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Supplementary Figure 1 from Pten Deficiency in Melanocytes Results in Resistance to Hair Graying and Susceptibility to Carcinogen-Induced Melanomagenesis

Supplementary Methods from Pten Deficiency in Melanocytes Results in Resistance to Hair Graying and Susceptibility to Carcinogen-Induced Melanomagenesis

Supplementary Figure 2 from Pten Deficiency in Melanocytes Results in Resistance to Hair Graying and Susceptibility to Carcinogen-Induced Melanomagenesis

Supplementary Figure 3 from Pten Deficiency in Melanocytes Results in Resistance to Hair Graying and Susceptibility to Carcinogen-Induced Melanomagenesis

Analyses using electrophoresis with accelerator mass spectrometry revealed that in vivo bioactivated radiolabeled troglitazone and flutamide, both known to be hepatotoxic in humans, bound nonspecifically to a variety of microsomal and cytosolic proteins in livers from chimeric mice with humanized liver. Unlike those of radiolabeled diazepam (rarely hepatotoxic) and previously reported 5-n-butyl-pyrazolo[1,5-a]pyrimidine (limited hepatotoxicity), some troglitazone and flutamide binding proteins were located in the top right area in a zone analysis, representing high covalent binding contents and high target protein concentrations. Among a variety of liver microsomal proteins bound, the binding target proteins of troglitazone and flutamide with the highest covalent binding contents (in terms of pmol equivalent per μg target protein) were 17β-hydroxysteroid dehydrogenase and 3β-hydroxysteroid dehydrogenase, respectively. Troglitazone and flutamide were activated to reactive metabolites and apparently bound to different target proteins in livers from chimeric mice with humanized liver. The highest covalent binding contents for troglitazone were higher than that for flutamide under the present conditions. These results indicate that the drug metabolism mediated by humanized livers (leading to binding in vivo) in combination with a zone analysis of covalent binding contents/target protein concentration data could be a good tool for evaluating the relationship between the nonspecific protein binding behavior of medicines and potential hepatotoxicity in humans. Thus, testing whether protein binding data of new medicines are unbalanced with respect to deviation from an inverse relationship or the presence of data points in the high covalent binding/high protein concentration zone might be an important concept in evaluating hepatotoxic potential.

The purpose of this study was to evaluate various types of meniscus scaffolds that mimic the meniscus structure, and to establish a novel cell-free meniscus scaffold with polyglycolic acid or poly-l-lactic acid. Four types of scaffolds were implanted into Japanese white rabbits: poly-l-lactic acid sponge poly-l-lactic acid, PGA-coated PLLA sponge, PGA lamination, and film-coated PGA lamination. Samples were harvested at 8 and 12 weeks after implantation, and a compression stress test was performed. The meniscus size and Ishida scores were evaluated for regenerated tissue. Immunohistochemistry was analyzed by anti-type I, II and X collagen antibodies to investigate the structure of the regenerated tissue, and by anti-iNOS antibody to investigate the inflammatory tissue of the meniscus. The cell nuclei of lymphocytes and foreign body multinucleated giant cells were counted in hematoxylin and eosin staining. Modified Mankin scores for cartilage degeneration were used for assessment after Safranin-O/Fast Green staining. The biomechanical test showed that l- and film-coated PGA lamination exhibited greater strength than s- and PGA-coated PLLA sponge. At 12 weeks, the size of meniscus and the Ishida score in implanted film-coated PGA lamination were improved significantly compared with the defect groups. The type II collagen staining intensity in the PGA lamination lamination is significantly higher than the defect at eight weeks. The staining intensity of iNOS and number of lymphocytes significantly increased in sponge poly-l-lactic acid at eight weeks, and increased in p-PLLA at 12 weeks. Foreign body multinucleated giant cells in implantation groups appeared, especially at eight weeks. The Mankin score for film-coated PGA lamination was significantly lower than for the defect at 12 weeks. Novel meniscal scaffolds especially PGA should possess not only biological but also biomechanical functions. In conclusions, film-coated PGA lamination was the beneficial property for meniscus scaffold from the points of better biomechanical function, good regeneration, and less inflammation with chondroprotective effects.

