In Vivo Imaging of Tumor Hypoxia by Maintaining Green Fluorescence of 9-Aminoanthracene Under Hypoxic Conditions.

In this study, 9-aminoanthracene (9AA) was used as a new fluorescence reagent for the in vivo imaging of tumor hypoxia by taking advantage of the maintenance of its green fluorescence under hypoxic conditions. As 9AA is insoluble in water, polyethylene glycol (PEG)-400 was used to dissolve 9AA in saline. Each organ was successfully stained with 9AA, as observed by green fluorescence using in vivo imaging, following intragastric administration of a 9AA PEG-saline solution in mice. Therefore, the intragastric administration of 9AA can be used for in vivo imaging of normal mice. Tumor hypoxia staining using the 9AA fluorescence method was evaluated by in vivo imaging of mice subcutaneously transplanted with Ehrlich ascites carcinoma cells and compared with conventional pimonidazole (PIMO) staining under hypoxic conditions. The tumor sections were stained with green fluorescence derived from 9AA and the same sections corresponded to hypoxic areas upon immunohistochemical staining with PIMO.

White matter imaging of ethanol-fixed rat brain by phase-contrast X-ray computed tomography.

BACKGROUND: Phase-contrast X-ray computed tomography imaging (PCI) based on crystal X-ray interferometry can detect minute density differences within biological soft tissues without contrast agents. Ethanol fixation yields increased tissue-background density differences due to the dehydrating and delipidifying effects of ethanol. PURPOSE: To obtain high image contrast of cerebral white matter structures in PCI, tissue fixation using ethanol and routinely used formalin have been examined. MATERIAL AND METHODS: Ethanol-fixed (EF) (n = 4) and formalin-fixed (FF) (n = 4) rat brains were imaged by crystal X-ray interferometry-based PCI. Tissue staining/microscopy was also performed for histological comparison and myelin density evaluation. Three-dimensional white matter tract images were reconstructed. RESULTS: Superior image contrast was obtained in the images of EF brains (EF images) compared to those of formalin-fixed brains (FF images), particularly for white matter structures. Significant density differences between the white matter structures and hippocampus (P < 0.01)/thalamus (P < 0.001) were observed in the EF, but not FF, images. Ethanol fixation enhanced the image contrast of white matter tracts by approximately sixfold compared to formalin fixation, and close agreement (r2 = 0.97; P < 0.05) between the density values on the CT images and the myelin density values in histological images was observed for the EF brains. Three-dimensional reconstruction of the white matter tracts was possible from the EF images, but not FF images. CONCLUSION: Ethanol fixation resulted in marked contrast enhancement of cerebral white matter structures in PCI. Thus, high-resolution PCI using ethanol for tissue fixation could be valuable for experimental neurological studies and postmortem neuropathology evaluation.

LRRK2 Inhibition Ameliorates Dexamethasone-Induced Glucose Intolerance via Prevents Impairment in GLUT4 Membrane Translocation in Adipocytes.

Mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are associated with Parkinson's disease. LRRK2 is a large protein with multiple functional domains, including a guanosine 5'-triphosphate (GTP)-binding domain and a protein kinase domain. Recent studies indicated that the members of the Rab GTPase family, Rab8a and Rab10, which are involved in the membrane transport of the glucose transporter type 4 (GLUT4) during insulin-dependent glucose uptake, are phosphorylated by LRRK2. However, the physiological role of LRRK2 in the regulation of glucose metabolism is largely unknown. In the present study, we investigated the role of LRRK2 using dexamethasone (DEX)-induced glucose intolerance in mice. LRRK2 knockout (KO) mice exhibited suppressed glucose intolerance, even after treatment with DEX. The phosphorylation of LRRK2, Rab8a and Rab10 was increased in the adipose tissues of DEX-treated wild-type mice. In addition, inhibition of the LRRK2 kinase activity prevented the DEX-induced inhibition of GLUT4 membrane translocation and glucose uptake in cultured 3T3-L1 adipocytes. These results suggest that LRRK2 plays an important role in glucose metabolism in adipose tissues.

