Identification of a prevalent nonsense mutation (W283X) and two novel mutations in the porphobilinogen deaminase gene of Swiss patients with acute intermittent porphyria

Acute intermittent porphyria (AIP) is an autosomal dominant disorder caused by decreased activity of porphobilinogen deaminase (PBGD), the third enzyme in the heme biosynthetic pathway. We report the first molecular analysis of PBGD gene mutations in AIP patients of Swiss origin. The PBGD gene of 18 Swiss AIP patients was analyzed by denaturing gradient gel electrophoresis screening of the genomic DNA and direct sequencing. Thirteen of the 18 patients (72%) carried a nonsense mutation G(849)-->A, W283X. In addition, 4 different mutations including 2 novel mutations (Q217L and Q292X), were identified in the 5 remaining AIP patients originating from both German- and Italian-speaking regions of Switzerland.     

Effects of 4wk of hindlimb suspension on skeletal muscle mitochondrial respiration in rats

Yajid, Fatima, Jacques G. Mercier, Béatrice M. Mercier, Hervé Dubouchaud, and Christian Préfaut.Effects of 4 wk of hindlimb suspension on skeletal musclemitochondrial respiration in rats. J. Appl.Physiol. 84(2): 479-485, 1998.We investigated inrats the effect of 4 wk of hypodynamia on the respiration of mitochondria isolated from four distinct muscles [soleus,extensor digitorum longus, tibial anterior, and gastrocnemius(Gas)] and from subsarcolemmal (SS) and intermyofibrillar (IMF)regions of mixed hindlimb muscles that mainly contained the four citedmuscles. With pyruvate plus malate as respiratory substrate, 4 wk ofhindlimb suspension produced an 18% decrease in state3 respiration for IMF mitochondria compared with thosein the control group (P < 0.05). TheSS mitochondria state 3 were notsignificantly changed. Concerning the four single muscles, themitochondrial respiration was significantly decreased in the Gasmuscle, which showed a 59% decrease in state3 with pyruvate + malate(P < 0.05). The other musclespresented no significant decrease in respiratory rate in comparisonwith the control group. With succinate + rotenone, there was nosignificant difference in the respiratory rate compared with therespective control group, whatever the mitochondrial origin (SS, orIMF, or from single muscle). We conclude that 4 wk of hindlimbsuspension alters the respiration of IMF mitochondria in hindlimbskeletal muscles and seems to act negatively on complex I of theelectron-transport chain or prior sites. The muscle mitochondria mostaffected are those isolated from the Gas muscle.

     

Projection maps of three members of the KdgM outer membrane protein family

We present the projection structures of the three outer membrane porins KdgM and KdgN from Erwinia chrysanthemi and NanC from Escherichia coli, based on 2D electron crystallography. A wide screening of 2D crystallization conditions yielded tubular crystals of a suitable size and quality to perform high-resolution electron microscopy. Data processing of untilted samples allowed us to separate the information of the two crystalline layers and resulted in projection maps to a resolution of up to 7 Å. All three proteins exhibit a similar putative β-barrel structure and the three crystal forms have the same symmetry. However, there are differences in the packing arrangements of the monomers as well as the densities of the projections. To interpret these projections, secondary structure prediction was performed using β-barrel specific prediction algorithms. The predicted transmembrane β-barrels have a high similarity in the arrangement of the putative β-strands and the loops, but do not match those of OmpG, a related protein porin whose structure was solved.     

Plasticity of polyubiquitin recognition as lysosomal targeting signals by the endosomal sorting machinery

Lysosomal targeting is fundamental for the regulated disposal of ubiquitinated membrane proteins from the cell surface. To elucidate ubiquitin (Ub) configurations that are necessary and sufficient as multivesicular body (MVB)/lysosomal-sorting motifs, the intraendosomal destination and transport kinetics of model transmembrane cargo molecules bearing monoubiquitinated, multi-monoubiquitinated, or polyubiquitinated cytoplasmic tails were determined. Monomeric CD4 chimeras with K63-linked poly-Ub chains and tetrameric CD4-mono-Ub chimeras were rapidly targeted to the lysosome. In contrast, lysosomal delivery of CD4 chimeras exposing K48-linked Ub chains was delayed, whereas delivery of monoubiquitinated CD4 chimeras was undetectable. Similar difference was observed in the lysosomal targeting of mono- versus polyubiquitinated invariant chain and CD4 ubiquitinated by the MARCH (membrane-associated RING-CH) IV Ub ligase. Consistent with this, Hrs (hepatocyte growth factor regulated tyrosine kinase phosphorylated substrate), an endosomal sorting adaptor, binds preferentially to K63-Ub chain and negligibly to mono-Ub. These results highlight the plasticity of Ub as a sorting signal and its recognition by the endosomal sorting machinery, and together with previous data, suggest a regulatory role for assembly and disassembly of Ub chains of specific topology in lysosomal cargo sorting.     

