Adhesive cell cultivation on polymer particle having grafted epoxy polymer chain

In this study, we synthesized a new cell immobilization support having poly(glycidyl methacrylate) as a graft polymer chain and used this support for cell cultivation. Base polymer particle was synthesized by suspension polymerization and epoxy polymer chain was extended from particle surface on graft polymerization. Produced polymer particles had broad particle size distribution ranging from 20 to 1000 μm and the degree of polymerization of grafted polymer chain was ranged from 500 to 1000. The effects of various factors, such as grafted polymer chain length and its surface density, composition of base polymer network and graft polymer chain, on the cell growth of murine fibroblast cell line (MS-5 cell) on polymer particle were studied. This polymer particle could cultivate not only fibroblast cell line but also epidermal cell line (HeLa cell), osteoblast cell line (MC3T3E1 cell), and chondrocyte cell line (ch-8 cell) on its surface. Growth rate is almost the same as that of cells using poly(styrene) tissue culture dish. To apply this cell cultivation system for examination of cell co-culture, HeLa cell immobilized on 100 μm of polymer particle was successfully co-cultured with MS-5 cell immobilized on 300 μm of polymer particle for four weeks.     

Global gene expression profiling in early-stage polycystic kidney disease in the Han:SPRD Cy rat identifies a role for RXR signaling

Han:SPRD Cy is a spontaneous rat model of polycystic kidney disease (PKD) caused by a missense mutation in Pkdr1. Cystogenesis in this model is not clearly understood. In the current study, we performed global gene expression profiling in early-stage PKD cyst development in Cy/Cy kidneys and normal (+/+) kidneys at 3 and 7 days of postnatal age. Expression profiles were determined by microarray analysis, followed by validation with real-time RT-PCR. Genes were selected with over 1.5-fold expression changes compared with age-matched +/+ kidneys for canonical pathway analysis. We found nine pathways in common between 3- and 7-day Cy/Cy kidneys. Three significantly changed pathways were designated "Vitamin D Receptor (VDR)/Retinoid X Receptor (RXR) Activation," "LPS/IL-1-Mediated Inhibition of RXR Function," and "Liver X Receptor (LXR)/RXR Activation." These results suggest that RXR-mediated signaling is significantly altered in developing kidneys with mutated Pkdr1. In gene ontology analysis, the functions of these RXR-related genes were found to be involved in regulating cell proliferation and organ morphogenesis. With real-time RT-PCR analysis, the upregulation of Ptx2, Alox15b, OSP, and PCNA, major markers of cell proliferation associated with the RXR pathway, were confirmed in 3- and 7-day Cy/Cy kidneys compared with 3-day +/+ kidneys. The increased RXR protein was observed in both the nucleus and cytoplasm of cystic epithelial cells in early-stage Cy/Cy kidneys, and the RXR-positive cells were strongly positive for PCNA staining. Taken together, cell proliferation and organ morphogenesis signals transduced by RXR-mediated pathways may have important roles for cystogenesis in early-stage PKD in this Pkdr1-mutated Cy rat.     

Microbial diversity with dominance of 16S rRNA gene sequences with high GC contents at 74 and 98 °C subsurface crude oil deposits in Japan

We prepared DNA from the production waters of oil deposits and wellheads of the high- and hypertemperature Japanese oil wells #AR39 (depth, 1230 m; temperature, 74 °C; pressure, 2.92 MPa) and #SR123 (depth, 1687 m; temperature, 98 °C; pressure, 11.3 MPa) to detect indigenous bacterial and archaeal microorganisms. We used PCR to amplify the 16S rRNA genes of microbial communities and characterized them based on their sequences. A few species of microorganisms with high GC contents were detected in samples from oil deposits, whereas the microbial constituents and their GC contents were diverse in wellhead samples. A comparison of the composition of the microbial communities found that the predominant indigenous populations in the #SR123 oil deposit were Thermotoga hypogea-, Thermotoga petrophila- and Thermodesulfobacterium commune-like bacteria with a 61-63% GC content in their 16S rRNA gene sequences, and Archaeoglobus fulgidus-like archaea with a 65% GC content, whereas the major population in #AR39 comprised Thermacetogenium phaeum- and Fervidobacterium pennavorans-like bacteria and Methanothermobacter thermautotrophicus-like archaea with a 60%, 60% and 61% GC content, respectively.     

