The Unique Non-Catalytic C-Terminus of P37delta-PI3K Adds Proliferative Properties In Vitro and In Vivo

The PI3K/Akt pathway is central for numerous cellular functions and is frequently deregulated in human cancers. The catalytic subunits of PI3K, p110, are thought to have a potential oncogenic function, and the regulatory subunit p85 exerts tumor suppressor properties. The fruit fly, Drosophila melanogaster, is a highly suitable system to investigate PI3K signaling, expressing one catalytic, Dp110, and one regulatory subunit, Dp60, and both show strong homology with the human PI3K proteins p110 and p85. We recently showed that p37δ, an alternatively spliced product of human PI3K p110δ, displayed strong proliferation-promoting properties despite lacking the catalytic domain completely. Here we functionally evaluate the different domains of human p37δ in Drosophila. The N-terminal region of Dp110 alone promotes cell proliferation, and we show that the unique C-terminal region of human p37δ further enhances these proliferative properties, both when expressed in Drosophila, and in human HEK-293 cells. Surprisingly, although the N-terminal region of Dp110 and the C-terminal region of p37δ both display proliferative effects, over-expression of full length Dp110 or the N-terminal part of Dp110 decreases survival in Drosophila, whereas the unique C-terminal region of p37δ prevents this effect. Furthermore, we found that the N-terminal region of the catalytic subunit of PI3K p110, including only the Dp60 (p85)-binding domain and a minor part of the Ras binding domain, rescues phenotypes with severely impaired development caused by Dp60 over-expression in Drosophila, possibly by regulating the levels of Dp60, and also by increasing the levels of phosphorylated Akt. Our results indicate a novel kinase-independent function of the PI3K catalytic subunit.     

Inhibition of mushroom tyrosinase by tropolone

Tropolone inhibits both mono- and o-dihydroxyphenolase activity of mushroom tyrosinase. Most of the inhibition exerted by tropolone was reversed by dialysis or by excess CU²⁺. The data indicate that tropolone and o-dihydroxyphenols compete for binding to the copper at the active site of the enzyme. Comparison between the effectiveness of various copper chelators showed that tropolone is one of the most potent inhibitors of mushroom tyrosinase; 50% inhibition was observed with 0.4 × 10^?6 M tropolone.     

Direct involvement of p53 in the base excision repair pathway of the DNA repair machinery

The p53 tumor suppressor that plays a central role in the cellular response to genotoxic stress was suggested to be associated with the DNA repair machinery which mostly involves nucleotide excision repair (NER). In the present study we show for the first time that p53 is also directly involved in base excision repair (BER). These experiments were performed with p53 temperature-sensitive (ts) mutants that were previously studied in in vivo experimental models. We report here that p53 ts mutants can also acquire wild-type activity under in vitro conditions. Using ts mutants of murine and human origin, it was observed that cell extracts overexpressing p53 exhibited an augmented BER activity measured in an in vitro assay. Depletion of p53 from the nuclear extracts abolished this enhanced activity. Together, this suggests that p53 is involved in more than one DNA repair pathway.     

A genome scan for familial combined hyperlipidemia reveals evidence of linkage with a locus on chromosome 11.

Familial combined hyperlipidemia (FCHL) is a common familial lipid disorder characterized by a variable pattern of elevated levels of plasma cholesterol and/or triglycerides. It is present in 10%-20% of patients with premature coronary heart disease. The genetic etiology of the disease, including the number of genes involved and the magnitude of their effects, is unknown. Using a subset of 35 Dutch families ascertained for FCHL, we screened the genome, with a panel of 399 genetic markers, for chromosomal regions linked to genes contributing to FCHL. The results were analyzed by use of parametric-linkage methods in a two-stage study design. Four loci, on chromosomes 2p, 11p, 16q, and 19q, exhibited suggestive evidence for linkage with FCHL (LOD scores of 1.3-2.6). Markers within each of these regions were then examined in the original sample and in additional Dutch families with FCHL. The locus on chromosome 2 failed to show evidence for linkage, and the loci on chromosome 16q and 19q yielded only equivocal or suggestive evidence for linkage. However, one locus, near marker D11S1324 on the short arm of human chromosome 11, continued to show evidence for linkage with FCHL, in the second stage of this design. This region does not contain any strong candidate genes. These results provide evidence for a candidate chromosomal region for FCHL and support the concept that FCHL is complex and heterogeneous.     

Stability of targeted metabolite profiles of urine samples under different storage conditions

Introduction

Few studies have investigated the influence of storage conditions on urine samples and none of them used targeted mass spectrometry (MS).

Objectives

We investigated the stability of metabolite profiles in urine samples under different storage conditions using targeted metabolomics.

Methods

Pooled, fasting urine samples were collected and stored at ?80 °C (biobank standard), ?20 °C (freezer), 4 °C (fridge), ~9 °C (cool pack), and ~20 °C (room temperature) for 0, 2, 8 and 24 h. Metabolite concentrations were quantified with MS using the AbsoluteIDQ? p150 assay. We used the Welch-Satterthwaite-test to compare the concentrations of each metabolite. Mixed effects linear regression was used to assess the influence of the interaction of storage time and temperature.

Results

The concentrations of 63 investigated metabolites were stable at ?20 and 4 °C for up to 24 h when compared to samples immediately stored at ?80 °C. When stored at ~9 °C for 24 h, few amino acids (Arg, Val and Leu/Ile) significantly decreased by 40% in concentration (P < 7.9E?04); for an additional three metabolites (Ser, Met, Hexose H1) when stored at ~20 °C reduced up to 60% in concentrations. The concentrations of four more metabolites (Glu, Phe, Pro, and Thr) were found to be significantly influenced when considering the interaction between exposure time and temperature.

Conclusion

Our findings indicate that 78% of quantified metabolites were stable for all examined storage conditions. Particularly, some amino acid concentrations were sensitive to changes after prolonged storage at room temperature. Shipping or storing urine samples on cool packs or at room temperature for more than 8 h and multiple numbers of freeze and thaw cycles should be avoided.