Human endometrial stem cells: High-yield isolation and characterization

Background

Menstrual blood is only recently and still poorly studied, but it is an abundant and noninvasive source of highly proliferative mesenchymal stromal cells (MSCs). However, no appropriate isolation method has been reported due to its high viscosity and high content of clots and desquamated epithelium.

Harmonizing Labeling and Analytical Strategies to Obtain Protein Turnover Rates in Intact Adult Animals

Changes in the abundance of individual proteins in the proteome can be elicited by modulation of protein synthesis (the rate of input of newly synthesized proteins into the protein pool) or degradation (the rate of removal of protein molecules from the pool). A full understanding of proteome changes therefore requires a definition of the roles of these two processes in proteostasis, collectively known as protein turnover. Because protein turnover occurs even in the absence of overt changes in pool abundance, turnover measurements necessitate monitoring the flux of stable isotope–labeled precursors through the protein pool such as labeled amino acids or metabolic precursors such as ammonium chloride or heavy water. In cells in culture, the ability to manipulate precursor pools by rapid medium changes is simple, but for more complex systems such as intact animals, the approach becomes more convoluted. Individual methods bring specific complications, and the suitability of different methods has not been comprehensively explored. In this study, we compare the turnover rates of proteins across four mouse tissues, obtained from the same inbred mouse strain maintained under identical husbandry conditions, measured using either [¹³C₆]lysine or [²H₂]O as the labeling precursor. We show that for long-lived proteins, the two approaches yield essentially identical measures of the first-order rate constant for degradation. For short-lived proteins, there is a need to compensate for the slower equilibration of lysine through the precursor pools. We evaluate different approaches to provide that compensation. We conclude that both labels are suitable, but careful determination of precursor enrichment kinetics in amino acid labeling is critical and has a considerable influence on the numerical values of the derived protein turnover rates.     

Response to hyperoxia is associated with similar ho-1 gene expression level in lungs of aging CBA mice of both sexes

Beside classical antioxidative enzymes, the response to hyperoxia might be mediated via regulation of other systems, such as heme oxygenase (HO). Ho-1 gene expression is found to be upregulated by hyperoxia in all groups of mice, while HO-1 protein isoform was increased only in 4 months old male mice. In steady-state conditions ho-1 and ho-2 gene expression remained unchanged irrespective of sex or age, which was not the case with protein level of both isoforms. This study suggests that in lungs of CBA mice the response to oxidative stress may be mediated through the interaction of other systems such as heme oxygenase, primarily via upregulation of ho-1 gene expression in both sexes. Contrary to our previous study in liver of hyperoxia treated mice, current results might imply that at conventional oxygen conditions lungs of female mice with the emphasis on aging females, are better prepared for oxidative stress conditions through the increase of HO-activity.     

The cold-active lipase of Pseudomonas fragi. Heterologous expression, biochemical characterization and molecular modeling.

A recombinant lipase cloned from Pseudomonas fragi strain IFO 3458 (PFL) was found to retain significant activity at low temperature. In an attempt to elucidate the structural basis of this behaviour, a model of its three-dimensional structure was built by homology and compared with homologous mesophilic lipases, i.e. the Pseudomonas aeruginosa lipase (45% sequence identity) and Burkholderia cepacia lipase (38%). In this model, features common to all known lipases have been identified, such as the catalytic triad (S83, D238 and H260) and the oxyanion hole (L17, Q84). Structural modifications recurrent in cold-adaptation, i.e. a large amount of charged residues exposed at the protein surface, have been detected. Noteworthy is the lack of a disulphide bridge conserved in homologous Pseudomonas lipases that may contribute to increased conformational flexibility of the cold-active enzyme.     

Mda-9/syntenin is expressed in uveal melanoma and correlates with metastatic progression

Uveal melanoma is an aggressive cancer that metastasizes to the liver in about half of the patients, with a high lethality rate. Identification of patients at high risk of metastases may provide indication for a frequent follow-up for early detection of metastases and treatment. The analysis of the gene expression profiles of primary human uveal melanomas showed high expression of SDCBP gene (encoding for syndecan-binding protein-1 or mda-9/syntenin), which appeared higher in patients with recurrence, whereas expression of syndecans was lower and unrelated to progression. Moreover, we found that high expression of SDCBP gene was related to metastatic progression in two additional independent datasets of uveal melanoma patients. More importantly, immunohistochemistry showed that high expression of mda-9/syntenin protein in primary tumors was significantly related to metastatic recurrence in our cohort of patients. Mda-9/syntenin expression was confirmed by RT-PCR, immunofluorescence and immunohistochemistry in cultured uveal melanoma cells or primary tumors. Interestingly, mda-9/syntenin showed both cytoplasmic and nuclear localization in cell lines and in a fraction of patients, suggesting its possible involvement in nuclear functions. A pseudo-metastatic model of uveal melanoma to the liver was developed in NOD/SCID/IL2Rγ null mice and the study of mda-9/syntenin expression in primary and metastatic lesions revealed higher mda-9/syntenin in metastases. The inhibition of SDCBP expression by siRNA impaired the ability of uveal melanoma cells to migrate in a wound-healing assay. Moreover, silencing of SDCBP in mda-9/syntenin-high uveal melanoma cells inhibited the hepatocyte growth factor (HGF)-triggered invasion of matrigel membranes and inhibited the activation of FAK, AKT and Src. Conversely syntenin overexpression in mda-9/syntenin-low uveal melanoma cells mediated opposite effects. These results suggest that mda-9/syntenin is involved in uveal melanoma progression and that it warrants further investigation as a candidate molecular marker of metastases and a potential therapeutic target.