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Parenchymal hepatocytes are responsible for most of the metabolic functions of the liver, but exhibit distinct functional properties depending on their localization within the hepatic lobule. Cytochrome P450 oxidases represent a family of drug-metabolizing enzymes, which are expressed predominantly in hepatocytes localized in the centrilobular area (zone 3). The present study describes a unique transgenic mouse strain that distinguishes zone 3 hepatocytes from periportal zone 1 hepatocytes by the intensity of EGFP fluorescence. Both zone 1 and zone 3 hepatocytes isolated from these mice showed the same zone-specific gene expression patterns as in liver tissue in vivo. Experiments using primary cultures of hepatocytes indicated that a combination of low oxygen concentration and activation of Wnt/β-catenin signaling maintained the expression of zone 3-specific P450 drug-metabolizing enzymes, which was characterized by their susceptibility to acetaminophen-induced mitochondrial dysfunction. These zone-specific hepatocytes provide a useful system in the research area of liver pathophysiology and drug development.

In the present study, we studied the effects of metformin and its interactions with the actions of bone morphogenetic proteins (BMPs) on ovarian steroidogenesis. It was revealed that metformin treatment enhanced progesterone production by human granulosa KGN cells and rat primary granulosa cells induced by forskolin and FSH, respectively. In human granulosa cells, it was found that metformin treatment suppressed phosphorylation of Smad1/5/9 activated by BMP-15 compared with that induced by other BMP ligands. Moreover, metformin treatment increased the expression of inhibitory Smad6, but not of that Smad7, in human granulosa cells, while metformin had no significant impact on the expression levels of BMP type-I and -II receptors. Thus, the mechanism by which metformin suppresses BMP-15-induced Smad1/5/9 phosphorylation is likely, at least in part, to be upregulation of inhibitory Smad6 expression in granulosa cells. The results suggest the existence of functional interaction between metformin and BMP signaling, in which metformin enhances progesterone production by downregulating endogenous BMP-15 activity in granulosa cells.

The involvement of orexins in reproductive function has been gradually uncovered. However, the functional role of orexins in ovarian steroidogenesis remains unclear. In the present study, we investigated the effects of orexin A on ovarian steroidogenesis by using rat primary granulosa cells that express both OX1 and OX2 receptors for orexins. Treatment with orexin A enhanced progesterone, but not estradiol, biosynthesis induced by FSH, whereas it did not affect basal levels of progesterone or estradiol. In accordance with the effects on steroidogenesis, orexin A increased the mRNA levels of progesterogenic enzymes, including StAR, P450scc and 3βHSD, but not P450arom, and cellular cAMP synthesis induced by FSH. Under the condition of blockage of endogenous BMP actions by noggin or BMP-signaling inhibitors, orexin A failed to increase levels of progesterone synthesis induced by FSH treatment, suggesting that endogenous BMP activity in granulosa cells might be involved in the enhancement of progesterone synthesis by orexin A. Treatment with orexin A impaired Smad1/5/9 activation as well as Id-1 mRNA expression stimulated by BMP-6 and BMP-7, the latter of which was reversed by treatment with an OX1 antagonist. It was also found that orexin A suppressed the mRNA expression of both type-I and -II receptors for BMPs and increased that of inhibitory Smad6 and Smad7 in granulosa cells. On the other hand, treatments with BMP-6 and -7 suppressed the expression of OX1 and OX2. Collectively, the results indicated that orexin A enhances FSH-induced progesterone production, at least in part, by downregulating BMP signaling in granulosa cells. Thus, a new role of orexin A in facilitating progesterone synthesis and functional interaction between the orexin and BMP systems in granulosa cells were revealed.

Summary Cell senescence plays a key role in age-associated organ dysfunction, but the in vivo pathogenesis is largely unclear. Here, we generated a p16-CreERT2-tdTomato mouse model to analyze the in vivo characteristics of p16high cells at a single-cell level. We found tdTomato-positive p16high cells detectable in all organs, which were enriched with age. We also found that these cells failed to proliferate and had half-lives ranging from 2.6 to 4.2 months, depending on the tissue examined. Single-cell transcriptomics in the liver and kidneys revealed that p16high cells were present in various cell types, though most dominant in hepatic endothelium and in renal proximal and distal tubule epithelia, and that these cells exhibited heterogeneous senescence-associated phenotypes. Further, elimination of p16high cells ameliorated nonalcoholic steatohepatitis-related hepatic lipidosis and immune cell infiltration. Our new mouse model and single-cell analysis provide a powerful resource to enable the discovery of previously unidentified senescence functions in vivo.

