Chromatin organization during spermiogenesis in Octopus vulgaris. II: DNA-interacting proteins

In this article we study the proteins responsible for chromatin condensation during spermiogenesis in the cephalopod Octopus vulgaris. The DNA of ripe sperm nuclei in this species is condensed by a set of five different proteins. Four of these proteins are protamines. The main protamine (Po2), a protein of 44 amino acid residues, is extraordinarily simple (composed of only three different amino acid types: arginine (R), serine (S), and glycine (G). It is a basic molecule consisting of 79.5 mol% arginine residues. The rest of the protamines (Po3, Po4, Po5) are smaller molecules (33, 28, and 30 amino acid residues, respectively) that are homologous among themselves and probably with the main Po2 protamine. The ripe sperm nucleus of O. vulgaris also contains a small quantity of a molecule (Po1) that is similar to Po2 protamine. This protein could represent a Po2 protamine-precursor in a very advanced step of its processing. We discuss the characteristics of these proteins, as well as the relation between the complexity of chromatin condensation and the transitions of nuclear proteins during spermiogenesis in O. vulgaris.     

Vitrification of pronuclear-stage mouse embryos on solid surface (SSV) versus in cryotube: comparison of the effect of equilibration time and different sugars in the vitrification solution

The cryopreservation of pronuclear-stage embryos has particular importance in transgenic technology and human assisted reproductive technology (ART). The objective of this study was to improve the efficiency of cryopreservation of pronuclear-stage mouse embryos. Two vitrification methods (solid surface vitrification (SSV) vs. vitrification in cryotube) have been compared with special emphasis on the effect of the exposure of the embryos to the solutions for various times and the sugar content (trehalose, sucrose, or raffinose) of the vitrification solutions. Pronuclear-stage embryos were either exposed to 1 M dimethyl sulfoxide (DMSO) + 1 M propylene-glycol (PG) solution for 2, 5, 10, or 15 min or not exposed to this "equilibration" solution. The vitrification solutions consisted of 2.75 M DMSO and 2.75 M PG in M2 medium supplemented with 1 M trehalose (DPT), 1 M sucrose (DPS), or 1 M raffinose (DPR). In the cryotube method, groups of 15-25 embryos were transferred into a 1.8 ml cryotube containing 30 microl of DPT, DPS, or DPR. After 30 sec, the cryotubes were directly plunged into liquid nitrogen (LN(2)) and stored for 1 day to 1 month. Vitrified samples were warmed by immersing the cryotubes in a 40 degrees C water bath and then immediately diluted with 300 microl of 0.3 M trehalose, sucrose, or raffinose in M2. In the SSV method, after equilibration 15-20 embryos were exposed to DPT, DPS, or DPR solutions for around 20 sec before being dropped in 2-microl drops onto a pre-cooled (-150 to -180 degrees C) metal surface. Vitrified droplets were stored in cryovials in LN(2). Warming was performed by transferring the vitrified droplets into 0.3 M solutions of trehalose, sucrose, or raffinose at 37 degrees C, respectively. Results showed that both SSV and cryotube vitrification methods can result in high rates of in vitro blastocyst development (up to 58.3 and 68.5% with DPR, respectively), not statistically different from that of the controls (58.3 and 64.4%). Even without the equilibration step prior to vitrification, relatively high-survival rates have been achieved, except for the DPS solution. In conclusion, vitrification of pronuclear-stage mouse embryos can result in high rates of in vitro development to blastocyst, and the use of raffinose in the vitrification solution is advantageous to improve cryosurvival.     

Origin of a new Reticulitermes termite (Isoptera, Rhinotermitidae) inferred from mitochondrial and nuclear DNA data

The Holoarctic termite genus Reticulitermes is widely distributed in Europe. A new Reticulitermes species, R. sp. nov, was recently found in France and Italy. Its phylogenetic position was investigated using a 743-bp fragment of mitochondrial 16S rRNA-ND1 genes and 382-bp of the nuclear ITS2 region. Phylogenies for these sequences were estimated by neighbor-joining, maximum-parsimony and maximum-likelihood analysis. The results strongly supported a relationship between R. sp. nov. and the termite species from the eastern Mediterranean area including Reticulitermes balkanensis from the Balkans, Reticulitermes lucifugus from Turkey and Reticulitermes clypeatus from Israel. The hypothesis of a relationship between R. sp. nov. and the Japanese Reticulitermes speratus was rejected by parametric bootstrap. The current distribution of R. sp. nov. could be linked to postglacial colonization routes between Balkan refuge and northern regions.     

Endocytosis and sorting of ErbB2 and the site of action of cancer therapeutics trastuzumab and geldanamycin

ErbB2 is a transmembrane tyrosine kinase whose surface overexpression is linked to tumorigenesis and poor prognosis in breast cancer patients. Two models have emerged that account for the high surface distribution of ErbB2. In one model, the surface pool is dynamic and governed by a balance between endocytosis and recycling, whereas in the other it is retained, static, and excluded from endocytosis. These models have contrasting implications for how ErbB2 exerts its biological function and how cancer therapies might down-regulate surface ErbB2, such as the antibody trastuzumab (Herceptin) or the Hsp90 inhibitor geldanamycin. Little is known, however, about how these treatments affect ErbB2 endocytic trafficking. To investigate this issue, we examined breast carcinoma cells by immunofluorescence and quantitative immunoelectron microscopy and developed imaging and trafficking kinetics assays using cell surface fluorescence quenching. Surprisingly, trastuzumab does not influence ErbB2 distribution but instead recycles passively with internalized ErbB2. By contrast, geldanamycin down-regulates surface ErbB2 through improved degradative sorting in endosomes exclusively rather than through increased endocytosis. These results reveal substantial dynamism in the surface ErbB2 pool and clearly demonstrate the significance of endosomal sorting in the maintenance of ErbB2 surface distribution, a critical feature of its biological function.     

