Transferrin, insulin, and progesterone modulate intracellular concentrations of coenzyme Q and cholesterol, products of the mevalonate pathway, in undifferentiated PC12 cells.

Coenzyme Q (CoQ) is important not only as an essential lipid for the mitochondrial electron transport system, but also as an antioxidant. CoQ levels decrease during aging and in various diseases. Orally administered CoQ is not readily taken up in the brain, so it is necessary to develop a method to increase the amount of CoQ in neurons. CoQ is synthesized via mevalonate pathway, like cholesterol. Transferrin, insulin, and progesterone are factors used in the culture of neurons. In this study, we determined the effect of these reagents on cellular CoQ and cholesterol levels. The administration of transferrin, insulin, and progesterone increased cellular CoQ levels in undifferentiated PC12 cells. When serum was removed and only insulin was administered, intracellular CoQ levels increased. This increase was even more pronounced with concurrent administration of transferrin, insulin, and progesterone. Cholesterol level decreased by the administration of transferrin, insulin, and progesterone. Progesterone treatment lowered intracellular cholesterol levels in a concentration-dependent manner. Our findings suggest that transferrin, insulin, and progesterone may be useful in regulating CoQ levels and cholesterol levels, which are products of the mevalonate pathway.

Cellular level of coenzyme Q increases with neuronal differentiation, playing an important role in neural elongations.

Deficiency of coenzyme Q has been reported in various neuro-logical diseases, and the behavior of this lipid in neurons has attracted attention. However, the behavior of this lipid in normal neurons remains unclear. In this study, we analyzed the concen-tration of coenzyme Q before and after neuronal differentiation. Nerve growth factor treatment of PC12 cells caused neurite outgrowth and neuronal differentiation, and the amount of intra-cellular coenzyme Q increased dramatically during this process. In addition, when the serum was removed from the culture medium of N1E-115 cells and the neurite outgrowth was confirmed, the intracellular coenzyme Q level also increased. To elucidate the role of the increased coenzyme Q, we administered nerve growth factor to PC12 cells with coenzyme Q synthesis inhibitors and found that coenzyme Q levels decreased, neurite outgrowth was impaired, and differentiation markers were reduced. These results indicate that coenzyme Q levels increase during neuronal differentiation and that this increase is important for neurite outgrowth.

Inositol hexakisphosphate kinase 2 promotes cell death of anterior horn cells in the spinal cord of patients with amyotrophic lateral sclerosis.

We have previously reported that inositol hexakisphosphate kinase (InsP6K)2 mediates cell death. InsP6K2 is abundantly expressed in anterior horn cells of the mammalian spinal cord. We investigated the role of InsP6K2 in spinal cords of patients with amyotrophic lateral sclerosis (ALS). Autopsy specimens of lumbar spinal cords from ten patients with sporadic ALS and five non-neurological disease patients (NNDPs) were obtained. We performed quantitative real-time PCR, immunostaining, and western blotting for InsP6K1, InsP6K2, InsP6K3, protein kinase B (Akt), casein kinase 2 (CK2), and 90-kDa heat-shock protein (HSP90). In contrast to InsP6K1 and InsP6K3 mRNA expression, InsP6K2 levels in anterior horn cells of the spinal cord were significantly increased in ALS patients compared to NNDPs. In ALS patients, InsP6K2 translocated from the nucleus to the cytoplasm. However, we observed a decrease in HSP90, CK2, and Akt activity in ALS patients compared to NNDPs. A previous study reported that InsP6K2 activity is suppressed after binding to HSP90 and subsequent phosphorylation and degradation by CK2, thus decreasing InsP6K2 activity. However, InsP7, which is generated by InsP6K2, can compete with Akt for PH domain binding. Consequently, InsP7 can inhibit Akt phosphorylation. Our results suggest that InsP6K2 is activated in the spinal cord of patients with ALS and may play an important role in ALS by inducing cell death mechanisms via Akt, CK2, and HSP90 pathways.

Hyperadrenocorticism Caused by a Pituitary Ganglioglioma in a Dog.

