Heterozygosity for a Hypomorphic Polβ Mutation Reduces the Expansion Frequency in a Mouse Model of the Fragile X-Related Disorders

The Fragile X-related disorders (FXDs) are members of the Repeat Expansion Diseases, a group of human genetic conditions resulting from expansion of a specific tandem repeat. The FXDs result from expansion of a CGG/CCG repeat tract in the 5’ UTR of the FMR1 gene. While expansion in a FXD mouse model is known to require some mismatch repair (MMR) proteins, our previous work and work in mouse models of another Repeat Expansion Disease show that early events in the base excision repair (BER) pathway play a role in the expansion process. One model for repeat expansion proposes that a non-canonical MMR process makes use of the nicks generated early in BER to load the MMR machinery that then generates expansions. However, we show here that heterozygosity for a Y265C mutation in Polβ, a key polymerase in the BER pathway, is enough to significantly reduce both the number of expansions seen in paternal gametes and the extent of somatic expansion in some tissues of the FXD mouse. These data suggest that events in the BER pathway downstream of the generation of nicks are also important for repeat expansion. Somewhat surprisingly, while the number of expansions is smaller, the average size of the residual expansions is larger than that seen in WT animals. This may have interesting implications for the mechanism by which BER generates expansions.     

Frequency of the cystic fibrosis ΔF508 mutation in a large sample of the French population

Summary We have determined the frequency of the cystic fibrosis (CF) ΔF508 mutation in a large sample of CF patients originating from different areas of France, including the greater Paris, Brittany, Alsace, Lorraine and Rh?ne-Alpes regions. A total of 422 CF chromosomes were studied, and the defect was found to account for 75% of the mutant alleles. In the course of the survey, a rare nucleotide sequence polymorphism leading to an isoleucine to valine substitution at position 506 of the CF transmembrane conductance regulator protein has been characterized in an unaffected individual. Our data enable the evaluation of the probabilities that a chromosome negative for the ΔF508 mutation carriers another CF defect.     

Frequency Dependence of Electrical Coupling in Deiters″ Cells of the Guinea Pig Cochlea

Immunolabeling with antibodies against connexins 26 and 30 showed that, in the guinea pig cochlea, supporting Deiters″ cells are massively interconnected and form an orderly network within the organ of Corti. In paired patch-clamp recordings the coupling ratio (CR) of adjacent Deiters″ cells at the apex of the cochlea (～0.31) was 3-fold smaller than in isolated cell pairs due to shunting afforded by multicellular connectivity. With sinusoidal current stimuli the delay in signal propagation between adjacent cells increased with increasing frequency whereas the amplitude did not change significantly up to 200 Hz (corner frequency F_c ～220 Hz). Depolarizing voltage commands applied to an outer hair cell (OHC) elicited outward potassium currents in the OHC and inward currents in the abutting Deiters″ cells, supplying direct evidence for potassium buffering in the organ of Corti. Computational analysis indicates that electrical signals injected into a Deiters″ cell are transmitted across a network segment spanning 8 cell diameters. Thus electrical coupling in the organ of Corti is unlikely to influence the selectivity of frequency filtering performed mechanically by the mammalian cochlea.     

Frequency of bird hybrids: does detectability make all the difference?

The number of hybrids recorded in the field may be biased because these data may simply reflect the detectability of a hybrid pairing. A questionnaire was constructed for assessing the detectability of natural avian hybrids. Ratings from 17 independent experienced observers were subjected to factor analysis and all observers loaded onto the same single factor. The reliability was extremely high (Cronbachs alpha=0.96). Initially, I found a weak positive and significant correlation between the number of hybrids and their detectability index (r_s=0.268; P=0.015; n=81). However, the respective correlations within three orders and one family containing enough hybrids for analysis revealed non-significant results. Significant differences existed among orders with respect to both detectability and hybrid frequency. At the family level, differences existed in detectability, but not in the number of hybrids recorded. Furthermore, I found no publication bias. These results suggest a general influence of detectability and on higher taxonomic levels, and that either detectability or the number of hybrids vary between orders possibly due to phylogenetic effects. Data about hybrids should be used in a cautious manner in analyses across a wide range of species, but seem unbiased when restricted to analyses within orders and families. As a rule of thumb, distinctness of hybrids seems to be a function of the plumage difference between the hybridising species.Communicated by F. Bairlein     

How Close Should the Outer Hair Cell RC Roll-Off Frequency Be to the Characteristic Frequency?

