The organellar peptidasome,PreP: A journey from Arabidopsis to Alzheimer's disease

The novel peptidasome, called presequence protease, PreP, was originally identified and characterized in Arabidopsis thaliana as a mitochondrial matrix and chloroplast stroma localized metalloprotease. PreP has a function as the organellar peptide clearing protease and is responsible for degrading free targeting peptides and also other unstructured peptides up to 65 amino acid residues that might be toxic to organellar functions. PreP contains an inverted Zn-binding motif and belongs to the pitrilysin protease family. The crystal structure of AtPreP refined at 2.1 Å demonstrated a unique totally enclosed large cavity of 10 000 Å³ that opens and closes in response to peptide binding, revealing a novel catalytic mechanism for proteolysis. Homologues of PreP have been found in yeast and human mitochondria. Interestingly, the human PreP, hPreP, is the protease that is responsible for clearing the human brain mitochondria from the toxic amyloid-β peptide (Aβ) associated with Alzheimer's disease (AD). Accumulation of Aβ has been shown in the brain mitochondria from AD patients and mutant transgenic mice overexpressing Aβ. Here, we present a review of our present knowledge on structural and functional characteristics of PreP and discuss its mitochondrial Aβ-degrading activity in the human brain mitochondria in relation to AD.     

A SPR strategy for high-throughput ligand screenings based on synthetic peptides mimicking a selected subdomain of the target protein: A proof of concept on HER2 receptor

The discovery of pharmaceutical agents is a complex, lengthy and costly process, critically depending on the availability of rapid and efficient screening methods. In particular, when targets are large, multidomain proteins, their complexity may affect unfavorably technical feasibility, costs and unambiguity of binding test interpretation.A possible strategy to overcome these problems relies on molecular design of receptor fragments that are: sensible targets for ligand screenings, conformationally stable also as standalone domains, easily synthesized and immobilized on chip for Biacore experiments.An additional desirable feature for new ligands is the ability of selectively targeting alternative conformational states typical of many proteins.To test the feasibility of such approach on a case with potential applicative interest, we developed a surface plasmon resonance (SPR)-based screening method for drug candidates toward HER2, a Tyr-kinase receptor targeted in anticancer therapies. HER2 was mimicked by HER2-DIVMP, a modified fragment of it immobilized onto the sensor surface specifically modeling HER2 domain IV in its bounded form, designed by structural comparison of HER2 alone and in complex with Herceptin, a monoclonal therapeutic anti-HER2 antibody.This design and its implementation in SPR devices was validated by investigating Herceptin- HER2-DIVMP affinity, measuring its dissociation constant (K_D = 19.2 nM). An efficient synthetic procedure to prepare the HER2-DIVMP peptide was also developed. The HER2-DIVMP conformational stability suggested by experimental and computational results, makes it also a valuable candidate as a mold to design new molecules selectively targeting domain IV of HER2.     

Effect of (+)-usnic acid on mitochondrial functions as measured by mitochondria-specific oligonucleotide microarray in liver of B6C3F1 mice

Usnic acid is a lichen metabolite used as a weight-loss dietary supplement due to its uncoupling action on mitochondria. However, its use has been associated with severe liver disorders in some individuals. Animal studies conducted thus far evaluated the effects of usnic acid on mitochondria primarily by measuring the rate of oxygen consumption and/or ATP generation. To obtain further insight into usnic acid-mediated effects on mitochondria, we examined the expression levels of 542 genes associated with mitochondrial structure and functions in liver of B6C3F₁ female mice using a mitochondria-specific microarray. Beginning at 8 weeks of age, mice received usnic acid at 0, 60, 180, and 600 ppm in ground, irradiated 5LG6 diet for 14 days. Microarray analysis showed a significant effect of usnic acid on the expression of several genes only at the highest dose of 600 ppm. A prominent finding of the study was a significant induction of genes associated with complexes I through IV of the electron transport chain. Moreover, several genes involved in fatty acid oxidation, the Krebs cycle, apoptosis, and membrane transporters were over-expressed. Usnic acid is a lipophilic weak acid that can diffuse through mitochondrial membranes and cause a proton leak (uncoupling). The up-regulation of complexes I–IV may be a compensatory mechanism to maintain the proton gradient across the mitochondrial inner membrane. In addition, induction of fatty acid oxidation and the Krebs cycle may be an adaptive response to uncoupling of mitochondria.     