An 11-year-old castrated male Chihuahua dog was presented with complaints of polyuria, polydipsia, abdominal enlargement, and alopecia. Hyperadrenocorticism was diagnosed on the basis of clinical signs, blood tests, adrenocorticotropin-stimulation test results, and an elevated serum adrenocorticotropin concentration. Contrast-enhanced magnetic resonance imaging showed that the pituitary gland was enlarged, compatible with a pituitary macroadenoma. Pituitary-dependent hyperadrenocorticism was suspected, and transsphenoidal hypophysectomy was thus performed for complete resection of the tumor. After surgery, the serum adrenocorticotropin concentration normalized and the hyperadrenocorticism resolved. Histological and immunocytochemical analyses revealed a benign tumor composed of mature neuronal cells and glial cells, suggestive of a ganglioglioma with immunolabeling for adrenocorticotropin. Careful analysis of the resected tumor revealed no pituitary adenoma tissue. The clinical and histopathologic findings indicated that the ganglioglioma was directly responsible for the hyperadrenocorticism. This is the first case of hyperadrenocorticism caused by a ganglioglioma in a dog.

Selenium and Glutathione-Depleted Rats as a Sensitive Animal Model to Predict Drug-Induced Liver Injury in Humans.

Drug-induced liver injury (DILI) is one of the most serious and frequent drug-related adverse events in humans. Selenium (Se) and glutathione (GSH) have a crucial role for the hepatoprotective effect against reactive metabolites or oxidative damage leading to DILI. The hepatoprotective capacity related to Se and GSH in rodents is considered to be superior compared to the capacity in humans. Therefore, we hypothesize that Se/GSH-depleted rats could be a sensitive animal model to predict DILI in humans. In this study, Se-deficiency is induced by feeding a Se-deficient diet and GSH-deficiency is induced by l-buthionine-S,R-sulfoxinine treatment via drinking water. The usefulness of this animal model is validated using flutamide, which is known to cause DILI in humans but not in intact rats. In the Se/GSH-depleted rats from the present study, decreases in glutathione peroxidase-1 protein expression and GSH levels and an increase in malondialdehyde levels in the liver are observed without any increase in plasma liver function parameters. Five-day repeated dosing of flutamide at 150 mg/kg causes hepatotoxicity in the Se/GSH-depleted rats but not in normal rats. In conclusion, Se/GSH-depleted rats are the most sensitive for detecting flutamide-induced hepatotoxicity in all the reported animal models.

Localization of SOX2-positive stem/progenitor cells in the anterior lobe of the common marmoset (Callithrix jacchus) pituitary.

Studies on mouse and rat pituitaries reported that Sox2-expressing cells play roles as stem/progenitor cells in the adult pituitary gland. The presence of cells with stem cell-like properties in the pituitary adenoma and SOX2-positive cells has been demonstrated in the human pituitary. However, considering the difficulty in fully examining the stem/progenitor cell properties in the human pituitary, in the present study, we analyzed the SOX2-positive cells in the pituitary of the adult common marmoset (Callithrix jacchus), which is used as a non-human primate model. Immunohistochemistry demonstrated that localization pattern of SOX2-positive cells in the common marmoset pituitary was similar to that observed in the rodent pituitary, i.e., in the two types of niches (marginal cell layer and parenchymal-niche) and as scattered single cells in the parenchyma of the anterior lobe. Furthermore, most of the SOX2-positive cells express S100 and were located in the center or interior of LAMININ-positive micro-lobular structures. Collectively, the present study reveals properties of SOX2-positive cells in the common marmoset pituitary and suggests that the common marmoset proves to be a useful tool for analyzing pituitary stem/progenitor cells in a non-human primate model.

Liposome-Encapsulated Hemoglobin Accelerates Skin Wound Healing in Diabetic dB/dB Mice.

