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We predicted the energy balance (EB) of Holstein cows in Japan from milk traits obtained by herd testing. Records covered 156 lactations of 102 cows. The number of artificial inseminations was highest, and the first conception rate was lowest in the low-EB group. Four prediction models were developed-for the whole lactation and for the early, middle, and late stages of lactation-with 20 variables, covering days in milk (DIM), milk yield, and milk composition traits. The actual and predicted EB means agreed well within DIM classes; the means of the residuals were smaller in the lactation stage models than in the all-lactation model, but the standard deviations (SDs) of the residuals were similar among models. After data reduction, the SDs of the residuals for 100 iterations were1 throughout lactation in both types of models when n = 100. After model reduction, including the daily change of milk yield as a variable minimized the SDs of the residuals. Our equations for herd-level EB prediction have potential for use in genetic evaluation.

TEA domain family transcription factor 4 (Tead4) is known to be important for the trophectoderm (TE) segregation in murine embryos. However, the role of TEAD4 in early development of porcine embryos is still unknown. We examined TEAD4 expression patterns and attempted to determine the functions of TEAD4 during porcine preimplantation development using RNA interference. TEAD4 mRNA was upregulated from the 2-4-cell to 8-16-cell stages and then decreased to the blastocyst stage. Nuclear localization of TEAD4 protein was detected at the 16-cell stage, as well as at subsequent developmental stages. In porcine embryos injected with TEAD4 siRNA, transformation from morula to blastocyst was inhibited. Although TEAD4 downregulation did not affect the expression levels of POU class 5 homeobox 1 (OCT-4), transcription of SRY-related HMG-box gene 2 (SOX2) was detected at high level in TEAD4-downregulated embryos. It is possible that TEAD4 contributes to blastocyst formation in porcine embryos through downregulation of SOX2 expression. Collectively, our results indicate that TEAD4 is an important factor for the preimplantation development of porcine embryos.

In mouse development, differentiation of the inner cell mass (ICM) and trophectoderm (TE) during the transition from the morula to blastocyst stage is regulated by the Hippo pathway; however, the functions of the Hippo pathway in porcine embryogenesis have not been investigated. In the present study, we examined the gene expression patterns of the Hippo pathway members yes-associated protein 1 (YAP1) and large tumor suppressor 2 (LATS2) and the functions of these genes during porcine preimplantation development using RNA interference. Both YAP1 and LATS2 mRNA levels were shown high in the in vitro matured oocytes and 1-cell stage embryos and fell progressively with development. YAP1 nuclear localization was detected at the morula and blastocyst stages. Downregulation of either YAP1 or LATS2 inhibited porcine preimplantation development and affected the expression levels of POU class 5 homeobox 1 (OCT-4) and SRY-related HMG-box gene 2 (SOX2), transcription factors necessary for the ICM/TE differentiation. Taken together, YAP1 and LATS2 are essential for porcine preimplantation development, and it is possible that the Hippo pathway has important roles in porcine ICM/TE segregation.

Introduction Various techniques are available for in vivo imaging, and precise understanding of their characteristics is essential for effective use of the imaging results. We established human mesothelioma cell lines expressing red fluorescent protein (RFP) and examined their fluorescence intensity and uptake of positron emission tomography (PET) tracer analogs to compare their characteristics and assess their usefulness in the evaluation of therapeutics. Method A human mesothelioma cell line was stably transfected to express RFP. Fluorescence, cell number and protein amount were measured during cell growth and treatment with cytotoxic reagents. In in vivo experiments, RFP-expressing cells were injected subcutaneously or into the pleural cavity of nude mice, and fluorescence images were taken with or without pemetrexed treatment. The uptake of [ 3 H]3′-deoxy-3′-fluorothymidine ([ 3 H]FLT) and [ 14 C]2-fluoro-2-deoxy-d-glucose ([ 14 C]FDG) under treatment with the above reagents in vitro and in vivo were examined. Results Strong correlation was observed between fluorescence intensity and total cell number with or without cytotoxic treatment. The uptake of [ 3 H]FLT and [ 14 C]FDG decreased rapidly after the initiation of treatment with actinomycin D or cycloheximide. When treated with pemetrexed, the uptake of [ 3 H]FLT temporarily increased. The cells formed subcutaneous and orthotopic tumors, with fluorescence intensity correlating with tumor volume. The correlation was sustained under pemetrexed treatment. The uptake of [ 3 H]FLT in vivo increased significantly early after pemetrexed treatment. Conclusion Fluorescence imaging could be used to semiquantitatively monitor tumor size, whereas PET could be used to monitor tumor response to therapeutic treatments, and especially, FLT might be a good marker of the response to anti-folate chemotherapeutics.