Mitochondrial calcium uniporter blocker effectively prevents brain mitochondrial dysfunction caused by iron overload

AimsAlthough iron overload induces oxidative stress and brain mitochondrial dysfunction, and is associated with neurodegenerative diseases, brain mitochondrial iron uptake has not been investigated. We determined the role of mitochondrial calcium uniporter (MCU) in brain mitochondria as a major route for iron entry. We hypothesized that iron overload causes brain mitochondrial dysfunction, and that the MCU blocker prevents iron entry into mitochondria, thus attenuating mitochondrial dysfunction.Main methodsIsolated brain mitochondria from male Wistar rats were used. Iron (Fe^2 + and Fe^3 +) at 0–286 μM were applied onto mitochondria at various incubation times (5–30 min), and the mitochondrial function was determined. Effects of MCU blocker (Ru-360) and iron chelator were studied.Key findingsBoth Fe^2 + and Fe^3 + entered brain mitochondria and caused mitochondrial swelling in a dose- and time-dependent manner, and caused mitochondrial depolarization and increased ROS production. However, Fe^2 + caused more severe mitochondrial dysfunction than Fe^3 +. Although all drugs attenuated mitochondrial dysfunction caused by iron overload, only an MCU blocker could completely prevent ROS production and mitochondrial depolarization.SignificanceOur findings indicated that iron overload caused brain mitochondrial dysfunction, and that an MCU blocker effectively prevented this impairment, suggesting that MCU could be the major portal for brain mitochondrial iron uptake.     

Enhanced parkin levels favor ER-mitochondria crosstalk and guarantee Ca2 + transfer to sustain cell bioenergetics

Loss-of-function mutations in PINK1 or parkin genes are associated with juvenile-onset autosomal recessive forms of Parkinson disease. Numerous studies have established that PINK1 and parkin participate in a common mitochondrial-quality control pathway, promoting the selective degradation of dysfunctional mitochondria by mitophagy. Upregulation of parkin mRNA and protein levels has been proposed as protective mechanism against mitochondrial and endoplasmic reticulum (ER) stress. To better understand how parkin could exert protective function we considered the possibility that it could modulate the ER–mitochondria inter-organelles cross talk. To verify this hypothesis we investigated the effects of parkin overexpression on ER–mitochondria crosstalk with respect to the regulation of two key cellular parameters: Ca^2 + homeostasis and ATP production. Our results indicate that parkin overexpression in model cells physically and functionally enhanced ER–mitochondria coupling, favored Ca^2 + transfer from the ER to the mitochondria following cells stimulation with an 1,4,5 inositol trisphosphate (InsP₃) generating agonist and increased the agonist-induced ATP production. The overexpression of a parkin mutant lacking the first 79 residues (ΔUbl) failed to enhance the mitochondrial Ca^2 + transients, thus highlighting the importance of the N-terminal ubiquitin like domain for the observed phenotype. siRNA-mediated parkin silencing caused mitochondrial fragmentation, impaired mitochondrial Ca^2 + handling and reduced the ER–mitochondria tethering. These data support a novel role for parkin in the regulation of mitochondrial homeostasis, Ca^2 + signaling and energy metabolism under physiological conditions.     