Since liposome-encapsulated hemoglobin with high O2 affinity (h-LEH, P50 O2  = 10 mm Hg) has been reported to accelerate skin wound healing in normal mice, it was tested in dB/dB mice with retarded wound healing, as seen in human diabetics. Two full-thickness dorsal wounds 6 mm in diameter encompassed by silicone stents were created in dB/dB mice. Two days later (day 2), the animals were randomly assigned to receive intravenous h-LEH (2 mL/kg, n = 7) or saline (2 mL/kg, n = 7). The same treatment was repeated 4 days after wounding (day 4), and the size of the skin lesions was analyzed by photography, surface perfusion was detected by Laser-Doppler imager, and plasma cytokines and chemokines were determined on days 0, 2, 4, and 7, when all animals were euthanized for morphological studies. The size of the ulcer compared to the skin defect or silicone stent became significantly reduced on days 4 and 7 in mice treated with h-LEH (47 ± 8% of original size), similar to the level in wild-type mice, compared to saline-treated dB/dB mice (68 ± 18%, P < 0.01). Mice treated with h-LEH had significantly attenuated inflammatory cytokines, increased surface perfusion, and increased Ki67 expression on day 7 in accordance with the ulcer size reduction, while there was no significant difference in chemokines, histological granulation, epithelial thickness, and granulocyte infiltration detected by immunohistochemical staining in the ulcer between the treatment groups. The results suggest that h-LEH (2 mL/kg) early after wounding may accelerate skin wound healing in dB/dB mice to levels equivalent to wild-type mice probably via mechanism(s) involving reduced hypoxia, increased surface perfusion, suppressed inflammation, accelerated in situ cell proliferation and protein synthesis.

Aberrant activation of atypical protein kinase C in carbon tetrachloride-induced oxidative stress provokes a disturbance of cell polarity and sealing of bile canalicular lumen.

Polarized hepatocytes contain tight junctions (TJs), which are among the most important junctions for sealing the bile canalicular lumen from the sinusoidal space. Alterations in TJs are implicated in chronic cholestatic liver diseases, such as primary biliary cirrhosis and primary sclerosing cholangitis, which have lipid peroxidation marker elevations or antioxidant vitamin decreases. However, the effect of oxidative stress on hepatocyte polarity or liver morphology is unknown. We found that carbon tetrachloride (CCl4)-induced oxidative stress resulted in disassembly of TJs. Ultrastructural analysis revealed disruption in TJs, Golgi morphology, and expansion of the bile canalicular lumen size in CCl4-treated hepatocytes. The Par complex [Par-3-atypical protein kinase C (aPKC) and Par-6 ternary complex] regulates TJs and lumen formation, and the Par-3-aPKC complex formation was inhibited by CCl4 treatment. Moreover, the antioxidant compound vitamin E prohibited a CCl4-induced disturbance in TJs and Par-3-aPKC complex formation. aPKC phosphorylates Par-3 and down-regulates its own affinity with Par-3. Importantly, aPKC kinase activity and Par-3 phosphorylation were significantly increased in CCl4-treated rat livers. These results indicate that the Par-3-aPKC complex plays a crucial role in the maintenance of hepatocyte polarity and sealing of the bile canalicular lumen. Our findings suggest that bile canalicular lumen expansion might explain the presence of cholestasis in patients with primary biliary cirrhosis and primary sclerosing cholangitis.

A method for estimating spatial resolution of real image in the Fourier domain.

Spatial resolution is a fundamental parameter in structural sciences. In crystallography, the resolution is determined from the detection limit of high-angle diffraction in reciprocal space. In electron microscopy, correlation in the Fourier domain is used for estimating the resolution. In this paper, we report a method for estimating the spatial resolution of real images from a logarithmic intensity plot in the Fourier domain. The logarithmic intensity plots of test images indicated that the full width at half maximum of a Gaussian point spread function can be estimated from the images. The spatial resolution of imaging X-ray microtomography using Fresnel zone-plate optics was also estimated with this method. A cross section of a test object visualized with the imaging microtomography indicated that square-wave patterns up to 120-nm pitch were resolved. The logarithmic intensity plot was calculated from a tomographic cross section of brain tissue. The full width at half maximum of the point spread function estimated from the plot coincided with the resolution determined from the test object. These results indicated that the logarithmic intensity plot in the Fourier domain provides an alternative measure of the spatial resolution without explicitly defining a noise criterion.