Whole cell recordings were made using midbrain slices to examine the effects of two different anaesthetics on ATP-sensitive K (K(ATP)) channels in principle neurons of rat substantia nigra pars compacta. When neurons were dialyzed with an ATP-free pipette solution during perfusion with a glucose-free external solution, a hyperpolarization and an outward current developed slowly in a tolbutamide-inhibitable manner. The volatile anaesthetic 3% isoflurane slightly depolarised the neurons in the presence of ATP in the pipette solution and glucose in the external solution, but it did not affect the hyperpolarization or outward current in response to omission of ATP and glucose. Ketamine, an intravenous anaesthetic, did not change the membrane potential when ATP and glucose were included; however, it reversibly inhibited the hyperpolarization and outward current induced by intracellular ATP depletion in a dose-dependent manner. These effects of ketamine were not mimicked by AP-5, an NMDA receptor antagonist, or indatraline, an inhibitor of catecholamine uptake. These findings suggest that these anaesthetics have no stimulatory action on K(ATP) channels in these neurons when intracellular ATP is preserved and that ketamine but not isoflurane inhibits K(ATP) channels when the channels were activated by low intracellular ATP.

Aquaglycero-aquaporins (agAQPs) are one of the water channel proteins located in the cell membrane that transport not only water but also some small solutes such as glycerol. Since agAQPs are involved in cancer proliferation and malignancy, it might be possible to utilize them as new targets for cancer molecular imaging. In this study, we investigated whether agAQPs can be specifically targeted by using [(14)C]-labeled glycerol ([(14)C]glycerol), which passes through agAQPs. In the in vitro experiments, comparing the cancer cell lines with different expression levels of AQP3 and AQP9, major agAQPs known to be expressed in cancers, and examining the effect of their inhibitors on these cells, the expression of AQP3 and AQP9 in cell lines was shown to be closely related to [(14)C]glycerol uptake. When [(14)C]glycerol was injected into tumor-bearing mice, Spearman's rank coefficient analysis revealed that radioactivity levels in tumor and in plasma were mutually correlated only in tumors expressing agAQPs at a high level. These results indicate the possibility of using agAQPs as new targets to characterize cancer using radiolabeled glycerol as a molecular probe.

18F-FDG PET is a promising tool for detecting vulnerable plaques, depending on the extent of macrophage infiltration; however, it is still not clear which stage of the lesion can be detected by 18F-FDG PET. Methods: In this study, we investigated the effect of foam cell formation on 18F-FDG uptake using cultured mouse peritoneal macrophages. Results:18F-FDG accumulation was increased by foam cell formation, but the uptake was decreased to the control level after complete differentiation to foam cells. Changes in hexokinase activity tended to accompany changes in 18F-FDG uptake. In contrast, changes in glucose-6-phosphatase activity and glucose transporter 1 expression did not parallel 18F-FDG uptake. Conclusion: Our results suggest that 18F-FDG PET detects the early stage of foam cell formation in atherosclerosis.

Epidermal growth factor receptor (EGFR) is one of the very attractive targets for cancer therapy. In this study, we generated fusion proteins containing one or two Src-homology 2 (SH2) domains of growth factor receptor bound protein 2 (Grb2), which bind to phosphorylated EGFR, added with HIV-1 transactivating transcription for cell membrane penetration (termed TSF and TSSF, respectively). We examined if they can interfere Grb2-mediated signaling pathway and suppress tumor growth as expected from the lack of SH3 domain, which is necessary to intermediate EGFR–Grb2 cell signaling, in the fusion proteins. The transduction efficiency of TSSF was similar to that of TSF, but the binding activity of TSSF to EGFR was higher than that of TSF. Treatment of EGFR-overexpressing cells showed that TSSF decreased p42-ERK phosphorylation, while TSF did not. Both the proteins delayed cell growth but did not induce cell death in culture. TSSF also significantly suppressed tumor growth in vivo under consecutive administration. In conclusion, TSSF showed an ability to inhibit EGFR–Grb2 signaling and could have a potential to treat EGFR-activated cancer.

