Loss of HR6B ubiquitin-conjugating activity results in damaged synaptonemal complex structure and increased crossing-over frequency during the male meiotic prophase

The ubiquitin-conjugating enzymes HR6A and HR6B are the two mammalian homologs of Saccharomyces cerevisiae RAD6. In yeast, RAD6 plays an important role in postreplication DNA repair and in sporulation. HR6B knockout mice are viable, but spermatogenesis is markedly affected during postmeiotic steps, leading to male infertility. In the present study, increased apoptosis of HR6B knockout primary spermatocytes was detected during the first wave of spermatogenesis, indicating that HR6B performs a primary role during the meiotic prophase. Detailed analysis of HR6B knockout pachytene nuclei showed major changes in the synaptonemal complexes. These complexes were found to be longer. In addition, we often found depletion of synaptonemal complex proteins from near telomeric regions in the HR6B knockout pachytene nuclei. Finally, we detected an increased number of foci containing the mismatch DNA repair protein MLH1 in these nuclei, reflecting a remarkable and consistent increase (20 to 25%) in crossing-over frequency. The present findings reveal a specific requirement for the ubiquitin-conjugating activity of HR6B in relation to dynamic aspects of the synaptonemal complex and meiotic recombination in spermatocytes.     

Genetic Models in Applied Physiology. Merosin deficiency leads to alterations in passive and active skeletal muscle mechanics.

The role of extracellular elements on the mechanical properties of skeletal muscles is unknown. Merosin is an essential extracellular matrix protein that forms a mechanical junction between the sarcolemma and collagen. Therefore, it is possible that merosin plays a role in force transmission between muscle fibers and collagen. We hypothesized that deficiency in merosin may alter passive muscle stiffness, viscoelastic properties, and contractile muscle force in skeletal muscles. We used the dy/dy mouse, a merosin-deficient mouse model, to examine changes in passive and active muscle mechanics. After mice were anesthetized and the diaphragm or the biceps femoris hindlimb muscle was excised, passive length-tension relationships, stress-relaxation curves, or isometric contractile properties were determined with an in vitro biaxial mechanical testing apparatus. Compared with controls, extensibility was smaller in the muscle fiber direction and the transverse fiber direction of the mutant mice. The relaxed elastic modulus was smaller in merosin-deficient diaphragms compared with controls. Interestingly, maximal muscle tetanic stress was depressed in muscles from the mutant mice during uniaxial loading but not during biaxial loading. However, presence of transverse passive stretch increases maximal contractile stress in both the mutant and normal mice. Our data suggest that merosin contributes to muscle passive stiffness, viscoelasticity, and contractility and that the mechanism by which force is transmitted between adjacent myofibers via merosin possibly in shear.     

The circadian clock genes affect reproductive capacity in the desert locust Schistocerca gregaria

The circadian clocks govern many metabolic and behavioral processes in an organism. In insects, these clocks and their molecular machinery have been found to influence reproduction in many different ways. Reproductive behavior including courtship, copulation and egg deposition, is under strong influence of the daily rhythm. At the molecular level, the individual clock components also have their role in normal progress of oogenesis and spermatogenesis. In this study on the desert locust Schistocerca gregaria, three circadian clock genes were identified and their expression profiles were determined. High expression was predominantly found in reproductive tissues. Similar daily expression profiles were found for period (per) and timeless (tim), while the clock (clk) mRNA level is higher 12 h before the first per and tim peak. A knockdown of either per or tim resulted in a significant decrease in the progeny produced by dsRNA treated females confirming the role of clock genes in reproduction and providing evidence that both PER and TIM are needed in the ovaries for egg development. Since the knockdown of clk is lethal for the desert locust, its function remains yet to be elucidated.     