Microinjection of serum-starved mitochondria derived from somatic cells affects parthenogenetic development of bovine and murine oocytes

Microinjection of isolated mitochondria into oocytes is an effective method to introduce exogenous mitochondrial DNA. In nuclear transfer procedures in which donor cell mitochondria are transferred with nuclei into recipient oocytes; development and survival rates of reconstructed embryos may be also directly influenced by mitochondrial viability. Mitochondrial viability is dramatically affected by cell culture conditions, such as serum starvation prior to nuclear transfer. This study was conducted to examine the influence of exogenous mitochondria using bovine and mouse parthenogenetic models. Mitochondria were isolated from primary cells at confluency and after serum starvation. The bovine oocytes injected with serum-starved mitochondria showed lower rates of morula and blastocyst formation when compared to uninjected controls (P < 0.05). However, the developmental rates between non-starved mitochondria injection and controls were not different (P > 0.05). The murine oocytes injected with serum-starved mitochondria showed lower rates of development when compared with non-starved mitochondria and controls (P < 0.01). In contrast to mitochondria transfer, ooplasm transfer did not affect murine or bovine parthenogenetic development (P > 0.05). The overall results showed that injection of serum-starved mitochondria influenced parthenogenetic development of both bovine and murine oocytes. Our results illustrate that the somatic mitochondria introduction accompanying nuclei has the capacity to affect reconstructed embryo development; particularly when using serum-starved cells as donor cells.     

Targeting the tumor and its associated stroma: One and one can make three in adoptive T cell therapy of solid tumors

Adoptive T cell therapy (ACT) has become a promising immunotherapeutic option for cancer patients. The proof for ACT therapeutic efficacy was first obtained with allogenic T cells and then reproduced with T cells isolated from patients’ tumor samples (i.e. tumor-infiltrating lymphocytes). It is now clear that specificity of ACT products can be educated by genetically engineering T cells with classical T Cell Receptors (TCR) or chimeric antigen receptors (CAR). To date a poor accessibility of the tumor mass and a hostile microenvironment, influenced by genetic and epigenetic instability, mainly limit ACT therapeutic efficacy in the case of solid tumors. Available data indicate that these hurdles might be overcome by combinatorial therapeutic strategies targeting the tumor and its associated stroma. Here we review some of the available dual targeting strategies focusing on given combination of TCR/CAR-redirected T cell products and their association with drugs targeting the tumor-vessel and/or epigenetic modifiers, with the ability to sensitize tumors to T cell recognition. Existing data have proven synergistic effects in combined settings (one and one can indeed make three) and suggest that further benefit might be achieved by additional combinatorial therapeutic approaches (could one + one + one make ten?) in ACT of solid tumor.     

Redox-Optimized ROS Balance and the relationship between mitochondrial respiration and ROS

The Redox-Optimized ROS Balance [R-ORB] hypothesis postulates that the redox environment [RE] is the main intermediary between mitochondrial respiration and reactive oxygen species [ROS]. According to R-ORB, ROS emission levels will attain a minimum vs. RE when respiratory rate (VO₂) reaches a maximum following ADP stimulation, a tenet that we test herein in isolated heart mitochondria under forward electron transport [FET]. ROS emission increased two-fold as a function of changes in the RE (~ 400 to ~ 900 mV·mM) in state 4 respiration elicited by increasing glutamate/malate (G/M). In G/M energized mitochondria, ROS emission decreases two-fold for RE ~ 500 to ~ 300 mV·mM in state 3 respiration at increasing ADP. Stressed mitochondria released higher ROS, that was only weakly dependent on RE under state 3. As a function of VO₂, the ROS dependence on RE was strong between ~ 550 and ~ 350 mV·mM, when VO₂ is maximal, primarily due to changes in glutathione redox potential. A similar dependence was observed with stressed mitochondria, but over a significantly more oxidized RE and ~ 3-fold higher ROS emission overall, as compared with non-stressed controls. We conclude that under non-stressful conditions mitochondrial ROS efflux decreases when the RE becomes less reduced within a range in which VO₂ is maximal. These results agree with the R-ORB postulate that mitochondria minimize ROS emission as they maximize VO₂ and ATP synthesis. This relationship is altered quantitatively, but not qualitatively, by oxidative stress although stressed mitochondria exhibit diminished energetic performance and increased ROS release.     

