Colocalization of MID1IP1 and c-Myc is Critically Involved in Liver Cancer Growth via Regulation of Ribosomal Protein L5 and L11 and CNOT2.

Though midline1 interacting protein 1 (MID1IP1) was known as one of the glucose-responsive genes regulated by carbohydrate response element binding protein (ChREBP), the underlying mechanisms for its oncogenic role were never explored. Thus, in the present study, the underlying molecular mechanism of MID1P1 was elucidated mainly in HepG2 and Huh7 hepatocellular carcinoma cells (HCCs). MID1IP1 was highly expressed in HepG2, Huh7, SK-Hep1, PLC/PRF5, and immortalized hepatocyte LX-2 cells more than in normal hepatocyte AML-12 cells. MID1IP1 depletion reduced the viability and the number of colonies and also increased sub G1 population and the number of TUNEL-positive cells in HepG2 and Huh7 cells. Consistently, MID1IP1 depletion attenuated pro-poly (ADP-ribose) polymerase (pro-PARP), c-Myc and activated p21, while MID1IP1 overexpression activated c-Myc and reduced p21. Furthermore, MID1IP1 depletion synergistically attenuated c-Myc stability in HepG2 and Huh7 cells. Of note, MID1IP1 depletion upregulated the expression of ribosomal protein L5 or L11, while loss of L5 or L11 rescued c-Myc in MID1IP1 depleted HepG2 and Huh7 cells. Interestingly, tissue array showed that the overexpression of MID1IP1 was colocalized with c-Myc in human HCC tissues, which was verified in HepG2 and Huh7 cells by Immunofluorescence. Notably, depletion of CCR4-NOT2 (CNOT2) with adipogenic activity enhanced the antitumor effect of MID1IP1 depletion to reduce c-Myc, procaspase 3 and pro-PARP in HepG2, Huh7 and HCT116 cells. Overall, these findings provide novel insight that MID1IP1 promotes the growth of liver cancer via colocalization with c-Myc mediated by ribosomal proteins L5 and L11 and CNOT2 as a potent oncogenic molecule.

Thalidomide targets EGFL6 to inhibit EGFL6/PAX6 axis-driven angiogenesis in small bowel vascular malformation.

BACKGROUND: Small bowel vascular malformation disease (SBVM) is the most common cause of obscure gastrointestinal bleeding (OGIB). Several studies suggested that EGFL6 was able to promote the growth of tumor endothelial cells by forming tumor vessels. To date, it remains unclear how EGFL6 promotes pathological angiogenesis in SBVM and whether EGFL6 is a target of thalidomide. METHODS: We took advantage of SBVM plasma and tissue samples and compared the expression of EGFL6 between SBVM patients and healthy people via ELISA and Immunohistochemistry. We elucidated the underlying function of EGFL6 in SBVM in vitro and by generating a zebrafish model that overexpresses EGFL6, The cycloheximide (CHX)-chase experiment and CoIP assays were conducted to demonstrate that thalidomide can promote the degradation of EGFL6 by targeting CRBN. RESULTS: The analysis of SBVM plasma and tissue samples revealed that EGFL6 was overexpressed in the patients compared to healthy people. Using in vitro and in vivo assays, we demonstrated that an EMT pathway triggered by the EGFL6/PAX6 axis is involved in the pathogenesis of SBVM. Furthermore, through in vitro and in vivo assays, we elucidated that thalidomide can function as anti-angiogenesis medicine through the regulation of EGFL6 in a proteasome-dependent manner. Finally, we found that CRBN can mediate the effect of thalidomide on EGFL6 expression and that the CRBN protein interacts with EGFL6 via a Lon N-terminal peptide. CONCLUSION: Our findings revealed a key role for EGFL6 in SBVM pathogenesis and provided a mechanism explaining why thalidomide can cure small bowel bleeding resulting from SBVM.

3Rs-friendly approach to exogenous metabolic activation that supports high-throughput genetic toxicology testing.