Recent experiments have shown a much larger conductance in outer hair cells, the central components of the mammalian cochlear amplifier. The report used only the cell's linear capacitance, which together with increased conductance, raised the cell's RC corner frequency so that voltage-dependent motility was better able to amplify high-frequency sounds. We construct transfer functions for a simple model of a high characteristic frequency (CF) local cochlear resonance. These show that voltage roll-off does not occur above the RC corner. Instead, it is countered by high-pass filtering that is intrinsic to the mammal's electromechanical resonance. Thus, the RC corner of a short outer hair cell used for high-frequency amplification does not have to be close to the CF, but depending on the drag, raised only above 0.1 CF. This high-pass filter, built in to the mammalian amplifier, allows for sharp frequency selectivity at very high CF.     

Frequency response of the electroreceptors (“small pit organs”) of the catfish,Ictalurus nebulosus LeS

Summary By electrophysiologically recording the spikes from the ramus lateralis ventralis nervi vagi of the catfishIctalurus nebulosus LeS., we determined the frequency response of the electroreceptors (small pit organs) in unanaesthetized, freely swimming specimens. The response frequency domain we were able to measure ranged from 0,03 to 25 Hz. We found a peak of greatest sensitivity at about 3 to 7 Hz (Fi. 5), using a stimulus with a current density of 5 × 10^–4A/mm² in water with a specific resistance of 20 Ohm. m. The threshold current density of the small pit organs in a homogeneous sinusoidally varying electric field (3 Hz) proved to be 3 × 10^–5 A/mm².

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Zusammenfassung Durch Ableitung von Nervenimpulsen vom ramus lateralis ventralis nervi vagi beim Zwergwels,Ictalurus nebulosus LeS., versuchten wir den Frequenzbereich der Electroreceptoren (small pit organs) bei frei herumschwimmenden, nicht narkotisierten Tieren zu bestimmen. Der wirksame Frequenzbereich, in dem Bestimmungen vorgenommen werden konnten, erstreckte sich von 0,03 bis 25 Hz. Die Empfindlichkeitskurve der small pit organs erreichte einen Gipfel bei Frequenzen von 3–7 Hz (Fig. 5). Die Reizstromdichte betrug dabei 5 × 10^–4A/mm²; der spezifische Wasserwiderstand war 20 Ohm. m. Es stellte sich heraus, daß die Schwellenstromdichte der small pit organs im homogenen elektrischen Wechselstromfeld (3 Hz) 3 × 10^–5 A/mm² betrug.

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We wish to thank Prof. S. Dijkgraaf for his stimulating interest, and Mr. W. J. G. Loos for making the drawings.     

Frequency of the ΔF508 mutation on cystic fibrosis chromosomes in Denmark

Summary We have investigated the frequency of the ΔF508 mutation on cystic fibrosis (CF) chromosomes in Denmark. Of 304 chromosome tested, 86.8% have the ΔF508 mutation. The majority of the chromosomes with this mutation are found on chromosomes with the XV2c/KM19 haplotype B (97.3%), whereas 15/16 chromosomes with haplotype C have another mutation, confirming that only very few mutations will account for the majority of CF genes in the Danish population.     

Effect of Long-Term Exposure to a Randomly Varied 50 Hz Power Frequency Magnetic Field on the Fertility of the Mouse

A double blind study on BALB/c mice was conducted to examine the effects of extremely low-frequency electromagnetic fields on fertility. The mice were continuously exposed or sham-exposed from conception for two generations to magnetic fields varying between 0.5 and 77?μT. Biological parameters related to fertility were evaluated. Serum testosterone levels and mass of testes and adrenals were determined. No significant difference was found between the sham-exposed and exposed groups for all the biological endpoints, except for sperm motility. A significant difference between the two groups was found prior to the swim-up test with quantitative analysis of sperm motility as well as after the swim-up test for quantitative and qualitative analysis of sperm motility. ELF-EMFs significantly decreased the number of living sperm and the quality of movement of sperm, although these adversities did not impact on the outcome of the other parameters investigated.     

