Effect of PSK on the maturation of dendritic cells derived from human peripheral blood monocytes

Dendritic cells (DCs) are powerful antigen-presenting cells (APCs) that have attracted attention in recent years from the viewpoint of DC vaccine therapy against cancer. However, the existence of a strongly immunosuppressed state in cancer-bearing individuals inhibits DC maturation, which is one of the problems facing anti-cancer DC vaccine therapy. Protein-bound polysaccharide K (PSK), which is extracted from the cultured mycelium of Coriolus versicolor (Fr.) Quél, is used as an anti-cancer agent in Japan. PSK is reported to improve the immunosuppressed state and might be associated with DC maturation directly. We examined the effect of PSK on the maturation of DC derived from CD14-positive cells obtained from human peripheral blood monocytes using a negative selection method. CD14-positive cells cultured in the presence of PSK significantly increased the expression of HLA class II antigen and CD40; significantly increased the number and expression of CD80-, CD86- and CD83-positive cells; decreased Fluorescein isothiocyanate (FITC)-dextran uptake, augmented IL-12 production; augmented the allogeneic mixed lymphocyte reaction; and induced antigen-specific cytotoxicity. These results indicate that PSK promotes both the phenotypic and functional maturation of DC derived from human CD14-positive mononuclear cells. The clinical significance of the combined use of PSK in DC vaccine therapy remains for study.     

Alteration of cell cycle regulators correlates with survival in epithelial ovarian cancer patients

The p16-cyclinD1/CDK4-pRb pathway (RB pathway) and p14ARF-MDM2-p53 pathway (p53 pathway) work at the G1-S checkpoint, and the ATM-chk2-CDC25-cyclinB1/cdk1 pathway works at the G2-M checkpoint. The disruption of these pathways is thought to be related to the prognosis of human cancer. In this study, we analyzed the status of these pathways in 107 epithelial ovarian cancer (EOC) patients by immunohistochemistry and evaluated the relationship of these results with chemotherapy response and the prognosis. Altered RB, p53, and G2 pathways were detected in 50.5% (54/107), 51.4% (55/107), and 33.6% (36/107) of cases, respectively. The overall survival (OS) of 77.3% for patients with a normal RB pathway was significantly higher than the OS of 50.0% for patients with an altered RB pathway (by Kaplan-Meier analysis, P = 0.0021). The OS of 66.2% for patients with a normal G2 pathway was significantly higher than the OS of 58.3% for patients with an altered G2 pathway (P = 0.0416). However, the status of the p53 pathway was not related to OS. By univariate and multivariate analyses, advanced stage, high histological grade, altered RB pathway, and altered G2 pathway were significant predictors of poor OS. However, there was no significant relationship between pathway status and chemotherapy response. The status of the RB pathway and of the G2 pathway were independent prognostic factors of EOC.     

Disruption of spine homeostasis causes dopaminergic compensatory up-regulation, resulting in schizophrenia

Schizophrenia is a complex psychiatric disorder clinically categorized into three main symptom domains: positive, negative and cognitive deficits. Many reports have shown that great reduction in spine number was observed in schizophrenia patients. In addition, genetic studies have identified mutations in numerous genes that encode synaptic proteins in schizophrenia patients. Furthermore, it is well known that antipsychotic drugs change the number of spines, indicating that disturbance in spine homeostasis is deeply involved in the pathogenesis of schizophrenia. On the other hand, it is commonly accepted that alteration in dopaminergic systems is also involved in the pathogenesis of schizophrenia. However, the relationships between the changes of spine homeostasis and those of the dopaminergic system are largely unknown. Recently, spine homeostasis is reported to be tightly regulated by dopamine D1 receptors. Thus, I will set a new notion that disturbed spine homeostasis results in compensatory up-regulation in the dopaminergic system to keep normal cognitive functions. The hypothesis is as follows. Disturbance in spine homeostasis based on genetic vulnerabilities is the main cause of schizophrenia, and this disturbance results in network deficiency, negative symptoms and cognitive deficits. Since spine homeostasis is deeply regulated by dopamine D1 receptors, disturbed spine homeostasis leads to compensatory hyperactivity in the dopamine system to keep normal cognitive functions. This dopaminergic hyperactivity stimulates dopamine D2 receptors, leading to positive symptoms. All current antipsychotic medications have antagonist actions at dopamine D2 receptors. However, these drugs are not so effective to negative symptoms, consistent with the hypothesis, where dopamine D2 receptor over-activation is secondary. Thus, this hypothesis can integrally explain three main symptom domains: positive, negative and cognitive deficits.     

