Humans but Not Chimpanzees Vary Face-Scanning Patterns Depending on Contexts during Action Observation.

Human and nonhuman primates comprehend the actions of other individuals by detecting social cues, including others' goal-directed motor actions and faces. However, little is known about how this information is integrated with action understanding. Here, we present the ontogenetic and evolutionary foundations of this capacity by comparing face-scanning patterns of chimpanzees and humans as they viewed goal-directed human actions within contexts that differ in whether or not the predicted goal is achieved. Human adults and children attend to the actor's face during action sequences, and this tendency is particularly pronounced in adults when observing that the predicted goal is not achieved. Chimpanzees rarely attend to the actor's face during the goal-directed action, regardless of whether the predicted action goal is achieved or not. These results suggest that in humans, but not chimpanzees, attention to actor's faces conveying referential information toward the target object indicates the process of observers making inferences about the intentionality of an action. Furthermore, this remarkable predisposition to observe others' actions by integrating the prediction of action goals and the actor's intention is developmentally acquired.

Comprehensive phosphoproteome analysis unravels the core signaling network that initiates the earliest synapse pathology in preclinical Alzheimer's disease brain.

Using a high-end mass spectrometry, we screened phosphoproteins and phosphopeptides in four types of Alzheimer's disease (AD) mouse models and human AD postmortem brains. We identified commonly changed phosphoproteins in multiple models and also determined phosphoproteins related to initiation of amyloid beta (Aß) deposition in the mouse brain. After confirming these proteins were also changed in and human AD brains, we put the proteins on experimentally verified protein-protein interaction databases. Surprisingly, most of the core phosphoproteins were directly connected, and they formed a functional network linked to synaptic spine formation. The change of the core network started at a preclinical stage even before histological Aß deposition. Systems biology analyses suggested that phosphorylation of myristoylated alanine-rich C-kinase substrate (MARCKS) by overactivated kinases including protein kinases C and calmodulin-dependent kinases initiates synapse pathology. Two-photon microscopic observation revealed recovery of abnormal spine formation in the AD model mice by targeting a core protein MARCKS or by inhibiting candidate kinases, supporting our hypothesis formulated based on phosphoproteome analysis.

A functional deficiency of TERA/VCP/p97 contributes to impaired DNA repair in multiple polyglutamine diseases.

It is hypothesized that a common underlying mechanism links multiple neurodegenerative disorders. Here we show that transitional endoplasmic reticulum ATPase (TERA)/valosin-containing protein (VCP)/p97 directly binds to multiple polyglutamine disease proteins (huntingtin, ataxin-1, ataxin-7 and androgen receptor) via polyglutamine sequence. Although normal and mutant polyglutamine proteins interact with TERA/VCP/p97, only mutant proteins affect dynamism of TERA/VCP/p97. Among multiple functions of TERA/VCP/p97, we reveal that functional defect of TERA/VCP/p97 in DNA double-stranded break repair is critical for the pathology of neurons in which TERA/VCP/p97 is located dominantly in the nucleus in vivo. Mutant polyglutamine proteins impair accumulation of TERA/VCP/p97 and interaction of related double-stranded break repair proteins, finally causing the increase of unrepaired double-stranded break. Consistently, the recovery of lifespan in polyglutamine disease fly models by TERA/VCP/p97 corresponds well to the improvement of double-stranded break in neurons. Taken together, our results provide a novel common pathomechanism in multiple polyglutamine diseases that is mediated by DNA repair function of TERA/VCP/p97.

Therapeutic efficacy of c-kit-targeted radioimmunotherapy using 90Y-labeled anti-c-kit antibodies in a mouse model of small cell lung cancer.