Aldosterone (Aldo) is involved in various cardiovascular diseases such as hypertension and heart failure. Aldo levels are known to be increased in patients with polycystic ovary syndrome, and expression of the mineralocorticoid receptor (MR) has also been detected in the ovary. However, the effect of Aldo on reproductive function has yet to be elucidated. Here, we examined the effects of Aldo on follicular steroidogenesis using primary culture of rat granulosa cells by focusing on the ovarian bone morphogenetic protein (BMP) system acting as a luteinizing inhibitor. We found that Aldo treatment increased FSH-induced progesterone production in a concentration-responsive manner. Consistent with the effects on steroidogenesis, Aldo increased mRNA levels of progesterogenic factor and enzymes including StAR and P450scc, whereas Aldo failed to change FSH-induced estradiol and cAMP synthesis or P450arom expression by granulosa cells. Progesterone production and StAR expression induced by FSH and Aldo were reversed by co-treatment with spironolactone, suggesting the involvement of geonomic MR action. Aldo treatment attenuated Smad1/5/9 phosphorylation and Id1 transcription induced by BMP-6. Furthermore, Aldo enhanced the expression of inhibitory Smad6 in the presence of BMP-6. In addition, BMP-6 downregulated MR expression, while Aldo modulated the mRNA levels of endogenous BMP-6 and BMP type-II receptors, indicating the existence of a feedback loop between the BMP system and MR in granulosa cells. Collectively, the results indicated that Aldo predominantly enhances FSH-induced progesterone production by inhibiting BMP-Smad signaling, suggesting a novel role of Aldo in ovarian steroidogenesis and a functional link between MR and BMP pathways in granulosa cells.

Patient initials or identifier number K.M. ### Relevant clinical history and physical exam A 57 year-old man was admitted to our hospital with effort angina pectoris. His coronary risk factors were diabetes mellitus, dyslipidemia and smoking. ### Relevant test results prior to

Although branch duct intraductal papillary mucinous neoplasms of the pancreas (BD-IPMN) are being diagnosed with increasing frequency, the incidence of concomitant pancreatic carcinoma (PC) is not well known. We investigated the incidence and clinical features of synchronous and metachronous PC in patients with BD-IPMN.We studied 168 BD-IPMN patients diagnosed by various imaging modalities, including endoscopic retrograde pancreatography, between 1990 and 2008. We reviewed the medical records and clinical features in both patients developing and not developing PC. The diagnosis of PC was histologically verified in all patients.PC was observed in 9 (5.4%) of 168 patients. Five were synchronously detected at the time of BD-IPMN diagnosis, whereas four were metachronously identified during the follow-up period. All PCs occurred in regions separate from the BD-IPMN lesion. All PCs represented histologically invasive ductal adenocarcinomas, whereas the BD-IPMN lesion was diagnosed as adenoma. Patients developing PC were significantly older than patients not developing PC (p = 0.017). The diameters of the BD-IPMN lesions and main pancreatic ducts were significantly smaller in patients developing PC than patients not developing PC (p = 0.013 and p0.001, respectively).It was not infrequent for PC to occur in the pancreas with BD-IPMN. Particular attention should therefore be paid to the development of PC, even in low-risk BD-IPMN, as well as to changes in BD-IPMN.

The Notch ligand Delta-like 1 (Dll1) is critical for the generation of marginal zone (MZ) B cells in the spleen. However, the precise mechanism underlying the differentiation of MZB cells is unclear. To determine whether hematopoietic cells or non-hematopoietic cells provides the Dll1-mediated signals to primitive hematopoietic cells, we transplanted lineage(-)c-kit(+)Sca-1(+) (KSL) bone marrow cells derived from wild-type (Dll1(+/+)) GFP-transgenic mice into lethally irradiated Dll1 conditional knockout (cKO) mice. After transplantation, we examined the kinetics of hematopoietic reconstitution and found that although the frequency of stem/progenitor subsets and of more mature lymphoid, myeloid, and erythroid lineages were normal, the donor-derived hematopoietic cells failed to differentiate into MZB cells. We further demonstrated that while the splenic stromal cells of wild-type mice expressed Dll1 molecule, the splenic stromal cells of recipient Dll1 cKO mice deleted the expression of Dll1. These results suggesting that the expression of Dll1 in splenic non-hematopoietic stromal cells, but not hematopoietic cells, is essential for the development of MZB cells.