Biochemical Characterization of Ferulic Acid and Caffeic Acid Which Effectively Inhibit Melanin Synthesis via Different Mechanisms in B16 Melanoma Cells.

In this study, we examined the inhibitory effects of ferulic acid and caffeic acid on melanin production using a murine B16 melanoma cell line. The mechanisms by which the two acids inhibit melanin production were investigated by evaluating their effects on the activity of tyrosinase, which is involved is the first step of melanin biosynthesis. Ferulic acid showed no toxicity against the melanoma cells at any dose, whereas caffeic acid exerted cellular toxicity at concentrations higher than 0.35 mM. Both ferulic and caffeic acids effectively inhibited melanin production in the B16 melanoma cells. Ferulic acid reduced tyrosinase activity by directly binding to the enzyme, whereas no binding was observed between caffeic acid and tyrosinase. Both ferulic acid and caffeic acid inhibited casein kinase 2 (CK2)-induced phosphorylation of tyrosinase in a dose-dependent manner in vitro. Ferulic acid was found to be a more effective inhibitor of melanin production than caffeic acid; this difference in the inhibitory efficacy between the two substances could be attributable to the difference in their tyrosine-binding activity. Our analysis revealed that both substances also inhibited the CK2-mediated phosphorylation of tyrosinase.

Deficiency in either COX-1 or COX-2 genes does not affect amyloid beta protein burden in amyloid precursor protein transgenic mice.

Epidemiologic studies indicate that chronic use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated with a lower risk for developing Alzheimer's disease (AD). Because the primary mode of action of NSAIDs is to inhibit cyclooxygenase (COX) activity, it has been proposed that perturbed activity of COX-1 or COX-2 contributes to AD pathogenesis. To test the role of COX-1 or COX-2 in amyloid deposition and amyloid-associated inflammatory changes, we examined amyloid precursor protein (APP) transgenic mice in the context of either COX-1 or COX-2 deficiency. Our studies showed that loss of either COX-1 or COX-2 gene did not alter amyloid burden in brains of the APP transgenic mice. However, one marker of microglial activation (CD45) was decreased in brains of COX-1 deficient/APP animals and showed a strong trend in reduction in COX-2 deficient/APP animals. These results suggest that COX activity and amyloid deposition in brain are likely independent processes. Further, if NSAIDs do causally reduce the risks of AD, then our findings indicate that the mechanisms are likely not due primarily to their inhibition on COX or γ-secretase modulation activity, the latter reported recently after acute dosing of ibuprofen in humans and nonhuman primates.

Suppressive Effect of Dietary Fucoidan on Proinflammatory Immune Response and MMP-1 Expression in UVB-Irradiated Mouse Skin.

It is well known that ultraviolet B irradiation leads to dermal inflammation. In this study, we found that Mekabu fucoidan suppressed edema, decreased the thickness of the prickle cell layer, and decreased matrix metalloproteinase 1 in the skin of mice irradiated with ultraviolet B. Moreover, we found that the mean level of interferon gamma of Mekabu fucoidan-treated, ultraviolet B-irradiated mice (approximately 2.2 ng/mL) was not significantly different from that in normal mice (approximately 2.5 ng/mL). In contrast, a significant decrease in the mean level of interferon gamma (approximately 1.3 ng/mL) in ultraviolet B-irradiated control mice was observed compared with that in Mekabu fucoidan-treated, ultraviolet B-irradiated mice. The mean thickness of the prickle cell layer in the skin of Mekabu fucoidan-treated, ultraviolet B-irradiated mice was less than that in the ultraviolet B-irradiated control mice. Metalloproteinase 1 activity was significantly higher in the skin of ultraviolet B-irradiated mice than in the skin of untreated, nonirradiated normal mice. Metalloproteinase 1 in the skin of ultraviolet B-irradiated, Mekabu fucoidan- or L(+)-ascorbic acid (vitamin C)-treated mice was significantly lower than that in the ultraviolet B-irradiated control mice. Mitigation of the morphological changes in Mekabu fucoidan-treated mice was correlated with a decrease in metalloproteinase 1 levels. These data indicate that Mekabu fucoidan is an effective suppressor of inflammation in an ultraviolet B-irradiated mouse model.