Effects of liposome-encapsulated hemoglobin on gastric wound healing in the rat.

Liposome-encapsulated hemoglobin (LEH) may improve microcirculation and oxygen (O2 ) metabolism at a surgical wound to accelerate its healing. Ten mL/kg of LEH with high (h-LEH) or low O2 -affinity (l-LEH), homologous red blood cells (RBC), empty liposome or saline as a control was infused before a 10-mm incision and interrupted suture closure of the gastric wall in a total of 110 rats. Two and 4 days later, the stomach was excised for bursting pressure determination and histological sampling. The dose-response relationship was examined in 70 additional rats receiving progressively reduced doses of h-LEH. Hypoxia-inducible factor-1α (HIF-1α) was stained immunohistochemically in 54 other rats to examine its accumulation at the anastomotic sites. Bursting pressure of the surgical wound was significantly higher 2 days after surgery only in the h-LEH-treated rats (P < 0.05), but not at 4 days after surgery, when other rats showed increased bursting pressure to a nonsignificant level. Histological examination revealed less granulocyte infiltration, better granulation, and more macrophage infiltration in h-LEH-treated rats at 2 days, but no longer at 4 days postsurgery. Dose-response study revealed that 0.4 mL/kg of h-LEH (hemoglobin 24 mg/kg) was effective for elevating bursting pressure at 2 days. h-LEH-treated rats had significantly suppressed HIF-1α accumulation in the wound 6, 24, and 48 h after surgery as compared with control animals treated with homologous RBC or saline. In conclusion, the results suggest that h-LEH, but not l-LEH or homologous transfusion, may accelerate wound healing early after gastric incision and anastomosis in the rat. The mechanism(s) appears to be related to improved O2 supply, aerobic metabolism, and suppressed inflammation in the wound.

Significance of ZO-1, an Intercellular Adhesion Molecule, as a Prognostic Marker in Lung Adenocarcinoma.

In epithelial tissues, intercellular adhesion structures are formed between adjacent cells via intercellular adhesion factors, such as zonula occludens (ZO-1), to maintain the structure and function of tissues and organs, thereby contributing to homeostasis. Epithelial cells are polarized into apical and basal regions by tight junctions (TJs), a type of intercellular adhesion structure, and thus, their intracellular organelles are asymmetrically distributed. Normal epithelial cells maintain their cellular function by controlling cytoskeletal reorganization, motility, and division by maintaining asymmetry in their intracellular organelles. Among the features common to many cancer tissues are abnormalities in cell polarity and intercellular adhesion. Lung adenocarcinoma consists of a mixture of five different histologic types that can be distinguished in the same section: lepidic, papillary, acinar, micropapillary, and solid patterns. Therefore, it is often difficult to accurately assess histological images because the staining differs according to the histological types. In the present study, we evaluated ZO-1 staining based on histological features observed in a single section and examined its relationship to clinicopathological features. In non-tumor areas, ZO-1 was expressed on the plasma membrane and in the cytoplasm of normal alveolar epithelial cells. However, in tumor areas, ZO-1 staining was mainly localized in the cytoplasm and on the plasma membrane only in a few cells. ZO-1-negative cases tended to have poorer prognoses in all histological types, with a poorer prognosis in the solid pattern. These results suggest that ZO-1 expression in solid-pattern lung adenocarcinoma may be a useful prognostic marker.

Carbon tetrachloride-induced hepatic injury through formation of oxidized diacylglycerol and activation of the PKC/NF-κB pathway.

