Mitochondrial DNA content of mature spermatozoa and oocytes in the genetic model Drosophila

Although crucial to the success of fertilization and embryogenesis, little is known about the mitochondrial DNA (mtDNA) content of mature spermatozoa and oocytes across taxa and across different fertilization systems. Oocytes are assumed to hold a large population of mtDNAs that populate emerging cells during early embryogenesis, whereas spermatozoa harbor only a limited pool of mtDNAs that is believed to sustain functionality but fails to contribute paternal mtDNA to the zygote. Recent work suggests that mature sperm of the genetic model Drosophila melanogaster lack mtDNA, questioning the significance of zygotic mechanisms for the selective elimination of paternal mtDNA and their necessity for fertilization success. This finding further contradicts previous observations of the inheritance of paternal mtDNA in drosophilids. Using quantitative polymerase chain reaction, we estimate the mtDNA content of several laboratory strains of D. melanogaster and D. simulans to shed light on this discrepancy and to describe the mitochondrial/mtDNA load of gametes within this system. These measurements led to an average estimate of 22.91±4.61 mtDNA molecules/copies per spermatozoon across both species and to 1.07E+07±2.71E+06 molecules/copies per oocyte for D. simulans. As a consequence, the ratio of paternal and maternal mtDNA in the zygote was estimated at 1:4.65E+05.     

Vaccination with anti-idiotype antibody ganglidiomab mediates a GD2-specific anti-neuroblastoma immune response

Purpose

Immunotherapy targeting disialoganglioside GD₂ emerges as an important treatment option for neuroblastoma, a pediatric malignancy characterized by poor outcome. Here, we report the induction of a GD₂-specific immune response with ganglidiomab, a new anti-idiotype antibody to anti-GD₂ antibodies of the 14.18 family.

Experimental design and results

Ganglidiomab was generated following immunization of Balb/c mice with 14G2a, and splenocytes were harvested to generate hybridoma cells. Clones were screened by ELISA for mouse antibody binding to hu14.18. One positive clone was selected to purify and characterize the secreted IgG protein (κ, IgG₁). This antibody bound to anti-GD₂ antibodies 14G2a, ch14.18/CHO, hu14.18, and to immunocytokines ch14.18-IL2 and hu14.18-IL2 as well as to NK-92 cells expressing scFv(ch14.18)-zeta receptor. Binding of these anti-GD₂ antibodies to the nominal antigen GD₂ as well as GD₂-specific lysis of neuroblastoma cells by NK-92-scFv(ch14.18)-zeta cells was competitively inhibited by ganglidiomab, proving GD₂ surrogate function and anti-idiotype characteristics. The dissociation constants of ganglidiomab from anti-GD₂ antibodies ranged from 10.8 ± 5.01 to 53.5 ± 1.92 nM as determined by Biacore analyses. The sequences of framework and complementarity-determining regions of ganglidiomab were identified. Finally, we demonstrated induction of a GD₂-specific humoral immune response after vaccination of mice with ganglidiomab effective in mediating GD₂-specific killing of neuroblastoma cells.

Conclusion

We generated and characterized a novel anti-idiotype antibody ganglidiomab and demonstrated activity against neuroblastoma.     

197 Modeling the maturation pathway of the HIV-1 5’-UTR RNA dimer

HIV-1 genomic RNA is packaged as a dimer into the virions. The initial metastable RNA dimer is believed to be formed by virtue of “kissing interactions” between two copies of the palindromic apical loops of stem-loop SL1 of the 5’-untranslated region (5’-UTR) of the genomic RNA. Viral nucleocapsid protein NCp7 promotes maturation of the RNA dimer into more stable form, which involves extended or linear form of SL1 dimer (reviewed in Paillart et al., 2004; Moore & Hu, 2009; Lu et al., 2011). In vitro experiments have shown that this conversion occurs at stoichiometric amounts of NCp7 without breaking interactions between the two copies of the SL1 apical loops (Mujeeb et al., 2007). We have proposed a hypothetical pathway and calculated models of the intermediate structures for the SL1 stem-loop dimer maturation that does not require simultaneous dissociation of all base pairs in SL1 stems; this pathway involves formation of an RNA analog of the Holliday junction intermediate between the two stems of the SL1 dimer and a following branch migration towards the palindromic duplex (Ulyanov et al., 2011). Here, we extend these models to the dimer of the 1–344 fragment of HIV-1 RNA, which includes all of the 5’-UTR and the gag start AUG codon region, and show that the branch-migration mechanism of the dimer maturation is also feasible for the full 5’-UTR RNA. All RNA models have been calculated with the miniCarlo program (Zhurkin et al., 1991).     

Can the Neural Basis of Repression Be Studied in the MRI Scanner? New Insights from Two Free Association Paradigms

Background

The psychodynamic theory of repression suggests that experiences which are related to internal conflicts become unconscious. Previous attempts to investigate repression experimentally were based on voluntary, intentional suppression of stimulus material. Unconscious repression of conflict-related material is arguably due to different processes, but has never been studied with neuroimaging methods.

Methods

We used functional magnetic resonance imaging (fMRI) in addition with skin conductance recordings during two free association paradigms to identify the neural mechanisms underlying forgetting of freely associated words according to repression theory.

Results

In the first experiment, free association to subsequently forgotten words was accompanied by increases in skin conductance responses (SCRs) and reaction times (RTs), indicating autonomic arousal, and by activation of the anterior cingulate cortex. These findings are consistent with the hypothesis that these associations were repressed because they elicited internal conflicts. To test this idea more directly, we conducted a second experiment in which participants freely associated to conflict-related sentences. Indeed, these associations were more likely to be forgotten than associations to not conflict-related sentences and were accompanied by increases in SCRs and RTs. Furthermore, we observed enhanced activation of the anterior cingulate cortex and deactivation of hippocampus and parahippocampal cortex during association to conflict-related sentences.

