Cardiac arrhythmia and late-onset muscle weakness caused by a myofibrillar myopathy with unusual histopathological features due to a novel missense mutation in FLNC.

Myofibrillar myopathies (MFM) are mostly adult-onset diseases characterized by progressive morphological alterations of the muscle fibers beginning in the Z-disk and the presence of protein aggregates in the sarcoplasm. They are mostly caused by mutations in different genes that encode Z-disk proteins, including DES, CRYAB, LDB3, MYOT, FLNC and BAG3. A large family of French origin, presenting an autosomal dominant pattern, characterized by cardiac arrhythmia associated to late-onset muscle weakness, was evaluated to clarify clinical, morphological and genetic diagnosis. Muscle weakness began during adult life (over 30 years of age), and had a proximal distribution. Histology showed clear signs of a myofibrillar myopathy, but with unusual, large inclusions. Subsequently, genetic testing was performed in MFM genes available for screening at the time of clinical/histological diagnosis, and desmin (DES), αB-crystallin (CRYAB), myotilin (MYOT) and ZASP (LDB3), were excluded. LMNA gene screening found the p.R296C variant which did not co-segregate with the disease. Genome wide scan revealed linkage to 7q.32, containing the FLNC gene. FLNC direct sequencing revealed a heterozygous c.3646T>A p.Tyr1216Asn change, co-segregating with the disease, in a highly conserved amino acid of the protein. Normal filamin C levels were detected by Western-blot analysis in patient muscle biopsies and expression of the mutant protein in NIH3T3 showed filamin C aggregates. This is an original FLNC mutation in a MFM family with an atypical clinical and histopathological presentation, given the presence of significantly focal lesions and prominent sarcoplasmic masses in muscle biopsies and the constant heart involvement preceding significantly the onset of the myopathy. Though a rare etiology, FLNC gene should not be excluded in early-onset arrhythmia, even in the absence of myopathy, which occurs later in the disease course.

[The choice of a pediatric anesthesia ventilator]. / Comment choisir un respirateur d'anesthésie pédiatrique?

The technology of anesthesia ventilators has substantially progressed during last years. The choice of a pediatric anesthesia ventilator needs to be led by multiple parameters: requirement, technical (pneumatic performance, velocity of halogenated or oxygen delivery), cost (purchase, in operation, preventive and curative maintenance), reliability, ergonomy, upgradability, and compatibility. The demonstration of the interest of pressure support mode during maintenance of spontaneous ventilation anesthesia makes this mode essential in pediatrics. In contrast, the financial impact of target controlled inhalation of halogenated has not be studied in pediatrics. Paradoxically, complex and various available technologies had not been much prospectively studied. Anesthesia ventilators performances in pediatrics need to be clarified in further clinical and bench test studies.

Cyclic stretch reveals a mechanical role for intermediate filaments in a desminopathic cell model.

Mechanics is now recognized as crucial in cell function. To date, the mechanical properties of cells have been inferred from experiments which investigate the roles of actin and microtubules ignoring the intermediate filaments (IFs) contribution. Here, we analyse myoblasts behaviour in the context of myofibrillar myopathy resulting from p.D399Y desmin mutation which disorganizes the desmin IF network in muscle cells. We compare the response of myoblasts expressing either mutated or wild-type desmin to cyclic stretch. Cells are cultivated on supports submitted to periodic uniaxial stretch of 20% elongation amplitude and 0.3 Hz frequency. We show that during stretching cycles, cells expressing mutated desmin reduce their mean amplitude both for the elongation and spreading area compared to those expressing wild-type desmin. Even more unexpected, the reorientation angles are altered in the presence of p.D399Y desmin. Yet, at rest, the whole set of those parameters are similar for the two cell populations. Thus, we demonstrate that IFs affect the mechanical properties and the dynamics of cell reorientation. Since these processes are known due to actin cytoskeleton, these results suggest the IFs implication in mechanics signal transduction. Further studies may lead to better understanding of their contribution to this process.

First Report of Bacterial Canker of Walnut Caused by Brenneria nigrifluens in France.

Since the summer of 2000, vertical oozing cankers have been observed on trunks and branches of Persian walnut trees (Juglans regia). Cvs. Fernor, Chandler, Mayette, and Hartley were the most frequently affected, but cvs. Lara and Franquette could also be affected. Brenneria nigrifluens (synonym Erwinia nigrifluens) (3) was isolated from diseased trees from 13 orchards and nurseries in southwestern (Aquitaine, Périgord, Charentes, and Quercy), southeastern (Grenoble areas), and western (near Angers) France. Cankers were observed on trunks and branches where brown-to-black exudates staining the bark appeared mainly in the summer. Isolations were performed primarily from exudates but also from infected tissues by using King's medium B. Colonies similar in appearance to Brenneria nigrifluens (1) were purified and characterized. Gram reaction, Kovac's oxidase, oxidative-fermentative metabolism, and urease activity were assayed for all isolates. API Biotype 100 kits (BioMérieux, Marcy l'Etoile, France) were used as recommended, except that incubations were at 28°C for 4 days. When compared with the reference strain (French Collection of Plant Pathogenic Bacteria (CFBP) 4998T = National Collection of Plant Pathogenic Bacteria (NCPPB) 564T = American Type Culture Collection (ATCC) 13028T) from California, 14 isolated strains were identified as B. nigrifluens on the basis of physiological and biochemical characteristics. These 14 strains were deposited in the CFBP under Accession Nos. 6746 to 6759. Pathogenicity of three selected strains (CFBP 6746, 6747, and 6758) was confirmed by inoculating branches of 7-year-old walnut trees with 108 CFU of each isolate introduced in wounds (2). The reference strain (CFBP 4998T) and water were similarly inoculated as controls. Two and five months later, necrotic lesions were observed in the inner bark and dark lines were observed in internal wood, but no external cankers were observed on any trees inoculated with the local and reference strains. B. nigrifluens was reisolated from the dark lines in internal wood up to approximately 10 cm from the inoculation site. To our knowledge, this is the first report of this bacterium in France. References: (1) L. Hauben et al. Syst. Appl. Microbiol. 21:384, 1998. (2) M. Ridé and S. Ridé. Proc. Int. Conf. Plant Pathogenic Bacteria, 4th, 2:957, 1978. (3) E. E. Wilson et al. Phytopathology 47:669, 1957.