Effect of CDP-choline on age-dependent modifications of energy- and glutamate-linked enzyme activities in synaptic and non-synaptic mitochondria from rat cerebral cortex

The effect of aging and CDP-choline treatment (20 mg kg⁻¹ body weight i.p. for 28 days) on the maximal rates (V_max) of representative mitochondrial enzyme activities related to Krebs’ cycle (citrate synthase, α-ketoglutarate dehydrogenase, malate dehydrogenase), glutamate and related amino acid metabolism (glutamate dehydrogenase, glutamate–oxaloacetate- and glutamate–pyruvate transaminases) were evaluated in non-synaptic and intra-synaptic “light” and “heavy” mitochondria from frontal cerebral cortex of male Wistar rats aged 4, 12, 18 and 24 months.During aging, enzyme activities vary in a complex way respect to the type of mitochondria, i.e. non-synaptic and intra-synaptic. This micro-heterogeneity is an important factor, because energy-related mitochondrial enzyme catalytic properties cause metabolic modifications of physiopathological significance in cerebral tissue in vivo, also discriminating pre- and post-synaptic sites of action for drugs and affecting tissue responsiveness to noxious stimuli.Results show that CDP-choline in vivo treatment enhances cerebral energy metabolism selectively at 18 months, specifically modifying enzyme catalytic activities in non-synaptic and intra-synaptic “light” mitochondrial sub-populations. This confirms that the observed changes in enzyme catalytic activities during aging reflect the bioenergetic state at each single age and the corresponding energy requirements, further proving that in vivo drug treatment is able to interfere with the neuronal energy metabolism.     

Mitochondriotropic Liposomes

Mitochondrial dysfunction contributes to a large variety of human disorders, ranging from neurodegenerative and neuromuscular diseases, obesity, and diabetes to ischemia-reperfusion injury and cancer. Increasing pharmacological efforts toward therapeutic interventions have been made leading to the emergence of “Mitochondrial Medicine” as a new field of biomedical research. The identification of molecular mitochondrial drug targets in combination with the development of methods for selectively delivering biologically active molecules to the site of mitochondria will eventually launch a multitude of new therapies for the treatment of mitochondria-related diseases, which are based either on the selective protection, repair, or eradication of cells. Yet, while tremendous efforts are being undertaken to identify new mitochondrial drugs and drug targets, the development of mitochondria-specific drug carrier systems is lagging behind. To ensure a high efficiency of current and future mitochondrial therapeutics, delivery systems need to be developed, which are able to selectively transport biologically active molecules to and into mitochondria within living human cells. In this study we present the first data demonstrating that conventional liposomes can be rendered mitochondria-specific via the attachment of known mitochondriotropic residues to the liposomal surface.     

The mitochondria-targeted anti-oxidant MitoQ reduces aspects of mitochondrial fission in the 6-OHDA cell model of Parkinson's disease

Parkinson's disease (PD) is a neurodegenerative disorder for which available treatments provide symptom relief but do not stop disease progression. Mitochondria, and in particular mitochondrial dynamics, have been postulated as plausible pharmacological targets. Mitochondria-targeted antioxidants have been developed to prevent mitochondrial oxidative damage, and to alter the involvement of reactive oxygen species (ROS) in signaling pathways. In this study, we have dissected the effect of MitoQ, which is produced by covalent attachment of ubiquinone to a triphenylphosphonium lipophilic cation by a ten carbon alkyl chain. MitoQ was tested in an in vitro PD model which involves addition of 6-hydroxydopamine (6-OHDA) to SH-SY5Y cell cultures. At sublethal concentrations of 50 μM, 6-OHDA did not induce increases in protein carbonyl, mitochondrial lipid peroxidation or mitochondrial DNA damage. However, after 3 h of treatment, 6-OHDA disrupts the mitochondrial morphology and activates the machinery of mitochondrial fission, but not fusion. Addition of 6-OHDA did not increase the levels of fission 1, mitofusins 1 and 2 or optic atrophy 1 proteins, but does lead to the translocation of dynamin related protein 1 from the cytosol to the mitochondria. Pre-treatment with MitoQ (50 nM, 30 min) results in the inhibition of the mitochondrial translocation of Drp1. Furthermore, MitoQ also inhibited the translocation of the pro-apoptotic protein Bax to the mitochondria. These findings provide mechanistic evidence for a role for redox events contributing to mitochondrial fission and suggest the potential of mitochondria-targeted therapeutics in diseases that involve mitochondrial fragmentation due to oxidative stress.     

