Establishment of novel meniscal scaffold structures using polyglycolic and poly-l-lactic acids.

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.

Validation of uPA/SCID mouse with humanized liver as a human liver model: protein quantification of transporters, cytochromes P450, and UDP-glucuronosyltransferases by LC-MS/MS.

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.

Simultaneous absolute protein quantification of transporters, cytochromes P450, and UDP-glucuronosyltransferases as a novel approach for the characterization of individual human liver: comparison with mRNA levels and activities.

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.

Absolute quantification and differential expression of drug transporters, cytochrome P450 enzymes, and UDP-glucuronosyltransferases in cultured primary human hepatocytes.

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.

Reliability and robustness of simultaneous absolute quantification of drug transporters, cytochrome P450 enzymes, and Udp-glucuronosyltransferases in human liver tissue by multiplexed MRM/selected reaction monitoring mode tandem mass spectrometry with nano-liquid chromatography.

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.

Activation of morphine glucuronidation by fatty acyl-CoAs and its plasticity: a comparative study in humans and rodents including chimeric mice carrying human liver.

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.

Effect of intrauterine undernutrition during late gestation on pancreatic beta cell mass.

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).

Ablation of C/EBPbeta alleviates ER stress and pancreatic beta cell failure through the GRP78 chaperone in mice.

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.

Approach for in vivo protein binding of 5-n-butyl-pyrazolo[1,5-a]pyrimidine bioactivated in chimeric mice with humanized liver by two-dimensional electrophoresis with accelerator mass spectrometry.

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.

Prediction of human disposition toward S-3H-warfarin using chimeric mice with humanized liver.

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.

CYP2C9-catalyzed metabolism of S-warfarin to 7-hydroxywarfarin in vivo and in vitro in chimeric mice with humanized liver.

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.

Aldehyde oxidase-catalyzed metabolism of N1-methylnicotinamide in vivo and in vitro in chimeric mice with humanized liver.

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.

Biphasic response of pancreatic beta-cell mass to ablation of tuberous sclerosis complex 2 in mice.

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.

1,5-Anhydroglucitol stimulates insulin release in insulinoma cell lines.

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.

Keratinocyte-specific Pten deficiency results in epidermal hyperplasia, accelerated hair follicle morphogenesis and tumor formation.

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.

Vasoactive intestinal peptide and cytokines enhance stem cell factor production from epidermal keratinocytes DJM-1.

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.

Alteration of mast cell proliferation/apoptosis and expression of stem cell factor in the regression of mastocytoma--report of a case and a serial immunohistochemical study.

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).

Activation of mast cells within a tumor of angiosarcoma: ultrastructural study of five cases.

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.