UNLABELLED: Small cell lung cancer (SCLC) is an aggressive tumor and prognosis remains poor. Therefore, the development of more effective therapy is needed. We previously reported that high levels of an anti-c-kit antibody (12A8) accumulated in SCLC xenografts. In the present study, we evaluated the efficacy of two antibodies (12A8 and 67A2) for radioimmunotherapy (RIT) of an SCLC mouse model by labeling with the (90)Y isotope. METHODS: (111)In- or (125)I-labeled antibodies were evaluated in vitro by cell binding, competitive inhibition and cellular internalization assays in c-kit-expressing SY cells and in vivo by biodistribution in SY-bearing mice. Therapeutic efficacy of (90)Y-labeled antibodies was evaluated in SY-bearing mice upto day 28 and histological analysis was conducted at day 7. RESULTS: [(111)In]12A8 and [(111)In]67A2 specifically bound to SY cells with high affinity (8.0 and 1.9 nM, respectively). 67A2 was internalized similar to 12A8. High levels of [(111)In]12A8 and [(111)In]67A2 accumulated in tumors, but not in major organs. [(111)In]67A2 uptake by the tumor was 1.7 times higher than for [(111)In]12A8. [(90)Y]12A8, but not [(90)Y]67A2, suppressed tumor growth in a dose-dependent manner. Tumors treated with 3.7 MBq of [(90)Y]12A8, and 1.85 and 3.7 MBq of [(90)Y]67A2 (absorbed doses were 21.0, 18.0 and 35.9 Gy, respectively) almost completely disappeared approximately 2 weeks after injection, and regrowth was not observed except for in one mouse treated with 1.85 MBq [(90)Y]67A2. The area of necrosis and fibrosis increased depending on the RIT effect. Apoptotic cell numbers increased with increased doses of [(90)Y]12A8, whereas no dose-dependent increase was observed following [(90)Y]67A2 treatment. Body weight was temporarily reduced but all mice tolerated the RIT experiments well. CONCLUSION: Treatment with [(90)Y]12A8 and [(90)Y]67A2 achieved a complete therapeutic response when SY tumors received an absorbed dose greater than 18 Gy and thus are promising RIT agents for metastatic SCLC cells at distant sites.

Comparison of 2-amino-[3-¹¹C]isobutyric acid and 2-deoxy-2-[¹8F]fluoro-D-glucose in nude mice with xenografted tumors and acute inflammation.

OBJECTIVE: 2-Deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) accumulates in tumors and also during active inflammation, including therapy-related inflammation. Additional PET tracers that are less avid to inflammation could be useful in differentiating cancer from inflammation and could complement the limitation of [18F]FDG-PET. 2-Amino-[3-11C]isobutyric acid ([3-11C]AIB) is a potential PET tracer for this purpose. We compared [3-11C]AIB and [18F]FDG uptakes in tumors and acute inflammation in a mouse model. METHODS: Acute inflammatory lesions were induced in the hind legs of tumor-bearing mice by intramuscular injection of turpentine, and we conducted biodistribution and dynamic PET studies on [3-11C]AIB and [18F]FDG. RESULTS: [3-11C]AIB tumor uptake increased with time and was statistically significantly higher than [18F]FDG uptake. In inflamed muscles, [3-11C]AIB uptake was statistically significantly lower than [18F]FDG uptake, and the tumor-to-inflammation ratio for [3-11C]AIB was statistically significantly higher than that for [18F]FDG. CONCLUSION: [3-11C]AIB accumulates more selectively in tumor tissue than does [18F]FDG and thus has the potential of discriminating between tumors and inflammatory lesions better and of complementing the limitation of [18F]FDG.

Novel human monoclonal antibody against epidermal growth factor receptor as an imaging probe for hepatocellular carcinoma.

OBJECTIVE: The epidermal growth factor receptor (EGFR) is overexpressed in many epithelial cancers, including hepatocellular carcinoma (HCC), and is an attractive target for cancer imaging and therapy. We attempted a novel noninvasive imaging method to evaluate anti-EGFR human monoclonal antibody clones for determining the uptake of therapeutic anti-EGFR antibody in HCC. METHODS: In-vitro cell binding of nine I-labeled antibody clones was compared in the human epidermoid cancer cell line A431, in three HCC cell lines Hep-G2, SK-Hep1, and HuH-7, and in the EGFR-negative control cell line A4. In-labeled or I-labeled 048-006 was subjected to cell binding, competitive inhibition, and internalization assays using A431, SK-Hep1, and HuH-7. Further, In-labeled 048-006 was evaluated in in-vivo biodistribution analysis and single-photon imaging in nude tumor-bearing mice. RESULTS: The 048-006 clone showed the highest binding to EGFR-expressing cells among the nine antibodies. In-labeled or I-labeled 048-006 specifically bound to EGFR-expressing cells with high affinity and was internalized after binding to EGFR. A431 and HuH-7 tumors showed high In-labeled 048-006 uptake, which was visualized by single-photon imaging. CONCLUSION: Radiolabeled human anti-EGFR monoclonal antibody 048-006 has the potential to be a safer imaging probe for predicting tumor uptake of anti-EGFR antibody therapeutic agents in HCC.