BN-PAGE analysis of the respiratory chain complexes in mitochondria of cucumber MSC16 mutant

Rearrangements of mitochondrial DNA in MSC16 mutant of cucumber (Cucumis sativus L.) affect mitochondrial functioning due to the alteration mainly of Complex I resulting in several metabolic changes. One-dimensional Blue-Native polyacrylamide gel electrophoresis (BN-PAGE) and densitometric measurements showed that the level and in-gel capacity of Complex I were lower in MSC16 leaf and root mitochondria as compared to wild-type (WT). The level and capacity of supercomplex I + III₂ were always lower in leaf but not in MSC16 root mitochondria. Two-dimensional BN/SDS-PAGE indicated that the band abundance for most of the subunits of Complex I was lower in MSC16 leaf and root mitochondria. Supercomplex I + III₂ level was only altered in MSC16 leaf mitochondria as measured after 2D BN/SDS-PAGE. No differences in the qualitative composition of the subunits of Complex I and supercomplex I + III₂ between MSC16 and WT mitochondria were observed. In MSC16 mitochondria Complex I impairment could be compensated to some extent by additional respiratory chain NADH dehydrogenases. A higher capacity and level of NDB-1 protein of external NADH dehydrogenase was observed in MSC16 leaf and root mitochondria as compared to WT. The level of COX II, mitochondrial-encoded subunit of Complex IV, was higher in MSC16 leaf and root mitochondria. However, the capacity of Complex IV was slightly higher only in MSC16 leaf mitochondria. The levels of complexes: III₂ and V and Complex V capacity did not differ in mitochondria between genotypes. An abundance of the subunits of respiratory complexes is one of the key factors determining not only their structure and functional stability but also a formation of the supercomplexes. We discuss here mitochondrial genome rearrangements in MSC16 mutant in a relation to assembly and/or stability (the lower level and capacity) of Complex I and supercomplex I + III₂.     

The anticancer agent doxorubicin disrupts mitochondrial energy metabolism and redox balance in skeletal muscle

The combined loss of muscle strength and constant fatigue are disabling symptoms for cancer patients undergoing chemotherapy. Doxorubicin, a standard chemotherapy drug used in the clinic, causes skeletal muscle dysfunction and premature fatigue along with an increase in reactive oxygen species (ROS). As mitochondria represent a primary source of oxidant generation in muscle, we hypothesized that doxorubicin could negatively affect mitochondria by inhibiting respiratory capacity, leading to an increase in H₂O₂-emitting potential. Here we demonstrate a biphasic response of skeletal muscle mitochondria to a single doxorubicin injection (20 mg/kg). Initially at 2 h doxorubicin inhibits both complex I- and II-supported respiration and increases H₂O₂ emission, both of which are partially restored after 24 h. The relationship between oxygen consumption and membrane potential (ΔΨ) is shifted to the right at 24 h, indicating elevated reducing pressure within the electron transport system (ETS). Respiratory capacity is further decreased at a later time point (72 h) along with H₂O₂-emitting potential and an increased sensitivity to mitochondrial permeability transition pore (mPTP) opening. These novel findings suggest a role for skeletal muscle mitochondria as a potential underlying cause of doxorubicin-induced muscle dysfunction.     