Chimeric mice with humanized liver (PXB mice) have been generated by transplantation of urokinase-type plasminogen activator/severe combined immunodeficiency mice with human hepatocytes. The purpose of the present study was to clarify the protein expression levels of metabolizing enzymes and transporters in humanized liver of PXB mice transplanted with hepatocytes from three different donors, and to compare their protein expressions with those of human livers to validate this human liver model. The protein expression levels of metabolizing enzymes and transporters were quantified in microsomal fraction and plasma membrane fraction, respectively, by means of liquid chromatography-tandem mass spectrometry. Protein expression levels of 12 human P450 enzymes, two human UDP-glucuronosyltransferases, eight human ATP binding cassette (ABC) transporters, and eight human solute carrier transporters were determined. The variances of protein expression levels among samples from mice humanized with hepatocytes from all donors were significantly greater than those from samples obtained from mice derived from each individual donor. Compared with the protein expression levels in human livers, all of the quantified metabolizing enzymes and transporters were within a range of 4-fold difference, except for CYP2A6, CYP4A11, bile salt export pump (BSEP), and multidrug resistance protein 3 (MDR3), which showed 4- to 5-fold differences between PXB mouse and human livers. The present study indicates that humanized liver of PXB mice is a useful model of human liver from the viewpoint of protein expression of metabolizing enzymes and transporters, but the results are influenced by the characteristics of the human hepatocyte donor.

Mass spectrometry (MS)-based multiplexed multiple reaction monitoring quantification of proteins has recently evolved as a versatile tool for accurate, absolute quantification of proteins. The purpose of this study was to examine the validity of the present method with regard to standard bioanalytical criteria for drug transporters, cytochrome P450 (CYP) enzymes and uridine 5′-diphospho-glucuronosyltransferases (UGTs). Membrane preparations from human liver tissue were used for target protein quantification. As a result, the determination coefficients (r2) of all targets were greater than 0.986. In the absence of matrix, inaccuracy values (expressed as % deviation) were −8.1% to 20.3%, whereas imprecision values (expressed as % coefficient of variation) were within 15.9%. In the presence of matrix, which consisted of digested plasma membrane fraction for transporters and digested microsomal membrane fraction for CYP enzymes and UGTs, respectively, the inaccuracy was −15.3%–8.1%, and the imprecision were within 18.9%. Sufficient sample stability of membrane fraction was shown for three freeze–thaw cycles, 32 days at −20°C, and in processed samples for 7 days at 10°C. In conclusion, this study demonstrated, for the first time, that the MS-based assay with nano-liquid chromatography provides adequate reliability and robustness for the quantification of selected drug transporters, P450 enzymes and UGTs. © 2011 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:4037–4043, 2011

Pancreatic beta cell failure is thought to underlie the progression from glucose intolerance to overt diabetes, and ER stress is implicated in such beta cell dysfunction. We have now shown that the transcription factor CCAAT/enhancer-binding protein beta (C/EBPbeta) accumulated in the islets of diabetic animal models as a result of ER stress before the onset of hyperglycemia. Transgenic overexpression of C/EBPbeta specifically in beta cells of mice reduced beta cell mass and lowered plasma insulin levels, resulting in the development of diabetes. Conversely, genetic ablation of C/EBPbeta in the beta cells of mouse models of diabetes, including Akita mice, which harbor a heterozygous mutation in Ins2 (Ins2WT/C96Y), and leptin receptor-deficient (Lepr-/-) mice, resulted in an increase in beta cell mass and ameliorated hyperglycemia. The accumulation of C/EBPbeta in pancreatic beta cells reduced the abundance of the molecular chaperone glucose-regulated protein of 78 kDa (GRP78) as a result of suppression of the transactivation activity of the transcription factor ATF6alpha, thereby increasing the vulnerability of these cells to excess ER stress. Our results thus indicate that the accumulation of C/EBPbeta in pancreatic beta cells contributes to beta cell failure in mice by enhancing susceptibility to ER stress.

Aldehyde oxidase-mediated oxidation of N(1)-methylnicotinamide to N(1)-methyl-2-pyridine-5-carboxamide (2-PY) and N(1)-methyl-4-pyridone-5-carboxamide (4-PY) in chimeric mice constructed by transplanting human hepatocytes into urokinase-type plasminogen activator-transgenic severe combined immunodeficient mice was examined in vivo and in vitro. The activity in liver cytosol of chimeric mice with a high replacement index was approximately 4-fold higher than that in control mice. Furthermore, the oxidation products in control mice were 2-PY and 4-PY, whereas, in chimeric mice, the major product was 2-PY, as in humans. The aldehyde oxidase in chimeric mouse liver was confirmed to be of human type by immunoblotting analysis. The ratio of pyridones (2-PY/4-PY) excreted in the urine of chimeric mice was closer to that of humans than to that of control mice. Thus, the aldehyde oxidase in chimeric mice has human-type functional characteristics.

