Lutz's spontaneous sedimentation technique and the paleoparasitological analysis of sambaqui (shell mound) sediments

Parasite findings in sambaquis (shell mounds) are scarce. Although the 121 shell mound samples were previously analysed in our laboratory, we only recently obtained the first positive results. In the sambaqui of Guapi, Rio de Janeiro, Brazil, paleoparasitological analysis was performed on sediment samples collected from various archaeological layers, including the superficial layer as a control. Eggs of Acanthocephala, Ascaridoidea and Heterakoidea were found in the archaeological layers. We applied various techniques and concluded that Lutz''s spontaneous sedimentation technique is effective for concentrating parasite eggs in sambaqui soil for microscopic analysis.     

Spermatogonial stem cell sensitivity to capsaicin: An in vitro study

Background

The placenta is an important site for iron metabolism in humans. It transfers iron from the mother to the fetus. One of the major iron transport proteins is transferrin, which is a blood plasma protein crucial for iron uptake. Its localization and expression may be one of the markers to distinguish placental dysfunction.

Methods

In the experimental study we used antibody preparation, mass spectrometric analysis, biochemical and immunocytochemical methods for characterization of transferrin expression on the human choriocarcinoma cell line JAR (JAR cells), placental lysates, and cryostat sections. Newly designed monoclonal antibody TRO-tf-01 to human transferrin was applied on human placentae from normal (n = 3) and abnormal (n = 9) pregnancies.

Results

Variations of transferrin expression were detected in villous syncytiotrophoblast, which is in direct contact with maternal blood. In placentae from normal pregnancies, the expression of transferrin in the syncytium was significantly lower (p < 0.001) when compared to placentae from abnormal ones (gestational diabetes, pregnancy induced hypertension, drug abuse).

Conclusion

These observations suggest that in the case of abnormal pregnancies, the fetus may require higher levels of transferrin in order to prevent iron depletion due to the stress from the placental dysfunction.     

Affinities of the nucleocapsid protein for variants of SL3 RNA in HIV-1

Efficient packaging of genomic RNA into new HIV-1 virus particles requires that nucleocapsid domains of precursor proteins bind the SL3 tetraloop (G317-G-A-G320) from the 5'-untranslated region. This paper presents the affinities of 35 RNA variants of SL3 for the mature 55mer NC protein, as measured by fluorescence quenching of tryptophan-37 in the protein by nucleobases. The 1:1 complexes that form in 0.2 M NaCl have dissociation constants ranging from 8 nM (GGUG) to 20 microM (GAUA). The highly conserved (GGAG) sequence for the wild type is not the most stable (K(d) = 28 nM), suggesting that other selective pressures beyond the stability of the complex must be satisfied. The leading requirement for strong interaction is for G320, followed closely by G318. Replacing either with U, A, or C reduces affinity by a factor of 15-120. NC-domains from multiple proteins combine to recognize unpaired G(2)-loci, where two guanines are in close proximity. We have previously measured affinities of the NC protein for the important stem-loops of the major packaging domain [Shubsda, M. F., Paoletti, A. C., Hudson, B. S., and Borer, P. N. (2002) Biochemistry 41, 5276-82]. Comparison with the present work shows that the nature of the stem also modulates NC-RNA interactions. Placing the G(2)-loci from the apical SL2 or SL1 loops on the SL3 stem increases affinity by a factor of 2-3, while placing the SL4 loop on the SL3 stem reduces affinity 50-fold. These results are interesting in the context of RNA-protein interaction, as well as for the discovery of antiNC agents for AIDS therapy.     

Binding to serine 65‐phosphorylated ubiquitin primes Parkin for optimal PINK1‐dependent phosphorylation and activation