Enhanced renal image contrast by ethanol fixation in phase-contrast X-ray computed tomography.

Phase-contrast X-ray imaging using a crystal X-ray interferometer can depict the fine structures of biological objects without the use of a contrast agent. To obtain higher image contrast, fixation techniques have been examined with 100% ethanol and the commonly used 10% formalin, since ethanol causes increased density differences against background due to its physical properties and greater dehydration of soft tissue. Histological comparison was also performed. A phase-contrast X-ray system was used, fitted with a two-crystal X-ray interferometer at 35 keV X-ray energy. Fine structures, including cortex, tubules in the medulla, and the vessels of ethanol-fixed kidney could be visualized more clearly than that of formalin-fixed tissues. In the optical microscopic images, shrinkage of soft tissue and decreased luminal space were observed in ethanol-fixed kidney; and this change was significantly shown in the cortex and outer stripe of the outer medulla. The ethanol fixation technique enhances image contrast by approximately 2.7-3.2 times in the cortex and the outer stripe of the outer medulla; the effect of shrinkage and the physical effect of ethanol cause an increment of approximately 78% and 22%, respectively. Thus, the ethanol-fixation technique enables the image contrast to be enhanced in phase-contrast X-ray imaging.

[18F]-FDG uptake in brain slices prepared from an aged mouse model of Alzheimer's disease using a dynamic autoradiography technique.

OBJECTIVE: 2-[18F]fluoro-2-deoxy-D-glucose positron emission tomography ([18F]-FDG-PET) is a imaging modality that has been used to measure of glucose metabolism in the brain in Alzheimer's disease (AD). Clinically, decreased glucose uptake has been reported in the brain of AD, although the precise underlying mechanisms have not yet been elucidated. To elucidate the mechanisms of decreased [18F]-FDG uptake in the AD by PET, [18F]-FDG uptake in the brain of aged model mouse of AD was investigated using a dynamic autoradiography technique "bioradiography". A X-ray phase-contrast imaging (X-PCI) and a histopathological evaluation were also investigated to elucidate the mechanisms underlying the relationships between decreased [18F]-FDG uptake and the pathological changes in the brain of AD mouse. METHODS: In this study, AD model mouse (5XFAD, APP+/PS1+) were used. [18F]-FDG-bioradiography was conducted in fresh slices of brain tissue under the condition of resting (slices immersed in 5 mM K+ solution) and metabolically active (in 50 mM K+ solution). Amyloid ß42 (Aß42) deposition in the brain of AD mouse was confirmed by X-PCI. In addition, the positive cells of phosphated tau protein (P-tau) and deposition of Aß42 were also examined by immunohistochemical staining. RESULTS: No significant differences were observed between the two groups in the resting condition. In the activate condition of the brain, [18F]-FDG uptake was significantly decreased in AD mice compared to WT mice. In X-PCI showed Aß deposition in the AD mouse, but not in the WT. The AD mouse also showed increased P-tau, accumulation of Aß42, increase in neuronal apoptosis, and decrease in the number of neurons than that of the WT mouse. CONCLUSION: Neuronal damage, and induction of neuronal apoptosis, decreased [18F]-FDG uptake, increased Aß accumulation and P-tau induced neurofibrillary degeneration are observed in AD mouse. In clinical diagnosis, reduction of [18F]-FDG uptake by PET is one of the means of diagnosing the onset of AD. Our results suggest that decreased uptake of [18F]-FDG in the brains of AD may be associated with neuronal dysfunction and cell death in the brain.

Amyloid precursor protein (APP) regulates synaptic structure and function.