The evaluation of spatially and temporally distributed records of translational shallow landslides in heterogeneous watersheds provides insights needed to understand disastrous processes. Recurrent slope instability events occurred between 1953 and 1998 in two watersheds of Mt. Aso, western Japan. This paper investigates (1) the spatio-temporal characteristics of translational shallow landslides (dimensions, numbers, densities, and area subjected to failure) observed at a particular location, (2) DEM based landform characteristics (elevation, slope angles, curvatures and their control on landslide distribution), and (3) rainfall characteristics. The evaluation of the landslide history, consequences and characteristics of spatially and temporally distributed landslides are based on the series of inventory maps for years 1954, 1977, 1990 and 1998. Geologically, the watersheds consist of pyroxene olivine andesite basalt lava, pyroclastics deposits, gravel, sand and clay deposits originated from Takadake, Nekodake, and Washigamine volcanoes. During 45 years (1953–1998), a total of 619 and 976 numbers of shallow landslides have been recognized in the Sakurakigawa and Furuegawa watersheds, respectively. Repeated sliding denuded a total surface area of 0.372 km 2 in the Sakurakigawa watershed representing 35% of the watershed area. Similarly slides denuded a total of 0.534 km 2 in the Furuegawa watershed representing 12% of the watershed area. For example, storm events of June 1953 and July 1990 with rainfall intensities of 49 and 61 mm h − 1 , respectively triggered numerous landslides. About 25% and 47% of Sakurakigawa and Furuegawa watersheds, respectively still bears the potential to produce landslides. Landslides were commonly observed where thick unconsolidated tephra layers and pyroclastics rocks overlain by thin tephra bed existed, and for a slope inclination range of 30–35°.

Concentrations of 1,5-anhydroglucitol (1,5-AG), which is a major circulating polyol, decrease in patients with diabetes mellitus. In both insulinoma-derived RINr and MIN6 cells, 1,5-AG stimulated insulin release within the range of 0.03-0.61 mM in a dose-dependent manner. Insulin release was maximally stimulated by 1,5-AG to levels that reached 25% and 100% greater than that of control (1,5-AG-free group) in RINr and MIN6 cells, respectively. A physiological concentration of 1,5-AG stimulated insulin release after a 5-min incubation and this action was maintained for 60 min. In addition, at approximately 1/200 the concentration of glucose, 1,5-AG had additive action with 20 mM glucose. The action of 1,5-AG on insulin secretion with other types of saccharides and polyol was similarly additive. Mannnoheptulose and diazoxide suppressed the stimulative action of 1,5-AG on insulin release. The secretagogue action of 1,5-AG seemed to be independent on an increase in the intracellular content of cAMP and ATP. These results suggest that 1,5-AG can stimulate insulin secretion through a mechanism that completely differs from that of glucose.

Stem cell factor can induce mast cell proliferation and melanocyte activation. Vasoactive intestinal peptide has been suggested to play a part in inflammatory dermatoses, such as atopic dermatitis. The aim of this study was to investigate the possible role of stem cell factor in atopic dermatitis by analyzing epidermal stem cell factor production induced by vasoactive intestinal peptide and cytokines. Full-length type stem cell factor transcript was detected in normal human epidermal keratinocytes, and a human epidermal keratinocyte cell line DJM-1, as well as normal human dermal fibroblasts, using reverse transcription-polymerase chain reaction. Spliced-type stem cell factor transcript was detected in both DJM-1 cells and normal human epidermal keratinocytes. Western blot analysis with stem cell factor antibody revealed a protein of the known molecular size of membrane-bound stem cell factor in the lysates of all three cell types. Stem cell factor immunoreactivity was found in the cytoplasm and the membrane of both DJM-1 cells and normal human epidermal keratinocytes using confocal laser scanning microscope. We examined the effects of vasoactive intestinal peptide and cytokines on stem cell factor production of DJM-1 cells using enzyme-linked immunosorbent assays. Stem cell factor contents significantly increased in culture supernatants of DJM-1 cells treated with 1000 nm vasoactive intestinal peptide and/or cytokines, including interleukins 4 and 13, tumor necrosis factor-alpha, and interferon-gamma. Overall, these results suggest that several inflammatory cytokines (T helper 1 and 2) and vasoactive intestinal peptide from mast cells and nerve endings are capable of inducing stem cell factor production from epidermal keratinocytes in atopic dermatitis.

The accumulation of mast cells around tumors is a well-recognized phenomenon in a number of malignancies, including basal cell carcinoma, melanoma, and breast cancer. However, little information exists regarding mast cells within tumor nests. To clarify the role of mast cells infiltrating in skin cancers, we examined the morphological features of mast cells within tumors of five cases of angiosarcoma, including two patients with Stewart-Treves syndrome, by electron microscopy. In light microscopy, mast cells were observed within tumor nests at various densities and exhibited weak staining intensity with toluidine blue. By electron microscopy, most of the tumor-infiltrating mast cells exhibited anaphylactic or piecemeal degranulation, indicating that the mast cells had been activated in situ. Some mast cells were noted in close apposition to tumor cells, suggesting the existence of direct cell-to-cell interactions. Tumor cells adjacent to mast cells showed no degenerative changes. In conclusion, these results suggest that careful histologic examination in combination with electron microscopy should enable us to identify more mast cells within cancer lesions with greater sensitivity than in a number of prior reports. Furthermore, the close proximity of mast cells and surrounding tumor cells suggests some biologically significant role of mast cells in the development of angiosarcoma, including tumor growth as well as host immunity and stromal reaction.