Localization of Mechanisms of Amplitude–Frequency Modulation of Pulse Blood Perfusion of the Soft Tissue Microvasculature. Pilot Study

Doklady Biochemistry and Biophysics - Peripheral blood flow analysis is used for noninvasive assessment of the cardiovascular system. The aim was to investigate the possibility of applying...     

Improved histochemical method for the demonstration of the activity of α-glucan phosphorylase

Summary The activity and localization of -glucan phosphorylase in experimental canine glycogen-depleted heart tissue has been investigated with biochemical and histochemical methods using dextran as enzyme acceptor. Only linear, essentially unbranched, dextrans exhibit acceptor properties; highly branched dextrans are not suitable acceptors for the enzyme. Results of Michaelis-Menten constant measurements for the linear essentially unbranched dextran fractions used, indicate that the affinity of the enzyme for the non-reducing end group of the dextran molecule increases with increasing molecular weight of the acceptor.In the glycogen-depleted tissue of anoxic and ischaemic cardiac musculature there is a gradual inactivation of the enzyme during the ischaemic period. Shortly before total inactivation the affinity of the enzyme, especially for the lower molecular dextran fractions, is greatly reduced. Therefore, for the histochemical demonstration of phosphorylase activity in infarcted areas of the heart it is essential to use as acceptor an unbranched dextran fraction with a high average molecular weight.This investigation was partially supported by a grant from the Netherlands Organization for the Advancement of Pure Research (Z.W.O.).     

Improved histochemical method for the demonstration of the activity of α-glucan phosphorylase

Summary A modified method for the histochemical demonstration of the activity of -glucan phosphorylase is described. In the histochemical system the enzyme catalyses the synthesis of glycogen by transglucosylation from -d-glucosylphosphate. The incubation medium contains dextran as glucosyl acceptor. Therefore, in contrast with the unmodified method, the new technique is able to demonstrate the activity of phosphorylase in ischaemic glycogen-depleted tissues.     

Integrated mechanism for the generation of the 5′ junctions of LINE inserts

To elucidate the molecular mechanism of the integration of long interspersed elements (LINEs), we characterized the 5′ ends of more than 200 LINE de novo retrotransposition events into chicken DT40 or human HeLa cells. Human L1 inserts produced 15-bp target-site duplications (TSDs) and zebrafish ZfL2-1 inserts produced 5-bp TSDs in DT40 cells, suggesting that TSD length depends on the LINE species. Further analysis of 5′ junctions revealed that the 5′-end-joining pathways of LINEs can be divided into two fundamental types—annealing or direct. We also found that the generation of 5′ inversions depends on host and LINE species. These results led us to propose a new model for 5′-end joining, the type of which is determined by the extent of exposure of 3′ overhangs generated after the second-strand cleavage and by the involvement of host factors.     

Extension of the Applicability of δJ-Values for the Configurational Assignment of Diastereomeric Phosphate-Modified Dideoxynucleotides

Abstract

The stereochemical assignment of dinucleoside-S-p-nitrobenzyl-phosphorothioates by NMR spectroscopy is reported. It was found that the method based on the difference of the vicinal phosphorus-carbon coupling constants (δJ =³J_C4′,p-³J_C2′,p) can widely be applied for the determination of the configuration at the pohosphorus atom in phosphate-modified dideoxynucleotides.     