The amyloid precursor protein (APP) plays a critical role in Alzheimer's disease (AD) pathogenesis. APP is proteolytically cleaved by ß- and γ-secretases to generate the amyloid ß-protein (Aß), the core protein component of senile plaques in AD. It is also cleaved by α-secretase to release the large soluble APP (sAPP) luminal domain that has been shown to exhibit trophic properties. Increasing evidence points to the development of synaptic deficits and dendritic spine loss prior to deposition of amyloid in transgenic mouse models that overexpress APP and Aß peptides. The consequence of loss of APP, however, is unsettled. In this study, we investigated whether APP itself plays a role in regulating synaptic structure and function using an APP knock-out (APP-/-) mouse model. We examined dendritic spines in primary cultures of hippocampal neurons and CA1 neurons of hippocampus from APP-/- mice. In the cultured neurons, there was a significant decrease (~35%) in spine density in neurons derived from APP-/- mice compared to littermate control neurons that were partially restored with sAPPα-conditioned medium. In APP-/- mice in vivo, spine numbers were also significantly reduced but by a smaller magnitude (~15%). Furthermore, apical dendritic length and dendritic arborization were markedly diminished in hippocampal neurons. These abnormalities in neuronal morphology were accompanied by reduction in long-term potentiation. Strikingly, all these changes in vivo were only seen in mice that were 12-15 months in age but not in younger animals. We propose that APP, specifically sAPP, is necessary for the maintenance of dendritic integrity in the hippocampus in an age-associated manner. Finally, these age-related changes may contribute to AD pathology independent of Aß-mediated synaptic toxicity.

Undaria pinnatifida (Wakame) Intake Ameliorates High-Fat Diet-Induced Glucose Intolerance via Promoting GLUT4 Expression and Membrane Translocation in Muscle.

Type 2 diabetes mellitus (T2DM), a lifestyle-related disease, is developed due to eating habits and decreased physical activity. Diabetes also increases the risk of cancer and major neurodegenerative diseases; controlling the onset of diabetes helps prevent various illnesses. Eating seaweed, such as Undaria pinnatifida (wakame), is a part of the Asian food culture. Therefore, we analyzed the antidiabetic effect of wakame intake using the high-fat diet-induced diabetes mouse model. Furthermore, we analyzed the effect of wakame extract on the cell membrane translocation of glucose transporter-4 (GLUT4) and activation of insulin signal molecules, such as AKT and AMPK, in insulin-sensitive tissues. Differentiated C2C12 cells were incubated with wakame components. The membrane translocation of GLUT4 and phosphorylation of AKT and AMPK were investigated with immunofluorescence staining and Western blotting, respectively. Also, male C57BL/6J mice were fed the normal diet (ND), high-fat diet (HFD), ND with 1% wakame powder (ND + W), or HFD with 1% wakame powder (HFD + W). We evaluated the effect of wakame intake on high-fat diet-induced glucose intolerance using an oral glucose tolerance test. Moreover, we analyzed insulin signaling molecules, such as GLUT4, AKT, and AMPK, in muscle using Western blotting. GLUT4 membrane translocation was promoted by wakame components. Also, GLUT4 levels and AKT and AMPK phosphorylation were significantly elevated by wakame components in C2C12 cells. In addition, the area under the curve (AUC) of the HFD + W group was significantly smaller than that of the HFD group. Furthermore, the level of GLUT4 in the muscle was increased in the wakame intake group. This study revealed that various wakame components exerted antidiabetic effects on the mice on a high-fat diet by promoting glucose uptake in the skeletal muscle, enhancing GLUT4 levels, and activating AKT and AMPK.

LRRK2 negatively regulates glucose tolerance via regulation of membrane translocation of GLUT4 in adipocytes.

