The roles of WRN and BLM RecQ helicases in the Alternative Lengthening of Telomeres

Approximately 10% of all cancers, but a higher proportion of sarcomas, use the recombination-based alternative lengthening of telomeres (ALT) to maintain telomeres. Two RecQ helicase genes, BLM and WRN, play important roles in homologous recombination repair and they have been implicated in telomeric recombination activity, but their precise roles in ALT are unclear. Using analysis of sequence variation present in human telomeres, we found that a WRN– ALT+ cell line lacks the class of complex telomere mutations attributed to inter-telomeric recombination in other ALT+ cell lines. This suggests that WRN facilitates inter-telomeric recombination when there are sequence differences between the donor and recipient molecules or that sister-telomere interactions are suppressed in the presence of WRN and this promotes inter-telomeric recombination. Depleting BLM in the WRN– ALT+ cell line increased the mutation frequency at telomeres and at the MS32 minisatellite, which is a marker of ALT. The absence of complex telomere mutations persisted in BLM-depleted clones, and there was a clear increase in sequence homogenization across the telomere and MS32 repeat arrays. These data indicate that BLM suppresses unequal sister chromatid interactions that result in excessive homogenization at MS32 and at telomeres in ALT+ cells.     

First report of a green toad (Bufo viridis sensu lato) in the Early Pleistocene of Spain: Palaeobiogeographical and palaeoecological implications

For the first time unequivocal fossil remains of a green toad (Bufo viridis s.l.) are described in the Iberian Peninsula. The fossils come from the Cueva Victoria site, a late Early Pleistocene (ca. 1.1–1.2 Ma) karstic filling in semi-arid southeastern Spain (Murcia region). By extension, other remains from two other Early Pleistocene Spanish localities, Barranco León D (ca. 1.3 Ma) and Almenara-Casablanca 3 (ca. 1.1 Ma), are cautiously attributed to the group B. viridis. The B. viridis group was previously reported with some uncertainty to the west of its current distribution area in Western Europe (Spain and France) in the Pliocene (Bufo cf. viridis) and less probably in the Early Miocene (Bufo aff. viridis). Since no osteological differences have been established between the recently described extant species of B. viridis s.l. (e.g. Bufo balearicus, Bufo siculus, Bufo boulengeri, B. viridis sensu stricto and Bufo variabilis) no precise palaeobiogeographical relationships can be drawn for the Spanish fossils. However, the occurrence of a third species of bufonid toad during the Pleistocene in the South of the Iberian Peninsula raises some interesting ecological questions in relation to the local disappearance of the green toad, which can be hypothetically linked to the intensification of the Pleistocene glacial/interglacial climate dynamic or to probable competition with another toad, Bufo calamita.     

The medial basal hypothalamus and luteinizing hormone release in the rat: Where are the LH-RH neurons responsible for tonic gonadotropin secretion?

Careful review of the literature demonstrates conflicting results concerning the ability of the deafferented medial basal hypothalamus to support gonadotropin release in the rat and thus one may question the existence of LH-RH neurons in the medial basal hypothalamus. The direct search for the LH-RH perikarya in the rat hypothalamus has not settled the question of whether these releasing hormone neurons are located in the medial basal hypothalamus. Most investigators do agree that following complete hypothalamic deafferentation there is a reduction of the immunoassayable LH-RH in the medial basal hypothalamus; however, these results do not necessarily prove that LH-RH originates outside the hypothalamus. It is argued that the completely deafferented medial basal hypothalamus may be so altered by the deafferentation procedure that it may be inadequate as a means to study neuroendocrine function.     

Purification of Pregnancy Zone Protein and Its Receptor Binding Domain from Human Plasma

A new significantly improved method for purification of pregnancy zone protein (PZP), α₂-macroglobulin (α₂M), and the C-terminal PZP receptor binding domain is presented. Several steps in an earlier procedure have been deleted, and modifications in the gradients in the DEAE step leave most of the contaminants bound to a DEAE-Sephacel gel. This procedure makes possible the rapid, simultaneous purification of both of these closely related unstable proteins in native form from human plasma, with no thiolester cleavage or formation of tetrameric PZP. The final preparations of both α₂M and PZP are pure as determined by nonreducing and reducing polyacrylamide gel electrophoresis following silver staining and no cross-contamination can be observed. The yield has been significantly improved and typically more than 500 mg PZP can be obtained from 1 liter pregnancy plasma. Furthermore, the stability of PZP at different temperatures on storage was studied. In liquid nitrogen PZP can be maintained in native dimeric form with intact thiolester for many years. The storage of native PZP with intact functional properties during and after purification is an obligatory prerequisite to elucidate the biological role of PZP. The receptor binding domain of PZP can be cleaved from the PZP–methylamine complex by papain and isolated from the other peptides by S-200 gel filtration. The cleavage site was determined and the C-terminal fragment was identified with several site-specific monoclonal antibodies against PZP.     