Protein kinase C (PKC) participates in signal transduction, and its overactivation is involved in various types of cell injury. PKC depends on diacylglycerol (DAG) for its activation in vivo We have previously reported that DAG peroxides (DAG-O(O)H) activate PKC in vitro more strongly than unoxidized DAG, suggesting that DAG-O(O)H, if generated in vivo under oxidative stress, would act as an aberrant signal transducer. The present study examined whether DAG-O(O)H are formed in carbon tetrachloride (CCl(4))-induced acute rat liver injury in association with activation of the PKC/nuclear factor (NF)-κB pathway. A single subcutaneous injection of CCl(4) resulted in a marked increase in hepatic DAG-O(O)H content. At the molecular level, immunohistochemistry and subcellular fractionation combined with immunoblotting localized PKCα, ßI, ßII and δ isoforms to cell membranes, while immunoblotting showed phosphorylation of the p65 subunit of NF-κB, and immunoprecipitation using isoform-specific anti-PKC antibodies revealed specific association of PKCα and p65. In addition, expression of tumor necrosis factor α (TNFα) and neutrophil invasion increased in the CCl(4)-treated rats. Furthermore, we demonstrated that Vitamin E, one of the most important natural antioxidants that suppresses peroxidation of membrane lipids, significantly inhibited the CCl(4)-induced increase in hepatic DAG-O(O)H content and TNFα expression as well as phosphorylation of PKCα and p65. These data demonstrate for the first time that DAG-O(O)H are generated in the process of CCl(4)-induced liver injury, resulting in activation of the PKC/NF-κB pathway and TNFα-mediated aggravation of liver injury.

Over-expression of BACH2 is related to ongoing somatic hypermutation of the immunoglobulin heavy chain gene variable region of de novo diffuse large B-cell lymphoma.

The basic region-leucine zipper (bZip) factor BTB, CNC homology 2 (BACH2) is known to have important roles in class switch recombination and somatic hypermutation (SHM) of the immunoglobulin (Ig) gene. In this study, we investigated the relationship between the expression of BACH2 and the status of SHM of the Ig heavy chain gene variable region (IgHV) for SHM in diffuse large B-cell lymphoma (DLBCL). We examined 20 cases of DLBCL, 13 of which were germinal center B-cell (GCB) DLBCL and 7 were non-GCB DLBCL. Seven cases were negative, 6 were positive (cytoplasmic expression) and 7 were strongly positive (both nuclear and cytoplasmic expression) for BACH2. Confirmed mutation (CM) was identified in 8 cases and the CM index (number of confirmed mutations per 10 subclones) was distributed from 0 to 5. A CM index of 7 strongly positive (over-expression) cases with BACH2 were distributed from 0 to 5, and that of 7 negative and 6 positive cases were distributed from 0 to 1. Over-expression of BACH2 was statistically related to CM index (P = 0.008). In conclusion, over-expression of BACH2 is critical for ongoing SHM of IgHV in DLBCL, and our data suggest that BACH2 may play an essential role for SHM of the Ig gene in B-cell lymphoma.

Structural aging of human neurons is opposite of the changes in schizophrenia.

Human mentality develops with age and is altered in psychiatric disorders, though their underlying mechanism is unknown. In this study, we analyzed nanometer-scale three-dimensional structures of brain tissues of the anterior cingulate cortex from eight schizophrenia and eight control cases. The distribution profiles of neurite curvature of the control cases showed a trend depending on their age, resulting in an age-correlated decrease in the standard deviation of neurite curvature (Pearson's r = -0.80, p = 0.018). In contrast to the control cases, the schizophrenia cases deviate upward from this correlation, exhibiting a 60% higher neurite curvature compared with the controls (p = 7.8 × 10-4). The neurite curvature also showed a correlation with a hallucination score (Pearson's r = 0.80, p = 1.8 × 10-4), indicating that neurite structure is relevant to brain function. This report is based on our 3D analysis of human brain tissues over a decade and is unprecedented in terms of the number of cases. We suggest that neurite curvature plays a pivotal role in brain aging and can be used as a hallmark to exploit a novel treatment of schizophrenia.

Enhanced expression of sulfatide, a sulfated glycolipid, in well-differentiated endometrial adenocarcinoma.

