Detection and characterization of two chimpanzee polyomavirus genotypes from different subspecies

The complete nucleotide sequences of three chimpanzee polyomavirus genetic variants were determined. Phylogenetic analysis indicated that the viruses form two different genotypes of ChPyV. Comparison with other primate polyomaviruses revealed a putative agnogene, and an unusually long VP1 open reading frame. The transcriptional control regions (TCR) of the viruses were extremely short (155 nucleotides), and highly conserved amongst the genotypes. Analysis of the TCR from different chimpanzee subspecies, and from a series of tissues from five individuals confirmed its genetic stability, and also indicates that double-infections with different genotypes can occur

Mammalian Target of Rapamycin Complex I (mTORC1) Activity in Ras Homologue Enriched in Brain (Rheb)-Deficient Mouse Embryonic Fibroblasts

<div><p>The Ras-like GTPase Rheb has been identified as a crucial activator of mTORC1. Activation most likely requires a direct interaction between Rheb and mTOR, but the exact mechanism remains unclear. Using a panel of Rheb-deficient mouse embryonic fibroblasts (MEFs), we show that Rheb is indeed essential for the rapid increase of mTORC1 activity following stimulation with insulin or amino acids. However, mTORC1 activity is less severely reduced in Rheb-deficient MEFs in the continuous presence of serum or upon stimulation with serum. This remaining mTORC1 activity is blocked by depleting the cells for amino acids or imposing energy stress. In addition, MEK inhibitors and the RSK-inhibitor BI-D1870 interfere in mTORC1 activity, suggesting that RSK acts as a bypass for Rheb in activating mTORC1. Finally, we show that this rapamycin-sensitive, Rheb-independent mTORC1 activity is important for cell cycle progression. In conclusion, whereas rapid adaptation in mTORC1 activity requires Rheb, a second Rheb-independent activation mechanism exists that contributes to cell cycle progression.</p> </div

Effect of mTOR inhibition on 4E-BP1.

<p><b>3a</b>. Asynchronously growing cells Rheb-deficient cells (N21) or control cells (N45) were either left untreated or treated with various concentrations of PP242 (2, 1 or 0.25 µM) or rapamycin (50 nM) for 60 minutes. Total cell lysates were probed with antibodies indicated on the right. <b>3B</b>. Rheb-deficient cells (N21) or control cells (N45) were serum starved overnight and stimulated with insulin, serum or rapamycin as indicated on the top. Alternatively cells were depleted for amino acids for two hours (-AA) after which the culture medium was reconstituted with amino acids (-AA +AA) for 30 minutes or serum (-AA +serum) for 90 minutes. Total lysates were analyzed by Western blot with the antibodies indicated on the right. <b>3C</b>. Association of 4E-BP1 with eIF4E from Rheb-deficient (N21, N23) or Rheb-positive (N45) cells. Cells serum starved overnight and then either treated with rapamycin for 1 hour or depleted for amino acids for two hours. Alternatively, they were grown in complete medium (lanes 10-12). Subsequently, cells were lysed and eIF4E plus associating proteins were pulled down using an ^m7GTP-Sepharose pull-down assay. Isolated proteins (upper two panels) and total lysates (lower three panels) were analyzed by Western blotting with the antibodies indicated on the right. In all cases the immunoblots shown are representative of observations for at least two experiments. </p

Characterization of Rheb-deficient cells (N21, N23) or control cells (N46, N45).

