Synthesis and biological evaluation of a novel 99mTc labeled 2-nitroimidazole derivative as a potential agent for imaging tumor hypoxia

Tumor hypoxia plays a major role in reducing the efficacy of therapeutic modalities like chemotherapy and radiation therapy in combating cancer. In order to target hypoxic tissues, a tripeptide ligand having a 2-nitroimidazole moiety, as a bioreductive species, was synthesized. The latter was radiolabeled with ^99mTc for imaging hypoxic regions of tumors and was characterized by means of its rhenium analogue. The biodistribution and scintigraphic image of the corresponding ^99mTc-complex showed accumulation in tumor and these results suggest that it could be a marker for imaging tumor hypoxia.     

Comparative study of the interaction of fullerenol nanoparticles with eukaryotic and bacterial model membranes using solid-state NMR and FTIR spectroscopy

Native fullerene is notoriously insoluble in water and forms aggregates toxic to cell membranes, thus limiting its use in nanomedicine. In contrast, water-soluble fullerenol is compatible with biological systems and shows low in vivo toxicity on human cell lines. The interaction mechanism between these hydrophilic nanoparticles and biological membranes is however not well understood. Therefore, in this work, the effect of fullerenol on model eukaryotic and bacterial membranes was investigated using (31)P- and (2)H solid-state NMR as well as FTIR spectroscopy. DPPC/cholesterol and DPPC/DPPG bilayers were used to mimic eukaryotic and bacterial cell membranes, respectively. Our results show low affinity of fullerenol for DPPC/cholesterol bilayers but a clear interaction with model bacterial membranes. A preferential affinity of fullerenol for the anionic phospholipids DPPG in DPPC/DPPG membranes is also observed. Our data suggest that fullerenol remains at the water/bilayer interface of eukaryote-like membranes. They also indicate that the presence of a polar group such as DPPG's hydroxyl moiety at the bilayer surface plays a key role in the interaction of fullerenol with membranes. Hydrogen bonding of fullerenol nanoparticles with DPPGs' OH groups is most likely responsible for inducing lipid segregation in the lipid bilayer. Moreover, the location of the nanoparticles in the polar region of DPPG-rich regions appears to disturb the acyl chain packing and increase the membrane fluidity. The preferential interaction of fullerenol with lipids mostly found in bacterial membranes is of great interest for the design of new antibiotics.     

Patterns of protein oxidation in Arabidopsis seeds and during germination

Increased cellular levels of reactive oxygen species are known to occur during seed development and germination, but the consequences in terms of protein degradation are poorly characterized. In this work, protein carbonylation, which is an irreversible oxidation process leading to a loss of function of the modified proteins, has been analyzed by a proteomic approach during the first stages of Arabidopsis (Arabidopsis thaliana) seed germination. In the dry mature seeds, the legumin-type globulins (12S cruciferins) were the major targets. However, the acidic alpha-cruciferin subunits were carbonylated to a much higher extent than the basic (beta) ones, consistent with a model in which the beta-subunits are buried within the cruciferin molecules and the alpha-subunits are more exposed to the outside. During imbibition, various carbonylated proteins accumulated. This oxidation damage was not evenly distributed among seed proteins and targeted specific proteins as glycolytic enzymes, mitochondrial ATP synthase, chloroplastic ribulose bisphosphate carboxylase large chain, aldose reductase, methionine synthase, translation factors, and several molecular chaperones. Although accumulation of carbonylated proteins is usually considered in the context of aging in a variety of model systems, this was clearly not the case for the Arabidopsis seeds since they germinated at a high rate and yielded vigorous plantlets. The results indicate that the observed specific changes in protein carbonylation patterns are probably required for counteracting and/or utilizing the production of reactive oxygen species caused by recovery of metabolic activity in the germinating seeds.     

The interaction between RPAP3 and TRBP reveals a possible involvement of the HSP90/R2TP chaperone complex in the regulation of miRNA activity

MicroRNAs silence mRNAs by guiding the RISC complex. RISC assembly occurs following cleavage of pre-miRNAs by Dicer, assisted by TRBP or PACT, and the transfer of miRNAs to AGO proteins. The R2TP complex is an HSP90 co-chaperone involved in the assembly of ribonucleoprotein particles. Here, we show that the R2TP component RPAP3 binds TRBP but not PACT. The RPAP3-TPR1 domain interacts with the TRBP-dsRBD3, and the 1.5 Å resolution crystal structure of this complex identifies key residues involved in the interaction. Remarkably, binding of TRBP to RPAP3 or Dicer is mutually exclusive. Additionally, we found that AGO(1/2), TRBP and Dicer are all sensitive to HSP90 inhibition, and that TRBP sensitivity is increased in the absence of RPAP3. Finally, RPAP3 seems to impede miRNA activity, raising the possibility that the R2TP chaperone might sequester TRBP to regulate the miRNA pathway.     

Site-specific attachment of polyethylene glycol-like oligomers to proteins and peptides

Modification of proteins with polymers is a viable method to tune protein properties, e.g., to render them more water-soluble by using hydrophilic polymers. We have utilized precision-length, polyethylene glycol-based oligomers carrying a thioester moiety in transthioesterification and native chemical ligation reactions with internal and N-terminal cysteine residues in proteins and peptides. These reactions lead to uniquely modified proteins with an increased solubility in chaotrope- and detergent-free aqueous systems. Polymer modification of internal cysteines is fully reversible and allows generation of stable protein-polymer conjugates for enzymatic manipulations as demonstrated by proteolytic cleavage of a protein construct that was only soluble in buffers incompatible with protease activity before polymer modification. The permanent polymer modification of a Rab protein at its N-terminal cysteine produced a fully active Rab variant that was efficiently prenylated. Thus, PEGylation of prenylated proteins might be a viable route to increase water solubility of such proteins in order to carry out experiments in detergent- and lipid-free systems.     

Proteome-wide characterization of seed aging in Arabidopsis: a comparison between artificial and natural aging protocols

A variety of mechanisms have been proposed to account for the extension of life span in seeds (seed longevity). In this work, we used Arabidopsis (Arabidopsis thaliana) seeds as a model and carried out differential proteomics to investigate this trait, which is of both ecological and agricultural importance. In our system based on a controlled deterioration treatment (CDT), we compared seed samples treated for different periods of time up to 7 d. Germination tests showed a progressive decrease of germination vigor depending on the duration of CDT. Proteomic analyses revealed that this loss in seed vigor can be accounted for by protein changes in the dry seeds and by an inability of the low-vigor seeds to display a normal proteome during germination. Furthermore, CDT strongly increased the extent of protein oxidation (carbonylation), which might induce a loss of functional properties of seed proteins and enzymes and/or enhance their susceptibility toward proteolysis. These results revealed essential mechanisms for seed vigor, such as translational capacity, mobilization of seed storage reserves, and detoxification efficiency. Finally, this work shows that similar molecular events accompany artificial and natural seed aging.