Molecular and genetic characterisation of German families with paramyotonia congenita and demonstration of founder effect in the Ravensberg families

Eighteen German families with a history of paramyotonia congenita (PC) were characterised by genetic und mutational analysis at the SCN4A locus, which encodes the -subunit of the adult skeletal muscle sodium channel. We concentrated our analysis primarily on these families to test the hypothesis that a predominance of one common mutation occurs in all German PC families and that this mutation arose in a common ancestor originating in the North-West of the country. The present eighteen PC families exhibit two different mutations (R1448C and R1448H) on various SCN4A dinucleotide repeat haplotypes and therefore the majority of the mutations probably occurred independently. However, the R1448H mutation is extremely frequent in the North-West of Germany (Ravensberger Land) on a specific SCN4A microsatellite haplotype, indicating a founder effect within this subpopulation. Our results suggest that the R1448C/R1448H mutations are by far the most common to be associated with the PC phenotype in the German population.This paper is dedicated to Professor Dr. Dr. Peter Emil Becker on the occasion of this 85th birthday     

Prevention of Carbon Tetrachloride-Induced Lipid Peroxidation in Liver Microsomes from Dehydroepiandrosterone-Pretreated Rats

Dehydroepiandrosterone (DHEA), a lipid soluble steroid, administered to rats (100 mg/kg b.wt) by a single intraperitoneal injection, increases to twice its normal level in the liver microsomes. Microsomes so enriched become resistant to lipid peroxidation induced by incubation with carbon tetrachloride in the presence of a NADPH-regenerating system: also the lipid peroxidation-dependent inactivation of glucose-6-phosphatase and gamma-glutamyl transpetidase due to the haloalkane are prevented. Noteworthy, the liver microsomal drug-metabolizing enzymes and in particular the catalytic activity of cytochrome P₄₅₀IIE1, responsible for the CCl₄-activation, are not impaired by the supplementation with the steroid. Consistently, in DHEA-pretreated microsomes the protein covalent binding of the trichloromethyl radical (CCl₃°), is similar to that of not supplemented microsomes treated with CCl₄. It thus seems likely that DHEA protects liver microsomes from oxidative damage induced by carbon tetrachloride through its own antioxidant properties rather than inhibiting the metabolism of the toxin.     

Cellular localization and cytochemical probing of resistance reactions to arbuscular mycorrhizal fungi in a ‘locus a’ myc− mutant of Pisum sativum L.

Pisum sativum L. myc^– mutants which fail to form arbuscular mycorrhiza have recently been identified amongst nod^– mutants (Duc et al., 1989, Plant Sci. 60, 215–222). The reason for this resistance to symbiotic fungi has been investigated in the case of a locus a mutant (P2) inoculated with Glomus mosseae (Nicol. and Gerd.) Gerd, and Trappe. The fungal symbiont formed viable appressoria in contact with the root surface but its development was stopped at the root epidermis. Abundant material was deposited on the inner face of root cell walls adjacent to the appressoria in the P2 mutant, but not in the wild-genotype parent cultivar (Frisson) forming a symbiotic mycorrhizal infection. Fluorescence, histochemical, cytochemical and immunocytological approaches were used to characterize the paramural deposits in epidermal and hypodermal cells of the mutant. Strong fluorescence under blue light indicated the accumulation of phenolic compounds although polymers like lignin or suberin were not localized. Proteins and glycoproteins were homogeneously distributed within the paramural deposits. In the latter, the periodic acid-thiocarbohydrazide-silver proteinate (PATAg) reaction for 1,4-polysaccharide detection showed a heterogeneous composition with electron-dense points surrounded by non-reactive material, but cytological tests for cellulose and pectin gave weak responses as compared to epidermal and hypodermal walls of the wild genotype. -1,3-Glucans indicative of callose were detected by in-situ immunolocalization in the paramural deposits below appressoria on mutant roots, but not in walls of the wild genotype. Thus, appressorium formation by G. mosseae on roots of the locus a P. sativum mutant elicits wall modifications usually associated with activation of defence responses to pathogens. It is proposed that this locus must be involved in a key event in symbiotic infection processes in P. sativum, and the possible role of complex regulatory interactions between symbiosis and defence genes in endomycorrhiza development is discussed.Abbreviations DAPI 4,6-diamino-2-phenylindole - FDA fluo-rescein diacetate - PATAg periodic acid-thiocarbohydrazide-silver proteinate The authors are grateful to C. Arnould for technical assistance, K. Niehaus for the purified Sirofluor, K. Roberts for the AFRC JIM5 antibody and J. Lherminier (INRA, Dijon, France), for useful discussion. This collaborative research programme was financially supported by MRT, INRA, EPR-Bourgogne (grant to A.G., Contrat de Plan project 3060A), EEC COST ACTION 8.10 (Endomycorrhizas) and the National Research Council of Italy, Special Project RAISA, Sub-project N.2, Paper N. 801     