Functional characterization of the D-Tyr-tRNATyr deacylase from Escherichia coli.

The yihZ gene of Escherichia coli is shown to produce a deacylase activity capable of recycling misaminoacylated D-Tyr-tRNATyr. The reaction is specific and, under optimal in vitro conditions, proceeds at a rate of 6 s-1 with a Km value for the substrate equal to 1 microM. Cell growth is sensitive to interruption of the yihZ gene if D-tyrosine is added to minimal culture medium. Toxicity of exogenous D-tyrosine is exacerbated if, in addition to the disruption of yihZ, the gene of D-amino acid dehydrogenase (dadA) is also inactivated. Orthologs of the yihZ gene occur in many, but not all, bacteria. In support of the idea of a general role of the D-Tyr-tRNATyr deacylase function in the detoxification of cells, similar genes can be recognized in Saccharomyces cerevisiae, Caenorhabditis elegans, Arabidopsis thaliana, mouse, and man.

tRNA anticodon recognition and specification within subclass IIb aminoacyl-tRNA synthetases.

Subclass IIb aminoacyl-tRNA synthetases (Asn-, Asp- and LysRS) recognize the anticodon triplet of their cognate tRNA (GUU, GUC and UUU, respectively) through an OB-folded N-terminal extension. In the present study, the specificity of constitutive lysyl-tRNA synthetase (LysS) from Escherichia coli was analyzed by cross-mutagenesis of the tRNA(Lys) anticodon, on the one hand, and of the amino acid residues composing the anticodon binding site on the other. From this analysis, a tentative model is deduced for both the recognition of the cognate anticodon and the rejection of non-cognate anticodons. In this model, the enzyme offers a rigid scaffold of amino acid residues along the beta-strands of the OB-fold for tRNA binding. Phe85 and Gln96 play a critical role in this spatial organization. This scaffold can recognize directly U35 at the center of the anticodon. Specification of the correct enzyme:tRNA complex is further achieved through the accommodation of U34 and U36. The binding of these bases triggers the conformationnal change of a flexible seven-residue loop between strands 4 and 5 of the OB-fold (L45). Additional free energy of binding is recovered from the resulting network of cooperative interactions. Such a mechanism would not depend on the modifications of the anticodon loop of tRNA(Lys) (mnm5s2U34 and t6A37). In the model, exclusion by the synthetase of non-cognate anticodons can be accounted for by a hindrance to the positioning of the L45 loop. In addition, Glu135 would repulse a cytosine base at position 35. Sequence comparisons show that the composition and length of the L45 loop are markedly conserved in each of the families composing subclass IIb aminoacyl-tRNA synthetases. The possible role of the loop is discussed for each case, including that of archaebacterial aspartyl-tRNA synthetases.

Structure-function relationship of the Lrp-binding region upstream of lysU in Escherichia coli.

In Escherichia coli, one of the two genes encoding lysyl-tRNA synthetase, lysU, belongs to the regulon controlled by the leucine-responsive regulatory protein (Lrp). To map the site of Lrp action, mutants escaping regulation in rich medium were generated through random mutagenesis of the lysU promoter region. The mutations showed parallel effects on the strength of Lrp-DNA association, as measured in vitro by gel retardation experiments, and on the degree of repression of lysU expression by Lrp in vivo. In addition, DNase I and hydroxyl radical footprinting experiments indicated that several Lrp molecules bind to a DNA region of over 110 bp in a highly cooperative manner. This region, which encompasses the -35 box of the lysU promoter, was the target of all the mutations affecting the strength of the Lrp-DNA association. These mutations are frequently located in short A + T-rich runs distributed along the Lrp binding region with a periodicity of one helix turn. Because we could find such a regular alternance of A + T runs upstream of several other Lrp-regulated genes, we suggest that this pattern is one feature indicative of the binding of Lrp.

Escherichia coli leucine-responsive regulatory protein (Lrp) controls lysyl-tRNA synthetase expression.

Using random Tn10 insertion mutagenesis, we isolated an Escherichia coli mutant strain affected in the regulation of lysU, the gene encoding the inducible form of lysyl-tRNA synthetase. The transposon giving rise to the altered expression of lysU was found inserted within lrp. The latter gene codes for the leucine-responsive regulatory protein (Lrp) which mediates a global response of the bacterium to leucine. An involvement of Lrp in the regulation of lysU was searched for by using a lysU-lacZ operon fusion. The following conclusions were reached: (i) inactivation of lrp causes an increased activity of the lysU promoter, whatever the growth conditions assayed, (ii) insertion of a wild-type lrp gene into a multi-copy plasmid significantly reduces lysU expression, and (iii) sensitivity of the lysU promoter to the presence of leucine in the growth medium is abolished in the lrp context.