Background: Spontaneous regression of solitary mastocytoma is a well-described phenomenon, but its mechanism is unknown. Methods: Serial-section immunohistochemical analyses were performed on biopsies of a mastocytoma from a Japanese child during the proliferation stage (PS, 7 months of age) and the regression stage (RS, 5 years old). Results: Mast cell (MC) density in RS was markedly decreased (406 cells/mm2) compared to that in PS (3554 cells/mm2). MCs in RS were larger than those in PS. With proliferative cell nuclear antigen (PCNA) staining, 1.7% MCs were positive in PS, whereas no positive MCs were seen in RS. TUNEL-labeling index (LI) in RS (2.8%) increased 1.5-fold in PS (1.9%). With stem cell factor (SCF) staining, 57% of lesional MCs in RS revealed strong cytoplasmic immunoreactivity, whereas only 9% of MCs were positive in PS. Epidermal SCF reactivity was found as intracellular and intercellular patterns in both PS and RS. Conclusions: Loss of MC proliferating activity, an increase in apoptotic MCs, and increased expression of SCF in remaining MCs in RS may play a role in the involution of mastocytomas.

Some Merkel cell carcinomas (MCC) have been reported to regress spontaneously. To clarify the mechanisms of spontaneous regression (SR) of MCC, we analyzed the TUNEL index, the labeling index of proliferating cell nuclear antigen (PCNA), the labeling index of bcl-2 protein, and the expression of p53 of the tumor cells. We also evaluated the number of infiltrating lymphocytes surrounding the tumor in the tissue specimens. Among seven patients with MCC (SR: n =4; non-regression (NR): n =3), the TUNEL index in the SR group was significantly higher than that in NR group (5.2 and 2.0%, respectively). In addition, the number of lymphocytes around the tumor nests was also significantly increased in the SR group compared to NR group (1576 and 663 cells/mm 2 , respectively). Most of the infiltrating lymphocytes were UCHL-1 positive T-cells. There were no significant differences of the PCNA labeling index, the bcl-2 protein labeling index, and the expression p53 between SR and NR group. These results indicate that apoptosis and local T-cell mediated immune response might be involved in spontaneous regression of MCC.

The serum concentration of 1,5-anhydroglucitol (1,5-AG), a polyol which originates mainly in the diet, is used in Japan as a new marker for glycemia. To evaluate the potential interference of 1, 5-AG measurements by traditional Chinese medicines (Kampo), we examined the 1,5-AG content in 32 types of concentrated dosage forms of Kampo using high performance liquid chromatography (HPLC). The 32 types of Kampo were the most frequently used in Japan, two of which, Ninjin-yoei-to (7030 microg/g dry weight) and Kami-kihi-to (6700 microg/g dry weight), contained large amounts of 1,5-AG. Six others contained small amounts of 1,5-AG. Both Ninjin-yoei-to and Kami-kihi-to contain the same ingredient, Polygalae radix, which is a crude form of polygalitol (1,5-AG). To confirm the effects of these Kampo medicines on the serum levels of 1,5-AG, we administered Ninjin-yoei-to (7.5 g/day) for 8 weeks to 18 patients with Type 2 diabetes mellitus (Type 2 DM). The serum level of 1,5-AG increased from 9.8+/-8.9 to 28.1+/-17.5 microg/ml by week 8. Hemoglobin A1c (HbA1c) had not changed by week 8. Thus, an abnormal serum 1,5-AG level may be present in patients taking Kampo which contains Polygalae radix.

The uptake of 1,5-anhydro-D-glucitol (1,5-AG) occurs by passive mechanisms in cells or tissues that have passive glucose transporters. It is known that serum 1,5-AG concentrations are reduced in patients with diabetes mellitus. To elucidate the metabolism of this substance and its physiological role in pancreatic beta-cells, we assayed 1,5-AG transport in the insulinoma-derived cell lines, RINr and MIN6. Both cell lines showed an insulin-insensitive, concentration-dependent uptake of 1,5-AG with a saturation time of approximately 120 min, and most of the 1,5-AG in the cytoplasm was in the free form. A biphasic saturation curve was obtained using a wide range of 1,5-AG concentrations, suggesting that accumulation was mediated by a high affinity and a low affinity transporter. The high affinity transporter had a K(m) of 10.4 in RINr cells and 13.0 mM in MIN6 cells, and the low affinity transporter had a K(m)100 times, being much higher than the physiological concentrations of 1,5-AG. These results indicate that the 1,5-AG carrier system in insulinoma cells is distinct from that in either the somatic cells or renal tubular cells. These findings also suggest that a unique 1,5-AG transport system is present in pancreatic beta-cells.