In this study, we investigated the involvement of Akt and members of the mitogen-activated protein kinase (MAPK) superfamily, including ERK, JNK, and p38 MAPK, in gemcitabine-induced cytotoxicity in human pancreatic cancer cells. We found that gemcitabine induces apoptosis in PK-1 and PCI-43 human pancreatic cancer cell lines. Gemcitabine specifically activated p38 MAPK in a dose- and time-dependent manner. A selective p38 MAPK inhibitor, SB203580, significantly inhibited gemcitabine-induced apoptosis in both cell lines, suggesting that phosphorylation of p38 MAPK may play a key role in gemcitabine-induced apoptosis in pancreatic cancer cells. A selective JNK inhibitor, SP600125, failed to inhibit gemcitabine-induced apoptosis in both cell lines. MKK3/6, an upstream activator of p38 MAPK, was phosphorylated by gemcitabine, indicating that the MKK3/6-p38 MAPK signaling pathway is indeed involved in gemcitabine-induced apoptosis. Furthermore, gemcitabine-induced cleavage of the caspase substrate poly(ADP-ribose) polymerase was inhibited by pretreatment with SB203580, suggesting that activation of p38 MAPK by gemcitabine induces apoptosis through caspase signaling. These results together suggest that gemcitabine-induced apoptosis in human pancreatic cancer cells is mediated by the MKK3/6-p38 MAPK-caspase signaling pathway. Further, these results lead us to suggest that p38 MAPK should be investigated as a novel molecular target for human pancreatic cancer therapies.

Patient initials or identifier number M.M. ### Relevant clinical history and physical exam A 56 year-old man was admitted to our hospital for effort angina. His coronary risk factors were smoking, hypertension and dyslipidemia. His ECG was normal, and there was no hypokinesis in echo

Patient initials or identifier number Y.S. ### Relevant clinical history and physical exam An 82 year-old man, who was underwent coronary artery bypass grafting (the left internal mammary artery [LIMA] to the left anterior descending coronary artery [LAD]) 4 years before, was admitted to our

Capillary flow properties of a concentrated N,N -dimethylacetamide (DMAc) solution of segmented poly(urethane-urea) (PUR) prepared from 4,4′-diphenylmethanediisocyanate (MDI), polytetramethlyleneoxide (PTMO), and ethylenediamine (EDA) (polymer concentration, 30.0 wt%; hereafter referred to as ‘PUR-solution’) as well as the dissolved state was investigated as functions of solution temperature ( T sol ) and shear rate (γ): non-Newtonian index n in the equation η s = κ γ −n decreased remarkably with increase in T sol in the range more than about 90°C. The logarithmic shear viscosity (log η s ) of the PUR-solution as a function of 1/ T sol gave two linear regions below and above 90°C, suggesting that the activation energy of flow of the PUR-solution estimated in the T sol range from 90 to 130°C ( ΔE h ) was higher than that ( ΔE 1 ) in the T sol range of 40–80°C. The ΔE h − ΔE 1 value was proved to become somewhat smaller as γ increased, indicating that the shear stress might control the dissolved state of PUR to some extent but this contribution is much smaller than that by T sol . 1 H and 13 C n.m.r. analysis on the dissolved states of PUR in the solution revealed existence of some hydrogen bondings mainly between NH and CO of urea groups, and suggested that the sudden decrease in η s of the PUR-solution at more than about 90°C is associated with a steep decrease in the average strength of the hydrogen bondings.