MultiFlow® DNA Damage-p53, γH2AX, Phospho-Histone H3 is a miniaturized, flow cytometry-based assay that provides genotoxic mode of action information by distinguishing clastogens, aneugens, and nongenotoxicants. Work to date has focused on the p53-competent human cell line TK6. While mammalian cell genotoxicity assays typically supply exogenous metabolic activation in the form of concentrated rat liver S9, this is a less-than-ideal approach for several reasons, including 3Rs considerations. Here, we describe our experiences with low concentration S9 and saturating co-factors which were allowed to remain in contact with cells and test chemicals for 24 continuous hours. We exposed TK6 cells in 96-well plates to each of 15 reference chemicals over a range of concentrations, both in the presence and absence of 0.25% v/v phenobarbital/ß-naphthoflavone-induced rat liver S9. After 4 and 24 hr of treatment cell aliquots were added to wells of a microtiter plate containing the working detergent/stain/antibody cocktail. After a brief incubation robotic sampling was employed for walk-away flow cytometric data acquisition. PROAST benchmark dose (BMD) modeling was used to characterize the resulting dose-response curves. For each of the 8 reference pro-genotoxicants studied, relative nuclei count, γH2AX, and/or p53 biomarker BMD values were order(s) of magnitude lower for 0.25% S9 conditions compared to 0% S9. Conversely, several of the direct-acting reference chemicals exhibited appreciably lower cytotoxicity and/or genotoxicity BMD values in the presence of S9 (eg, resorcinol). These results prove the efficacy of the low concentration S9 system, and indicate that an efficient and highly scalable multiplexed assay can effectively identify chemicals that require bioactivation to exert their genotoxic effects.

Mitochondrial Genome Variation Affects Multiple Respiration and Nonrespiration Phenotypes in Saccharomyces cerevisiae.

Mitochondrial genome variation and its effects on phenotypes have been widely analyzed in higher eukaryotes but less so in the model eukaryote Saccharomyces cerevisiae Here, we describe mitochondrial genome variation in 96 diverse S. cerevisiae strains and assess associations between mitochondrial genotype and phenotypes as well as nuclear-mitochondrial epistasis. We associate sensitivity to the ATP synthase inhibitor oligomycin with SNPs in the mitochondrially encoded ATP6 gene. We describe the use of iso-nuclear F1 pairs, the mitochondrial genome equivalent of reciprocal hemizygosity analysis, to identify and analyze mitochondrial genotype-dependent phenotypes. Using iso-nuclear F1 pairs, we analyze the oligomycin phenotype-ATP6 association and find extensive nuclear-mitochondrial epistasis. Similarly, in iso-nuclear F1 pairs, we identify many additional mitochondrial genotype-dependent respiration phenotypes, for which there was no association in the 96 strains, and again find extensive nuclear-mitochondrial epistasis that likely contributes to the lack of association in the 96 strains. Finally, in iso-nuclear F1 pairs, we identify novel mitochondrial genotype-dependent nonrespiration phenotypes: resistance to cycloheximide, ketoconazole, and copper. We discuss potential mechanisms and the implications of mitochondrial genotype and of nuclear-mitochondrial epistasis effects on respiratory and nonrespiratory quantitative traits.

Protein synthesis inhibitors induce both memory impairment and its recovery.

The involvement of protein synthesis in the mechanisms of conditioned food aversion memory impairment and recovery in grape snails was studied. It was found that protein synthesis inhibitor (cycloheximide) injections before a reminder by the conditioned stimulus (CS) caused amnesia development. Three days after amnesia induction, injections of cycloheximide or another protein synthesis inhibitor, anisomycin, combined with a reminder by four CSs resulted in memory retrieval, which was saved for 24 h. Cycloheximide injections and the administration of one CS as a reminder to an amnestic animals caused the memory expression only in response to this CS, while it was absent the next day. The isolated administration of a reminder or inhibitor injections without a reminder was not effective. It is suggested that amnesia is an active process and that one of its mechanisms may be a protein-dependent amnesia reactivation caused by a reminder. The administration of protein synthesis inhibitors led to impairment of amnesia reactivation and to recovery of the state formed before amnesia induction and thus to the recovery of conditioned food aversion memory.

Induction of Apoptosis in TNF-Treated L929 Cells in the Presence of Necrostatin-1.