Mitochondrial trafficking of APP and alpha synuclein: Relevance to mitochondrial dysfunction in Alzheimer's and Parkinson's diseases

Mitochondrial dysfunction is an important intracellular lesion associated with a wide variety of diseases including neurodegenerative disorders. In addition to aging, oxidative stress and mitochondrial DNA mutations, recent studies have implicated a role for the mitochondrial accumulation of proteins such as plasma membrane associated amyloid precursor protein (APP) and cytosolic alpha synuclein in the pathogenesis of mitochondrial dysfunction in Alzheimer's disease (AD) and Parkinson's disease (PD), respectively. Both of these proteins contain cryptic mitochondrial targeting signals, which drive their transport across mitochondria. In general, mitochondrial entry of nuclear coded proteins is assisted by import receptors situated in both outer and inner mitochondrial membranes. A growing number of evidence suggests that APP and alpha synclein interact with import receptors to gain entry into mitochondrial compartment. Additionally, carboxy terminal cleaved product of APP, ～ 4 kDa Abeta, is also transported into mitochondria with the help of mitochondrial outer membrane import receptors. This review focuses on the mitochondrial targeting and accumulation of these two structurally different proteins and the mode of mechanism by which they affect the physiological functions of mitochondria.     

Release of targeted p53 from the mitochondrion as an early signal during mitochondrial dysfunction

Increased accumulation of p53 tumor suppressor protein is an early response to low-level stressors. To investigate the fate of mitochondrial-sequestered p53, mouse embryonic fibroblast cells (MEFs) on a p53-deficient genetic background were transfected with p53-EGFP fusion protein led by a sense (m53-EGFP) or antisense (c53-EGFP) mitochondrial import signal. Rotenone exposure (100 nM, 1 h) triggered the translocation of m53-EGFP from the mitochondrion to the nucleus, thus shifting the transfected cells from a mitochondrial p53 to a nuclear p53 state. Antibodies for p53 serine phosphorylation or lysine acetylation indicated a different post-translational status of recombinant p53 in the nucleus and mitochondrion, respectively. These data suggest that cycling of p53 through the mitochondria may establish a direct pathway for p53 signaling from the mitochondria to the nucleus during mitochondrial dysfunction. PK11195, a pharmacological ligand of mitochondrial TSPO (formerly known as the peripheral-type benzodiazepine receptor), partially suppressed the release of mitochondria-sequestered p53. These findings support the notion that p53 function mediates a direct signaling pathway from the mitochondria to nucleus during mitochondrial dysfunction.     

A cell-based single-stranded DNA aptamer specifically targets gastric cancer

Gastric cancer is one of the most prevailing cancers with high morbidity and mortality. Limitations in the current diagnosis and therapy, specially lacking of specific molecular therapeutic targets, ask for the development of new strategies. Aptamer, a newly developed adaptive molecule, could be used in clinical detection and therapy because of its high affinity and specificity. As no aptamer has ever been developed in preventing gastric cancer so far, we were the first who cloned such an aptamer specifically targeting gastric cancer. The aptamer was selected by systematic evolution of ligands by exponential enrichment with gastric cancer cell-line HGC-27 as target cell line and immortalized gastric epithelial cell-line GES-1 as control cell line. The affinity and specificity of candidate aptamers were examined by flow cytometry, confocal imagining and aptamer-based histochemistry staining. After 19 cycles of systematic evolution of ligands by exponential enrichment and subsequent cloning and sequencing, an aptamer with the highest affinity and specificity (nominated as AGC03) among candidates was screened out from a random single-stranded DNA pool. Moreover, AGC03 could not only specifically bind to gastric cancer cells (the equilibrium dissociation constant value was 16.49 ± 0.40 nM) in vitro, but also recognize cancer cells in human cancer tissue. Our most important finding is that AGC03 could even be internalized into cells automatically. In conclusion, we obtained a novel aptamer specifically targeting gastric cancer, which is an effective tool for both gastric cancer diagnosis and drug delivery.     