Using Sculptor and Situs for simultaneous assembly of atomic components into low-resolution shapes

We describe an integrated software system called Sculptor that combines visualization capabilities with molecular modeling algorithms for the analysis of multi-scale data sets. Sculptor features extensive special purpose visualization techniques that are based on modern GPU programming and are capable of representing complex molecular assemblies in real-time. The integration of graphics and modeling offers several advantages. The user interface not only eases the usually steep learning curve of pure algorithmic techniques, but it also permits instant analysis and post-processing of results, as well as the integration of results from external software. Here, we implemented an interactive peak-selection strategy that enables the user to explore a preliminary score landscape generated by the colors tool of Situs. The interactive placement of components, one at a time, is advantageous for low-resolution or ambiguously shaped maps, which are sometimes difficult to interpret by the fully automatic peak selection of colors. For the subsequent refinement of the preliminary models resulting from both interactive and automatic peak selection, we have implemented a novel simultaneous multi-body docking in Sculptor and Situs that softly enforces shape complementarities between components using the normalization of the cross-correlation coefficient. The proposed techniques are freely available in Situs version 2.6 and Sculptor version 2.0.     

Developing a denoising filter for electron microscopy and tomography data in the cloud

The low radiation conditions and the predominantly phase-object image formation of cryo-electron microscopy (cryo-EM) result in extremely high noise levels and low contrast in the recorded micrographs. The process of single particle or tomographic 3D reconstruction does not completely eliminate this noise and is even capable of introducing new sources of noise during alignment or when correcting for instrument parameters. The recently developed Digital Paths Supervised Variance (DPSV) denoising filter uses local variance information to control regional noise in a robust and adaptive manner. The performance of the DPSV filter was evaluated in this review qualitatively and quantitatively using simulated and experimental data from cryo-EM and tomography in two and three dimensions. We also assessed the benefit of filtering experimental reconstructions for visualization purposes and for enhancing the accuracy of feature detection. The DPSV filter eliminates high-frequency noise artifacts (density gaps), which would normally preclude the accurate segmentation of tomography reconstructions or the detection of alpha-helices in single-particle reconstructions. This collaborative software development project was carried out entirely by virtual interactions among the authors using publicly available development and file sharing tools.     

Bacteriophage MS2 RNA: A correlation between the stability of the codon: Anticodon interaction and the choice of code words

Summary The non-random distribution of degenerate code words in Bacteriophage MS2 RNA can be explained partially by considerations of the stability of the codon-anticodon complex in prokaryotic systems. Supporting this hypothesis we note that wobble codons are positively selected in codons having G and/or C in the first two positions. In contrast, wobble codons are statitically less likely in codons composed of A and U in the first two positions. Analyses of nucleotides adjacent to 5 and 3 ends of codons indicate a nonrandom distribution as well. It is thus likely that some elements of RNA evolution are independent of the structural needs of the RNA itself and of the translated protein product.This work was supported by grants from the Belgian Government Actions concertées - Gekon-certéerde Acties, N.F.W.O. and F.K.F.O. as well as from le Ministère de l'éducation du Québec. A preliminary report of this work was given at the EMBO ribosome workshop, Brussels 1976     

Impaired enzyme-to-enzyme channelling between hexokinase isoenzyme(s) and phosphoglucoisomerase in rat pancreatic islets incubated at a low concentration of D-glucose

It was recently proposed that in rat pancreatic islets exposed to 8.3 mM D-glucose, alpha-D-glucose-6-phosphate undergoes enzyme-to-enzyme channelling between hexokinase isoenzyme(s) and phosphoglucoisomerase. To explore the identity of the hexokinase isoenzyme(s) involved in such a tunnelling process, the generation of 3HOH from the alpha- and beta-anomers of either D-[2-3H]glucose or D-[5-3H]glucose was now measured over 60 min incubation at 4 degrees C in pancreatic islets exposed only to 2.8 mM D-glucose, in order to decrease the relative contribution of glucokinase to the phosphorylation of the hexose. Under these experimental conditions, the ratio for 3HOH production from D-[2-3H]glucose/D-[5-3H]glucose at anomeric equilibrium (39.7 +/- 11.6%) and the beta/alpha ratios for the generation of 3HOH from either the D-[2-3H]glucose anomers (70.9 +/- 12.6%) or the D-[5-3H]glucose anomers (59.6 +/- 12.4%) indicated that a much greater fraction of alpha-D-glucose-6-phosphate escapes from the process of enzyme-to-enzyme channelling in the islets exposed to 2.8 mM, rather than 8.3 mM D-glucose. These findings suggest, therefore, that the postulated process of enzyme-to-enzyme channelling involves mainly glucokinase.     