Plasma esterase activities in rats fed magnesium-deficient diets

In a study with rats it was determined whether dietary magnesium concentration affects plasma esterase activities. The feeding of a diet with 0.01% (w/w) instead of 0.04% magnesium reduced plasma magnesium concentration by 50%. Plasma total esterase, arylesterase and butyrylcholinesterase activities were significantly decreased in the magnesium-deficient rats. In rats fed a diet containing 0.02% magnesium, plasma magnesium concentration was lowered by 30%, and group mean plasma total esterase activity was decreased, but not the activities of arylesterase and butyrylcholinesterase.     

Tetanus toxin selectively impairs anti-tumoral but not anti-microbial macrophage-mediated effector functions

Abstract The present study was designed to establish the susceptibility of macrophage-mediated effector functions to tetanus toxin (TT). Using the murine macrophage cell line, GG2EE, generated in vitro by v- raf /v- myc oncogenes, we have previously provided evidence that TT selectively inhibits interferon gamma (IFN-γ), but not basal, lysozyme activity. Here we show that while neither phagocytic nor candidacidal activities are affected by TT treatment, antitumoral activity is significantly impaired after exposure to TT. This phenomenon, which is dose-dependent, is fully ascribed to the holotoxin, as heat inactivated TT, C or A-B fragments result ineffective. Furthermore, C but not A-B fragment competes with TT in abrogating its inhibitory effects. Overall, these data indicate that TT is not a broad-spectrum, down-regulating signal on macrophage-mediated functions, thus implying that its toxic action is exerted on specific molecular targets.     

Compatible and Incompatible Interactions Between Callus Tissue and Mycorrhizal or Pathogenic Fungi

Tissue cultures of Nicotiana tabacum were utilized to investigate the mechanisms associated with host specificity and non-host incompatibility in mycorrhizal and pathogenic fungi. They were tested for expression of resistance to different species of mycorrhizal fungi and to a fungal pathogen of tobacco, Thielaviopsis basicola , by monitoring the production of callose, phenolic compounds and peroxidases in dual cultures. Tobacco cells reacted to the presence of all the mycorrhizal fungi with callose deposits, whereas callose was nearly always absent in tobacco cells inoculated with their pathogen T. basicola. The broad-host range ectomycorrhizal fungi Hebeloma crustuliniforme, Lac-caria laccata and Suilhis granulatus elicited less intense responses than did Hymenoscyphus ericae. The results obtained for phenolic production and peroxidase activity were consistently similar to those obtained for callose deposition. They showed that H. ericae , an endomycorrhizal symbiont of Ericaceae, was highly incompatible with tobacco cells and that the tobacco pathogen T. basicola did not elicit strong reactions in the cells of its host. In this paper, the possibility of utilizing callus cultures as a simple model system to study both the different degrees of compatibility and the early events of recognition between mycorrhizal fungi and their host or non-host plants is discussed.     

Molecular and chromosomal evolution in anoas (Bovidae: Bubalus spec.)

As a contribution to their taxonomy, population genetic data on zoo-living anoas are reported, and a review of the history of the captive stock is provided. Four different chromosome numbers of 44, 45, 47 and 48 chromosomes have been found, respectively, when karyotyping captive anoas descending from three breeding lines. The number of chromosome arms is 60 throughout, indicating that Robertsonian rearrangements are responsible for this cytogenetic variation. An electrophoretic comparison of isozymes and blood proteins representing 21 genetic loci revealed polymorphism in seven loci: haemoglobin, glyoxalase, superoxide dismutase, phosphoglucomutase, carbonic anhydrase, glucose phosphate isomerase, and an unidentified acid serum protein. Considering the small number of founder specimens and subsequent inbreeding, allozyme variability appears fairly high in anoas. Genetic distances between zoo populations amount to 0.0505 or less. Southern blot hybridizations of restricted DNA from anoas and African buffaloes with a probe from the DRB-like region of the chimpanzee's MHC class II genes also indicate a low degree of genetic differentiation between mountain and lowland anoas. The relevance of these genetic data for the taxonomic classification of mountain and lowland anoas, and for the conservation of anoas by captive breeding is discussed.     

