40 kilobase deletion (CF 40 kb del 4-10) removes exons 4 to 10 of the Cystic Fibrosis Transmembrane Conductance Regulator gene.
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Blotting, Southern↗
Biomedical subjects
M Mathieu
Publications and source records attributed to M Mathieu.
Dystonin is essential for maintaining neuronal cytoskeleton organization.
The mouse neurological mutant dystonia musculorum (dt) suffers from a hereditary sensory neuropathy. We have previously described the cloning and characterization of the dt gene, which we named dystonin (Dst). We had shown that dystonin is a neural isoform of bullous pemphigoid antigen 1 (Bpag1) with an N-terminal actin-binding domain. It has been shown previously that dystonin is a cytoskeletal linker protein, forming a bridge between F-actin and intermediate filaments. Here, we have used two different antibody preparations against dystonin and detected a high-molecular-weight protein in immunoblot analysis of spinal cord extracts. We also show that this high-molecular-weight protein was not detectable in the nervous system of all dt alleles tested. Immunohistochemical analysis revealed that dystonin was present in different compartments of neurons--cell bodies, dendrites, and axons, regions which are rich in the three elements of the cytoskeleton (F-actin, neurofilaments, and microtubules). Ultrastructural analysis of dt dorsal root axons revealed disorganization of the neurofilament network and surprisingly also of the microtubule network. In this context it is of interest that we observed altered levels of the microtubule-associated proteins MAP2 and tau in spinal cord neurons of different dt alleles. Finally, dt dorsal root ganglion neurons formed neurites in culture, but the cytoskeleton was disorganized within these neurites. Our results demonstrate that dystonin is essential for maintaining neuronal cytoskeleton integrity but is not required for establishing neuronal morphology.
Animals↗
Organization of atrial natriuretic factor-like immunoreactive system in the brain of the frog Rana esculenta during development.
Immunocytochemical distribution of the atrial natriuretic factor (ANF) has been studied in the brain and pituitary of the anuran Rana esculenta during development and in juvenile animals. Using human ANF and rat alpha-ANF antisera, immunoreactive cell bodies and nerve fibers were revealed in stage II-III tadpoles and in successive larval stages. Soon after hatching, stages II-III, the ANF-like-immunoreactive elements were confined to the preoptic area-median eminence complex. During successive stages of development, new groups of ANF-immunoreactive cell bodies appeared. In larval stage VI, immunoreactive perikarya were found in the rostral part of the anteroventral area of the thalamus and numerous ANF-like-immunoreactive cells appeared in the pars distalis of the pituitary. In larval stages XIV and XVIII, the distribution of ANF immunoreactivity was virtually similar. The ANF-immunoreactive cells in the preoptic nucleus and in the pituitary pars distalis were comparatively more abundant than in stage VI. During the metamorphic climax (stages XXI-XXII), a new group of ANF-immunoreactive cell bodies appeared in the rostral part of the ventrolateral area of the thalamus. During this stage, ANF-immunoreactive fiber projections were found in the pars intermedia for the first time. However, the pars distalis cells were very weakly immunofluorescent. The pattern of ANF immunoreactivity in the brain of juvenile animals was very similar to that described for stages XXI and XXII, whereas the pars distalis cells showed no immunoreactivity. It is conceivable that, early during development, ANF-related peptides may be involved in the regulation of pituitary secretion by means of autocrine mechanisms or may act as a classic pituitary hormone.
Animals↗
The cell cycle gene SKP1 is regulated by light in postnatal rat brain.