Epidemiological studies have shown that abnormalities of glucose metabolism are involved in leucine-rich repeat kinase 2 (LRRK2)-associated Parkinson's disease (PD). However, the physiological significance of this association is unclear. In the present study, we investigated the effect of LRRK2 on high-fat diet (HFD)-induced glucose intolerance using Lrrk2-knockout (KO) mice. We found for the first time that HFD-fed KO mice display improved glucose tolerance compared with their wild-type (WT) counterparts. In addition, high serum insulin and leptin, as well as low serum adiponectin resulting from HFD in WT mice were improved in KO mice. Using western blotting, we found that Lrrk2 is highly expressed in adipose tissues compared with other insulin-related tissues that are thought to be important in glucose tolerance, including skeletal muscle, liver, and pancreas. Lrrk2 expression and phosphorylation of its kinase substrates Rab8a and Rab10 were significantly elevated after HFD treatment in WT mice. In cell culture experiments, treatment with a LRRK2 kinase inhibitor stimulated insulin-dependent membrane translocation of glucose transporter 4 (Glut4) and glucose uptake in mouse 3T3-L1 adipocytes. We conclude that increased LRRK2 kinase activity in adipose tissue exacerbates glucose tolerance by suppressing Rab8- and Rab10-mediated GLUT4 membrane translocation.

Substrate sequence influences γ-secretase modulator activity, role of the transmembrane domain of the amyloid precursor protein.

A subset of non-steroidal anti-inflammatory drugs modulates the γ cleavage site in the amyloid precursor protein (APP) to selectively reduce production of Aß42. It is unclear precisely how these γ-secretase modulators (GSMs) act to preferentially spare Aß40 production as well as Notch processing and signaling. In an effort to determine the substrate requirements in NSAID/GSM activity, we determined the effects of sulindac sulfide and flurbiprofen on γ-cleavage of artificial constructs containing several γ-secretase substrates. Using FLAG-tagged constructs that expressed extracellularly truncated APP, Notch-1, or CD44, we found that these substrates have different sensitivities to sulindac sulfide. γ-Secretase cleavage of APP was altered by sulindac sulfide, but CD44 and Notch-1 were either insensitive or only minimally altered by this compound. Using chimeric APP constructs, we observed that the transmembrane domain (TMD) of APP played a pivotal role in determining drug sensitivity. Substituting the APP TMD with that of APLP2 retained the sensitivity to γ-cleavage modulation, but replacing TMDs from Notch-1 or ErbB4 rendered the resultant molecules insensitive to drug treatment. Specifically, the GXXXG motif within APP appeared to be critical to GSM activity. Consequently, the modulatory effects on γ-cleavage appears to be substrate-dependent. We hypothesize that the substrate present in the γ-secretase complex influences the conformation of the complex so that the binding site of GSMs is either stabilized or less favorable to influence the cleavage of the respective substrates.

Lysine 624 of the amyloid precursor protein (APP) is a critical determinant of amyloid ß peptide length: support for a sequential model of γ-secretase intramembrane proteolysis and regulation by the amyloid ß precursor protein (APP) juxtamembrane region.

γ-Secretase is a multiprotein intramembrane cleaving aspartyl protease (I-CLiP) that catalyzes the final cleavage of the amyloid ß precursor protein (APP) to release the amyloid ß peptide (Aß). Aß is the primary component of senile plaques in Alzheimer's disease (AD), and its mechanism of production has been studied intensely. γ-Secretase executes multiple cleavages within the transmembrane domain of APP, with cleavages producing Aß and the APP intracellular domain (AICD), referred to as γ and ε, respectively. The heterogeneous nature of the γ cleavage that produces various Aß peptides is highly relevant to AD, as increased production of Aß 1-42 is genetically and biochemically linked to the development of AD. We have identified an amino acid in the juxtamembrane region of APP, lysine 624, on the basis of APP695 numbering (position 28 relative to Aß) that plays a critical role in determining the final length of Aß peptides released by γ-secretase. Mutation of this lysine to alanine (K28A) shifts the primary site of γ-secretase cleavage from 1-40 to 1-33 without significant changes to ε cleavage. These results further support a model where ε cleavage occurs first, followed by sequential proteolysis of the remaining transmembrane fragment, but extend these observations by demonstrating that charged residues at the luminal boundary of the APP transmembrane domain limit processivity of γ-secretase.

Rapid and specific alterations of goblet cell mucin in rat airway and small intestine associated with resistance against Nippostrongylus brasiliensis reinfection.