Development of positron emission tomography probe of 64Cu-labeled anti-C-kit 12A8 Fab to measure protooncogene C-kit expression.

INTRODUCTION: C-kit is an important diagnostic and therapeutic target molecule for several malignancies, and c-kit-targeted drugs have been used clinically. Because abundant c-kit expression in tumors is a prerequisite for successful c-kit-targeted therapy, imaging of c-kit expression is expected to play a pivotal role in the therapeutic decision for each patient. We evaluated (64)Cu-labeled Fab of anti-c-kit antibody 12A8 as a positron emission tomography (PET) imaging probe. METHODS: (111)In- or (125)I-Labeled 12A8 Fab was evaluated in vitro by cell binding, competitive inhibition and cellular internalization assays, and in vivo by biodistribution in mice bearing c-kit-expressing and -non-expressing tumors. Next, Fab fragment was labeled with the positron emitter (64)Cu and evaluated by PET. RESULTS: Radiolabeled 12A8 Fab showed specific binding to c-kit-expressing cells with high affinity and internalized into cells after binding to c-kit on cell surface. Although tumor accumulation of [(111)In]Fab was lower than that of [(111)In]IgG, the faster blood clearance of [(111)In]Fab provided higher tumor-to-blood ratio at 6 h postinjection onwards. Blood clearance of (64)Cu-labeled 12A8 Fab was slower than that of [(111)In]Fab, but PET using [(64)Cu]Fab clearly visualized the tumor at 6 h postinjection onwards. CONCLUSION: The (64)Cu-labeled 12A8 Fab could be used for c-kit-specific PET imaging and might help in selecting appropriate patients for c-kit-targeted treatments.

Analysis of inhibition of topoisomerase IIalpha and cancer cell proliferation by ingenolEZ.

We previously reported that many ingenol compounds derived from Euphorbia kansui exhibit topoisomerase inhibitory activity and/or inhibitory activity of cell proliferation. The inhibitory effects of 20-O-(2'E,4'Z-decadienoyl) ingenol and 3-O-(2'E,4'Z-decadienoyl)-ingenol among these compounds on topoisomerase II activity and on the cell proliferative activity and arrest phase of the cell cycle were studied using a mouse breast cancer (MMT) cell line. Although 20-O-ingenolEZ exerted inhibitory effects on both topoisomerase II activity and cell proliferative activity, 3-O-ingenolEZ exerted inhibitory activity on neither. The 20-O-ingenolEZ-induced cell arrest of MMT-cell proliferation led to a cell cycle arrest in the G2/M phase. Topoisomerase II inhibition can be divided into the poison and catalytic inhibitor types. A checkpoint mechanism is activated when cells are treated with these topoisomerase II inhibitors. Poison-type inhibition occurs via induction of the DNA damage checkpoint and the catalytic-type inhibition occurs via induction of the DNA-decatenation checkpoint, suggestive of distinct checkpoint reactions. 20-O-ingenolEZ inhibited topoisomerase IIalpha activity through inhibition of ATPase, and induced DNA-decatenation checkpoint without signaling for phosphorylation of H2AX.

Knockdown of COPA, identified by loss-of-function screen, induces apoptosis and suppresses tumor growth in mesothelioma mouse model.