A revised annotation and comparative analysis of Helicobacter pylori genomes

Huge amounts of genomic information are currently being generated. Therefore, biologists require structured, exhaustive and comparative databases. The PyloriGene database (http://genolist.pasteur.fr/PyloriGene) was developed to respond to these needs, by integrating and connecting the information generated during the sequencing of two distinct strains of Helicobacter pylori. This led to the need for a general annotation consensus, as the physical and functional annotations of the two strains differed significantly in some cases. A revised functional classification system was created to accommodate the existing data and to make it possible to classify coding sequences (CDS) into several functional categories to harmonize CDS classification. The annotation of the two complete genomes was revised in the light of new data, allowing us to reduce the percentage of hypothetical proteins from approximately 40 to 33%. This resulted in the reassignment of functions for 108 CDS (approximately 7% of all CDS). Interestingly, the functions of only approximately 13% of CDS (222 out of 1658 CDS) were annotated as a result of work done directly on H.pylori genes. Finally, comparison of the two published genomes revealed a significant amount of size variation between corresponding (orthologous) CDS. Most of these size variations were due to natural polymorphisms, although other sources of variation were identified, such as pseudogenes, new genes potentially regulated by slipped-strand mispairing mechanism, or frame-shifts. 113 of these differences were due to different start codon assignments, a common problem when constructing physical annotations.     

Identification of 13 novel human modification guide RNAs

Members of the two expanding RNA subclasses termed C/D and H/ACA RNAs guide the 2'-O-methylations and pseudouridylations, respectively, of rRNA and spliceosomal RNAs (snRNAs). Here, we report on the identification of 13 novel human intron-encoded small RNAs (U94-U106) belonging to the two subclasses of modification guides. Seven of them are predicted to direct 2'-O-methylations in rRNA or snRNAs, while the remainder represent novel orphan RNA modification guides. From these, U100, which is exclusively detected in Cajal bodies (CBs), is predicted to direct modification of a U6 snRNA uridine, U(9), which to date has not been found to be pseudouridylated. Hence, within CBs, U100 might function in the folding pathway or other aspects of U6 snRNA metabolism rather than acting as a pseudouridylation guide. U106 C/D snoRNA might also possess an RNA chaperone activity only since its two conserved antisense elements match two rRNA sequences devoid of methylated nucleotides and located remarkably close to each other within the 18S rRNA secondary structure. Finally, we have identified a retrogene for U99 snoRNA located within an intron of the Siat5 gene, supporting the notion that retro-transposition events might have played a substantial role in the mobility and diversification of snoRNA genes during evolution.     

Structural motifs in the RNA encoded by the early nodulation gene enod40 of soybean

The plant gene enod40 is highly conserved among legumes and also present in various non-legume species. It is presumed to play a central regulatory role in the Rhizobium–legume interaction, being expressed well before the initiation of cortical cell divisions resulting in nodule formation. Two small peptides encoded by enod40 mRNA as well as its secondary structure have been shown to be key elements in the signalling processes underlying nodule organogenesis. Here results concerning the secondary structure of mRNA of enod40 in soybean are presented. This study combined a theoretical approach, involving structure prediction and comparison, as well as structure probing. Our study indicates five conserved domains in enod40 mRNA among numerous leguminous species. Structure comparison suggests that some domains are also conserved in non-leguminous species and that an additional domain exists that was found only in leguminous species developing indeterminate nodules. Enzymatic and chemical probing data support the structure for three of the domains, and partially for the remaining two. The rest of the molecule appears to be less structured. Some of the domains include motifs, such as U-containing internal loops and bulges, which seem to be conserved. Therefore, they might be involved in the regulatory role of enod40 RNA.     

Reloading of atrophied rat soleus muscle induces tenascin-C expression around damaged muscle fibers

The hypothesis was tested that mechanical loading, induced by hindlimb suspension and subsequent reloading, affects expression of the basement membrane components tenascin-C and fibronectin in the belly portion of rat soleus muscle. One day of reloading, but not the previous 14 days of hindlimb suspension, led to ectopic accumulation of tenascin-C and an increase of fibronectin in the endomysium of a proportion (8 and 15%) of muscle fibers. Large increases of tenascin-C (40-fold) and fibronectin (7-fold) mRNA within 1 day of reloading indicates the involvement of pretranslational mechanisms in tenascin-C and fibronectin accumulation. The endomysial accumulation of tenascin-C was maintained up to 14 days of reloading and was strongly associated with centrally nucleated fibers. The observations demonstrate that an unaccustomed increase of rat soleus muscle loading causes modification of the basement membrane of damaged muscle fibers through ectopic endomysial expression of tenascin-C.