OBJECTIVE This prospective study was designed to elucidate the relationship between the serum level of 1,5-anhydroglucitol (1,5AG) and the urinary excretion of N-acetylglucosaminidase (NAG) and albumin in patients who were in the early stages of diabetes. RESEARCH DESIGN AND METHODS A total of 1,062 male nondiabetic subjects with impaired glucose tolerance were monitored for blood glucose level once every 2–3 months, and the values were evaluated. Of these 1,062 subjects, 112 showed a worsening of glycemia during the observation period to the level seen in diabetes. We began to monitor the glycemia and parameters of renal damage in the 112 patients from the onset of diabetes. RESULTS The urinary excretion of NAG and albumin were elevated even at the onset of diabetes. The abnormal excretion of NAG and albumin was associated with a change in serum 1,5AG and was quickly reversible when the serum 1,5AG improved. In the 3 years after the onset of diabetes, we obtained at least 18 measurements of one parameter for each patient and calculated the mean. Urinary NAG was found to be significantly correlated with the fasting plasma level of glucose (FPG; r = 0.512, P < 0.0001), the level of HbA1 (r = 0.351, P = 0.001), and the level of 1,5AG (r = −0.790, P < 0.0001). The urinary excretion of albumin was weakly but significantly correlated with levels of FPG (r = 0.383, P < 0.0001) and HbA1 (r = 0.337, P = 0.0001), but it was more strongly correlated with 1,5AG (r = −0.632, P < 0.0001). The level of 1,5AG was significantly correlated with FPG (r = −0.681, P < 0.0001) and HbA1 (r = −0.609, P < 0.0001). CONCLUSIONS When the renal damage is not severe, the serum level of 1,5AG appeared to be an indicator of the reversible renal damage caused by hyperglycemia, as well as of the severity of the glycemia itself.

We report a case of 59-year-old male with malignant schwannoma of the back associated with von Recklinghausen's disease.In 1995, the first surgical resection was performed. The tumor was diagnosed as cutaneus neurofibroma. But in 1997, the tumor rapidly recurred and the second resection was performed. The histopathological findings were consistent with malignant schwannoma. Six months after the second resection, he has no evidence of recurrence or metastasis.

The purpose of the present study was to determine the absolute protein expression levels of multiple drug-metabolizing enzymes and transporters in 17 human liver biopsies, and to compare them with the mRNA expression levels and functional activities to evaluate the suitability of the three measures as parameters of hepatic metabolism. Absolute protein expression levels of 13 cytochrome P450 (P450) enzymes, NADPH-P450 reductase (P450R) and 6 UDP-glucuronosyltransferase (UGT) enzymes in microsomal fraction, and 22 transporters in plasma membrane fraction were determined using liquid chromatography/tandem mass spectrometry. CYP2C9, CYP2E1, CYP3A4, CYP2A6, UGT1A6, UGT2B7, UGT2B15, and P450R were abundantly expressed (more than 50 pmol/mg protein) in human liver microsomes. The protein expression levels of CYP3A4, CYP2B6, and CYP2C8 were each highly correlated with the corresponding enzyme activity and mRNA expression levels, whereas for other P450s, the protein expression levels were better correlated with the enzyme activities than the mRNA expression levels were. Among transporters, the protein expression level of organic anion-transporting polypeptide 1B1 was relatively highly correlated with the mRNA expression level. However, other transporters showed almost no correlation. These findings indicate that protein expression levels determined by the present simultaneous quantification method are a useful parameter to assess differences of hepatic function between individuals.

The levels of metabolizing enzymes and transporters expressed in hepatocytes are decisive factors for hepatobiliary disposition of most drugs. Induction via nuclear receptor activation can significantly alter those levels, with the coregulation of multiple enzymes and transporters occurring to different extents. Here, we report the use of a targeted liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) method for concurrent quantification of multiple cytochrome P450 (P450), UDP-glucuronosyltransferase (UGT), and transporter proteins in cultured primary human hepatocytes. The effects of culture format (i.e., sandwich culture versus conventional culture) and of dexamethasone (DEX) media concentrations on mRNA, protein, and activity levels were determined for three donors, and protein expression was compared with that in liver. In general, P450 and UGT expression was lower in hepatocyte cultures than that in liver, and CYP2C9 was found to be the most abundant P450 isoform expressed in cultured hepatocytes. The sandwich culture format and 0.1 μM DEX in media retained the protein expression in the hepatocytes closest to the levels found in liver. However, higher in vitro expression was observed for drug transporters, especially for multidrug resistance protein 1 and breast cancer resistance protein. Direct protein quantification was applied successfully to study in vitro induction in sandwich cultured primary hepatocytes in a 24-well format using the prototypical inducers rifampicin, omeprazole, and phenobarbital. We conclude that targeted absolute LC-MS/MS quantification of drug-metabolizing enzymes and transporters can broaden the scope and significantly increase the impact of in vitro drug metabolism studies, such as induction, as an important supplement or future alternative to mRNA and activity data.