During the early postnatal phase of high neuronal plasticity, an altered visual input leads to great modifications of visual cortex organization [Y. Frégnac, M. Imbert, Development of neuronal selectivity in primary visual cortex of cat, Physiol. Rev., 64 (1984) 375-434; D.H. Hubel, T.N. Wiesel, S. LeVay, Plasticity of ocular dominance columns in monkey striate cortex, Philos. Trans. R. Soc. London, Ser. B, 278 (1977) 377-409.]. We used refined differential screening of an organized cDNA library to identify the genes that may participate in this plasticity. We isolated a candidate plasticity gene encoding for a 163 aa protein that is closely related to the human and yeast Skp1p, a key factor in cell cycle progression [C. Baï, K. Hofman, L. Ma, M. Goebl, J.W. Harper, S.J. Elledge, SKP1 connects cell cycle regulators to the ubiquitin proteolysis machinery through a novel motif, the F-box, Cell, 86 (1996) 263-274; C. Connelly, P. Hieter, Budding yeast SKP1 encodes an evolutionary conserved kinetochore protein required for cell cycle progression, Cell, 86 (1996) 275-285; H. Zhang, R. Kobayashi, K. Galaktionov, D. Beach, p19Skp1 and p45Skp2 are essential elements of the cyclin A-CDK2 S phase kinase, Cell, 82 (1995) 915-925.]. Northern blot analysis showed that the expression of SKP1 (Skp1p gene) dramatically decreased after 2 h of light stimulation in the visual cortex of young dark-reared rats. This down regulation lasted at least 72 h. It was specific for the critical period as we did not observe any significant regulation of SKP1 mRNA by light in adult dark-reared rat brain. The down regulation was observed in the superior colliculus but also in the frontal cortex and in the hippocampus. The fact that this down regulation was not restricted to the visual system, suggested that it could be produced by dark rearing-induced hormonal changes. The significance of SKP1 expression in the brain and its regulation are discussed.
Animals↗
Crystallization and preliminary X-Ray analysis of rotavirus protein VP6.
As a first step to gain insight into the structure of the rotavirus virion at atomic resolution, we report here the expression, purification, and crystallization of recombinant rotavirus protein VP6. This protein has the property of polymerizing in the form of tubular structures in solution which have hindered crystallization thus far. Using a combination of electron microscopy and small-angle X-ray scattering, we found that addition of Ca2+ at concentrations higher than 100 mM results in depolymerization of the tubes, leading to an essentially monodisperse solution of trimeric VP6 even at high protein concentrations (higher than 10 mg/ml), thereby enabling us to search for crystallization conditions. We have thus obtained crystals of VP6 which diffract to better than 2.4 A resolution and belong to the cubic space group P4132 with a cell dimension a of 160 A. The crystals contain a trimer of VP6 lying along the diagonal of the cubic unit cell, resulting in one VP6 monomer per asymmetric unit and a solvent content of roughly 70%.
Animals↗
Glucocorticoid insensitive asthma: a one year clinical follow up pilot study.
BACKGROUND: Glucocorticoid resistant or insensitive asthmatic subjects are usually defined as patients whose baseline pre-bronchodilation forced expiratory volume in one second (FEV1) of less than 70-80% predicted improves significantly in response to beta 2 agonists but by less than 15% following 1-2 weeks of 40 mg prednisolone daily. Since there is little long term clinical information on these patients, a one year prospective study was performed to assess whether glucocorticoid sensitivity may vary over time. METHODS: Nineteen severe asthmatic subjects were studied and received 40 mg prednisolone daily for seven days. Prednisolone was given for a further seven days in glucocorticoid insensitive asthmatics and then stopped. Patients were followed up for one year and the glucocorticoid test was repeated on five patients in each group six months later. RESULTS: Eleven patients were classified as glucocorticoid insensitive and eight as glucocorticoid sensitive on day 7. The demographic characteristics of the patients were similar in both groups. Four glucocorticoid insensitive patients became responsive after one further week of prednisolone treatment. Six months later, four of five glucocorticoid sensitive patients and three of five previously glucocorticoid insensitive patients were glucocorticoid sensitive. CONCLUSIONS: Glucocorticoid sensitivity varies over time.
Adult↗
Dystonin is an essential component of the Schwann cell cytoskeleton at the time of myelination.