OBJECTIVES: It is well known that a poorly differentiated endometrial adenocarcinoma shows more rapid progression and a worse response to therapy than a well-differentiated endometrial adenocarcinoma. Qualitative and quantitative changes of cell surface glycolipids occur during neoplastic transformation. Sulfatide is one of the sulfated glycolipids in the cell membrane that may have an important role in various functions such as cell adhesion. To examine the molecular background of the morphological and biological features of well-differentiated and poorly differentiated cancer, we measured the levels of lipids, especially glycolipids, in tumor tissues from patients with endometrial carcinoma. MATERIALS AND METHODS: We determined the composition of lipids and glycolipids in tumor tissues, investigated glycosyltransferase messenger RNA expression by the reverse transcription-polymerase chain reaction, and assessed the localization of galactosylceramide sulfotransferase (an enzyme involved in sulfatide biosynthesis) by immunohistochemical staining. RESULTS: No significant differences were observed between well-differentiated and poorly differentiated cancer with respect to the levels of cholesterol ester, cholesterol, phospholipids, cholesterol sulfate, ceramides, neutral glycolipids of the globo series, and GM3 ganglioside. However, the amount of sulfatides in well-differentiated tumors was significantly greater than that in poorly differentiated tumors, which was confirmed by thin-layer chromatography and immunostaining with a monoclonal antisulfatide antibody. Altered expression of sulfatide was found to be secondary to a change of galactosylceramide sulfotransferase messenger RNA expression. Immunohistochemical staining revealed that galactosylceramide sulfotransferase expression was characteristically observed in glandular areas but not in solid areas. CONCLUSION: These findings suggest that sulfatide contributes to the well-differentiated phenotype of endometrial adenocarcinoma and that it is being expressed in normal uterine endometrium at sites of gland formation during the luteal phase, as we have previously reported.

Liposome-encapsulated hemoglobin accelerates skin wound healing in mice.

Effects of liposome-encapsulated hemoglobin with high O2 affinity (m-LEH, P50O2 = 17 mm Hg) on skin wound healing in mice were examined. Two full-thickness dorsal wounds 6 mm in diameter encompassed by silicone stents were created in Balb/c mice. Two days later (day 2), the animals randomly received intravenous m-LEH (2 mL/kg, n = 12), homologous blood transfusion (red blood cell [RBC], n = 11), or saline (n = 12). The same treatment was repeated 4 days after wounding (day 4), and the sizes of the skin defects and ulcers were monitored on days 0, 2, 4, and 7, when all animals were euthanized for morphological studies. While the size of the skin defect in relation to the stent ring remained the same in all groups, the size of the ulcer compared with the skin defect (or silicone stent) became significantly reduced on days 4 and 7 in mice treated with m-LEH (46 ± 10% of pretreatment size, P < 0.01) compared with mice treated with RBC transfusion (73 ± 6%) or saline (76 ± 7%). m-LEH treatment significantly accelerated granulation, increased epithelial thickness, suppressed early granulocyte infiltration, and increased Ki67 expression in accordance with the ulcer size reduction, while there was no difference in surface blood flow or CD31 expression among the groups. The results suggest that m-LEH (2 mL/kg) may accelerate skin wound healing in Balb/c mice via mechanism(s) involving reduced inflammation and increased metabolism, but not by improved hemodynamics or endothelial regeneration.

Possible Glutathione Peroxidase 4-Independent Reduction of Phosphatidylcholine Hydroperoxide: Its Relevance to Ferroptosis.

Ferroptosis is mainly caused by iron-mediated peroxidation of phospholipids and has recently attracted attention due to its involvement in various diseases. At the center of it is supposedly the inability of glutathione peroxidase 4 (GPX4) to reduce excess peroxidized phospholipids (e.g., phosphatidylcholine hydroperoxide (PCOOH)) that trigger ferroptosis. However, the involvement of enzymes other than GPX4 in ferroptosis is scarcely known. To elucidate this matter, we evaluated the uptake of PCOOH in a GPX4 knockout (KO) human hepatoma cell line HepG2 generated using CRISPR-Cas9. After confirming that GPX4 expression in the KO cells was below the detection limit, we cultured both wild-type (WT) and GPX4 KO HepG2 cells in a medium containing 50 µM PCOOH for 1-8 hours. By analyzing the level of PCOOH and its reduction product (phosphatidylcholine hydroxide, PCOH) in cells using liquid chromatography-tandem mass spectrometry, we detected the cellular uptake of PCOOH. On top of this, we detected a large amount of PCOH not only in WT HepG2 but also in GPX4 KO HepG2, thus indicating the notable involvement of enzymes other than GPX4 (e.g., other GPX family, glutathione S-transferase, thioredoxin, or peroxiredoxin) in reducing PCOOH. Further corroboration of these findings hopefully leads to the development of novel methods to prevent ferroptosis-related diseases by targeting enzymes other than GPX4.

