Relationships between EGFR signaling-competent and endocytosis-competent membrane microdomains

Membrane microdomains, the so-called lipid rafts, function as platforms to concentrate receptors and assemble the signal transduction machinery. Internalization, in most cases, is carried out by different specialized structures, the clathrin-coated pits. Here, we show that several endocytic proteins are efficiently recruited to morphologically identified plasma membrane lipid rafts, upon activation of the epidermal growth factor (EGF) receptor (EGFR), a receptor tyrosine kinase. Analysis of detergent-resistant membrane fractions revealed that the EGF-dependent association of endocytic proteins with rafts is as efficient as that of signaling effector molecules, such as Grb2 or Shc. Finally, the EGFR, but not the nonsignaling transferrin receptor, could be localized in nascent coated pits that almost invariably contained raft membranes. Thus, specialized membrane microdomains have the ability to assemble both the molecular machineries necessary for intracellular propagation of EGFR effector signals and for receptor internalization.     

Characterization of Schizosaccharomyces pombe ER alpha-mannosidase: a reevaluation of the role of the enzyme on ER-associated degradation

It has been postulated that creation of Man8GlcNAc2 isomer B (M8B) by endoplasmic reticulum (ER) alpha-mannosidase I constitutes a signal for driving irreparably misfolded glycoproteins to proteasomal degradation. Contrary to a previous report, we were able to detect in vivo (but not in vitro) an extremely feeble ER alpha-mannosidase activity in Schizosaccharomyces pombe. The enzyme yielded M8B on degradation of Man9GlcNAc2 and was inhibited by kifunensin. Live S. pombe cells showed an extremely limited capacity to demannosylate Man9GlcNAc2 present in misfolded glycoproteins even after a long residence in the ER. In addition, no preferential degradation of M8B-bearing species was detected. Nevertheless, disruption of the alpha-mannosidase encoding gene almost totally prevented degradation of a misfolded glycoprotein. This and other conflicting reports may be best explained by assuming that the role of ER mannosidase on glycoprotein degradation is independent of its enzymatic activity. The enzyme, behaving as a lectin binding polymannose glycans of varied structures, would belong together with its enzymatically inactive homologue Htm1p/Mnl1p/EDEM, to a transport chain responsible for delivering irreparably misfolded glycoproteins to proteasomes. Kifunensin and 1-deoxymannojirimycin, being mannose homologues, would behave as inhibitors of the ER mannosidase or/and Htm1p/Mnl1p/EDEM putative lectin properties.     

EGCG, a major component of green tea, inhibits VEGF production by swine granulosa cells

Vascular Endothelial Growth Factor (VEGF) plays a pivotal role in the physiological ovarian angiogenic process: its production appears to be stimulated by the hypoxic environment which takes place during follicle development. Recently, epigallocatechin-3-gallate (EGCG) from green tea has been used in livestock nutrition as an alternative to antibiotics. However, despite many potential benefits of EGCG consumption, it is also important to get an insight on the possible reproductive-related consequences of feeding supplementation: in fact this substance has been found to inhibit angiogenesis, a process fundamental for follicle development. Therefore, we evaluated the effect of EGCG (5 and 50 microg/ml) on the production of the main angiogenetic factor, VEGF, by swine granulosa cells cultured in normoxia (19% O2), partial (5% O2) or total hypoxia (1% O2). In addition, we studied the effect of the catechin on cell proliferation. Our data demonstrate that both partial and total hypoxia stimulated VEGF production. EGCG reduced VEGF production independently of the O2 condition: 50 microM was the most effective doses. Granulosa cell proliferation was inhibited by EGCG even if only by the highest concentration. This effect might possibly be due to the decrease induced in VEGF production. Therefore feeding supplementation with EGCG should be carefully considered.     

Human locomotion on snow: determinants of economy and speed of skiing across the ages

We explore here the evolution of skiing locomotion in the last few thousand years by investigating how humans adapted to move effectively in lands where a cover of snow, for several months every year, prevented them from travelling as on dry ground. Following historical research, we identified the sets of skis corresponding to the 'milestones' of skiing evolution in terms of ingenuity and technology, built replicas of them and measured the metabolic energy associated to their use in a climate-controlled ski tunnel.Six sets of skis were tested, covering a span from 542 AD to date. Our results show that: (i) the history of skiing is associated with a progressive decrease in the metabolic cost of transport, (ii) it is possible today to travel at twice the speed of ancient times using the same amount of metabolic power and (iii) the cost of transport is speed-independent for each ski model, as during running. By combining this finding with the relationship between time of exhaustion and the sustainable fraction of metabolic power, a prediction of the maximum skiing speed according to the distance travelled is provided for all past epochs, including two legendary historical journeys (1206 and 1520 AD) on snow. Our research shows that the performances in races originating from them (Birkebeiner and Vasaloppet) and those of other modern competitions (skating versus classical techniques) are well predicted by the evolution of skiing economy. Mechanical determinants of the measured progression in economy are also discussed in the paper.     