Malignant mesothelioma is a highly aggressive tumor arising from serosal surfaces of the pleura and is triggered by past exposure to asbestos. Currently, there is no widely accepted treatment for mesothelioma. Development of effective drug treatments for human cancers requires identification of therapeutic molecular targets. We therefore conducted a large-scale functional screening of mesothelioma cells using a genome-wide small interfering RNA library. We determined that knockdown of 39 genes suppressed mesothelioma cell proliferation. At least seven of the 39 genes-COPA, COPB2, EIF3D, POLR2A, PSMA6, RBM8A, and RPL18A-would be involved in anti-apoptotic function. In particular, the COPA protein was highly expressed in some mesothelioma cell lines but not in a pleural mesothelial cell line. COPA knockdown induced apoptosis and suppressed tumor growth in a mesothelioma mouse model. Therefore, COPA may have the potential of a therapeutic target and a new diagnostic marker of mesothelioma.

C-kit-targeted imaging of gastrointestinal stromal tumor using radiolabeled anti-c-kit monoclonal antibody in a mouse tumor model.

INTRODUCTION: Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor arising from the gastrointestinal tract and highly expresses mutated c-kit. We aimed to develop a specific and sensitive method for detecting GISTs using radiolabeled anti-c-kit monoclonal antibody. METHODS: A mutated c-kit-expressing cell clone was established by transfecting an expressing vector of mutated c-kit gene into HEK293 human embryonic kidney cells. The tumors were developed by inoculating c-kit-expressing cells into nude mice. (125)I- and (111)In-labeled anti-c-kit antibodies (12A8 and 41A11) were evaluated in vitro by cell binding, competitive inhibition and cellular internalization assays, and in vivo by biodistribution and imaging studies in tumor-bearing mice. RESULTS: Both (125)I- and (111)In-labeled antibodies showed specific binding with c-kit-expressing cells with high affinity (dissociation constants = 2.2-7.1x10(9) M(-1)). Internalization assay showed that (125)I-labeled antibodies were rapidly internalized and dehalogenated, with the release of (125)I from the cells, resulting in reduction of cell-associated radioactivity with time. In contrast, (111)In-labeled antibody was internalized but did not result in the reduced radioactivity associated with tumor cells. Reflecting this phenomenon, the in vivo tumor uptake of (125)I-labeled antibody was low on Day 1, further decreasing with time, while tumor uptake of (111)In-labeled antibody was high on Day 1, further increasing with time. The xenografted tumor was clearly visualized by scintigraphy after injection of (111)In-labeled antibody. CONCLUSION: The anti-c-kit monoclonal antibody labeled with a metal radionuclide would be promising for c-kit-targeted imaging of GISTs.

Development of positron emission tomography imaging by 64Cu-labeled Fab for detecting ERC/mesothelin in a mesothelioma mouse model.

BACKGROUND: Malignant mesothelioma is a highly aggressive form of cancer. Curative surgery is the only effective therapy for mesothelioma, and therefore early diagnosis is important. However, early diagnosis is difficult using current diagnostic imaging techniques, and a new imaging method for early diagnosis is urgently required. We evaluated the affinity of radiolabeled monoclonal antibodies to the C-terminal fragment of ERC/mesothelin for this purpose. METHODS: In-labeled or I-labeled IgG against C-terminal fragment of ERC and its Fab fragment were evaluated in vitro by cell binding, competitive inhibition, and cellular internalization assays, and in vivo by biodistribution in mice bearing ERC-expressing tumors. Next, the Fab fragment was labeled with the positron emitter Cu and evaluated by positron emission tomography (PET). RESULTS: Radiolabeled IgG and Fab showed specific binding to ERC-expressing mesothelioma cells with high affinity. Both radiolabeled IgG and Fab internalized into cells after binding to ERC on the cell surface. In-labeled IgG accumulated in ERC-expressing tumors and resulted in a moderate tumor-to-blood ratio at 4 days after injection. Furthermore, PET using Cu-labeled Fab visualized the tumor at 6 h after injection. CONCLUSION: Cu-labeled Fab can be useful for ERC-specific PET imaging, and can thus facilitate improved diagnosis of patients with early-stage mesothelioma.

Oxidative-inflammatory damage in cirrhosis: effect of vitamin E and a fermented papaya preparation.