A central role for the Schwann cell cytoskeleton in the process of peripheral nerve myelination has long been suggested. However, there is no genetic or biological evidence as yet to support this assumption. Here we show that dystonia musculorum (dt) mice, which carry mutations in dystonin, a cytoskeletal crosslinker protein, have hypo/amyelinated peripheral nerves. In neonatal dt mice, Schwann cells were arrested at the promyelinating stage and had multiple myelinating lips. Nerve graft experiments and primary cultures of Schwann cells demonstrated that the myelination abnormality in dt mice was autonomous to Schwann cells. In culture, dt Schwann cells showed abnormal polarization and matrix attachment, and had a disorganized cytoskeleton. Finally, we show that the dt mutation was semi-dominant, heterozygous animals presenting hypo- and hyper-myelinated peripheral nerves. Altogether, our results suggest that dt Schwann cells are deficient for basement membrane interaction and demonstrate that dystonin is an essential component of the Schwann cell cytoskeleton at the time of myelination.
Animals↗
Prognostic significance of supraclavicular lymph nodes in small cell lung cancer: a study from four consecutive clinical trials, including 1,370 patients. "Petites Cellules" Group.
STUDY OBJECTIVE: To determine the prognostic significance of supraclavicular lymph node (SCLN) involvement in small cell lung cancer. MATERIALS AND METHODS: Patients (1,370) with small cell lung cancer were included in four consecutive clinical trials and classified as having either limited or extensive forms of disease using the Veterans Administration staging system. RESULTS: SCLN was present in 17% of patients (n = 234). Median survival was 258 days for patients with SCLN (n = 234) and 297 days for patients without SCLN (n = 1136) (p = 0.002). SCLN involvement was correlated with the presence of distant metastases at baseline (169 vs 65, p = < 0.001). Median survival was 375 days for patients with limited forms without SCLN (n = 529), 332 days for those with limited forms with SCLN (n = 65) (p = 0.12), 244 days for those with extensive forms without SCLN (n = 604), and 228 days for those with extensive forms with SCLN (n = 169) (p = 0.94). The 2-year survival rates were 17%, 12%, 2%, and 4%, respectively. Cox models confirmed that SCLN did not provide any significant additional prognostic information. CONCLUSION: SCLN is highly correlated with extensive forms explaining its overall prognostic value. In limited disease, SCLN is only a minor poor prognostic factor, not justifying any amendment to the staging system currently used.
Adult↗
[Evaluation of clinical efficacy of a venotonic drug: lessons of a therapeutic trial with hemisynthesis diosmin in "heavy legs syndrome"].
Venous-type symptoms, i.e. painful sensation of heavy, swollen or restless legs, influenced by orthostatism and warm environment, significantly alters quality of life of a large proportion of women. Although the condition is frequently associated with chronic venous insufficiency, no demonstrable hemodynamic abnormality of the superficial as well as deep venous systems of the lower limbs can be found in many patients. The pathogenesis of this syndrome remains unknown, and there is no objective measurement of any biological nor hemodynamic parameters that can be used for its assessment. Diosmine and other flavane derivatives have been shown beneficial in this condition using various discomfort indexes. The aim of this work was to compare the therapeutic efficacy of two formulations of the same compound diosmine. In the analysis, particular attention was paid to the signification and usefulness of discomfort scales. This study was a double-blind, placebo-controlled therapeutic trial, comparing the efficacy of a new formulation, hemisynthesis diosmine 600 mg, one tablet per day taken in the morning, versus the usual tablet formulation of 300 mg taken twice a day (morning and evening). Treatment blindness was assured by the double placebo method. Two parallel groups were treated 28 days with one or the other treatment. Randomization was performed with stratification by center. The main evaluation criteria were a composite scale of venous type symptoms (i.e. the sum of individual score 0-4 of each symptom), and a visual analog auto-evaluation scale quoted each week by the patient. The global opinion of the physician on treatment adequacy to the clinical situation, and the degree of patient satisfaction (four grade scales) were used as subsidiary criteria. In order to increase the homogeneity of the study sample, inclusion was restricted to non-menopaused women aged 18 years and over, having given written informed consent, complaining of distressing sensation of heavy legs, without history of venous thrombosis, varicose veins, superficial or deep venous reflux at the duplex-scan examination. Patients with other causes of pain in the lower limbs, taking analgesic medications or requiring elastic stocking were not included. 