Genetic Diversity of Isolates of Glomus mosseae from Different Geographic Areas Detected by Vegetative Compatibility Testing and Biochemical and Molecular Analysis

We detected, for the first time, the occurrence of vegetative incompatibility between different isolates of the arbuscular mycorrhizal fungal species Glomus mosseae. Vegetative compatibility tests performed on germlings belonging to the same isolate showed that six geographically different isolates were capable of self-anastomosing, and that the percentage of hyphal contacts leading to fusions ranged from 60 to 85%. Successful anastomoses were characterized by complete fusion of hyphal walls, protoplasm continuity and occurrence of nuclei in the middle of hyphal bridges. No anastomoses could be detected between hyphae belonging to different isolates, which intersected without any reaction in 49 to 68% of contacts. Microscopic examinations detected hyphal incompatibility responses in diverse pairings, consisting of protoplasm retraction from the tips and septum formation in the approaching hyphae, even before physical contact with neighboring hyphae. Interestingly, many hyphal tips showed precontact tropism, suggesting that specific recognition signals may be involved during this stage. The intraspecific genetic diversity of G. mosseae revealed by vegetative compatibility tests was confirmed by total protein profiles and internal transcribed spacer-restriction fragment length polymorphism profiles, which evidenced a higher level of molecular diversity between the two European isolates IMA1 and BEG25 than between IMA1 and the two American isolates. Since arbuscular mycorrhizal fungi lack a tractable genetic system, vegetative compatibility tests may represent an easy assay for the detection of genetically different mycelia and an additional powerful tool for investigating the population structure and genetics of these obligate symbionts.     

Insertion mutations at the maizeOpaque2 locus induced by transposable element familiesAc, En/Spm andBg

Eight independently isolated unstable alleles of theOpaque2 (O2) locus were analysed genetically and at the DNA level. The whole series of mutations was isolated from a maize strain carrying a wild-typeO2 allele and the transposable elementActivator (Ac) at thewx-m7 allele. Previous work with another unstable allele of the same series has shown that it was indeed caused by the insertion of anAc element. Unexpectedly, the remaining eight mutations were not caused by the designatedAc element, but by other insertions that are structurally similar or identical to one of two different autonomous transposable elements. Six mutations were caused by the insertion of a transposable element of theEnhancer/Suppressor-Mutator (En/Spm) family. Two mutations were the result of the insertion of a transposable element of theBergamo (Bg) family. Genetic tests carried out with plants carrying the unstable mutations demonstrated that all were caused by the insertion of an autonomous transposable element.     

Novel nuclear ribonucleoprotein structural components in the dormouse adrenal cortex during hibernation

Adrenocortical cell nuclei of the dormouse Muscardinus avellanarius were investigated by electron microscopic immunocytochemistry in hibernating, arousing and euthermic individuals. While the basic structural constituents of the cell nucleus did not significantly were found in nuclei of hibernating dormice. Lattice-like bodies (LBs), clustered granules (CGs), fibrogranular material (FGM) and granules associated with bundles of nucleoplasmic fibrils (NF) all contained ribonucleoproteins (RNPs), as shown by labeling with anti-snRNP (small nuclear RNP), anti-m₃G-capped RNA and anti-hnRNP (heterogeneous nuclear RNP) antibodies. Moreover, the FGM also showed immunoreactivity for the proliferation associated nuclear antigen (PANA) and the non-snRNP splicing factor SC-35. All these nuclear structural components disappeared early during arousal and were not found in euthermic animals. These novel RNP-containing structures, which have not been observed in other tissues investigated so far in the same animal model, could represent storage and/or processing sites for pre-mRNA during the extreme metabolic condition of hibernation, to be quickly released upon arousal. NFs, which had been sometimes found devoid of associated granules in nuclei of brown adipose tissue from hibernating dormice, were present in much higher amouts in adrenocortical cell nuclei; they do not contain RNPs and their role remains to be elucidated. The possible roles of these structures are discussed in the frame of current knowledge of morpho-functional relationships in the cell nucleus.