It has been shown that necroptosis-caspase-independent programmed necrotic cell death-can be induced by treatment with tumor necrosis factor (TNF) in the L929 murine fibrosarcoma cell line, even in the absence of a caspase inhibitor. Although it was reported that necrostatin-1-a specific inhibitor of necroptosis-inhibited TNF-induced necroptosis in L929 cells, it has not been elucidated whether the cells eventually die by apoptosis in the presence of necrostatin-1. In this paper, induction of apoptosis was demonstrated in TNF-treated L929 cells in the presence of necrostatin-1. Co-treatment with cycloheximide expedited apoptosis induction in necrostatin-1/TNF-treated L929 cells: typical apoptotic morphological changes, including membrane blebbing and nuclear fragmentation, induction of caspase-3 activity, proteolytic activation of caspases-3, -8, and -9, and cleavage of poly(ADP-ribose) polymerase (PARP) (a well-known substrate of caspase-3) were observed. Moreover, co-treatment with Z-VAD-fmk (a pan-caspase inhibitor) inhibited apoptosis by completely inhibiting caspases, resulting in a shift from apoptosis to necroptosis. In contrast, co-treatment with Z-Asp-CH2-DCB (a caspase inhibitor preferential to caspase-3) inhibited apoptosis without expediting necroptosis. These results indicate that apoptosis can be induced in TNF-treated L929 cells when the cells are protected from necroptosis, and support the notion that partial activation of caspase-8 in the presence of a caspase inhibitor preferential to caspase-3 suppresses both apoptosis and necroptosis.

NLRP3 regulates a non-canonical platform for caspase-8 activation during epithelial cell apoptosis.

Nod-like receptor, pyrin containing 3 (NLRP3) is characterized primarily as a canonical caspase-1 activating inflammasome in macrophages. NLRP3 is also expressed in the epithelium of the kidney and gut; however, its function remains largely undefined. Primary mouse tubular epithelial cells (TEC) lacking Nlrp3 displayed reduced apoptosis downstream of the tumor necrosis factor (TNF) receptor and CD95. TECs were identified as type II apoptotic cells that activated caspase-8, tBid and mitochondrial apoptosis via caspase-9, responses that were reduced in Nlrp3-/- cells. The activation of caspase-8 during extrinsic apoptosis induced by TNFα/cycloheximide (TNFα/CHX) was dependent on adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) and completely independent of caspase-1 or caspase-11. TECs and primary human proximal tubular epithelial cells (HPTC) did not activate a canonical inflammasome, caspase-1, or IL-1ß secretion in response to TNFα/CHX or NLRP3-dependent triggers, such as ATP or nigericin. In cell fractionation studies and by confocal microscopy, NLRP3 colocalized with ASC and caspase-8 in speck-like complexes at the mitochondria during apoptosis. The formation of NLRP3/ASC/caspase-8 specks in response to TNFα/CHX was downstream of TNFR signaling and dependent on potassium efflux. Epithelial ASC specks were present in enteroids undergoing apoptosis and in the injured tubules of wild-type but not Nlrp3-/- or ASC-/- mice following ureteric unilateral obstruction in vivo. These data show that NLRP3 and ASC form a conserved non-canonical platform for caspase-8 activation, independent of the inflammasome that regulates apoptosis within epithelial cells.

Presence of the Paternal Pronucleus Assists Embryo in Overcoming Cycloheximide Induced Abnormalities in Zygotic Mitosis.

After fertilization, the maternal and paternal chromosomes independently proceed through pronuclear formation. These chromatin reconfigurations occur within a shared cytoplasm thus exposing both gametes to the same factors. Here, we report that continuous cycloheximide [40 µg/mL] treatment of parthenogenotes, androgenotes, and ICSI embryos reveals ORC2 pronuclear instability in the maternal (MPN) but not the paternal pronucleus (PPN). When released from CHX after 8 h, the MPN can recover ORC2 and proceed through replication, however, parthenogenotes encounter severe mitotic defects while both ICSI embryos and androgenotes are able to recover and develop at significantly higher rates. Taken together, these data suggest cycloheximide treatment promotes an environment that asymmetrically affects the stability of ORC2 on the MPN, and the ability of the MPN to develop. Furthermore, the presence of the PPN in the zygote can ameliorate both effects. These data suggest further evidence for crosstalk between the two pronuclei during the first cell cycle of the embryo. J. Cell. Biochem. 117: 1806-1812, 2016. © 2016 Wiley Periodicals, Inc.

A Versatile Cell Death Screening Assay Using Dye-Stained Cells and Multivariate Image Analysis.

A novel dye-based method for measuring cell death in image-based screens is presented. Unlike conventional high- and medium-throughput cell death assays that measure only one form of cell death accurately, using multivariate analysis of micrographs of cells stained with the inexpensive mix, red dye nonyl acridine orange, and a nuclear stain, it was possible to quantify cell death induced by a variety of different agonists even without a positive control. Surprisingly, using a single known cytotoxic agent as a positive control for training a multivariate classifier allowed accurate quantification of cytotoxicity for mechanistically unrelated compounds enabling generation of dose-response curves. Comparison with low throughput biochemical methods suggested that cell death was accurately distinguished from cell stress induced by low concentrations of the bioactive compounds Tunicamycin and Brefeldin A. High-throughput image-based format analyses of more than 300 kinase inhibitors correctly identified 11 as cytotoxic with only 1 false positive. The simplicity and robustness of this dye-based assay makes it particularly suited to live cell screening for toxic compounds.