Mutations in the mitochondrial protein kinase PINK1 are associated with autosomal recessive Parkinson disease (PD). We and other groups have reported that PINK1 activates Parkin E3 ligase activity both directly via phosphorylation of Parkin serine 65 (Ser⁶⁵)—which lies within its ubiquitin‐like domain (Ubl)—and indirectly through phosphorylation of ubiquitin at Ser⁶⁵. How Ser⁶⁵‐phosphorylated ubiquitin (ubiquitin^{Phospho‐Ser65}) contributes to Parkin activation is currently unknown. Here, we demonstrate that ubiquitin^{Phospho‐Ser65} binding to Parkin dramatically increases the rate and stoichiometry of Parkin phosphorylation at Ser⁶⁵ by PINK1 in vitro. Analysis of the Parkin structure, corroborated by site‐directed mutagenesis, shows that the conserved His302 and Lys151 residues play a critical role in binding of ubiquitin^{Phospho‐Ser65}, thereby promoting Parkin Ser⁶⁵ phosphorylation and activation of its E3 ligase activity in vitro. Mutation of His302 markedly inhibits Parkin Ser⁶⁵ phosphorylation at the mitochondria, which is associated with a marked reduction in its E3 ligase activity following mitochondrial depolarisation. We show that the binding of ubiquitin^{Phospho‐Ser65} to Parkin disrupts the interaction between the Ubl domain and C‐terminal region, thereby increasing the accessibility of Parkin Ser⁶⁵. Finally, purified Parkin maximally phosphorylated at Ser⁶⁵ in vitro cannot be further activated by the addition of ubiquitin^{Phospho‐Ser65}. Our results thus suggest that a major role of ubiquitin^{Phospho‐Ser65} is to promote PINK1‐mediated phosphorylation of Parkin at Ser⁶⁵, leading to maximal activation of Parkin E3 ligase activity. His302 and Lys151 are likely to line a phospho‐Ser⁶⁵‐binding pocket on the surface of Parkin that is critical for the ubiquitin^{Phospho‐Ser65} interaction. This study provides new mechanistic insights into Parkin activation by ubiquitin^{Phospho‐Ser65}, which could aid in the development of Parkin activators that mimic the effect of ubiquitin^{Phospho‐Ser65}.     

Functional variation among frugivorous birds: implications for rainforest seed dispersal in a fragmented subtropical landscape

Seed dispersal plays a critical role in rainforest regeneration patterns, hence loss of avian seed dispersers in fragmented landscapes may disrupt forest regeneration dynamics. To predict whether or not a plant will be dispersed in fragmented forests, it is necessary to have information about frugivorous bird distribution and dietary composition. However, specific dietary information for frugivorous birds is often limited. In such cases, information on the seed-crushing behaviour, gape width and relative dietary dominance by fruit may be used to describe functional groups of bird species with respect to their potential to disperse similar seeds. We used this information to assess differences in the seed dispersal potential of frugivorous bird assemblages in a fragmented rainforest landscape of southeast Queensland, Australia. The relative abundance of frugivorous birds was surveyed in extensive, remnant and regrowth rainforest sites (16 replicates of each). Large-gaped birds with mixed diets and medium-gaped birds with fruit-dominated diets were usually less abundant in remnants and regrowth than in continuous forest. Small-gaped birds with mixed diets and birds with fruit as a minor dietary component were most abundant in regrowth. We recorded a similar number of seed-crushing birds and large-gaped birds with fruit-dominated diets across site types. Bird species that may have the greatest potential to disperse a large volume and wide variety of plants, including large-seeded plants, tended to be less abundant outside of extensive forests, although one species, the figbird Sphecotheres viridis, was much more abundant in these areas. The results suggest that the dispersal of certain plant taxa would be limited in this fragmented landscape, although the potential for the dispersal of large-seeded plants may remain, despite the loss of several large-gaped disperser species.     

A recent insertion of an alu element on the Y chromosome is a useful marker for human population studies

A member of the Alu family of repeated DNA elements has been identified on the long arm of the human Y chromosome, Yq11. This element, referred to as the Y Alu polymorphic (YAP) element, is present at a specific site on the Y chromosome in some humans and is absent in others. Phylogenetic comparisons with other Alu sequences reveal that the YAP element is a member of the polymorphic subfamily-3 (PSF-3), a previously undefined subfamily of Alu elements. The evolutionary relationships of PSF-3 to other Alu subfamilies support the hypothesis that recently inserted elements result from multiple source genes. The frequency of the YAP element is described in 340 individuals from 14 populations, and the data are combined with those from other populations. There is both significant heterogeneity among populations and a clear pattern in the frequencies of the insertion: sub-Saharan Africans have the highest frequencies, followed by northern Africans, Europeans, Oceanians, and Asians. An interesting exception is the relatively high frequency of the YAP element in Japanese. The greatest genetic distance is observed between the African and non-African populations. The YAP is especially useful for studying human population history from the perspective of male lineages.      

Quantitative footprinting analysis

This review outlines the steps for obtaining relative constants for drugs from footprinting data. After correcting the autoradiographic spot intensities for differing amounts of radioactive DNA loaded into the lanes of a sequencing gel, footprinting plots, showing individual spot intensities as a function of drug concentration, are constructed. The initial relative slopes of footprinting plots are proportional to the binding constant of the drug for its DNA sites. Slopes of plots outside the drug binding sites can be used to identify locations of altered DNA structure. It illustrates the power of quantitative footprinting analysis by analyzing the binding of the antiviral agent netrospin to a 139-base pair restriction fragment in the presence of the antitumor agent actinomycin D. While two netrospin binding regions are unaffected by actinomycin D a third region experiences enhanced binding in the presence of the antitumor agent.