411 research outputs found
Preferential exchange: strengthening connections in complex networks
No full textMany social, technological, and biological interactions involve network relationships whose outcome intimately depends on the structure of the network and on the strengths of the connections. Yet, although much information is now available concerning the structure of many networks, the strengths are more difficult to measure. Here we show that, for one particular social network, notably the e-mail network, a suitable measure of the strength of the connections can be available. We also propose a simple mechanism, based on positive feedback and reciprocity, that can explain the observed behavior and that hints toward specific dynamics of formation and reinforcement of network connections. Network data from contexts different from social sciences indicate that power-law, and generally broad, distributions of the connection strength are ubiquitous, and the proposed mechanism has a wide range of applicability
Analysis of the Secondary Structure of the Catalytic Domain of Mouse Ras Exchange Factor CDC25Mm
No full textThe minimal active domain GEF domain. of the mouse Ras exchange factor CDC25Mm was purified to homogeneity
from recombinant Escherichia coli culture. The 256 amino acids polypeptide shows high activity in vitro and forms a stable
complex with H-ras p21 in absence of guanine nucleotides. Circular dichroism CD. spectra in the far UV region indicate
that this domain is highly structured with a high content of a-helix 42%.. Near UV CD spectra evidenced good signal due
to phenylalanine and tyrosine while a poor contribution was elicited by the three tryptophan residues contained in this
domain. The tryptophan fluorescence signal was scarcely affected by denaturation of the protein or by formation of the
binary complex with H-ras p21, suggesting that the Trp residues, which are well conserved in the GEF domain of several
Ras-exchange factors, were exposed to the surface of the protein and they are not most probably directly involved in the
interaction with Ras proteins. q1998 Elsevier Science B.
Mass spectrometry-based metabolomics : convergence of Snf1/AMPK and methionine metabolism to control mitochondrial respiration
No full textMass spectrometry-based metabolomics has become increasingly popular in biology and molecular medicine. Analysis of the key metabolites in biological fluids has become an important part of improving disease diagnosis. In this context, chromatography coupled with mass spectrometry represents a valid tool to characterize and quantify numerous compounds present in biological matrices. In the present work, we have explored the role of methionine metabolism on cellular functions of a prototrophic yeast strain during exponential growth phase and determining how this affects the metabolic state upon SNF1 deletion.
Experimental
Yeast strains and growth conditions S. cerevisiae strains used in this study are reported in Table 1. Synthetic medium (SD)
contains 2% glucose, 6.7 g/L of Yeast Nitrogen Base (Difco). Methionine was added to the concentrations indicated in figure legends. In these conditions, cells exhibit exponential growth between OD600nm = 0.1 per ml (equivalent to 2*106 cells/ml) and OD600nm = 2.5 per ml (5*107 cells/ml); all experiments were performed in exponential phase of growth (OD600nm/ml 0.5-1) or post-log phase (24 h, 48 h and 72 h after the beginning of exponential phase). Antimycin A was added to a final concentration of 1 μg/ml from 2 mg/ml stock in 100% ethanol; the same volume of ethanol was added in the control culture.
Cells were properly manupulated in order to extract RNA and carried out all the necessary biological investigation in addition to metabolites analysis by GC-MS.
Metabolites analysis
13C-labelling of proteinogenic amino acids was achieved by growth on 20 g/L glucose as a mixture of 80% (w/w) unlabelled and 20% (w/w) uniformly labeled [U-13C]glucose (13C, 99 %; Cambridge Isotope Laboratories, Inc). Cells from an overnight minimal medium culture were washed and used for inoculation below an OD600 of 0.03. 13C-labelled biomass aliquots were harvested by centrifugation during the mid-exponential growth phase at an OD600 of ≤1. The cells (about 0.3 mg of dry biomass) were washed once with sterile water, and hydrolysed in 150 μL 6 M HCl at 105 oC for 6 h. The hydrolysate was dried in a heating block at 80 oC under a constant airflow.