The intestinal parasitic nematode Nippostrongylus brasiliensis is expelled rapidly from the rat in reinfection challenge compared with that of the primary infection owing to the host defense mechanisms raised against the pre-intestinal- and intestinal-stage larvae. We examined the relationship between the mucin alterations in airway and jejunal mucosae and the worm expulsion after third-stage larva reinfection. When rats had been inoculated with fourth-stage larvae and immunized with only the intestinal-stage worms for more than 8 days, the challenge larvae were expelled during the intestinal stage along with a rapid increase of the specific sialomucin in jejunal mucosa, without any effect on the bronchial mucus. When rats had been infected with third-stage larvae and immunized with only the pre-intestinal stage larvae by killing with antihelminthic, the challenge larvae were rejected during the pre-intestinal stage along with marked goblet cell hyperplasia and Muc5AC mucin hyperproduction on the bronchial mucosa, but not as a result of jejunal mucin alteration. Taking these finding together, immunization with pre-intestinal- and intestinal-stage worms independently increases the airway and intestinal goblet cell mucins, respectively, and in both cases, the mucin alterations may contribute to rapid worm expulsion upon reinfection.

Fucose-containing sulfated polysaccharides from brown seaweeds inhibit proliferation of melanoma cells and induce apoptosis by activation of caspase-3 in vitro.

Fucose-containing sulfated polysaccharides (FCSPs) extracted from seaweeds, especially brown macro-algae, are known to possess essential bioactive properties, notably growth inhibitory effects on tumor cells. In this work, we conducted a series of in vitro studies to examine the influence of FCSPs products from Sargassumhenslowianum C. Agardh (FSAR) and Fucus vesiculosus (FVES), respectively, on proliferation of melanoma B16 cells and to investigate the underlying apoptosis promoting mechanisms. Cell viability analysis showed that both FCSPs products, i.e., FSAR and FVES, decreased the proliferation of the melanoma cells in a dose-response fashion, with FSAR being more potent at lower dosages, and FVES being relatively more anti-proliferative than FSAR at higher dosages. Flow cytometric analysis by Annexin V staining of the melanoma cells exposed to the FCSPs products confirmed that both FSAR and FVES induced apoptosis. The FCSPs-induced apoptosis was evidenced by loss of plasma membrane asymmetry and translocation of the cell membrane phospholipids and was accompanied by the activation of caspase-3. The FCSPs bioactivity is proposed to be attributable to distinct structural features of the FCSPs, particularly the presence of sulfated galactofucans (notably in S.henslowianum) and sulfated fucans (notably in F. vesiculosus). This study thus indicates that unfractionated FCSPs may exert bioactive effects on skin cancer cells via induction of apoptosis through cascades of reactions that involve activation of caspase-3.

Suppression of inflammatory responses after onset of collagen-induced arthritis in mice by oral administration of the Citrus flavanone naringin.

Rheumatoid arthritis (RA) is closely related to the pathogenesis of tumor necrosis factor α in lesions. We investigated the suppressive effects of a Citrus flavanone naringin on inflammatory responses in mice with collagen-induced arthritis (CIA), a mouse model for RA. To investigate potential preventive and therapeutic effects of naringin, mice were given naringin orally three times a week from the second immunization with collagen (day 21) and from day 31, when symptoms of CIA had reached a plateau, respectively. In both cases, inflammation-related clinical scores for knee joints were significantly reduced by administration of naringin. Histological analyses demonstrated that representative phenomena, such as damage to interchondral joints, infiltration of inflammatory cells and pannus formation, were significantly depressed by treatment with naringin. In addition, increases in the expression of high-mobility group box-1 protein in the joints of mice with CIA were suppressed by naringin. These results suggest that oral administration of naringin might be effective for treating human patients with RA.

Visualization Ability of Phase-Contrast Synchrotron-Based X-Ray Imaging Using an X-Ray Interferometer in Soft Tissue Tumors.