We generated a mouse strain lacking protein kinase Cα (PKCα) and evaluated the significance of the enzyme in epithelial hyperplasia and tumor formation. PKCα-deficient mice exhibited increased susceptibility to tumor formation in two-stage skin carcinogenesis by single application of 7,12-dimethylbenz(a)anthracene (DMBA) for tumor initiation and repeated applications of 12-O-tetradecanoylphorbol-13-acetate (TPA) for tumor promotion. Tumor formation was not enhanced by DMBA or TPA treatment alone, suggesting that PKCα suppresses tumor promotion. However, the severity of epidermal hyperplasia induced by topical TPA treatment was markedly reduced. In mutant mice, the number of 5-bromo-2′-deoxyuridine–labeled epidermal basal keratinocytes increased 16 to 24 hours after topical TPA treatment as in the case of wild-type mice, but significantly decreased at 36 and 48 hours. Furthermore, the regenerating epithelium induced by skin wound significantly decreased in thickness but was not structurally impaired. The enhanced tumor formation may not be associated with epidermal hyperplasia. The induction levels of epidermal growth factor (EGF) receptor ligands, tumor growth factor α (TGF-α), and heparin-binding EGF-like growth factor, in the skin of mutant mice by TPA treatment were significantly lower than those in the skin of wild-type mice. PKCα may regulate the supply of these EGF receptor ligands in basal keratinocytes, resulting in a reduced epidermal hyperplasia severity in the mutant mice. We propose that PKCα positively regulates epidermal hyperplasia but negatively regulates tumor formation in two-stage skin carcinogenesis.

Diacylglycerol modulates cell functions primarily through activation of protein kinase C (PKC). In a previous study, however, we found that a diacylglycerol analogue, 1-oleoyl-2-acetylglycerol (OAG), accelerated desensitization of neuronal nicotinic acetylcholine receptors (nAchRs) independently of PKC activation in PC12 cells. In the present study, we investigated whether other analogues and endogenous diacylglycerol exert similar effects on neuronal nAchRs and characterized the modulation by diacylglycerol. We measured the nicotine-induced whole-cell current in the absence and presence of diacylglycerol analogues in PC12 cells. We also investigated the effects of a blockade of metabolic pathways of diacylglycerol by inhibiting diacylglycerol lipase and kinase. We found that all four diacylglycerol analogues studied promoted desensitization and depressed the nondesensitized component of the nicotine-induced current. These effects seemed independent of PKC activation because they were not antagonized by the PKC inhibitors staurosporine or bisindolylmaleimide I; one analogue that lacks the PKC-stimulating action was also effective. The effects of diacylglycerol analogues were not antagonized by high doses of nicotine and were independent of the membrane potential. Similar modulatory effects were observed by treatment with RHC80267, a blocker of diacylglycerol lipase, and R59949, an inhibitor of diacylglycerol kinase, in the presence of staurosporine. These results suggest that diacylglycerol, both exogenously applied and endogenously produced, modulates neuronal nAchRs independently of PKC activation in PC12 cells; further, these effects seemed consistent with a noncompetitive and voltage-independent block. They raised the possibility that PKC-independent inhibition of neuronal nAchRs by diacylglycerol may be a novel modulatory process.

As cancer stem cells (CSCs) are postulated to play critical roles in cancer development, including metastasis and recurrence, CSC imaging would provide valuable information for cancer treatment and lead to CSC-targeted therapy. To assess the possibility of in vivo CSC targeting, we conducted basic studies on radioimmunotargeting of cancer cells positive for CD133, a CSC marker recognized in various cancers. Antibodies against CD133 were labeled with 125I, and their in vitro cell binding properties were tested. Using the same isotype IgG as a control, in vivo biodistribution of the labeled antibody retaining immunoreactivity was examined in mice bearing an HCT116 xenograft in which a population of the cancer cells expressed CD133. Intratumoral distribution of the labeled antibody was examined and compared to the CD133 expression pattern. The 125I-labeled anti-CD133 antibody showed a modest but significantly higher accumulation in the HCT116 xenograft compared to the control IgG. The intratumoral distribution of the labeled antibody mostly overlapped with the CD133 expression, whereas the control IgG was found in the area close to the necrotic tumor center. Our results indicate that noninvasive in vivo targeting of CSCs could be possible with radiolabeled antibodies against cell membrane markers.