Cellular Uptake of Pyrroloquinoline Quinone in Its Intact and Derivatized Forms from the Cell Culture Medium of 3T3-L1.

Following a growing interest in the physiological effects of pyrroloquinoline quinone (PQQ), more cell culture experiments have begun to elucidate its mechanism of action. However, to our knowledge, no reports have used instrumental analysis, such as liquid chromatography-tandem mass spectrometry (LC-MS/MS), to study cellular uptake of PQQ. In addition, despite the propensity of PQQ to react with amino acids and other compounds, only a handful of cell culture experiments have been conducted on PQQ derivatives. In the present study, we prepared PQQ derivatives by reacting PQQ with various amino acids and used them as reference standards for optimizing the LC-MS/MS analysis conditions to detect PQQ and its derivatives. Using this method, we evaluated the uptake of PQQ into mouse 3T3-L1 cells and found that most PQQ added to the medium was taken up by the cells in its unchanged form, while some PQQ reacted with amino acids in the medium and was taken up by the cells as PQQ derivatives. These results suggest that PQQ derivatives may contribute to the physiological effects of PQQ. To further elucidate the function of PQQ, it is necessary for future studies to clarify the activity of PQQ derivatives and to evaluate the types of PQQ present in food, animal, and cell samples in more detail.

Protective Effect of Ebselen on Ischemia-reperfusion Injury in Epigastric Skin Flaps in Rats.

The purpose of this study was to determine the role of oxidized diacylglycerol (DAG) and the molecular mechanism underlying ischemia-reperfusion (I/R) injury in rat skin flaps. The protective effect of ebselen on the viability of rat skin flaps with I/R injury was investigated. Flaps were designed and raised in the left inguinal region. Then, a microvascular clamp was applied to the vascular pedicle and reperfused after 6 hr. After 7 days of I/R (I/R group), the skin flap survival area ratio was significantly reduced compared to the normal skin. The administration of ebselen significantly improved the ratio compared to the I/R group. The flap survival area ratio of the I/R + ebselen group was significantly improved compared to the I/R + vehicle group. In the I/R + ebselen group, the oxidized DAG content and intensity of phosphorylated PKCα and PKCδ were significantly lower compared to the I/R + vehicle group. Furthermore, the inflammatory response was suppressed in the I/R + ebselen group compared to the I/R + vehicle group. These results indicate that ebselen is useful as a preventive and therapeutic agent for skin flap necrosis caused by I/R, because of reduction and elimination of oxidized DAG.

Administration of mucuna beans (Mucuna pruriences (L.) DC. var. utilis) improves cognition and neuropathology of 3 × Tg-AD mice.

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the accumulation of extracellular amyloid-beta peptides (Aß) resulting in senile plaques and intracellular hyperphosphorylated tau protein resulting in neurofibrillary tangles (NFTs). Mucuna beans (Mucuna pruriences (L.) DC. var. utilis) are unique plants containing 3-9% L-3,4-dihydroxyphenylalanine (L-DOPA). Here we investigated the effect of the administration of Mucuna beans on AD prevention by feeding triple-transgenic mice (3 × Tg-AD mice) with a diet containing Mucuna beans for 13 months. The levels of Aß oligomers and detergent-insoluble phosphorylated tau decreased in the brain of mice fed with Mucuna beans (Mucuna group) compared to those of the Control group. Aß accumulation and phosphorylated tau accumulation in the brain in the Mucuna group were also reduced. In addition, administration of Mucuna beans improved cognitive function. These results suggest that administration of Mucuna beans may have a preventive effect on AD development in 3 × Tg-AD mice.