Impairment of thioredoxin reductase activity by oxidative stress in human rheumatoid synoviocytes

The thioredoxin/thioredoxin reductase system is strongly induced in patients with rheumatoid arthritis (RA). We have investigated the impact on TR activity of doses of superoxide anion generated by the hypoxanthine (HX)/xanthine oxidase (XO) system and by hydrogen peroxide, H(2)O(2), for various times and compared the findings with synoviocytes obtained from osteoarthritis (OA) patients. At baseline, TR activity in RA cells was significantly higher than in OA cells (2.31 +/- 0.65 versus 0.74 +/- 0.43 mUnit/mg protein, p < 0.01). HX/XO and H(2)O(2) in RA cells decreased TR activity, which was found to be unchanged in OA cells. H(2)O(2) and superoxide anion caused a time-dependent accumulation of oxidized TR and induced the formation of carbonyl groups in TR protein in RA cells rather than OA cells, and oxidized the selenocysteine of the active site. The oxidation in TR protein was irreversible in RA cells but not in OA cells. In conclusion, we report that the oxidative aggression generates modifications in the redox status of the active site of the TR and induces an alteration of the Trx/TR system, concomitant with those of the other antioxidant systems that could explain the causes of oxidative stress related to RA disease.     

Differential regulation of imprinting in the murine embryo and placenta by the Dlk1-Dio3 imprinting control region

Genomic imprinting is an epigenetic mechanism controlling parental-origin-specific gene expression. Perturbing the parental origin of the distal portion of mouse chromosome 12 causes alterations in the dosage of imprinted genes resulting in embryonic lethality and developmental abnormalities of both embryo and placenta. A 1 Mb imprinted domain identified on distal chromosome 12 contains three paternally expressed protein-coding genes and multiple non-coding RNA genes, including snoRNAs and microRNAs, expressed from the maternally inherited chromosome. An intergenic, parental-origin-specific differentially methylated region, the IG-DMR, which is unmethylated on the maternally inherited chromosome, is necessary for the repression of the paternally expressed protein-coding genes and for activation of the maternally expressed non-coding RNAs: its absence causes the maternal chromosome to behave like the paternally inherited one. Here, we characterise the developmental consequences of this epigenotype switch and compare these with phenotypes associated with paternal uniparental disomy of mouse chromosome 12. The results show that the embryonic defects described for uniparental disomy embryos can be attributed to this one cluster of imprinted genes on distal chromosome 12 and that these defects alone, and not the mutant placenta, can cause prenatal lethality. In the placenta, the absence of the IG-DMR has no phenotypic consequence. Loss of repression of the protein-coding genes occurs but the non-coding RNAs are not repressed on the maternally inherited chromosome. This indicates that the mechanism of action of the IG-DMR is different in the embryo and the placenta and suggests that the epigenetic control of imprinting differs in these two lineages.     

GABA<Subscript>A</Subscript> Receptor and Glycine Receptor Activation by Paracrine/Autocrine Release of Endogenous Agonists: More Than a Simple Communication Pathway

It is a common and widely accepted assumption that glycine and GABA are the main inhibitory transmitters in the central nervous system (CNS). But, in the past 20 years, several studies have clearly demonstrated that these amino acids can also be excitatory in the immature central nervous system. In addition, it is now established that both GABA receptors (GABARs) and glycine receptors (GlyRs) can be located extrasynaptically and can be activated by paracrine release of endogenous agonists, such as GABA, glycine, and taurine. Recently, non-synaptic release of GABA, glycine, and taurine gained further attention with increasing evidence suggesting a developmental role of these neurotransmitters in neuronal network formation before and during synaptogenesis. This review summarizes recent knowledge about the non-synaptic activation of GABA_ARs and GlyRs, both in developing and adult CNS. We first present studies that reveal the functional specialization of both non-synaptic GABA_ARs and GlyRs and we discuss the neuronal versus non-neuronal origin of the paracrine release of GABA_AR and GlyR agonists. We then discuss the proposed non-synaptic release mechanisms and/or pathways for GABA, glycine, and taurine. Finally, we summarize recent data about the various roles of non-synaptic GABAergic and glycinergic systems during the development of neuronal networks and in the adult.     

Potent mGluR5 antagonists: pyridyl and thiazolyl-ethynyl-3,5-disubstituted-phenyl series

We report the synthesis of four series of 3,5-disubstituted-phenyl ligands targeting the metabotropic glutamate receptor subtype 5: (2-methylthiazol-4-yl)ethynyl (1a-j,), (6-methylpyridin-2-yl)ethynyl (2a-j), (5-methylpyridin-2-yl)ethynyl (3a-j,), and (pyridin-2-yl)ethynyl (4a-j,). The compounds were evaluated for antagonism of glutamate-mediated mobilization of internal calcium in an mGluR5 in vitro assay. All compounds were found to be full antagonists and exhibited low nanomolar to subnanomolar activity.