Importance of the Sphingoid Base Length for the Membrane Properties of?Ceramides

The sphingoid bases of sphingolipids, including ceramides, can vary in length from 12 to >20 carbons. To study how such length variation affects the bilayer properties of ceramides, we synthesized ceramides consisting of a C12-, C14-, C16-, C18-, or C20-sphing-4-enin derivative coupled to palmitic acid. The ceramides were studied in mixtures with palmitoyloleoylphosphocholine (POPC) and/or palmitoylsphingomyelin (PSM), and in more complex bilayers also containing cholesterol. The trans-parinaric acid lifetimes showed that 12:1- and 14:1-PCer failed to increase the order of POPC bilayers, whereas 16:1-, 18:1-, and 20:1-PCer induced ordered- or gel-phase formation. Nevertheless, all of the analogs were able to thermally stabilize PSM, and a chain-length-dependent increase in the main phase transition temperature of equimolar PSM/Cer bilayers was revealed by differential scanning calorimetry. Similar thermal stabilization of PSM-rich domains by the ceramides was observed in POPC bilayers with a trans-parinaric acid-quenching assay. A cholestatrienol-quenching assay and sterol partitioning experiments showed that 18:1- and 20:1-PCer formed sterol-excluding gel phases with PSM, reducing the overall bilayer affinity of sterol. The effect of 16:1-PCer on sterol distribution was less dramatic, and no displacement of sterol from the PSM environment was observed with 12:1- and 14:1-PCer. The results are discussed in relation to other structural features that affect the bilayer properties of ceramides.     

Evaluation of the Redesigned Enterotube—a System for the Identification of Enterobacteriaceae

The redesigned Enterotube has been evaluated with 414 unknown Enterobacteriaceae cultures from the stock culture collection of the Center for Disease Control. When the Enterotube was used as recommended by the manufacturer, an average of 96.4% of these cultures were correctly identified. Only two groups (Salmonella and Edwardsiella) were identified with less than 90% accuracy (89.2 and 87.5%, respectively). The Enterotube now provides a convenient, rapid, and accurate test system for the identification of typically reacting enteric bacteria.     

Significance of the hydrophobic residues 225–230 of apoA-I for the biogenesis of HDL

We studied the significance of four hydrophobic residues within the 225–230 region of apoA-I on its structure and functions and their contribution to the biogenesis of HDL. Adenovirus-mediated gene transfer of an apoA-I[F225A/V227A/F229A/L230A] mutant in apoA-I^−/− mice decreased plasma cholesterol, HDL cholesterol, and apoA-I levels. When expressed in apoA-I^−/− × apoE^−/− mice, approximately 40% of the mutant apoA-I as well as mouse apoA-IV and apoB-48 appeared in the VLDL/IDL/LDL. In both mouse models, the apoA-I mutant generated small spherical particles of pre-β- and α4-HDL mobility. Coexpression of the apoA-I mutant and LCAT increased and shifted the-HDL cholesterol peak toward lower densities, created normal αHDL subpopulations, and generated spherical-HDL particles. Biophysical analyses suggested that the apoA-I[225–230] mutations led to a more compact folding that may limit the conformational flexibility of the protein. The mutations also reduced the ability of apoA-I to promote ABCA1-mediated cholesterol efflux and to activate LCAT to 31% and 66%, respectively, of the WT control. Overall, the apoA-I[225–230] mutations inhibited the biogenesis of-HDL and led to the accumulation of immature pre-β- and α4-HDL particles, a phenotype that could be corrected by administration of LCAT.     

Characterization of the insertase for β-barrel proteins of the outer mitochondrial membrane

The TOB–SAM complex is an essential component of the mitochondrial outer membrane that mediates the insertion of β-barrel precursor proteins into the membrane. We report here its isolation and determine its size, composition, and structural organization. The complex from Neurospora crassa was composed of Tob55–Sam50, Tob38–Sam35, and Tob37–Sam37 in a stoichiometry of 1:1:1 and had a molecular mass of 140 kD. A very minor fraction of the purified complex was associated with one Mdm10 protein. Using molecular homology modeling for Tob55 and cryoelectron microscopy reconstructions of the TOB complex, we present a model of the TOB–SAM complex that integrates biochemical and structural data. We discuss our results and the structural model in the context of a possible mechanism of the TOB insertase.