Endothelial microsomal prostaglandin E synthase-1 facilitates neurotoxicity by elevating astrocytic Ca2+ levels

Recurrent seizures may cause neuronal damage in the hippocampus. As neurons form intimate interactions with astrocytes via glutamate, this neuron-glia circuit may play a pivotal role in neuronal excitotoxicity following such seizures. On the other hand, astrocytes contact vascular endothelia with their endfeet. Recently, we found kainic acid (KA) administration induced microsomal prostaglandin E synthase-1 (mPGES-1) and prostaglandin E(2) (PGE(2)) receptor EP3 in venous endothelia and on astrocytes, respectively. In addition, mice deficient in mPGES-1 exhibited an improvement in KA-induced neuronal loss, suggesting that endothelial PGE(2) might modulate neuronal damage via astrocytes. In this study, we therefore investigated whether the functional associations between endothelia and astrocytes via endothelial mPGES-1 lead to neuronal injury using primary cultures of hippocampal slices. We first confirmed the delayed induction of endothelial mPGES-1 in the wild-type (WT) slices after KA-treatment. Next, we examined the effects of endothelial mPGES-1 on Ca(2+) levels in astrocytes, subsequent glutamate release and neuronal injury using cultured slices prepared from WT and mPGES-1 knockout mice. Moreover, we investigated which EP receptor on astrocytes was activated by PGE(2). We found that endothelial mPGES-1 produced PGE(2) that enhanced astrocytic Ca(2+) levels via EP3 receptors and increased Ca(2+)-dependent glutamate release, aggravating neuronal injury. This novel endothelium-astrocyte-neuron signaling pathway may be crucial for neuronal damage after repetitive seizures, and hence could be a new target for drug development.     

Residual stress distribution in rabbit limb bones

The presence of the residual stresses in bone tissue has been noted and the authors have reported that there are residual stresses in bone tissue. The aim of our study is to measure the residual stress distribution in the cortical bone of the extremities of vertebrates and to describe the relationships with the osteon population density. The study used the rabbit limb bones (femur, tibia/fibula, humerus, and radius/ulna) and measured the residual stresses in the bone axial direction at anterior and posterior positions on the cortical surface. The osteons at the sections at the measurement positions were observed by microscopy. As a result, the average stresses at the hindlimb bones and the forelimb bones were 210 and 149 MPa, respectively. In the femur, humerus, and radius/ulna, the residual stresses at the anterior position were larger than those at the posterior position, while in the tibia, the stress at the posterior position was larger than that at the anterior position. Further, in the femur and humerus, the osteon population densities in the anterior positions were larger than those in the posterior positions. In the tibia, the osteon population density in the posterior position was larger than that in the anterior position. Therefore, tensile residual stresses were observed at every measurement position in the rabbit limb bones and the value of residual stress correlated with the osteon population density (r=0.55, P<0.01).     

Fatigue and tensile properties of radicular dentin substrate

The purpose of this study was to compare the fatigue and tensile strengths of radicular dentin. Forty bovine lower central incisors were used, twenty teeth for the fatigue test and twenty teeth for the tensile test. Bovine teeth were each sectioned into coronal and radicular portions. Dentin slabs of 1mm thickness were prepared along the radicular tooth using a low-speed cutting machine and trimmed into dumbbell-shaped specimens. A dentin slab was harvested from each tooth. Subsequently, fatigue and tensile tests were performed in Hank's balanced saline solution at 37 °C. The staircase method was employed to determine fatigue strength and its standard deviation. Fracture surfaces were observed by scanning electron microscopy. Mean fatigue strength and tensile strength were 44.3±5.0 and 84.4±8.3 MPa, respectively. The fatigue strength of radicular dentin was significantly lower than the tensile strength. The fatigue strength of radicular dentin was only approximately one half of the tensile strength.     