A high-throughput direct fluorescence resonance energy transfer-based assay for analyzing apoptotic proteases using flow cytometry and fluorescence lifetime measurements.

Cytometry is a versatile and powerful method applicable to different fields, particularly pharmacology and biomedical studies. Based on the data obtained, cytometric studies are classified into high-throughput (HTP) or high-content screening (HCS) groups. However, assays combining the advantages of both are required to facilitate research. In this study, we developed a high-throughput system to profile cellular populations in terms of time- or dose-dependent responses to apoptotic stimulations because apoptotic inducers are potent anticancer drugs. We previously established assay systems involving protease to monitor live cells for apoptosis using tunable fluorescence resonance energy transfer (FRET)-based bioprobes. These assays can be used for microscopic analyses or fluorescence-activated cell sorting. In this study, we developed FRET-based bioprobes to detect the activity of the apoptotic markers caspase-3 and caspase-9 via changes in bioprobe fluorescence lifetimes using a flow cytometer for direct estimation of FRET efficiencies. Different patterns of changes in the fluorescence lifetimes of these markers during apoptosis were observed, indicating a relationship between discrete steps in the apoptosis process. The findings demonstrate the feasibility of evaluating collective cellular dynamics during apoptosis.

Antioxidant potential of CORM-A1 and resveratrol during TNF-α/cycloheximide-induced oxidative stress and apoptosis in murine intestinal epithelial MODE-K cells.

Targeting excessive production of reactive oxygen species (ROS) could be an effective therapeutic strategy to prevent oxidative stress-associated gastrointestinal inflammation. NADPH oxidase (NOX) and mitochondrial complexes (I and II) are the major sources of ROS production contributing to TNF-α/cycloheximide (CHX)-induced apoptosis in the mouse intestinal epithelial cell line, MODE-K. In the current study, the influence of a polyphenolic compound (resveratrol) and a water-soluble carbon monoxide (CO)-releasing molecule (CORM-A1) on the different sources of TNF-α/CHX-induced ROS production in MODE-K cells was assessed. This was compared with H2O2-, rotenone- or antimycin-A-induced ROS-generating systems. Intracellular total ROS, mitochondrial-derived ROS and mitochondrial superoxide anion (O2(-)) production levels were assessed. Additionally, the influence on TNF-α/CHX-induced changes in mitochondrial membrane potential (Ψm) and mitochondrial function was studied. In basal conditions, CORM-A1 did not affect intracellular total or mitochondrial ROS levels, while resveratrol increased intracellular total ROS but reduced mitochondrial ROS production. TNF-α/CHX- and H2O2-mediated increase in intracellular total ROS production was reduced by both resveratrol and CORM-A1, whereas only resveratrol attenuated the increase in mitochondrial ROS triggered by TNF-α/CHX. CORM-A1 decreased antimycin-A-induced mitochondrial O2(-) production without any influence on TNF-α/CHX- and rotenone-induced mitochondrial O2(-) levels, while resveratrol abolished all three effects. Finally, resveratrol greatly reduced and abolished TNF-α/CHX-induced mitochondrial depolarization and mitochondrial dysfunction, while CORM-A1 only mildly affected these parameters. These data indicate that the cytoprotective effect of resveratrol is predominantly due to mitigation of mitochondrial ROS, while CORM-A1 acts solely on NOX-derived ROS to protect MODE-K cells from TNF-α/CHX-induced cell death. This might explain the more pronounced cytoprotective effect of resveratrol.

E2F1 and E2F2 induction in response to DNA damage preserves genomic stability in neuronal cells.

E2F transcription factors regulate a wide range of biological processes, including the cellular response to DNA damage. In the present study, we examined whether E2F family members are transcriptionally induced following treatment with several genotoxic agents, and have a role on the cell DNA damage response. We show a novel mechanism, conserved among diverse species, in which E2F1 and E2F2, the latter specifically in neuronal cells, are transcriptionally induced after DNA damage. This upregulation leads to increased E2F1 and E2F2 protein levels as a consequence of de novo protein synthesis. Ectopic expression of these E2Fs in neuronal cells reduces the level of DNA damage following genotoxic treatment, while ablation of E2F1 and E2F2 leads to the accumulation of DNA lesions and increased apoptotic response. Cell viability and DNA repair capability in response to DNA damage induction are also reduced by the E2F1 and E2F2 deficiencies. Finally, E2F1 and E2F2 accumulate at sites of oxidative and UV-induced DNA damage, and interact with γH2AX DNA repair factor. As previously reported for E2F1, E2F2 promotes Rad51 foci formation, interacts with GCN5 acetyltransferase and induces histone acetylation following genotoxic insult. The results presented here unveil a new mechanism involving E2F1 and E2F2 in the maintenance of genomic stability in response to DNA damage in neuronal cells.

