Finally, the molecular basis of GABP beta dimerization was resolved. Carboxy-terminal regions of the two GABP beta polypeptides, which mediate dimerization, bear highly related primary amino acid sequences. Both sequences are free of alpha-helix destabilizing residues and, when displayed on idealized alpha-helical projections, reveal marked amphipathy. Two observations indicate that these regions adopt an alpha-helical conformation and intertwine as coiled-coils. First, the dimer-forming region of GABP beta 2-1 can functionally replace the leucine zipper of a bZIP transcription factor. Second, a synthetic peptide corresponding to this region shows distinctive helical properties when examined by circular dichroism spectroscopy. Finally, evidence is presented showing that GABP beta 1-1 and GABP beta 2-1 can heterodimerize through this carboxy-terminal domain, but neither protein can heterodimerize via the dimer-forming region of the bZIP protein C/EBP beta.

Localized expression of a myogenic regulatory gene, qmf1, in the somite dermatome of avian embryos.

qmf1 is a quail myogenic regulatory gene that is transcribed in skeletal myoblasts and differentiated muscle and shows sequence homology to MyoD1 and Myf5. We used the qmf1 transcript as an in situ hybridization marker for determined myogenic cells to study myogenic lineages in developing embryos. We present evidence for the temporal and spatial regulation of qmf1 mRNA expression and slow cardiac troponin C (TnC), fast skeletal troponin T (TnT), and alpha-cardiac actin contractile protein mRNA expression in the somite myotome and limb buds. Our results show that qmf1 is a marker for myogenic lineages during both somite formation and limb development and that qmf1 mRNAs, but not contractile protein mRNAs, localize in dorsal medial lip (DML) cells of the somite dermatome. We propose that the DML is a site of myogenic lineage determination.

Developmental and muscle-specific regulation of avian fast skeletal troponin T isoform expression by mRNA splicing.

We have investigated the developmental regulation of the avian fast skeletal muscle troponin T (TnTf) gene of the Japanese quail. Sequence analysis of troponin T mRNA, cDNA clones, and a genomic DNA segment demonstrate that the avian, fast skeletal TnTf protein isoforms are produced from a single gene. This TnTf gene is expressed in skeletal muscle, but not in adult cardiac muscles or in non-muscle tissues. In addition to known TnT isoforms, three new isoforms of TnT are described. These isoforms arise by regulated alternative RNA splicing of exons in the 5' and 3' regions of TnTf transcripts. Alternative splicing of the 5' TnTf exons involves splicing of multiple exons in different combinations (i.e. not mutually exclusive), whereas 3' alternative splicing involves mutually exclusive splice choices between two exons (alpha or beta exons). S1 nuclease protection and primer extension analyses show that alternative splicing of both 5' and 3' exons is precisely regulated and coordinated in physiologically different striated muscles, which express distinct, restricted combinations of 5' and 3' alternatively spliced exons in mRNA transcripts. In contrast, different embryonic muscles and clonal embryonic myoblast cultures coexpress the 3' alternative splice choices. This indicates that alternative splicing of TnTf mRNAs is controlled in different adult muscles by specific trans factors, and not by the restricted expression of different spliced forms in different embryonic myoblast lineages. Comparison of TnTf isoform expression in quail and chicken flight muscle (Wilkinson, J. M., Moir, A. J., and Waterfield, M. D. (1984) Eur. J. Biochem. 143, 47-56) to TnTf isoforms of the rat (Breitbart, R. E., and Nadal-Ginard, B. (1986) J. Mol. Biol. 188, 313-324), and rabbit (Pearlstone, J. R., Carpenter, M. R., and Smillie, M. B. (1976) Proc. Natl. Acad. Sci. U. S. A. 73, 1902-1906) indicates that the avian gene contains an additional exon(s) not present in mammalian genes. The alternative exon sequences TnTf mRNAs expressed in anatomically distinct quail muscles can be correlated with sequences in TnTf protein isoforms in these chicken muscles. Thus, the regulated splicing of alternative exons in TnT transcripts, and not selective translation of stochastically spliced TnT mRNAs, regulates TnTf isoform expression in specific muscles.