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}.     

DNA methylation pattern of CALCA in preterm neonates with bacterial sepsis as a putative epigenetic biomarker

Diagnosis of bacterial sepsis in preterm neonates can be difficult when using serum markers that rely on physiological changes because these changes may not necessarily be the result of bacterial infections alone. This retrospective investigation explores the potential use of the DNA methylation pattern of CpG sites in the promoter region of the calcitonin-related polypeptide α (CALCA) gene as an epigenetic biomarker for bacterial sepsis in preterm newborns. Four novel changes in the DNA methylation of eight CpG sites were detected in this gene and are present only in neonates with bacterial sepsis: (1) partial methylation at -769 CpG in gram-negative or gram-positive early onset sepsis (EOS) and late onset sepsis (LOS) episodes; (2) demethylation of 8 CpGs in gram-negative EOS followed by LOS (ELS) and in gram-negative EOS; (3) demethylation of 7 CpGs in gram-positive ELS and gram-positive EOS; (4) -771 C:G > T:A; 5′ de novo -778 CpG mutation on both alleles in EOS. These changes were not detected in birth weight and gestational age matched controls or in newborns with isolated infections. Our results indicate that the DNA methylation pattern of the promoter region of the CALCA gene varies in different types of bacterial preterm sepsis, thus suggesting a potential use as an epigenetic biomarker. A prospective confirmation of these results is essential.     

The four hexamerin genes in the honey bee: structure,molecular evolution and function deduced from expression patterns in queens,workers and drones

Background  

Hexamerins are hemocyanin-derived proteins that have lost the ability to bind copper ions and transport oxygen; instead, they became storage proteins. The current study aimed to broaden our knowledge on the hexamerin genes found in the honey bee genome by exploring their structural characteristics, expression profiles, evolution, and functions in the life cycle of workers, drones and queens.     

Status of complete proteome analysis by mass spectrometry: SILAC labeled yeast as a model system

Background  

Mass spectrometry has become a powerful tool for the analysis of large numbers of proteins in complex samples, enabling much of proteomics. Due to various analytical challenges, so far no proteome has been sequenced completely. O'Shea, Weissman and co-workers have recently determined the copy number of yeast proteins, making this proteome an excellent model system to study factors affecting coverage.     

Optical imaging of luminescence for in vivo quantification of gene electrotransfer in mouse muscle and knee

Background  

Optical imaging is an attractive non-invasive way to evaluate the expression of a transferred DNA, mainly thanks to its lower cost and ease of realization. In this study optical imaging was evaluated for monitoring and quantification of the mouse knee joint and tibial cranial muscle electrotransfer of a luciferase encoding plasmid. Optical imaging was applied to study the kinetics of luciferase expression in both tissues.     

Genome-wide association study of reproductive traits in Nellore heifers using Bayesian inference

Background

An important goal of Zebu breeding programs is to improve reproductive performance. A major problem faced with the genetic improvement of reproductive traits is that recording the time for an animal to reach sexual maturity is costly. Another issue is that accurate estimates of breeding values are obtained only a long time after the young bulls have gone through selection. An alternative to overcome these problems is to use traits that are indicators of the reproductive efficiency of the herd and are easier to measure, such as age at first calving. Another problem is that heifers that have conceived once may fail to conceive in the next breeding season, which increases production costs. Thus, increasing heifer’s rebreeding rates should improve the economic efficiency of the herd. Response to selection for these traits tends to be slow, since they have a low heritability and phenotypic information is provided only later in the life of the animal. Genome-wide association studies (GWAS) are useful to investigate the genetic mechanisms that underlie these traits by identifying the genes and metabolic pathways involved.

Results

Data from 1853 females belonging to the Agricultural Jacarezinho LTDA were used. Genotyping was performed using the BovineHD BeadChip (777 962 single nucleotide polymorphisms (SNPs)) according to the protocol of Illumina - Infinium Assay II ® Multi-Sample HiScan with the unit SQ ™ System. After quality control, 305 348 SNPs were used for GWAS. Forty-two and 19 SNPs had a Bayes factor greater than 150 for heifer rebreeding and age at first calving, respectively. All significant SNPs for age at first calving were significant for heifer rebreeding. These 42 SNPs were next or within 35 genes that were distributed over 18 chromosomes and comprised 27 protein-encoding genes, six pseudogenes and two miscellaneous noncoding RNAs.

Conclusions

The use of Bayes factor to determine the significance of SNPs allowed us to identify two sets of 42 and 19 significant SNPs for heifer rebreeding and age at first calving, respectively, which explain 11.35 % and 6.42 % of their phenotypic variance, respectively. These SNPs provide relevant information to help elucidate which genes affect these traits.