Before the GC/MS analyses all samples were subjected to a derivatization step as follows. Each sample was resuspended in 30 μL of acetonitrile, followed by 30 μL of MBDSTFA (N-methyl-N-ter-butyldimethylsilyl-trifluoroacetamide). The resulting mixture, contained in a closed vial, was stirred for 10 min and centrifuged for 15 sec. Then, the vial was incubated at 85° C. After 1 h, the sample slowly reached room temperature and was analysed by GC-MS (single quadrupole).
A METAFOR (metabolic flux ratio) analysis was perfomed: the mass isotopomer distribution of proteinogenic amino acids was used to calculate the split ratios of key branching points of yeast central metabolism using the software FIAT FLUX [3].
The results show that methionine addition leads to Snf1 and Acc1 phosphorylations as well as a general slow-down of proliferation, boosting mtDNA synthesis, mitochondrial pyruvate uptake and TCA cycle activity. Remarkably, mitochondrial proteomic analysis highlight that methionine upregulates proteins of the pyruvate dehydrogenase complex, TCA cycle and respiratory chain complexes.
Thus, Snf1/AMPK and methionine metabolism converge to control mitochondrial respiration in glucose repressed conditions.
References:
[1] Kushnirov, V. V. (2000). Yeast 16, 857–860. [2] Pessina, S., Tsiarentsyeva, V., Busnelli, S., Vanoni, M., Alberghina, L., and Coccetti, P. (2010). Cell Cycle 9, 2189–2200. [3] Zamboni, N., Fischer, E., and Sauer, U. (2005). BMC Bioinformatics 6, 209
Synthesis and biological evaluation of 1,4-diaryl-2-azetidinones as specific anticancer agents: activation of adenosine monophosphate activated protein kinase and induction of apoptosis
No full textA series of 1,4-diaryl-2-azetidinones, e.g., I, were synthesized and evaluated for antiproliferative activity, cell cycle effects, and apoptosis induction. Strong cytotoxicity was obsd. with the best compds. (±)-trans-20, (±)-trans-21, and enantiomers (+)-trans-20 and (+)-trans-21, which exhibited IC50 values of 3-13 nM against duodenal adenocarcinoma cells. They induced inhibition of tubulin polymn. and subsequent G2/M arrest. This effect was accompanied by activation of AMP-activated protein kinase, activation of caspase-3, and induction of apoptosis. Addnl., the most potent compds. displayed antiproliferative activity against different colon cancer cell lines, opening the route to a new class of potential therapeutic agents against colon cancer
1,4-Diaril-2-azetidinoni ad attivita' antitumorale
No full textVengono descritti nuovi analoghi della combretastatina A4 aventi una struttura 1,4-diaril-2-azetidinonica sostituita nella posizione 3 da un gruppo idrossile. I composti sono utili come agenti antitumorali
CK2 and GSK3 phosphorylation on S29 controls wild-type ATXN3 nuclear uptake
No full textAbstractIn the present work we show that murine ATXN3 (ATXN3Q6) nuclear uptake is promoted by phosphorylation on serine 29, a highly conserved residue inside the Josephin domain. Both casein kinase 2 (CK2) and glycogen synthase kinase 3 (GSK3) are able to carry out phosphorylation on this residue. S29 phosphorylation was initially assessed in vitro on purified ATXN3Q6, and subsequently confirmed in transfected COS-7 cells, by MS analysis. Site-directed mutagenesis of S29 to an alanine was shown to strongly reduce nuclear uptake, in COS-7 transiently transfected cells overexpressing ATXN3Q6, while substitution with phospho-mimic aspartic acid restored the wild-type phenotype. Finally, treatment with CK2 and GSK3 inhibitors prevented S29 phosphorylation and strongly inhibited nuclear uptake, showing that both kinases are involved in ATXN3Q6 subcellular sorting. Although other authors have previously addressed this issue, we show for the first time that ATXN3 is phosphorylated inside the Josephin domain and that S29 phosphorylation is involved in nuclear uptake of ATXN3
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