The transcription factor GATA4 is essential for heart morphogenesis. Heterozygous mutation of GATA4 causes familial septal defects. However, the phenotypic spectrum of heterozygous GATA4 mutation is not known. In this study, we defined the cardiac phenotypes that result from heterozygous mutation of murine Gata4. We then asked if GATA4 mutation occurs in humans with these forms of congenital heart disease (CHD). In mice, heterozygous Gata4 mutation was associated with atrial and ventricular septal defect (ASD, VSD), endocardial cushion defect (ECD), RV hypoplasia, and cardiomyopathy. Genetic background strongly influenced the expression of ECD and cardiomyopathy, indicating the presence of important genetic modifiers. In humans, nonsynonymous GATA4 sequence variants were associated with ECD (2/43), ASD (1/8), and RV hypoplasia in the context of double inlet left ventricle (1/9), forms of CHD that overlapped with abnormalities seen in the mouse model. These variants were not found in at least 500 control chromosomes, and encode proteins with non-conservative amino acid substitutions at phylogenetically conserved positions, suggesting that they are disease-causing mutations. Cardiomyopathy was not associated with GATA4 mutation in humans. These data establish the phenotypic spectrum of heterozygous Gata4 mutation in mice, and suggest that heterozygous GATA4 mutation leads to partially overlapping phenotypes in humans. Additional studies will be

The degeneration of substantia nigra (SN) dopamine (DA) neurons in sporadic Parkinson’s disease (PD) is characterized by disturbed gene expression networks. Micro(mi)RNAs are post-transcriptional regulators of gene expression and we recently provided evidence that these molecules may play a functional role in the pathogenesis of PD. Here, we document a comprehensive analysis of miRNAs in SN DA neurons and PD, including sex differences. Our data show that miRNAs are dysregulated in disease-affected neurons and differentially expressed between male and female samples with a trend of more up-regulated miRNAs in males and more down-regulated miRNAs in females. Unbiased Ingenuity Pathway Analysis (IPA) revealed a network of miRNA/target-gene associations that is consistent with dysfunctional gene and signaling pathways in PD pathology. Our study provides evidence for a general association of miRNAs with the cellular function and identity of SN DA neurons, and with deregulated gene expression networks and signaling pathways related to PD pathogenesis that may be sex-specific.

Yeast mitochondrial phosphate transport activity has been reconstituted from the import receptor (MIR) expressed as inclusion bodies in Escherichia coli. This result undermines the suggestion [Murakami, H., et al. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 3358-3362] that the MIR has been misidentified as the phosphate transport protein (PTP). PTP was solubilized with N-lauroylsarcosinate and Triton X-100 and purified with a yield of about 2 mg/L of induced bacterial culture. This PTP, reconstituted in liposomes, catalyzes phosphate uptake with a Vmax [24.5 degrees C, net (zero trans), pHi = 8.0, pHe = 6.8] of 0.61 mmol of phosphate min-1 (mg of PTP)-1 and a Km of 1.30 mM. This Vmax is higher and the Km about the same as that obtained with PTP purified from mitochondria. Replacement of Thr43 and Ile141 by other amino acids results in three types of PTP: (a) 2.5-5.0% Vmax of wild-type PTP (PTPwt) (Thr43Cys; Thr43Ser; Ile141Cys), (b) < 0.1% Vmax (detection limit of assay) of PTPwt (Thr43Ala; Thr43Asp), and (c) proton transport uncoupled from phosphate transport (Ile141Cys). Km changes are not significant. Activity of Thr43Cys confirms results obtained with mitochondrially expressed protein. Thus, yeast PTP requires Thr43 and mammalian PTP the similarly located Cys42 for high transport activity. Thr43 and Ile141 are each situated between two basic residues (LysThrArg vs ArgIleArg). Cys substitutions in either of these positions confer the same high N-ethylmaleimide sensitivity to the yeast PTPwt as displayed by the mammalian PTP.(ABSTRACT TRUNCATED AT 250 WORDS)

The homodimeric mitochondrial phosphate transport protein (PTP), which has six transmembrane helices per subunit, catalyzes inorganic phosphate transport in an electroneutral and pH gradient-dependent manner across the inner membrane. We have replaced the Glu, Asp, and His residues of the yeast PTP to assess their role in the transport mechanism. Mutants with physiologically relevant transport activity were identified by their ability to rescue the PTP null mutant yeast from glycerol medium. Five residues appear critical for transport: His-32 in helix A, Glu-126 and -137 in helix C, and Asp-39 and -236 at the matrix ends of helices A and E. These mutant PTPs are expressed at near normal levels in yeast. This yeast PTP and the mutants were expressed in Escherichia coli as inclusion bodies, solubilized, purified, and reconstituted. Their transport activities correlate well with the physiological assays. None of the transport inactivating mutations appear to be due to major protein conformation changes as assayed by the efficiency of PTP incorporation into liposomes. Only the Glu95Gln (cytosolic helices B and C-connecting segment), Glu163Gln and Glu164Gln (matrix helices C and D-connecting segment), and Glu126Asp (helix C) show a near 70% decrease in liposome incorporation efficiency. In addition, mutations at either end of helix D increase phosphate transport 2-fold. We would like to suggest that Glu-126, His-32, and Glu-137 (similar to Asp-96, Lys-216, and Asp-85 of bacteriorhodopsin) form a proton cotransport pathway that is coupled in an as yet undefined manner (possibly via His-32) to a phosphate transport pathway, which may include helix D.