In vitro synthesis of lectins in cell-free extracts from dry wheat and rye embryos

Cell-free extracts from dry wheat (Triticum vulgare L.) and rye (Secale cereale L.) embryos do not synthesize their corresponding lectins when incubated under conditions optimalized for translation of either exogenous or endogenous mRNA. Only when the extracts are prepared and incubated in the complete absence of sulphydryl containing compounds lectins are synthesized in vitro. Since immunoprecipitation techniques could not demonstrate unequivocally the presence of lectin among the cell-free translation products a specific affinity purification procedure has been developed in order to proof the presence of stored lectin mRNAs in dry wheat and rye embryos.Abbreviations DTT dithiothreitol - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - TCA trichloracetic acid     

Isolation and characterization of an angiotensin converting enzyme substrate from vitellogenic ovaries of Neobellieria bullata

Vitellogenic ovaries of the gray fleshfly Neobellieria bullata contain a variety of unidentified substances that interact, either as a substrate or as an inhibitor, with angiotensin converting enzyme (ACE). We here report the isolation and characterization of the first ACE interactive compound hereof. This 1312.7 Da peptide with the sequence NKLKPSQWISL, is substrate to both insect and human ACE. It is a novel peptide that shows high sequence similarity to a sequence at the N-terminal part of dipteran yolk polypeptides (YPs). We propose to call it N. bullata ovary-derived ACE interactive factor or Neb-ODAIF. Both insect and human ACE hydrolyze Neb-ODAIF by sequentially cleaving off two C-terminal dipeptides. K(m) values of Neb-ODAIF and Neb-ODAIF(1-9) (NKLKPSQWI) for human somatic ACE (sACE) are 17 and 81 microM, respectively. Additionally, Neb-ODAIF(1-7) (NKLKPSQ) also interacts with sACE (K(m/i)=90 microM). These affinity-constants are in range with those of the physiological ACE substrates and suggest the importance of Neb-ODAIF and its cleavage products in the elucidation of the physiological role of insect ACE. Alternatively, they can serve as lead compounds in the development of new drugs against ACE-related diseases in humans.     

Targeted Inactivation of Mouse RAD52 Reduces Homologous Recombination but Not Resistance to Ionizing Radiation

The RAD52 epistasis group is required for recombinational repair of double-strand breaks (DSBs) and shows strong evolutionary conservation. In Saccharomyces cerevisiae, RAD52 is one of the key members in this pathway. Strains with mutations in this gene show strong hypersensitivity to DNA-damaging agents and defects in recombination. Inactivation of the mouse homologue of RAD52 in embryonic stem (ES) cells resulted in a reduced frequency of homologous recombination. Unlike the yeast Scrad52 mutant, MmRAD52^−/− ES cells were not hypersensitive to agents that induce DSBs. MmRAD52 null mutant mice showed no abnormalities in viability, fertility, and the immune system. These results show that, as in S. cerevisiae, MmRAD52 is involved in recombination, although the repair of DNA damage is not affected upon inactivation, indicating that MmRAD52 may be involved in certain types of DSB repair processes and not in others. The effect of inactivating MmRAD52 suggests the presence of genes functionally related to MmRAD52, which can partly compensate for the absence of MmRad52 protein.