The formation of morphine-3-glucuronide (M-3-G, pharmacologically inactive) and morphine-6-glucuronide (M-6-G, active metabolite) by liver microsomes from humans and rodents, including chimeric mice carrying human liver, was evaluated in the presence of fatty acyl-CoAs. Medium- to long-chain fatty acyl-CoAs, including oleoyl-CoAs, at a physiologic level (around 15 microM) markedly enhanced M-3-G formation catalyzed by rat liver microsomes. A separate experiment indicated that 15 microM oleoyl-CoA enhanced (14)C-UDP-glucuronic acid (UDPGA) uptake by microsomes. The activation by acyl-CoAs disappeared or was greatly reduced by either pre-treating microsomes with detergent or freezing/thawing the rat liver before preparation. Many of the microsomes prepared from frozen human livers (N=14) resisted oleoyl-CoA-mediated activation of UDP-glucuronosyltransferase (UGT) activity, including M-6-G formation, which is highly specific to humans. In sharp contrast, the activity of M-6-G and M-3-G formation in freshly-prepared hepatic microsomes from chimeric mice with humanized liver was potently activated by oleoyl-CoA. Thus, acyl-CoAs activate morphine glucuronidation mediated by human as well as rat UGTs. This activation is assumed to be due to the acyl-CoA-facilitated transportation of UDPGA, and microsomes need to maintain the intact conditions required for the activation. The function of UGT appears to be dynamically changed depending on the cellular acyl-CoA level in many species.

We analyzed the effect of low birth weight on pancreatic beta cell mass. We used pregnant C57BL6J mice, and we reduced their food supply by 30% during the late gestational period and examined the changes in the metabolism and pancreatic beta cell mass. Pancreatic beta cell mass at birth was greatly decreased in the mice of the food restriction group (RG) as compared to the mice of the control group (CG). The body weight of RG mice exhibited a "catch-up growth" pattern and became equivalent to that of CG mice 7 days after birth, and thereafter exceeded that of CG mice; however, the pancreatic beta cell mass in RG mice remained lower than that in CG mice at the age of 4 weeks. A high-fat diet significantly increased the pancreatic beta cell mass in RG mice as compared to that in CG mice at 12 weeks of age. However, RG mice fed on high-fat diets tended to exhibit a decrease in the pancreatic beta cell mass at approximately 20 weeks of age. The plasma insulin concentrations also tended to be decreased in RG mice after 24 weeks of age as compared to those of CG mice. These results thus indicate that the growth of pancreatic beta cells is insufficient in RG mice, and pancreatic beta cell failure can easily develop as a consequence of insulin resistance.

Drug development of a potential analgesic agent 5-n-butyl-7-(3,4,5-trimethoxybenzoylamino)pyrazolo[1,5-a]pyrimidine was withdrawn because of its limited hepatotoxic effects in humans that could not be predicted from regulatory animal or in vitro studies. In vivo formation of glutathione conjugates and covalent binding of a model compound 5-n-butyl-pyrazolo[1,5-a]pyrimidine were investigated in the present study after intravenous administration to chimeric mice with a human or rat liver because of an interesting capability of human cytochrome P450 1A2 in forming a covalently bound metabolite in vitro. Rapid distribution and elimination of radiolabeled 5-n-butyl-pyrazolo[1,5-a]pyrimidine in plasma or liver fractions were seen in chimeric mice after intravenous administration. However, similar covalent binding in liver was detected over 0.17-24 h after intravenous administration. Radio-LC analyses revealed that the chimeric mice with humanized liver preferentially gave the 3-hydroxylated metabolite and its glutathione conjugate in the plasma and liver. On the contrary, chimeric mice with a rat liver had some rat-specific metabolites in vivo. Analyses by electrophoresis with accelerator mass spectrometry of in vivo radiolabeled liver proteins in chimeric mice revealed that bioactivated 5-n-butyl-pyrazolo[1,5-a]pyrimidine bound nonspecifically to a variety of microsomal proteins including human P450 1A2 as well as cytosolic proteins in the livers from chimeric mice with humanized liver. These results suggest that the hepatotoxic model compound 5-n-butyl-pyrazolo[1,5-a]pyrimidine was activated by human liver microsomal P450 1A2 to reactive intermediate(s) in vivo in humanized chimeric mice and could relatively nonspecifically bind to biomolecules such as P450 1A2 and other proteins.