Substantivity of zinc salts used as rinsing solutions and their effect on the inhibition of Streptococcus mutans

The antimicrobial efficacy of zinc (Zn) salts (sulfate and acetate) against Streptococcus mutans (S. mutans) present in the oral cavity was tested in this study. The substantivity of Zn salts was assessed by determining the concentration of Zn in whole, unstimulated saliva and by measuring the magnitude of suppression of salivary S. mutans, 2h after rinsing. The concentration of Zn was measured by atomic absorption spectrometry (AAS) with electrothermal atomization (ET AAS) in saliva sampled before (basal) and 24h after mouth rinsing with different concentrations of Zn (0.1%, 0.5% and 1%) administrated as sulfate and acetate. The estimation of Zn levels in samples collected 30, 60, 90 and 120 min after rinsing was carried out by AAS with flame atomization (FAAS). Immediately after rinsing, the concentration of Zn in saliva sharply increased with respect to the baseline values (0.055+/-0.017 mg/L), followed by a sustained decrease, probably due to clearance of salivary flow or swallowing during sampling. A significant reduction (>87%) in the total mean S. mutans counts was found 2h after rinsing either with sulfate or acetate solutions, as evidence of the high substantivity and effectiveness of the Zn salts tested. A statistically significant inverse relationship (p<0.001 and the Pearson correlation coefficients between -34% and -50%) was found between Zn levels and the respective pH values measured in the samples collected 60 and 120 min after rinsing, sustaining the theory of bacterial glycolysis inhibition.     

Paleomicrobiology: Revealing Fecal Microbiomes of Ancient Indigenous Cultures

Coprolites are fossilized feces that can be used to provide information on the composition of the intestinal microbiota and, as we show, possibly on diet. We analyzed human coprolites from the Huecoid and Saladoid cultures from a settlement on Vieques Island, Puerto Rico. While more is known about the Saladoid culture, it is believed that both societies co-existed on this island approximately from 5 to 1170 AD. By extracting DNA from the coprolites, followed by metagenomic characterization, we show that both cultures can be distinguished from each other on the basis of their bacterial and fungal gut microbiomes. In addition, we show that parasite loads were heavy and also culturally distinct. Huecoid coprolites were characterized by maize and Basidiomycetes sequences, suggesting that these were important components of their diet. Saladoid coprolite samples harbored sequences associated with fish parasites, suggesting that raw fish was a substantial component of their diet. The present study shows that ancient DNA is not entirely degraded in humid, tropical environments, and that dietary and/or host genetic differences in ancient populations may be reflected in the composition of their gut microbiome. This further supports the hypothesis that the two ancient cultures studied were distinct, and that they retained distinct technological/cultural differences during an extended period of close proximity and peaceful co-existence. The two populations seemed to form the later-day Taínos, the Amerindians present at the point of Columbian contact. Importantly, our data suggest that paleomicrobiomics can be a powerful tool to assess cultural differences between ancient populations.     

The Identification of Congeners and Aliens by Drosophila Larvae

We investigated the role of Drosophila larva olfactory system in identification of congeners and aliens. We discuss the importance of these activities in larva navigation across substrates, and the implications for allocation of space and food among species of similar ecologies. Wild type larvae of cosmopolitan D. melanogaster and endemic D. pavani, which cohabit the same breeding sites, used species-specific volatiles to identify conspecifics and aliens moving toward larvae of their species. D. gaucha larvae, a sibling species of D. pavani that is ecologically isolated from D. melanogaster, did not respond to melanogaster odor cues. Similar to D. pavani larvae, the navigation of pavani female x gaucha male hybrids was influenced by conspecific and alien odors, whereas gaucha female x pavani male hybrid larvae exhibited behavior similar to the D. gaucha parent. The two sibling species exhibited substantial evolutionary divergence in processing the odor inputs necessary to identify conspecifics. Orco (Or83b) mutant larvae of D. melanogaster, which exhibit a loss of sense of smell, did not distinguish conspecific from alien larvae, instead moving across the substrate. Syn ^97CS and rut larvae of D. melanogaster, which are unable to learn but can smell, moved across the substrate as well. The Orco (Or83b), Syn ^97CS and rut loci are necessary to orient navigation by D. melanogaster larvae. Individuals of the Trana strain of D. melanogaster did not respond to conspecific and alien larval volatiles and therefore navigated randomly across the substrate. By contrast, larvae of the Til-Til strain used larval volatiles to orient their movement. Natural populations of D. melanogaster may exhibit differences in identification of conspecific and alien larvae. Larval locomotion was not affected by the volatiles.