Mechanical and histological investigations on pressureless sintered SiC dental implants

The effect of sinterability manufacturing conditions, such as Si/C ratio, additives, firing temperature on mechanical properties of pressureless sintered SiC having electrical resistivity and oxidation behaviour in moist were studied. In order to evaluate the possibility of dental implant application of SiC, the implant study using rat femurs was performed. SiC was implanted in the drilled hole of the femurs and the histologic reaction to SiC was investigated under light microscopy and CMR. SiC provoked endosteal as bone formation. At 1-3 weeks, it was observed the formation of callus around the SiC implant. Finally, the intervening fibrous tissue between the encapsulating bone and SiC was replaced by bone tissue which was outgowing from the encapsulating bone surface. The observed interface between bone and SiC was similar to the bone-HA interface as to morphology. These results indicate the clinical application of SiC as a dental implant.     

A flexible and wearable biosensor for tear glucose measurement

A flexible and wearable amperometoric glucose sensor was fabricated and tested. Also, the sensor was utilized to tear glucose monitoring. The sensor was constructed by immobilizing GOD onto a flexible oxygen electrode (Pt working electrode and Ag/AgCl counter/reference electrode), which was fabricated using “Soft-MEMS” techniques onto a functional polymer membrane. In purpose of bioinstrumentation, adhesive agents were not used for constructing the flexible biosensor. Linear relationship between glucose concentration and output current was obtained in a range of 0.025–1.475 mmol/l, with a correlation coefficient of 0.998. Current dependences on pH and temperature were also evaluated. The current was largest at pH 7.0 and the current increased when temperature increased. This indicates that the output current depends on enzyme activity. Based on the basic characteristics investigation, the glucose sensor was applied to measurement of glucose in tear fluids on an eye site of a Japan white rabbit. The change of tear glucose level induced by oral-administration of glucose was monitored as a current change of the sensor attached on the eye site. In this investigation, the tear glucose level varied from 0.16 to 0.46 mmol/l. Although there was a delay of several tens of minutes towards blood sugar level, it is considered to be possible that non-invasive continuous glucose monitoring can be realized using the flexible biosensor.     

The inhibitory effects of isoflavonoids and resveratrol on oxdized low-density lipoprotein uptake in macrophage cell line J774.1

The interaction between oxidized low-density lipoprotein (LDL) and macrophages are known to be important in the development of arteriosclerosis. Macrophages take up oxidized LDL, becoming foam cells, which contribute to the thickening of the blood vessel wall. The antioxidant properties of polyphenols found in vegetables and other foods have been shown to have a protective effect against arteriosclerosis. To elucidate the effect of flavonoids from fruits, vegetables and cereals on oxidized LDL uptake in macrophages, the inhibitory activity of various flavonoids on DiI-ac-LDL uptake reaction in mouse macrophage cell line J774.1 was measured. We found significant uptake of DiI-ac-LDL, but not DiI-LDL, into the J774.1 cells. In addition, polyinosin and dextran sulphate inhibited uptake, but apigenin, kaempferol, and naringenin, did not. Isoflavones and resveratrol significantly inhibit uptake of DiI-ac-LDL into macrophages, and have a protective effect against arteriosclerosis.     

Differential regulation of the nitric oxide-cGMP pathway exacerbates postischaemic heart injury in stroke-prone hypertensive rats