Phase-contrast synchrotron-based X-ray imaging using an X-ray interferometer provides high sensitivity and high spatial resolution, and it has the ability to depict the fine morphological structures of biological soft tissues, including tumors. In this study, we quantitatively compared phase-contrast synchrotron-based X-ray computed tomography images and images of histopathological hematoxylin-eosin-stained sections of spontaneously occurring rat testicular tumors that contained different types of cells. The absolute densities measured on the phase-contrast synchrotron-based X-ray computed tomography images correlated well with the densities of the nuclear chromatin in the histological images, thereby demonstrating the ability of phase-contrast synchrotron-based X-ray imaging using an X-ray interferometer to reliably identify the characteristics of cancer cells within solid soft tissue tumors. In addition, 3-dimensional synchrotron-based phase-contrast X-ray computed tomography enables screening for different structures within tumors, such as solid, cystic, and fibrous tissues, and blood clots, from any direction and with a spatial resolution down to 26 µm. Thus, phase-contrast synchrotron-based X-ray imaging using an X-ray interferometer shows potential for being useful in preclinical cancer research by providing the ability to depict the characteristics of tumor cells and by offering 3-dimensional information capabilities.

Biochemical characterization of tau protein and its associated syndapin 1 and protein kinase Cepsilon for their functional regulation in rat brain.

BACKGROUND: We recently reported that both sulfatide and cholesterol-3-sulfate (SCS) function as potent stimulators for the GSK-3beta-mediated phosphorylation of tau protein (TP) in vitro [J. Biochem. 143 (2008) 359-367]. METHODS: By means of successive gel filtration on a Superdex 200 pg column and three distinct ion-exchange column chromatographies, TP and its associated proteins were highly purified from the extract of rat brain. RESULTS: We found that (i) syndapin 1 and novel protein kinase Cepsilon (nPKCepsilon) were identified as the TP-associated proteins; (ii) SCS highly stimulated the phosphorylation of TP and syndapin 1 by nPKCepsilon as well as CK1; (iii) the full phosphorylation of TP and syndapin 1 by nPKCepsilon in the presence of sulfatide resulted in their dissociation; (iv) TP primed by CK1 functioned as an effective phosphate acceptor for GSK-3beta; (v) syndapin 1 highly stimulated the GSK-3beta-mediated phosphorylation of TP; and (vi) TP isoforms were highly expressed in aged brain, whereas syndapin 1 was consistently detected in adult brain, but not in newborn brain. GENERAL SIGNIFICANCE: These results provided here suggest that (i) TP-associated nPKCepsilon suppresses the GSK-3beta-mediated phosphorylation of TP through the phosphorylation of GSK-3beta by the kinase in vitro; and (ii) SCS act as effective sole mediators to induce the GSK-3beta-mediated high phosphorylation of both TP and its associated syndapin 1 involved in the biochemical processes of neuronal diseases, including Alzheimer's disease.

Evaluation of cell viability and metabolic activity of a 3D cultured human epidermal model using a dynamic autoradiographic technique with a PET radiopharmaceutical.

Quality control of tissues and organs for transplant is important to confirm their safety and effectiveness for regenerative medicine. However, quality evaluation is only carried out using a limited range of inspection criteria, because many of the available evaluation tests are invasive. In order to explore the potential of 2-[18F]fluoro-2-deoxy-D-glucose ([18F]FDG)-bioradiography as a non-invasive test for estimation of the safety, soundness, and effectiveness of tissues for transplantation, [18F]FDG uptake and cell viability or metabolism were investigated using a reconstructed human epidermal model (RHEM). We developed an imaging system, and suitable bioradiographic image acquisition conditions and its effectiveness were investigated. [18F]FDG uptake increased in agreement with DNA content as a marker of cell numbers and for histological assessment during cell proliferation and keratinization. [18F]FDG uptake was significantly decreased in good agreement with the viability of tissues used with various hazardous chemical treatments. [18F]FDG uptake by the tissues was decreased by hypothermia treatment and increased by hypoxia treatment while maintaining cell viability in the tissue. Therefore, [18F]FDG-bioradiography can be useful to estimate cell viability or metabolism in this RHEM. This method might be utilized as a non-invasive test for quality evaluation of tissues for transplantation.