<p><b>1a</b>. PCR on decreasing amounts of genomic DNA isolated from N46 cells (Rheb+/-), in which exon 3 from a single allele has been excised and N23 cells (Rheb-/-), in which exon 3 from both Rheb alleles have been removed. In the positive control lane (+) a mixture of DNA isolated from wild type animals, animals carrying the <i>loxP</i> sites surrounding exon 3 and Rheb-deficient embryos was used. In the negative control lane (-) no input DNA was used. <b>1b</b>. Q-PCR on mRNA isolated from Rheb-positive (N45; first and third bar) and Rheb-deficient (N23; second and fourth bar) cells using primers from exon 2 and 3 (first and second bar) or from exon 2 and 5 (third and fourth bar). <b>1c</b>. Analysis of mTORC1 activity in total lysates by Western blotting. Cells were serum starved overnight. and left untreated, stimulated with insulin for 30 minutes (Ins) or depleted for amino acids for two hours and then replenished with amino acids for 30 minutes (AA). Blots were probed with antibodies indicated on the right. The upper three panels represent reprobes of the same blot. GAPDH and Rap1 were used as loading controls. The immunoblots shown are representative for at least four experiments. Numbers on top of immunoblots indicate ratio Rap1 over pS6K T389. <b>1d</b>. Cells were serum starved overnight. and left untreated, stimulated with insulin for 30 minutes (Ins) or with serum. The immunoblots shown are representative of observations for at least two experiments. Rap1 was used as loading control. Numbers on top of immunoblots indicate ratio Rap1 over pS6K T389. <b>1e</b>. Overview of the PI3K and ERK pathway components converging on mTORC1 and indication of the inhibitors and stimuli used in this study. Arrows represent activation, squares indicate inhibition. <b>1f</b>. Asynchronously growing cells were either left untreated or treated with various concentrations of PP242 (2, 1 or 0.25 µM) or rapamycin (50 nM) for 60 minutes. Total cell lysates were probed with antibodies indicated on the right. The immunoblots shown are representative for at least two experiments. GAPDH was used as loading control. Numbers on top of immunoblots indicate the ratio of pS6K T389 relative to GAPDH.</p

Analysis of Raptor phosphorylation.

<p><b>5a</b>. Rheb-deficient (N21, N23) or control cells (N45) were serum starved overnight and stimulated for 30 minutes with insulin or 90 minutes with serum. Endogenous Raptor was immuno-precipitated and Western blots were probed for S863 phosphorylation (upper panel). Hereafter, blots were stripped and probed for total Raptor levels. A representative example from three experiments is shown. <b>5b</b> Rheb-deficient (N21, N23) or control cells (N45) were serum starved overnight and stimulated for 30 minutes with insulin or 90 minutes with serum or pretreated with 10 µM U0126 before serum stimulation. Endogenous Raptor was immuno-precipitated and Western blots were probed with a pS863 Raptor antibody (upper panel) and reprobed for Raptor (lower panel). A representative example from two experiments is shown. In all panels numbers above the blot indicate the ratio of Raptor pS863 relative to Raptor. <b>5c</b>. Rheb-deficient (N21, N23) or control cells (N45) were serum starved overnight and either left untreated or stimulated for 90 minutes with serum. Where indicated, cells were pretreated with rapamycin (50 nM), PP242 (2 µM) or the PKB-inhibitor AKT_VIII (10 µM) for 60 minutes. Hereafter, endogenous Raptor was immuno-precipitated and analyzed under 5a. The immunoblots shown are representative of observations for at least two experiments. A representative example from two experiments is shown. <b>5d</b>. Schematic representation of the mechanism of Rheb-independent mTORC1 activation. Arrows represent activation, squares indicate inhibition.</p

Genetic association analysis of 13 nuclear-encoded mitochondrial candidate genes with type II diabetes mellitus: the DAMAGE study

Mitochondria play an important role in many processes, like glucose metabolism, fatty acid oxidation and ATP synthesis. In this study, we aimed to identify association of common polymorphisms in nuclear-encoded genes involved in mitochondrial protein synthesis and biogenesis with type II diabetes mellitus (T2DM) using a two-stage design. In the first stage, we analyzed 62 tagging single nucleotide polymorphisms (SNPs) in the Hoorn study (n=999 participants) covering all common variation in 13 biological candidate genes. These 13 candidate genes were selected from four clusters regarded essential for correct mitochondrial protein synthesis and biogenesis: aminoacyl tRNA synthetases, translation initiation factors, tRNA modifying enzymes and mitochondrial DNA transcription and replication. SNPs showing evidence for association with T2DM were measured in second stage genotyping (n=10164 participants). After a meta-analysis, only one SNP in SIRT4 (rs2522138) remained significant (P=0.01). Extending the second stage with samples from the Danish Steno Study (n=1220 participants) resulted in a common odds ratio (OR) of 0.92 (0.85–1.00), P=0.06. Moreover, in a large meta-analysis of three genome-wide association studies, this SNP was also not associated with T2DM (P=0.72). In conclusion, we did not find evidence for association of common variants in 13 nuclear-encoded mitochondrial proteins with T2DM