255 patients participated in the trial. Eighteen withdrew, equally distributed in both groups (6 lost, 5 interfering diseases, and two dropouts for side effects, namely headache and gastric pain). Twenty additional patients complained of detrimental events not requiring treatment withdrawal, equally distributed between both groups, and mainly involving digestive functions. The results confirmed a similar efficacy of the two drug regimens, and a small but significantly better improvement of the patients' auto-evaluation of their discomfort on the analogic scale (p = 0.021) for hemisynthesis diosmine 600 mg, mainly during the first two weeks; for all four criteria, the gamma risk showed that the once-a-day 600 mg preparation at least as effective as 300 mg twice daily (p < 0.001). On a methodological point of view, the comparison of evaluation criteria showed that the composite scale, although giving the feeling of a comprehensive and quantitative appraisal of the discomfort in the legs, was almost equivalent to a standard four grade rating of heaviness, which appeared as the central symptom of this condition. Auto-evaluation through an analogic scale proved to be more informative, more sensitive, less influenced by the physician's feelings and allow easier assessment of the time-course of the drug's effects. Global evaluations by the patient and the physician did not give additional information but could be used as quality criteria, assessing the coherence of the results obtained with the scales. This study demonstrated a similar efficacy of the two drug regimens, with a more rapid effectiveness of the 600 mg preparation taken once a day. Auto-evaluation on the analog scale proved to be the most informative and effe
Adult↗
[Corticoid therapy and bronchial inflammation in asthma. The use of bronchial biopsy].
The development of fibreoptic bronchoscopy has enabled significant progress in the understanding of the pathogenesis of asthma. It has brought to the fore the importance of bronchial inflammation even in asymptomatic patients and/or in patients who have only mild disease. The practice of bronchial biopsy in vivo is an excellent method of studying bronchial inflammation. The purpose of this general review is to recall the value of bronchial biopsies in the understanding of the effects of steroids on asthma: effects on the epithelium, the basement membrane and the blood vessels. Their cellular contents consist equally of cytokines, enzymes and adhesion molecules. At the level of the bronchial epithelium steroid therapy engenders a diminution in eosinophils, mast cells an lymphocytes. It restores the ratio of ciliated to other cells back to normal and increases the number of nerve synapses. Regarding the interstitium the corticoids diminish the number of eosinophils, mast cells and T lymphocytes. The effect on different lymphocyte subtypes is controversial, as is the effect of the basal membrane. Steroid therapy diminishes the proteins, GM-CSF.RANTES and IL-8 as well as the messengers IL-4, IL-13 and IL-5. It seems to increase the messengers for IFN-gamma and IL-12 and favourably modulates the vascular composition to inflammation in asthma. Nevertheless it is to be regretted that too few studies have looked at the correlations between histological changes and clinical and respiratory function improvement engendered by steroid therapy.
Adrenal Cortex Hormones↗
[Contributions and expectations of molecular genetics in perinatal neurologic diagnosis].
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Central Nervous System Diseases↗
The 1.8 A crystal structure of the dimeric peroxisomal 3-ketoacyl-CoA thiolase of Saccharomyces cerevisiae: implications for substrate binding and reaction mechanism.