Conclusions

These two experiments demonstrate that high autonomic arousal during free association predicts subsequent memory failure, accompanied by increased activation of conflict-related and deactivation of memory-related brain regions. These results are consistent with the hypothesis that during repression, explicit memory systems are down-regulated by the anterior cingulate cortex.     

Mitochondrial Phylogenomics of Modern and Ancient Equids

The genus Equus is richly represented in the fossil record, yet our understanding of taxonomic relationships within this genus remains limited. To estimate the phylogenetic relationships among modern horses, zebras, asses and donkeys, we generated the first data set including complete mitochondrial sequences from all seven extant lineages within the genus Equus. Bayesian and Maximum Likelihood phylogenetic inference confirms that zebras are monophyletic within the genus, and the Plains and Grevy’s zebras form a well-supported monophyletic group. Using ancient DNA techniques, we further characterize the complete mitochondrial genomes of three extinct equid lineages (the New World stilt-legged horses, NWSLH; the subgenus Sussemionus; and the Quagga, Equus quagga quagga). Comparisons with extant taxa confirm the NWSLH as being part of the caballines, and the Quagga and Plains zebras as being conspecific. However, the evolutionary relationships among the non-caballine lineages, including the now-extinct subgenus Sussemionus, remain unresolved, most likely due to extremely rapid radiation within this group. The closest living outgroups (rhinos and tapirs) were found to be too phylogenetically distant to calibrate reliable molecular clocks. Additional mitochondrial genome sequence data, including radiocarbon dated ancient equids, will be required before revisiting the exact timing of the lineage radiation leading up to modern equids, which for now were found to have possibly shared a common ancestor as far as up to 4 Million years ago (Mya).     

Specific features of telomerase RNA from Hansenula polymorpha

Telomerase, a ribonucleoprotein, is responsible for the maintenance of eukaryotic genome integrity by replicating the ends of chromosomes. The core enzyme comprises the conserved protein TERT and an RNA subunit (TER) that, in contrast, displays large variations in size and structure. Here, we report the identification of the telomerase RNA from thermotolerant yeast Hansenula polymorpha (HpTER) and describe its structural features. We show further that the H. polymorpha telomerase reverse transcribes the template beyond the predicted boundary and adds a nontelomeric dT in vitro. Sequencing of the chromosomal ends revealed that this nucleotide is specifically present as a terminal nucleotide at the 3′ end of telomeres. Mutational analysis of HpTER confirmed that the incorporation of dT functions to limit telomere length in this species.     

Off-pathway aggregation can inhibit fibrillation at high protein concentrations

Ribosomal protein S6 fibrillates readily at slightly elevated temperatures and acidic pH. We find that S6 fibrillation is retarded rather than favored when the protein concentration is increased above a threshold concentration of around 3.5 mg/mL. We name this threshold concentration C_FR, the concentration at which fibrillation is retarded. Our data are consistent with a model in which this inhibition is due to the formation of an off-pathway oligomeric species with native-like secondary structure. The oligomeric species dominates at high protein concentrations but exists in dynamic equilibrium with the monomer so that seeding with fibrils can overrule oligomer formation and favors fibrillation under C_FR conditions. Thus, fibrillation competes with formation of off-pathway oligomers, probably due to a monomeric conversion step that is required to commit the protein to the fibrillation pathway. The S6 oligomer is resistant to pepsin digestion. We also report that S6 forms different types of fibrils dependent on protein concentration. Our observations highlight the multitude of conformational states available to proteins under destabilizing conditions.     

The structure of helix 89 of 23S rRNA is important for peptidyl transferase function of Escherichia coli ribosome

Helix 89 of the 23S rRNA connects ribosomal peptidyltransferase center and elongation factor binding site. Secondary structure of helix 89 determined by X-ray structural analysis involves less base pairs then could be drawn for the helix of the same primary structure. It can be that alternative secondary structure might be realized at some stage of translation. Here by means of site-directed mutagenesis we stabilized either the "X-ray" structure or the structure with largest number of paired nucleotides. Mutation UU2492-3C which aimed to provide maximal pairing of the helix 89 of the 23S rRNA was lethal. Mutant ribosomes were unable to catalyze peptide transfer independently either with aminoacyl-tRNA or puromycin.     

Developing transgenic Anopheles mosquitoes for the sterile insect technique

In the last 10 years the availability of the genome sequence of Anopheles gambiae and the development of a transgenic technology for several species of Anopheles mosquitoes have, in combination, helped in enabling us to gain several insights into the biology of these mosquitoes that is relevant to their capacity as vectors of the malaria parasite. While this information is anticipated to inform many novel vector control strategies, the technique most likely to benefit in the near future from the availability of a reliable transgenic technology is the sterile insect technique (SIT), which relies on releasing large numbers of sterile insects to compete for mates in the wild, leading to population suppression. Although SIT has been proven to work reliably for many insects, the construction of suitable strains, and induction of sterility, has until now been a laborious process, combining classical genetics with radiation-induced sterility. Using transgenesis to create strains of Anopheles suitable for SIT could potentially offer several advantages over current approaches, in that the basic design of transgenic constructs designed for other insects should be rapidly transferable to mosquitoes, and induction of sterility as a product of the transgenic modification could obviate the requirement for radiation and its associated deleterious effects. In this paper the progress of different transgenic approaches in constructing tools for SIT will be reviewed.