Transferability and inter-laboratory variability assessment of the in vitro bovine oocyte fertilization test.

The dramatic increase in the number of animals required for reproductive toxicity testing imposes the validation of alternative methods to reduce the use of laboratory animals. As we previously demonstrated for in vitro maturation test of bovine oocytes, the present study describes the transferability assessment and the inter-laboratory variability of an in vitro test able to identify chemical effects during the process of bovine oocyte fertilization. Eight chemicals with well-known toxic properties (benzo[a]pyrene, busulfan, cadmium chloride, cycloheximide, diethylstilbestrol, ketoconazole, methylacetoacetate, mifepristone/RU-486) were tested in two well-trained laboratories. The statistical analysis demonstrated no differences in the EC50 values for each chemical in within (inter-runs) and in between-laboratory variability of the proposed test. We therefore conclude that the bovine in vitro fertilization test could advance toward the validation process as alternative in vitro method and become part of an integrated testing strategy in order to predict chemical hazards on mammalian fertility.

Development of cytotoxicity-sensitive human cells using overexpression of long non-coding RNAs.

Biosensors using live cells are analytical devices that have the advantage of being highly sensitive for their targets. Although attention has primarily focused on reporter gene assays using functional promoters, cell viability assays are still efficient. We focus on long non-coding RNAs (lncRNAs) that are involved in the molecular mechanisms associated with responses to cellular stresses as a new biological material. Here we have developed human live cells transfected with lncRNAs that can be used as an intelligent sensor of cytotoxicity for a broad range of environmental stresses. We identified three lncRNAs (GAS5, IDI2-AS1, and SNHG15) that responded to cycloheximide in HEK293 cells. Overexpression of these lncRNAs sensitized human cells to cell death in response to various stresses (cycloheximide, ultraviolet irradiation, mercury II chloride, or hydrogen peroxide). In particular, dual lncRNA (GAS5 plus IDI2-AS1, or GAS5 plus SNHG15) overexpression sensitized cells to cell death by more cellular stresses. We propose a method for highly sensitive biosensors using overexpression of lncRNAs that can potentially measure the cytotoxicity signals of various environmental stresses.

Functional characterization of dosage-dependent lethal mutation of ubiquitin in Saccharomyces cerevisiae.

Ubiquitin is a eukaryotic protein with 96% sequence conservation from yeast to human. Ubiquitin plays a central role in protein homeostasis and regulation of protein function. We have reported on the generation of variants of ubiquitin by in vitro evolution in Saccharomyces cerevisiae to advance our understanding of the role of the invariant amino acid residues of ubiquitin in relation to its function. One of the mutants generated, namely UbEP42, was a dosage-dependent lethal form of the ubiquitin gene, causing lethality to UBI4-deficient cells but not to ubiquitin wild-type cells. In the present study we investigated the functional reasons for the observed lethality. Expression of UbEP42 in a UBI4-deleted stress-sensitive strain resulted in an increased generation time due to a delayed S phase caused by decreased levels of Cdc28 protein kinase. Cells expressing UbEP42 displayed heightened sensitivity towards heat stress and exposure to cycloheximide. Furthermore, its expression had a negative effect on the degradation of substrates of the ubiquitin fusion degradation pathway. However, UbEP42 is incorporated into polyubiquitin chains. Collectively, our results establish that the effects seen with the mutant ubiquitin protein UbEP42 are not due to malfunction at the stage of polyubiquitination.

Metaxins 1 and 2, two proteins of the mitochondrial protein sorting and assembly machinery, are essential for Bak activation during TNF alpha triggered apoptosis.