The dimeric, peroxisomal 3-ketoacyl-CoA thiolase catalyses the conversion of 3-ketoacyl-CoA into acyl-CoA, which is shorter by two carbon atoms. This reaction is the last step of the beta-oxidation pathway. The crystal structure of unliganded peroxisomal thiolase of the yeast Saccharomyces cerevisiae has been refined at 1.8 A resolution. An unusual feature of this structure is the presence of two helices, completely buried in the dimer and sandwiched between two beta-sheets. The analysis of the structure shows that the sequences of these helices are not hydrophobic, but generate two amphipathic helices. The helix in the N-terminal domain exposes the polar side-chains to a cavity at the dimer interface, filled with structured water molecules. The central helix in the C-terminal domain exposes its polar residues to an interior polar pocket. The refined structure has also been used to predict the mode of binding of the substrate molecule acetoacetyl-CoA, as well as the reaction mechanism. From previous studies it is known that Cys125, His375 and Cys403 are important catalytic residues. In the proposed model the acetoacetyl group fits near the two catalytic cysteine residues, such that the oxygen atoms point towards the protein interior. The distance between SG(Cys125) and C3(acetoacetyl-CoA) is 3.7 A. The O2 atom of the docked acetoacetyl group makes a hydrogen bond to N(Gly405), which would favour the formation of the covalent bond between SG(Cys125) and C3(acetoacetyl-CoA) of the intermediate complex of the two-step reaction. The CoA moiety is proposed to bind in a groove on the surface of the protein molecule. Most of the interactions of the CoA molecule are with atoms of the loop domain. The three phosphate groups of the CoA moiety are predicted to interact with side-chains of lysine and arginine residues, which are conserved in the dimeric thiolases.
Acetyl-CoA C-Acyltransferase↗
N-terminal arm exchange is observed in the 2.15 A crystal structure of oxidized nitrite reductase from Pseudomonas aeruginosa.
BACKGROUND: Nitrite reductase from Pseudomonas aeruginosa (NiR-Pa) is a dimer consisting of two identical 60 kDa subunits, each of which contains one c and one d1 heme group. This enzyme, a soluble component of the electron-transfer chain that uses nitrate as a source of energy, can be induced by the addition of nitrate to the bacterial growth medium. NiR-Pa catalyzes the reduction of nitrite (NO2-) to nitric oxide (NO); in vitro, both cytochrome c551 and azurin are efficient electron donors in this reaction. NiR is a key denitrification enzyme, which controls the rate of the production of toxic nitric oxide (NO) and ultimately regulates the release of NO into the atmosphere. RESULTS: The structure of the orthorhombic form (P2(1)2(1)2) of oxidized NiR-Pa was solved at 2.15 A resolution, using molecular replacement with the coordinates of the NiR from Thiosphaera pantotropha (NiR-Tp) as the starting model. Although the d1-heme domains are almost identical in both enzyme structures, the c domain of NiR-Pa is more like the classical class I cytochrome-c fold because it has His51 and Met88 as heme ligands, instead of His17 and His69 present in NiR-Tp. In addition, the methionine-bearing loop, which was displaced by His17 of the NiR-Tp N-terminal segment, is back to normal in our structure. The N-terminal residues (5/6-30) of NiR-Pa and NiR-Tp have little sequence identity. In Nir-Pa, this N-terminal segment of one monomer crosses the dimer interface and wraps itself around the other monomer. Tyr10 of this segment is hydrogen bonded to an hydroxide ion--the sixth ligand of the d1-heme Fe, whereas the equivalent residue in NiR-Tp, Tyr25, is directly bound to the Fe. CONCLUSIONS: Two ligands of hemes c and d1 differ between the two known NiR structures, which accounts for the fact that they have quite different spectroscopic and kinetic features. The unexpected domain-crossing by the N-terminal segment of NiR-Pa is comparable to that of 'domain swapping' or 'arm exchange' previously observed in other systems and may explain the observed cooperativity between monomers of dimeric NiR-Pa. In spite of having similar sequence and fold, the different kinetic behaviour and the spectral features of NiR-Pa and NiR-Tp are tuned by the N-terminal stretch of residues. A further example of this may come from another NiR, from Pseudomonas stutzeri, which has an N terminus very different from that of the two above mentioned NiRs.
Amino Acid Sequence↗
Screening infants for neuroblastoma: discovery of alkaptonuria in one case.
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Alkaptonuria↗
[Study of plasma acylcarnitines using tandem mass spectrometry. Application to the diagnosis of metabolism hereditary diseases].