Chimeric mice, constructed by transplanting human hepatocytes, are useful for predicting the human metabolism of drug candidates. In this study, we investigated whether these mice show similar metabolic profile to humans by examining the hydroxylation of S-warfarin reported to be mainly metabolized to S-7-hydroxywarfarin (7-OH-warfarin), catalyzed by CYP2C9, in humans. When S-(3)H-warfarin was administered to chimeric mice and control (uPA(+/+)/SCID(wt/wt)) mice, the blood concentration-time curve was higher in chimeric than control mice. Plasma protein binding of S-(3)H-warfarin of chimeric and control mice amounted to 98.1 and 92.1%, respectively. When S-(3)H-warfarin was administered to these mice, radioactivity was mainly recovered in urine (81.7% in chimeric mice and 65.9% in control mice). After S-(3)H-warfarin was administered to these mice, the radioactivity was recovered in the bile of chimeric and control mice at 5.1 and 17.9%, respectively. The main urinary metabolite in chimeric mice was 7-OH-warfarin. the main urinary metabolite in control mice was S-4'-hydroxywarfarin. These results show that mass balance, metabolic disposition of S-(3)H-warfarin in chimeric mice with humanized liver were similar to reported human data.

Keratinocyte-specific Pten-null mice revealed distinct phenotypes, including epidermal and sebaceous gland hyperplasia. To determine the candidate genes that contribute to their phenotypes, we analyzed a comprehensive gene expression of Pten-null keratinocytes using microarray technology. Consequently, it was demonstrated that the most induced gene was adipocyte-specific fatty acid-binding protein (FABP4). Collectively, it is conceivable that the FABP4 pathway mediates the sebaceous gland hyperplasia in keratinocyte-specific Pten-null mice.

Chimeric mice having humanized livers were constructed by transplantation of human hepatocytes. In this study, we investigated whether these mice have a capacity for drug metabolism similar to that of humans by examining hydroxylation of S-warfarin, which is predominantly metabolized to S-7-hydroxywarfarin, catalyzed by CYP2C9, in humans but not mice. The 7-hydroxylating activity of chimeric mouse liver microsomes toward S-warfarin was approximately 10-fold higher than that of control (urokinase-type plasminogen activator-transgenic severe combined immunodeficient) mice. The 7-hydroxylase activity of chimeric mouse liver microsomes was markedly inhibited by sulfaphenazole, as was that of human liver microsomes, whereas the activity of control mice was unaffected. The CYP2C isoform in chimeric mouse liver was also confirmed to be the human isoform, CYP2C9, by immunoblot analysis. In the present in vivo study, the level of S-7-hydroxywarfarin in plasma of chimeric mice was approximately 7-fold higher than that in control mice, in agreement with the in vitro data. Thus, the CYP2C isoform in chimeric mice functions in vivo and in vitro as a human isoform, CYP2C9. These results suggest that chimeric mice with humanized liver could be useful for predicting drug metabolism in humans, at least regarding CYP2C9-dependent metabolism.

Phosphate and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor gene inactivated in numerous sporadic cancers, including melanomas. To analyze Pten functions in melanocytes, we used the Cre-loxP system to delete Pten specifically in murine pigment-producing cells and generated DctCrePtenflox/flox mice. Half of DctCrePtenflox/flox mice died shortly after birth with enlargements of the cerebral cortex and hippocampus. Melanocytes were increased in the dermis of perinatal DctCrePtenflox/flox mice. When the mutants were subjected to repeated depilations, melanocyte stem cells in the bulge of the hair follicle resisted exhaustion and the mice were protected against hair graying. Although spontaneous melanomas did not form in DctCrePtenflox/flox mice, large nevi and melanomas developed after carcinogen exposure. DctCrePtenflox/flox melanocytes were increased in size and exhibited heightened activation of Akt and extracellular signal–regulated kinases, increased expression of Bcl-2, and decreased expression of p27Kip1. Our results show that Pten is important for the maintenance of melanocyte stem cells and the suppression of melanomagenesis. [Cancer Res 2008;68(14):5760–8]

Recent studies have demonstrated the importance of insulin or insulin-like growth factor 1 (IGF-1) for regulation of pancreatic beta-cell mass. Given the role of tuberous sclerosis complex 2 (TSC2) as an upstream molecule of mTOR (mammalian target of rapamycin), we examined the effect of TSC2 deficiency on beta-cell function. Here, we show that mice deficient in TSC2, specifically in pancreatic beta cells (betaTSC2(-/-) mice), manifest increased IGF-1-dependent phosphorylation of p70 S6 kinase and 4E-BP1 in islets as well as an initial increased islet mass attributable in large part to increases in the sizes of individual beta cells. These mice also exhibit hypoglycemia and hyperinsulinemia at young ages (4 to 28 weeks). After 40 weeks of age, however, the betaTSC2(-/-) mice develop progressive hyperglycemia and hypoinsulinemia accompanied by a reduction in islet mass due predominantly to a decrease in the number of beta cells. These results thus indicate that TSC2 regulates pancreatic beta-cell mass in a biphasic manner.