The functional interaction between abaecin and pore-forming peptides indicates a general mechanism of antibacterial potentiation

Long-chain proline-rich antimicrobial peptides such as bumblebee abaecin show minimal activity against Gram-negative bacteria despite binding efficiently to specific intracellular targets. We recently reported that bumblebee abaecin interacts with Escherichia coli DnaK but shows negligible antibacterial activity unless it is combined with sublethal doses of the pore-forming peptide hymenoptaecin. These two bumblebee peptides are co-expressed in vivo in response to a bacterial challenge. Here we investigated whether abaecin interacts similarly with pore-forming peptides from other organisms by replacing hymenoptaecin with sublethal concentrations of cecropin A (0.3 μM) or stomoxyn (0.05 μM). We found that abaecin increased the membrane permeabilization effects of both peptides, confirming that it can reduce the minimal inhibitory concentrations of pore-forming peptides from other species. We also used atomic force microscopy to show that 20 μM abaecin combined with sublethal concentrations of cecropin A or stomoxyn causes profound structural changes to the bacterial cell surface. Our data indicate that the potentiating functional interaction between abaecin and pore-forming peptides is not restricted to specific co-expressed peptides from the same species but is likely to be a general mechanism. Combination therapies based on diverse insect-derived peptides could therefore be used to tackle bacteria that are recalcitrant to current antibiotics.     

Predicted ionisation in mitochondria and observed acute changes in the mitochondrial transcriptome after gamma irradiation: A Monte Carlo simulation and quantitative PCR study

It is a widely accepted that the cell nucleus is the primary site of radiation damage while extra-nuclear radiation effects are not yet systematically included into models of radiation damage.We performed Monte Carlo simulations assuming a spherical cell (diameter 11.5 μm) modelled after JURKAT cells with the inclusion of realistic elemental composition data based on published literature. The cell model consists of cytoplasm (density 1 g/cm³), nucleus (diameter 8.5 μm; 40% of cell volume) as well as cylindrical mitochondria (diameter 1 μm; volume 0.5 μm³) of three different densities (1, 2 and 10 g/cm³) and total mitochondrial volume relative to the cell volume (10, 20, 30%). Our simulation predicts that if mitochondria take up more than 20% of a cell's volume, ionisation events will be the preferentially located in mitochondria rather than in the cell nucleus.Using quantitative polymerase chain reaction, we substantiate in JURKAT cells that human mitochondria respond to gamma radiation with early (within 30 min) differential changes in the expression levels of 18 mitochondrially encoded genes, whereby the number of regulated genes varies in a dose-dependent but non-linear pattern (10 Gy: 1 gene; 50 Gy: 5 genes; 100 Gy: 12 genes).The simulation data as well as the experimental observations suggest that current models of acute radiation effects, which largely focus on nuclear effects, might benefit from more systematic considerations of the early mitochondrial responses and how these may subsequently determine cell response to ionising radiation.     

Development of a bifunctional chelating agent containing isothiocyanate residue for one step F-18 labeling of peptides and application for RGD labeling

We report herein a novel isothiocyanate active ligand for fluorine-18 labeling prepared by four step synthesis. It can be conjugated to a target molecule containing an amino functional group under weak basic conditions by way of thiourea bond formation. We explored the application of synthesized ligand by conjugating to well known α_vβ₃ integrin targeting peptide, c(RGDyK). The conjugated peptide showed good radiochemical yield and efficiency with an excellent radiochemical purity (97.1 ± 1.2%) in a short reaction time (10 min). Labeled peptide showed excellent in vitro and in vivo stability (>95%). α_vβ₃ integrin specific tumor uptake was observed both in biodistribution and small animal microPET studies on α_vβ₃-positive U87MG (human glioma cells) xenograft bearing mice. In general, successful application of synthesized ligand for labeling of RGD peptide could facilitate the possibility of using this ligand for labeling peptides containing an amino functional group.