BACKGROUND: L-carnitine is known to transport long chain fatty acids through the mitochondrial membrane but also to export accumulated acyl-CoA's as acylcarnitine esters. Acylcarnitine identification in body fluids allows the diagnosis of mitochondrial inborn errors especially fatty oxidation defects. Tandem mass spectrometry represents a new method for isolation and identification of acylcarnitines in plasma or in blood spotted onto filter paper (Guthrie cards). MATERIAL AND METHODS: In order to validate our method, we studied 30 plasmas from children affected with 15 different inborn errors of metabolism and five amniotic fluids from fetuses affected with several organic acidurias. Fourty-six samples from children at risk for mitochondrial fatty oxidation disorders have been analyzed. We developed a method of tandem mass spectrometry with liquid secondary ion mass spectrometry using deuterated acylcarnitines as internal standards. RESULTS: This method is very sensitive (detection limit = 2 microM). In all affected patients specific acylcarnitine signals corresponding to the metabolic block were constantly found. This confirms the diagnosis and validates the method. Among the 46 at risk children, four defects of long chain fatty acid oxidation were identified. CONCLUSION: This new method is of great interest especially for the long chain fatty acid oxidation defects. These defects are very difficult to diagnose with classical methods as urinary organic acid profiling. A small amount of plasma (100 microL) or blood spotted onto paper is required. The acylcarnitine profile allows a rapid diagnosis if a dedicated apparatus is available.
Acyl Coenzyme A↗
Diagnosis of inborn errors of metabolism by acylcarnitine profiling in blood using tandem mass spectrometry.
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Carnitine↗
Gene therapy strategies for asthma.
Asthma is a disease which is increasing in frequency and severity despite the incontestable advances in the understanding of its physiology and treatment. New therapeutic strategies are therefore required and at the time when a new treatment, that of gene therapy, is giving hope for the treatment of so far incurable illnesses, it would seem to us to be useful to discuss the possible use of this treatment in severe asthma and in particular in the case of the most severe asthmatic patients requiring continuous oral steroid treatment (referred to as steroid-dependent). These patients are those who require particular medical attention, they often need to be admitted to hospital and they represent over 50% of the total costs associated with asthma. No satisfactory alternative therapy is currently available. In this regard, a variety of gene-based strategies have recently been proposed for inflammatory and immunologic disorders. It is thus the purpose of this review to consider its application to asthma therapy, although there are very few published works that directly bear on gene therapy linked to asthma. After a brief outline of the epidemiology of asthma, its genetics and the available animal models, we will focus on its immunopathology slanting the discussion towards the possibility of a gene-based treatment.
Asthma↗
Clustering of mutations responsible for branchio-oto-renal (BOR) syndrome in the eyes absent homologous region (eyaHR) of EYA1.
Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder, characterised by the association of branchial, otic and renal anomalies with variable degrees of severity. We have recently identified EYA1 , a human homologue of the Drosophila eyes absent gene, as the gene underlying this syndrome. The products of both genes share a highly conserved 271 amino acid C-terminal region (eyaHR). The eyaHR was also found in the products of two other human genes (EYA2 and EYA3), demonstrating the existence of a novel gene family. We report here on the complete genomic structure of EYA1. This gene consists of 16 coding exons and extends over 156 kb. It encodes various alternatively spliced transcripts differing only in their 5' regions. Sequence analysis of the entire EYA1 coding region was performed for 20 unrelated patients affected by BOR syndrome, and six novel mutations were identified. Among these mutations, two are missense mutations, highlighting amino acid residues essential for the function of the EYA1 protein, and one mutation comprises a de novo Alu insertion into an exon. This insertion presumably occurs by retrotransposition, and the mobile Alu element has a poly(A) tail that is unstable throughout generations. To date, 14 mutations have been detected in BOR patients, all of which are different. However, all the mutations are located within or in the immediate vicinity of the eyaHR; the significance of this clustering is discussed.
Amino Acid Sequence↗