ERK2 shows a restrictive and locally selective mechanism of recognition by its tyrosine phosphatase inactivators not shared by its activator MEK1

The two regulatory residues that control the enzymatic activity of the mitogen-activated protein (MAP) kinase ERK2 are phosphorylated by the unique MAP kinase kinases MEK1/2 and dephosphorylated by several tyrosine-specific and dual specificity protein phosphatases. Selective docking interactions facilitate these phosphorylation and dephosphorylation events, controlling the specificity and duration of the MAP kinase activation-inactivation cycles. We have analyzed the contribution of specific residues of ERK2 in the physical and functional interaction with the ERK2 phosphatase inactivators PTP-SL and MKP-3 and with its activator MEK1. Single mutations in ERK2 that abrogated the dephosphorylation by endogenous tyrosine phosphatases from HEK293 cells still allowed efficient phosphorylation by endogenous MEK1/2. Discrete ERK2 mutations at the ERK2 docking groove differentially affected binding and inactivation by PTP-SL and MKP-3. Remarkably, the cytosolic retention of ERK2 by its activator MEK1 was not affected by any of the analyzed ERK2 single amino acid substitutions. A chimeric MEK1 protein, containing the kinase interaction motif of PTP-SL, bound tightly to ERK2 through its docking groove and behaved as a gain-of-function MAP kinase kinase that hyperactivated ERK2. Our results provide evidence that the ERK2 docking groove is more restrictive and selective for its tyrosine phosphatase inactivators than for MEK1/2 and indicate that distinct ERK2 residues modulate the docking interactions with activating and inactivating effectors.     

The DNA damage checkpoint response requires histone H2B ubiquitination by Rad6-Bre1 and H3 methylation by Dot1

The cellular response to DNA lesions entails the recruitment of several checkpoint and repair factors to damaged DNA, and chromatin modifications may play a role in this process. Here we show that in Saccharomyces cerevisiae epigenetic modification of histones is required for checkpoint activity in response to a variety of genotoxic stresses. We demonstrate that ubiquitination of histone H2B on lysine 123 by the Rad6-Bre1 complex, is necessary for activation of Rad53 kinase and cell cycle arrest. We found a similar requirement for Dot1-dependent methylation of histone H3. Loss of H3-Lys(79) methylation does not affect Mec1 activation, whereas it renders cells checkpoint-defective by preventing phosphorylation of Rad9. Such results suggest that histone modifications may have a role in checkpoint function by modulating the interactions of Rad9 with chromatin and active Mec1 kinase.     

miR-15a and miR-16-1 down-regulation in pituitary adenomas

Micro RNAs (miRs) are small noncoding RNAs, functioning as antisense regulators of other RNAs. miR-15a and miR-16-1 genes are located at chromosome 13q14, a region which is frequently deleted in pituitary tumors. An inverse correlation has been shown in B cell chronic lymphocytic leukemia (B-CLL) between miR-15a and miR-16-1 expression and the expression levels of arginyl-tRNA synthetase (RARS), an enzyme which associates with the cofactor p43 in the aminoacyl-tRNA synthetase complex. When secreted, p43 regulates local inflammatory response and macrophage chemotaxis, and seems to have anti-neoplastic properties in mice. We explored miR-15a and miR-16-1 expression in 10 GH-secreting and in 10 PRL-secreting pituitary macroadenomas by Northern blot, and investigated the possible correlation with in vivo and in vitro characteristics. We found that miR-15a and miR-16-1 are expressed at lower levels in pituitary adenomas as compared to normal pituitary tissue. Moreover, their expression inversely correlates with tumor diameter and with RARS expression (P < 0.05), but directly correlates with p43 secretion (P < 0.02). Therefore, miR15 and miR16 down-regulation in pituitary adenomas correlates with a greater tumor diameter and a lower p43 secretion, suggesting that these genes may, at least in part, influence tumor growth.     

Heritability of the timing of autumn migration in a natural bird population

In recent decades, global temperature has increased at an unprecedented rate. This has been causing rapid environmental shifts that have altered the selective regimes determining the annual organization of birds. In order to assess the potential for adaptive evolution in the timing of autumn migration, we estimated heritabilities of the onset of migratory activity in a southern German blackcap (Sylvia atricapilla) population. Heritabilities (h2=0.34-0.45) and coefficients of additive genetic variation (CV(A)=4.7-5.7) were significant and consistent when estimated by different methods, irrespective of whether they were derived from birds hatched in the wild or bred in captivity. In an artificial selection experiment, we selected for later onset of migratory activity, simulating expected natural selection on this trait. We obtained a significant delay in the mean onset of migratory activity by more than one week after two generations of selection. Realized heritability (h2=0.55) was in agreement with expected heritability in the cohort that the selection line was derived from. Our results suggest that evolutionary changes in the timing of autumn migration may take place over a very short time period and will most probably be unconstrained by the lack of additive genetic variation.