Using a working perfused heart model, we investigated the hypothesis that alterations in the NO-cGMP pathway may exacerbate postischaemic mechanical dysfunction in the hypertrophied heart. Ischaemia for 25 min followed by reperfusion for 30 min produced marked cardiac mechanical dysfunction in both stroke-prone spontaneously hypertensive rats (SHRSP) and normotensive Wistar Kyoto rats (WKY). Exogenous treatment with S-nitroso-N-acetyl-dl-penicillamine (SNAP), a NO donor, had beneficial effects on the cardiac dysfunction induced by ischaemia-reperfusion (I/R) in the WKY heart, but the cardioprotective effect of SNAP was eliminated by guanylyl cyclase inhibitor. Cardiac cGMP levels were increased by SNAP or ischaemia in WKY. In contrast, in SHRSP hearts, SNAP could not alleviate the cardiac dysfunction caused by I/R. Pre-ischaemia, the cardiac cGMP level was significantly higher in SHRSP than in WKY; however, no significant difference was found after SNAP and ischaemia. The myocardial Ca(2+)-dependent NO synthase (NOS) activity increased at the end of ischaemia in WKY. Conversely, the Ca(2+)-independent NOS activity and protein levels were upregulated by I/R in the SHRSP myocardium. In the SHRSP hearts, non-selective NOS and selective Ca(2+)-independent NOS inhibitors or antioxidant treatment alleviated cardiac dysfunction caused by I/R. Moreover, mRNA expression and Western blotting analysis of cGMP-dependent protein kinase type I showed more deterioration of SHRSP hearts compared with WKY. These results suggest that: (1) the NO-dependent cardioprotective effect is depressed; and (2) overproduction of NO derived from Ca(2+)-independent NOS contributes to postischaemic heart injury in the hypertrophied heart of hypertensive status.     

Erythrophagocytosis by liver macrophages (Kupffer cells) promotes oxidative stress, inflammation, and fibrosis in a rabbit model of steatohepatitis: implications for the pathogenesis of human nonalcoholic steatohepatitis

Nonalcoholic steatohepatitis (NASH) is a progressive fibrotic disease, the pathogenesis of which has not been fully elucidated. Here, we report a molecular aspect of this disease elucidated using rabbits fed a cholesterol-rich high-fat diet and exhibiting insulin resistance. The liver in this model showed steatohepatitis with fibrosis and high mRNA expression for some cytokines, heme oxygenase-1, transforming growth factor-beta1, and collagen alpha1(I). Erythrocytes isolated from the model showed marked fragility and the externalization of phosphatidylserine (PS) on the outer leaflet of the membrane and were frequently engulfed by Kupffer cells/macrophages in the hepatic sinusoids. Expression of milk fat globule-epidermal growth factor (EGF)-factor 8, a PS-binding protein, was augmented in the liver. In culture, RAW 264.7 cells engulfed erythrocytes oxidized by tert-butyl hydroperoxide, a process that was inhibited by anti-milk fat globule-EGF-factor 8 antibody. In addition, PS-positive erythrocytes appeared entrapped in the model liver in ex vivo perfusion experiments. Finally, in specimens from NASH patients, the aggregation of erythrocytes in inflammatory hepatic sinusoids was notable. These results indicate that the engulfment of PS-externalized, apoptotic signal-positive, erythrocytes by hepatic macrophages may lead to the deposition of iron derived from hemoglobin in the liver and be involved in the pathogenesis of steatohepatitis.     

Concerted interaction between origin recognition complex (ORC), nucleosomes and replication origin DNA ensures stable ORC–origin binding

Chromosomal replication origins, where DNA replication is initiated, are determined in eukaryotic cells by specific binding of a six‐subunit origin recognition complex (ORC). Many biochemical analyses have showed the detailed properties of the ORC–DNA interaction. However, because of the lack of in vitro analysis, the molecular architecture of the ORC–chromatin interaction is unclear. Recently, mainly from in vivo analyses, a role of chromatin in the ORC–origin interaction has been reported, including the existence of a specific pattern of nucleosome positioning around origins and of a specific interaction between chromatin—or core histones—and Orc1, a subunit of ORC. Therefore, to understand how ORC establishes its interaction with origin in vivo, it is essential to know the molecular mechanisms of the ORC–chromatin interaction. Here, we show that ORC purified from yeast binds more stably to origin‐containing reconstituted chromatin than to naked DNA and forms a nucleosome‐free region at origins. Molecular imaging using atomic force microscopy (AFM) shows that ORC associates with the adjacent nucleosomes and forms a larger complex. Moreover, stable binding of ORC to chromatin requires linker DNA. Thus, ORC establishes its interaction with origin by binding to both nucleosome‐free origin DNA and neighboring nucleosomes.