BACKGROUND AND AIM: Oxidative DNA damage occurs as an early event in hepatitis C virus (HCV) infection and is an indication of the potential for carcinogenesis. The aim of this study was to test a novel antioxidant/immunomodulator in patients with HCV-related cirrhosis. METHODS: The study group consisted of 50 patients with HCV-related cirrhosis with transaminase values less than twofold increased (alanine aminotransferase [ALT] < 80 IU/L). Patients underwent a standardized food-vitamin composition assessment and were assessed for dietary intake, nutritional status and iron level. Patients were randomly allocated into two groups and then given either alpha-tocopherol 900 IU/day or 9 g/day of a fermented papaya preparation (FPP, Immun-Age, Osato Research Institute, Gifu, Japan) at bedtime for 6 months. Ten healthy subjects served as controls. Patients were checked monthly for: routine tests, redox status (reduced glutathione, glutathione peroxidase, oxidized glutathione, malondialdehyde), plasma alpha-tocopherol, 8-hydroxy-deoxy-guanidine (8-OHdG) level in circulating leukocyte DNA and serum levels of cytokines. RESULTS: Patients with cirrhosis showed a significant imbalance of redox status (low antioxidants/high oxidative stress markers) (P < 0.005 vs controls). Neither treatment regimen affected transaminases as a whole. However, vitamin E supplementation almost normalized ALT only in the limited vitamin-E-deficient subgroup. A significant improvement of redox status was obtained by both regimens. However, only FPP significantly decreased 8-OHdG and the improvement of cytokine balance with FPP was significantly better than with vitamin E treatment (P < 0.05). CONCLUSIONS: Although the present data seem to suggest a potential supportive role of antioxidants/immunomodulators as FPP in HCV patients, more studies are needed to substantiate their effect on the natural history of the disease.

Nutraceutical supplementation: effect of a fermented papaya preparation on redox status and DNA damage in healthy elderly individuals and relationship with GSTM1 genotype: a randomized, placebo-controlled, cross-over study.

Our study group consisted of 54 elderly patients without major invalidating diseases who were randomly divided into two fully matched groups. Group A was given a certified fermented papaya preparation 9 g/day by mouth, while group B received placebo. Treatment was carried out in a cross-over manner with a 3-month supplementation followed by a 6-week washout period. Blood samples were drawn at entry and on a monthly basis to check routine parameters, redox status, and 8-OHdG in circulating leukocyte DNA. Polymorphism analysis of GSTM1 was carried out as well. The glutathune-S transferase M1 (GSTM1) genotype was null (-) in 40% and 46% of groups A and B, respectively. GSTM1 (-) smokers had a significantly higher level of plasma DNA adducts and leukocytes level of 8-OHdG than their GSTM1 (+) counterparts (P < 0.01). There was a weak correlation between cigarettes smoked/day and DNA adduct (r: 0.61, P < 0.05), which also correlated with antioxidant concentrations, but only in GSTM1 (-) smokers (P < 0.01). The fermented papaya preparation (FPP)-supplemented group showed a significant enhancement of the antioxidant protection (P < 0.01 vs. A) within the subgroups with GSTM1 (-) and of plasma DNA adduct, irrespective of the GSTM1 genotype. Only the GSTM1 (-) subgroup was the one that, under FPP treatment, increased lymphocyte 8-OHdG (P < 0.01). Such preliminary data show that FPP is a promising nutraceutical for improving antioxidant-defense in elderly patients even without any overt antioxidant-deficiency state while helping explain some inconsistent results of prior interventional studies.

Inhibition of cellular proliferation by diterpenes, topoisomerase II inhibitor.

We examined the effects of 12 terpene compounds derived from the roots of Euphorbia kansui on the proliferative activity of Xenopus embryo cells. Eight of these compounds showed significant inhibition of cellular proliferation even at low concentrations, while four of them needed to be present at higher concentrations to inhibit cellular proliferation. In order to define the mechanism of inhibition of cellular proliferation by these compounds, the effects of diterpene compounds on the activity of topoisomerase II were measured. Most of the diterpene compounds that inhibited cellular proliferation also inhibited topoisomerase II activity.