Targeted disruption in mice of the gene encoding D-3-phosphoglycerate dehydrogenase (Phgdh) results in embryonic lethality associated with a striking reduction in free L-serine and growth retardation including severe brain malformation. We previously observed a severe impairment in neurogenesis of the central nervous system of Phgdh knockout (KO) embryos and a reduction in the protein content of their brains. Although these findings suggest that L-serine deficiency links attenuation of mRNA translation to severe developmental malformation of the central nervous system, the underlying key molecular event remains unexplored. Here we demonstrate that mRNA of Eif4ebp1 encoding eukaryotic initiation factor 4 binding protein 1 and its protein, 4E-BP1, are markedly induced in the central nervous system of Phgdh KO embryos, whereas a modest induction is observed in the liver. The increase in 4E-BP1 was associated with a decrease in the cap initiation complex in the brain, as shown by lower levels of eukaryotic translation initiation factor 4G bound to eukaryotic translation initiation factor 4E (eIF4E) and increased eIF4E interaction with 4E-BP1 based on 7-methyl-GTP chromatography. eIF4E protein and polysomes were also diminished in Phgdh KO embryos. Induction of Eif4ebp1 mRNA and of 4E-BP1 was reproduced in mouse embryonic fibroblasts established from Phgdh KO embryos under the condition of L-serine deprivation. Induction of Eif4ebp1 mRNA was suppressed only when L-serine Abbreviations 3-PG, 3-phosphoglycerate; 4E-BP1, Eif4ebp1 protein; 7mGTP, 7-methyl-GTP; ActD, actinomycin D; CNS, central nervous system; eIF2a, a subunit of eukaryotic translation initiation factor 2; eIF4A, eukaryotic translation initiation factor 4A; eIF4E, eukaryotic translation initiation factor 4E; Eif4ebp1, eukaryotic initiation factor 4E binding protein 1; eIF4G, eukaryotic translation initiation factor 4G; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GCN2, general control nonderepressible 2 kinase; GLAST, astrocyte/radial glia-specific L-glutamate/L-aspartate transporter; HT, heterozygote; KO, knockout; MEF, mouse embryonic fibroblast; NMDA, N-methyl-D-aspartate; Phgdh, D-3-phosphoglycerate dehydrogenase; Psat, phosphoserine aminotransferase; Psph, phosphoserine phosphatase; QRT-PCR, quantitative real-time PCR; Shmt, serine hydroxymethyltransferase; WT, wild type.

The formation of zinc protoporphyrin IX (ZnPP) and protoporphyrin IX (PP) in porcine heart mitochondria was investigated by fluorescent spectral analysis. The time-course study at -1῍C and pH /./ with +** mM ZnCl, showed a linear formation of ZnPP from oxymyoglobin after a lag-period (3 h) at the formation rate of *..0 nmol/h. The formed amount of ZnPP is dependent upon the ZnCl, concentration. In the incubation without ZnCl,, the new peak derived from PP appeared at 0--nm after +, h, and then its intensity gradually increased. The formation of PP is believed to be due to the release of Fe ,ῌ in porphyrin ring of oxymyoglobin during the incubation. This suggests that the mitochondria have the ability to form PP from oxymyoglobin, and therefore, are directly related to the release of Fe ,ῌ from porphyrin ring in myoglobin.

So gut wie unschuldig: In photobiophysikalischen Studien zu Häm‐Eisen‐Nitrosylkomplexen ähnelten die Absorptionsspektren der sechsfach koordinierten Komplexe mit NO und Met oder Cys denjenigen des fünffach koordinierten Fe‐Häm(NO)+‐Komplexions. Weil die Absorptionsspektren verwandter Proteine mit Histidin als proximalem Ligand denen der gasförmigen Komplexe ähnlich sind, übt die Protein‐Mikroumgebung nur einen geringen Effekt auf den energieärmsten Übergang des Porphyrinmakrocyclus aus.

D-3-Phosphoglycerate dehydrogenase (Phgdh) is a necessary enzyme for de novo L-serine biosynthesis. Mutations in the human PHGDH cause serine deficiency disorders characterized by severe neurological symptoms including congenital microcephaly and psychomotor retardation. We showed previously that targeted disruption of Phgdh in mice causes overall growth retardation with severe brain microcephaly and leads to embryonic lethality. Here, amino acid analysis of Phgdh knockout (KO) mouse embryos demonstrates that free serine and glycine concentrations are decreased markedly in head samples, reflecting the metabolic changes of serine deficiency found in human patients. To understand the pathogenesis of serine deficiency disorders at the molecular level, we have exploited this animal model to identify altered gene expression patterns using a microarray technology. Comparative microarray analysis of the Phgdh KO and wild-type head at gestational day 13.5 revealed an upregulation of genes involved in transfer RNA aminoacylation, amino acid metabolism, amino acid transport, transcriptional regulation, and translation, and a downregulation of genes involved in transcription in neuronal progenitors and muscle and cartilage development. A computational network analysis software was used to construct transcriptional regulatory networks operative in the Phgdh KO embryos in vivo. These observations suggest that Phgdh inactivation alters transcriptional programs in several regulatory networks.

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