Natural History of Branch-Duct Type Intraductal Papillary-Mucinous Tumor (IPMT) of the Pancreas: ‘Prospective’ Long-Term Follow-Up By MR Cholangiopancreatography (MRCP) and EUS Madoka Minoguchi, Yusuke Mizukami, Kazumasa Nakamura, Junpei Sasajima, Tsutomu Izawa, Yasuhiro Nakano, Nobuyuki Yanagawa, Satoshi Tanno, Tsuneshi Fujii, Toshikatsu Okumura, Takeshi Obara, Yutaka Kohgo Background/Aims: In patients with branch-duct type IPMT, we previously demonstrated that MRCP can identify features suggestive of malignancy, such as cystic lesions larger than 30 mm and/or presence of a mural nodule (Gastrointest Endosc supple.53; AB175,2001). In this study, we examined the role of MRCP and EUS in the follow-up of these patients and focused on changes in the size of the cystic branch and emergence of mural nodules. Methods: After providing informed consent, 37 patients with branch-duct type IPMT diagnosed by ERCP were enrolled in this prospective study. Five patients with cystic lesions larger than 30 mm and 2 patients suspected to have mural nodule(s) at an initial examination who refused surgical intervention were also included in this study. Patients were surveyed by MRCP, EUS, and CT at least once a year. Results: An increase in the size of cystic lesions by either MRCP or EUS was demonstrated in 7 patients (18.9%), which was consistent with findings on CT. However, there were discrepancies in other cases; the size of cystic lesions was overestimated by EUS in 2 cases when compared to CT and MRCP, and underestimated in 6 cases because of inadequate visualization of entire lesion. Emergence of a new mural nodule was identified in 2 cases by both MRCP and EUS. The 2 cases of mural nodules that were present at initial examination did not increase in size on serial MRCP and EUS examinations during a 103.0 C/ 2.0 month follow-up. Surgical resection was performed because of an increase in size of the cystic lesion in 2 cases and emergence of a new mural nodule in 2 cases after 11 to 25 months of follow-up (mean 16.8 months). These were histologically diagnosed as adenoma in 3 patients and non-invasive adenocarcinoma in 1. The remaining 33 patients who did not have surgery were monitored for 36 to 138 months (mean 64.3 C/ 39.4 months). In 2 of 5 patients (40%) with a cystic lesion larger than 30 mm and 4 of 32 (12.5%) patients with lesions !30 mm, the size increased more than 20%. MRCP identified new or multiple IPMT lesions in 13 patients (21 lesions), but EUS missed 4 (19%) of them. No metastatic lesions associated with IPMTwere identified, but two patients died of other causes. Conclusions: Branch-duct type IPMTs without mural nodules usually grow slowly and can be followed by serial imaging studies, even in cases with a large cystic lesion. MRCP is a useful modality to monitor the changes in size of the cystic lesions, as well as to identify multicentric lesions. We conclude that MRCP may be equivalent to EUS for surveillance in patients with branch-duct type IPMT.

Natural History of Branch Duct Type Intraductal Papillary-Mucinous Neoplasms of the Pancreas: A Role of Endoscopic Ultrasonography in Follow-Up Satoshi Tanno, Yasuhiro Nakano, Tsutomu Izawa, Yusuke Mizukami, Toshikatsu Okumura, Yutaka Kohgo Background/Aim: The aim of this study was to elucidate the natural history and serial changes of the pancreatic duct system in the patients with branch duct type intraductal papillary-mucinous neoplasms of the pancreas (IPMNs). Methods: Sixtyfive patients (forty-seven men and eighteen women) with branch duct type IPMNs were selected for follow-up and examined by endoscopic ultrasonography (EUS) two or more times (mean, 3 times). All patients who had apparent mural nodules were excluded from follow-up study. The observation periods ranged from 14 to 131 months (mean, 61 months), and all patients underwent initial endoscopic retrograde pancreatography (ERP) and computed tomography (CT). Serial changes of the pancreatic duct system, such as maximum cystic diameter and the presence of mural nodule, were studied. Results: Five (8%) of 65 patients exhibited obvious progression of cystic dilatation. New apparent mural nodules were detected in 5 cases (8%) during the observation periods. These obvious changes were observed after a 19 to 112 months follow-up period (mean, 74 months). Of five patients with mural nodules, four cases underwent surgery after follow-up study, and the pathologic diagnosis was adenoma in two and non-invasive carcinoma in two. Mural nodules were accurately diagnosed in 5 cases (100%) by EUS, whereas in one (20%) by CT and in 0 (0%) by MRCP. Five patients with cystic size enlargement were followed up without surgical resection. None of the remaining 55 patients (84%) showed increases in cystic diameter. Conclusions: Most of branch duct type IPMNs without apparent mural nodules remained unchanged. Mural nodule is an important factor affecting the follow-up. EUS is the most sensitive method to detect mural nodules in follow-up. Abstracts