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.     

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}.     

The effect of nutrients on carbon and nitrogen fixation by the UCYN-A–haptophyte symbiosis

Symbiotic relationships between phytoplankton and N₂-fixing microorganisms play a crucial role in marine ecosystems. The abundant and widespread unicellular cyanobacteria group A (UCYN-A) has recently been found to live symbiotically with a haptophyte. Here, we investigated the effect of nitrogen (N), phosphorus (P), iron (Fe) and Saharan dust additions on nitrogen (N₂) fixation and primary production by the UCYN-A–haptophyte association in the subtropical eastern North Atlantic Ocean using nifH expression analysis and stable isotope incubations combined with single-cell measurements. N₂ fixation by UCYN-A was stimulated by the addition of Fe and Saharan dust, although this was not reflected in the nifH expression. CO₂ fixation by the haptophyte was stimulated by the addition of ammonium nitrate as well as Fe and Saharan dust. Intriguingly, the single-cell analysis using nanometer scale secondary ion mass spectrometry indicates that the increased CO₂ fixation by the haptophyte in treatments without added fixed N is likely an indirect result of the positive effect of Fe and/or P on UCYN-A N₂ fixation and the transfer of N₂-derived N to the haptophyte. Our results reveal a direct linkage between the marine carbon and nitrogen cycles that is fuelled by the atmospheric deposition of dust. The comparison of single-cell rates suggests a tight coupling of nitrogen and carbon transfer that stays balanced even under changing nutrient regimes. However, it appears that the transfer of carbon from the haptophyte to UCYN-A requires a transfer of nitrogen from UCYN-A. This tight coupling indicates an obligate symbiosis of this globally important diazotrophic association.     

Populations Microbiennes Des Bois

Resumé Les auteurs ont étudié la composition chimique (N, substances humiques, lignine) de copeaux de bois blanc exposés à l'air libre depuis 2 à 10 ans, ainsi que leur type de peuplement fongique. En l'absence de lignivore, le pH, les taux de lignine et d'N s'élèvent et il se forme des quantités modérées d'humus à forte capacité d'échange. Dans le cas contraire, on voit apparaître, en abondance, des substances humiques peu condensées et la matière organique subit une évolution rappelant celle du mor.Avec la collaboration technique de Melle. M. Clet.Kononova, dans sa monographie⁵, signale de Troussov (1916) une étude que nous n'avons pas eu en mains.     

Studies of esterase 6 in Drosophila melanogaster. XVIII. Biochemical differences between the slow and fast allozymes

Most natural populations of Drosophila melanogaster are polymorphic for two major electrophoretic variants at the esterase-6 locus. The frequency of the EST 6F allozyme is greatest in populations in warmer latitudes, whereas the EST 6S allozyme is predominant in colder latitudes. Latitudinal clines in electromorph frequencies are found on three continents. Purified preparations of the allozymes have been characterized for their pH optimum, substrate specificity, organophosphate inhibition, alcohol activation, thermal stability, and kinetic parameters. These and previous analyses of the EST 6 allozymes reveal that the two variants have differences in their physical and kinetic properties that may provide a basis for the selective maintenance of the polymorphisms and an explanation of the clinal variation observed in natural populations.      

Seropositivity is associated with insulin resistance in patients with early inflammatory polyarthritis: results from the Norfolk Arthritis Register (NOAR): an observational study

Introduction  

Cardiovascular disease (CVD) is the leading cause of death in patients with inflammatory polyarthritis (IP), especially in seropositive disease. In established rheumatoid arthritis (RA), insulin resistance (IR) is increased and associated with CVD. We investigated factors associated with IR in an inception cohort of patients with early IP.     

Phosphorylation of Parkin at Serine65 is essential for activation: elaboration of a Miro1 substrate-based assay of Parkin E3 ligase activity

Mutations in PINK1 and Parkin are associated with early-onset Parkinson''s disease. We recently discovered that PINK1 phosphorylates Parkin at serine65 (Ser⁶⁵) within its Ubl domain, leading to its activation in a substrate-free activity assay. We now demonstrate the critical requirement of Ser⁶⁵ phosphorylation for substrate ubiquitylation through elaboration of a novel in vitro E3 ligase activity assay using full-length untagged Parkin and its putative substrate, the mitochondrial GTPase Miro1. We observe that Parkin efficiently ubiquitylates Miro1 at highly conserved lysine residues, 153, 230, 235, 330 and 572, upon phosphorylation by PINK1. We have further established an E2-ubiquitin discharge assay to assess Parkin activity and observe robust discharge of ubiquitin-loaded UbcH7 E2 ligase upon phosphorylation of Parkin at Ser⁶⁵ by wild-type, but not kinase-inactive PINK1 or a Parkin Ser65Ala mutant, suggesting a possible mechanism of how Ser⁶⁵ phosphorylation may activate Parkin E3 ligase activity. For the first time, to the best of our knowledge, we report the effect of Parkin disease-associated mutations in substrate-based assays using full-length untagged recombinant Parkin. Our mutation analysis indicates an essential role for the catalytic cysteine Cys431 and reveals fundamental new knowledge on how mutations may confer pathogenicity via disruption of Miro1 ubiquitylation, free ubiquitin chain formation or by impacting Parkin''s ability to discharge ubiquitin from a loaded E2. This study provides further evidence that phosphorylation of Parkin at Ser⁶⁵ is critical for its activation. It also provides evidence that Miro1 is a direct Parkin substrate. The assays and reagents developed in this study will be important to uncover new insights into Parkin biology as well as aid in the development of screens to identify small molecule Parkin activators for the treatment of Parkinson''s disease.