The proteins Bax and Bak are central in the execution phase of apoptosis; however, little is known about the partners involved in the control of this complex process. Here, we show that mitochondrial Bak is incorporated into a VDAC2/Mtx1/Mtx2 multi-protein complex in both resting and dying cells. VDAC2 is a porin that has previously been described as a partner of Bak while Mtx1 and Mtx2 are two proteins of the mitochondrial sorting and assembly machinery (SAM) that have been implicated in TNF-induced apoptosis. We show that, after the induction of apoptosis, Bak switches from its association with Mtx2 and VDAC2 to interact with Mtx1.

Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo.

The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero.

Compounds used to produce cloned animals are genotoxic and mutagenic in mammalian assays in vitro and in vivo

The compounds 6-dimethylaminopurine and cycloheximide promote the successful production of cloned mammals and have been used in the development of embryos produced by somatic cell nuclear transfer. This study investigated the effects of 6-dimethylaminopurine and cycloheximide in vitro, using the thiazolyl blue tetrazolium bromide colorimetric assay to assess cytotoxicity, the trypan blue exclusion assay to assess cell viability, the comet assay to assess genotoxicity, and the micronucleus test with cytokinesis block to test mutagenicity. In addition, the comet assay and the micronucleus test were also performed on peripheral blood cells of 54 male Swiss mice, 35 g each, to assess the effects of the compounds in vivo. The results indicated that both 6-dimethylaminopurine and cycloheximide, at the concentrations and doses tested, were cytotoxic in vitro and genotoxic and mutagenic in vitro and in vivo, altered the nuclear division index in vitro, but did not diminish cell viability in vitro. Considering that alterations in DNA play important roles in mutagenesis, carcinogenesis, and morphofunctional teratogenesis and reduce embryonic viability, this study indicated that 6-dimethylaminopurine and cycloheximide utilized in the process of mammalian cloning may be responsible for the low embryo viability commonly seen in nuclear transfer after implantation in utero.

Visualization of acute liver damage induced by cycloheximide in rats using PET with [(18)F]FEDAC, a radiotracer for translocator protein (18 kDa).

Liver damage induced by drug toxicity is an important concern for both medical doctors and patients. The aim of this study was to noninvasively visualize acute liver damage using positron emission tomography (PET) with N-benzyl-N-methyl-2-[7,8-dihydro-7-(2-[(18)F]fluoroethyl)-8-oxo-2-phenyl-9H-purin-9-yl]acetamide ([(18)F]FEDAC), a radiotracer specific for translocator protein (18 kDa, TSPO) as a biomarker for inflammation, and to determine cellular sources enriching TSPO expression in the liver. A mild acute liver damage model was prepared by a single intraperitoneal injection of cycloheximide (CHX) into rats. Treatment with CHX induced apoptosis and necrotic changes in hepatocytes with slight neutrophil infiltration. The uptake of radioactivity in the rat livers was measured with PET after injection of [(18)F]FEDAC. The uptake of [(18)F]FEDAC increased in livers damaged from treatment with CHX compared to the controls. Presence of TSPO was examined in the liver tissue using quantitative reverse transcriptase-polymerase chain reaction and immunohistochemical assays. mRNA expression of TSPO was elevated in the damaged livers compared to the controls, and the level was correlated with the [(18)F]FEDAC uptake and severity of damage. TSPO expression in the damaged liver sections was mainly found in macrophages (Kupffer cells) and neutrophils, but not in hepatocytes. The elevation of TSPO mRNA expression was derived from the increase of the number of macrophages with TSPO and neutrophils with TSPO in damaged livers. From this study we considered that PET imaging with [(18)F]FEDAC represented the mild liver damage through the enhanced TSPO signal in inflammatory cells. We conclude that this method may be a useful tool for diagnosis in early stage of acute liver damage.

[Possibility of memory reparation: conditioned freezing disrupted by protein synthesis inhibition can be restored by reminder stimulation in mice].

Memory consolidation is a term used to describe the process of memory stabilization from labile, easily disrupted state into disruption-resistant long-term form. Protein synthesis inhibitors injected before or immediately after learning episode, produce significant amnesia. However in a limited number of studies the possibility of memory recovery after such pharmacologically-produced amnesia was shown. The aim of present study was to investigate the possibility of memory recovery in single-session fear conditioning paradigm in mice. Mice were injected with protein synthesis inhibitor cycloheximide twenty minutes prior learning to induce amnesia. Twenty four hours after training mice were subjected to reminder shock, similar to one used during training. Amnestic animals have demonstrated complete recovery of cued fear memory to the level of normal animals when tested 24h after reminder presentation. Thus our data indicate that specific type of memory might be restored using nonspecific stimulus as a reminder. Possible mechanisms of memory reparation after PSI injection during learning are discussed.