Specific In Situ Visualization of Plasma Cells Producing Antibodies against Porphyromonas gingivalis in Gingival Radicular Cyst: Application of the Enzyme-Labeled Antigen Method

The enzyme-labeled antigen method was applied to visualize plasma cells producing antibodies to Porphyromonas gingivalis, flora of the human oral cavity. Antibodies to P. gingivalis have reportedly been detected in sera of patients with periodontitis. Biotinylated bacterial antigens, Ag53, and four gingipain domains (Arg-pro, Arg-hgp, Lys-pro, and Lys-hgp) were prepared by the cell-free protein synthesis system using the wheat germ extract. In paraformaldehyde-fixed frozen sections of rat lymph nodes experimentally immunized with Ag53-positive and Ag53-negative P. gingivalis, plasma cells were labeled with biotinylated Arg-hgp and Lys-hgp. Antibodies to Ag53 were detected only in the nodes immunized with Ag53-positive bacteria. In two of eight lesions of gingival radicular cyst with inflammatory infiltration, CD138-positive plasma cells in frozen sections were signalized for Arg-hgp and Lys-hgp. An absorption study using unlabeled antigens confirmed the specificity of staining. The AlphaScreen method identified the same-type antibodies in tissue extracts but not in sera. Antibodies to Ag53, Arg-pro, and Lys-pro were undetectable. In two cases, serum antibodies to Arg-hgp and Lys-hgp were AlphaScreen positive, whereas plasma cells were scarcely observed within the lesions. These findings indicate the validity of the enzyme-labeled antigen method. This is the very first application of this novel histochemical technique to human clinical samples.     

Role for E-cadherin as an inhibitory receptor on epidermal gammadelta T cells

E-cadherin is a homophilic adhesion molecule that maintains homotypic intercellular adhesion between epithelial cells such as epidermal keratinocytes. E-cadherin is also expressed on resident murine epidermal γδ T cells, known as dendritic epidermal T cells (DETCs), but they express another receptor for E-cadherin, α(E)(CD103)β(7) integrin, as well. In this study, we analyzed functional differences between E-cadherin-mediated homophilic binding and heterophilic binding of α(E)β(7) integrin to E-cadherin in heterotypic intercellular adhesion of DETCs to keratinocytes. E-cadherin, but not α(E)β(7) integrin, was downregulated on activation of DETCs in vivo and in vitro. Short-term (1-h) adhesion of DETCs to keratinocytes in vitro was primarily mediated by α(E)β(7) integrin, and blocking of the binding of α(E)β(7) integrin to E-cadherin inhibited the lysis of keratinocytes by DETCs. Stable binding of E-cadherin on DETCs to plate-bound recombinant E-cadherin was observed only after 24-h culture in vitro. Cytokine production and degranulation by DETCs in response to suboptimal TCR cross-linking and mitogen stimulation were augmented by coligation of α(E)β(7) integrin. In contrast, engagement of E-cadherin on DETCs with immobilized anti-E-cadherin Ab, plate-bound recombinant E-cadherin, and E-cadherin on keratinocytes inhibited DETC activation. Therefore, E-cadherin acts as an inhibitory receptor on DETCs, whereas α(E)β(7) integrin acts as a costimulatory receptor. Differential expression of E-cadherin and α(E)β(7) integrin on resting and activated DETCs, as well as their opposite functions in DETC activation, suggests that E-cadherin and α(E)β(7) integrin on DETCs regulate their activation threshold through binding to E-cadherin on keratinocytes.