PTEN is a tumor suppressor gene mutated in many human cancers. We used the Cre-loxP system to generate a keratinocyte-specific null mutation of Pten in mice (k5Pten(flox/flox) mice). k5Pten(flox/flox) mice exhibit wrinkled skin because of epidermal hyperplasia and hyperkeratosis and ruffled, shaggy, and curly hair. Histological examination revealed that skin morphogenesis is accelerated in k5Pten(flox/flox) mice. Within 3 weeks of birth, 90% of k5Pten(flox/flox) mice die of malnutrition possibly caused by hyperkeratosis of the esophagus. All k5Pten(flox/flox) mice develop spontaneous tumors within 8.5 months of birth, and chemical treatment accelerates the onset of tumors. k5Pten(flox/flox) keratinocytes are hyperproliferative and resistant to apoptosis and show increased activation of the Pten downstream signaling mediators Akt/protein kinase B (PKB) and extracellular signal-regulated kinase. Pten is thus an important regulator of normal development and oncogenesis in the skin.

Spontaneous regression of solitary mastocytoma is a well-described phenomenon, but its mechanism is unknown.Serial-section immunohistochemical analyses were performed on biopsies of a mastocytoma from a Japanese child during the proliferation stage (PS, 7 months of age) and the regression stage (RS, 5 years old).Mast cell (MC) density in RS was markedly decreased (406 cells/mm2) compared to that in PS (3554 cells/mm2). MCs in RS were larger than those in PS. With proliferative cell nuclear antigen (PCNA) staining, 1.7% MCs were positive in PS, whereas no positive MCs were seen in RS. TUNEL-labeling index (LI) in RS (2.8%) increased 1.5-fold in PS (1.9%). With stem cell factor (SCF) staining, 57% of lesional MCs in RS revealed strong cytoplasmic immunoreactivity, whereas only 9% of MCs were positive in PS. Epidermal SCF reactivity was found as intracellular and intercellular patterns in both PS and RS.Loss of MC proliferating activity, an increase in apoptotic MCs, and increased expression of SCF in remaining MCs in RS may play a role in the involution of mastocytomas.

Using both cross-sectional and longitudinal methods, we investigated the relationship between post-load serum glucose concentration in a 75 g oral glucose tolerance test (OGTT) and overall glycaemic state in subjects with impaired glucose tolerance (IGT). Glycaemic state was assessed by measuring glycated haemoglobin (HbA1c) and the serum concentration of 1,5-anhydroglucitol (1,5-AG). In the cross-sectional study, the concentration of 1,5-AG, while remaining within a normal range, was reduced to a degree proportional to the post-load glycaemic level. Although the correlation between HbA1c and post-load plasma glucose was relatively weak (r = 0.281, P < 0.001), a significant inverse correlation (r =-0.824, P < 0.0001) was found between 1,5-AG and mean post-load plasma glucose concentration in 211 subjects with IGT. Fasting plasma glucose (r =-0.539, P < 0.0001) and 2 h plasma glucose (r =-0.621, P < 0.0001) were correlated with 1,5-AG less strongly than was post-load glycaemia. Both 1,5-AG and HbA1c were correlated weakly but significantly with the fasting insulin concentration. In the longitudinal study we measured 1,5-AG and mean post-load plasma glucose with an OGTT once yearly for 10 years in 15 subjects with IGT. Strong inverse correlations were seen between 1,5-AG and mean post-load plasma glucose in each subject (range of r values among subjects of -0.584 to -0.978). These findings suggest a close relationship between post-load plasma glucose concentration measured by OGTT and overall glycaemic state in subjects with IGT.

We investigated the effects of the continuous infusion of various steroids in rats on renal tubular reabsorption of glucose in vivo to elucidate the pathogenesis of steroid-induced glucosuria. Urinary glucose excretion increased 60 min after administration of dexamethasone (2.38 mM). By 120 min, urinary excretion of glucose was three times higher in the dexamethasone group than in the control group (24.1 +/- 4.6 versus 72.4 +/- 16.7 micromol); the plasma level of glucose did not increase. Dexamethasone had no effect on the resorption of 1,5-anhydro-D-glucitol, which is a glucose-resembling polyol that is actively absorbed by the renal tubules as glucose. Neither estradiol nor progesterone increased urinary excretion of glucose. These findings suggest that continuous administration of a high-dose glucocorticoid selectively influences the glucose reabsorption system in the kidney.