Increased ghrelin signaling prolongs survival in mouse models of human aging through activation of sirtuin1.

Caloric restriction (CR) is known to retard aging and delay functional decline as well as the onset of diseases in most organisms. Ghrelin is secreted from the stomach in response to CR and regulates energy metabolism. We hypothesized that in CR ghrelin has a role in protecting aging-related diseases. We examined the physiological mechanisms underlying the ghrelin system during the aging process in three mouse strains with different genetic and biochemical backgrounds as animal models of accelerated or normal human aging. The elevated plasma ghrelin concentration was observed in both klotho-deficient and senescence-accelerated mouse prone/8 (SAMP8) mice. Ghrelin treatment failed to stimulate appetite and prolong survival in klotho-deficient mice, suggesting the existence of ghrelin resistance in the process of aging. However, ghrelin antagonist hastened death and ghrelin signaling potentiators rikkunshito and atractylodin ameliorated several age-related diseases with decreased microglial activation in the brain and prolonged survival in klotho-deficient, SAMP8 and aged ICR mice. In vitro experiments, the elevated sirtuin1 (SIRT1) activity and protein expression through the cAMP-CREB pathway was observed after ghrelin and ghrelin potentiator treatment in ghrelin receptor 1a-expressing cells and human umbilical vein endothelial cells. Furthermore, rikkunshito increased hypothalamic SIRT1 activity and SIRT1 protein expression of the heart in the all three mouse models of aging. Pericarditis, myocardial calcification and atrophy of myocardial and muscle fiber were improved by treatment with rikkunshito. Ghrelin signaling may represent one of the mechanisms activated by CR, and potentiating ghrelin signaling may be useful to extend health and lifespan.

Expression level of P2X7 receptor is a determinant of ATP-induced death of mouse cultured neurons.

Activation of P2X7 receptor (P2X7R), a purinergic receptor, expressed by neurons is well-known to induce their death, but whether or not their sensitivity to ATP depends on its expression levels remains unclear. Here, we examined the effect of the expression level of P2X7Rs on cell viability using pure neuron cultures, co-cultures with astrocytes derived from SJL- and ddY-strain mice, and mouse P2X7R-expressing HEK293T cell systems. Treatment of pure neuron cultures with 5mM ATP for 2h, followed by 3-h incubation in fresh medium, resulted in death of both types of neurons, and their death was prevented by administration of P2X7R-specific antagonists. In both SJL- and ddY-neurons, ATP-induced neuronal death was inhibited by a mitochondrial permeability transition pore inhibitor cyclosporine A, mitochondrial dysfunction being involved in their death. The ATP-induced neuronal death was greater for SJL-neurons than for ddY-ones, this being correlated with the expression level of P2X7R in them, and the same results were obtained for the HEK293T cell systems. Co-culture of neurons with astrocytes increased the ATP-induced neuronal death compared to the case of pure neuron cultures. Overall, we reveal that neuronal vulnerability to ATP depends on the expression level of P2X7R, and co-existence of astrocytes exacerbates ATP-induced neuronal death.

On-tip photodetection: a simple and universal platform for optoelectronic screening.

A novel platform for transient photodetector component screening has been developed whereby an optical fiber tip serves as the counter electrode when placed in a variety of dielectric media, connected to a photoresponsive working electrode. The soft processing conditions allow for ubiquitous photodetection for organic and biological systems.

Phase I/II study of intraperitoneal docetaxel plus S-1 for the gastric cancer patients with peritoneal carcinomatosis.

PURPOSE: We designed a phase I/II trial of intraperitoneal (IP) docetaxel plus S-1 to determine the maximum tolerated dose (MTD) and recommended dose (RD) and to evaluate its efficacy and safety in gastric cancer patients with peritoneal carcinomatosis (PC). METHODS: Patients with PC confirmed by laparoscopy or laparotomy received IP docetaxel on days 1 and 15 and S-1 (80 mg/m(2)) on days 1-14 every 4 weeks. RESULTS: In the phase I part (n = 12), each cohort received escalating doses of docetaxel (35-50 mg/m(2)); the MTD was determined to be 50 mg/m(2) and the RD was determined to be 45 mg/m(2). Dose-limiting toxicities included grade 3 febrile neutropenia and grade 3 diarrhea. In the phase II part (n = 27), the median number of courses was 4 (range 2-11). The 1-year overall survival (OS) rate was 70 % (95 % confidence interval 53-87 %). The overall response rate was 22 % and peritoneal cytology turned negative in 18 of 22 (81 %) patients. The most frequent grade 3/4 toxicities included anorexia (19 %), neutropenia (7 %), and leukopenia (7 %). CONCLUSION: IP docetaxel plus S-1 is active and safety in gastric cancer patients with PC.

FK330, a novel inducible nitric oxide synthase inhibitor, prevents ischemia and reperfusion injury in rat liver transplantation.

Nitric oxide (NO), produced via inducible NO synthase (iNOS), is implicated in the pathophysiology of liver ischemia/reperfusion injury (IRI). We examined the effects of a novel iNOS inhibitor, FK330 (FR260330), in well-defined rat liver IRI models. In a model of liver cold ischemia followed by ex vivo reperfusion, treatment with FK330 improved portal venous flow, increased bile production and decreased hepatocellular damage. FK330 prevented IRI in rat model of 40-h cold ischemia followed by syngeneic orthotopic liver transplantation (OLT), as evidenced by: (1) increased OLT survival (from 20% to 80%); (2) decreased hepatocellular damage (serum glutamic oxaloacetic transaminase/glutamic pyruvic transaminase levels); (3) improved histological features of IRI; (4) reduced intrahepatic leukocyte infiltration, as evidenced by decreased expression of P-selectin/intracellular adhesion molecule 1, ED-1/CD3 cells and neutrophils; (5) depressed lymphocyte activation, as evidenced by expression of pro-inflammatory cytokine (TNF-alpha, IL-1beta, IL-6) and chemokine (IP-10, MCP-1, MIP-2) programs; (6) prevented hepatic apoptosis and down-regulated Bax/Bcl-2 ratio. Thus, by modulating leukocyte trafficking and cell activation patterns, treatment of rats with FK330, a specific iNOS inhibitor, prevented liver IRI. These results provide the rationale for novel therapeutic approaches to maximize organ donor pool through the safer use of liver grafts despite prolonged periods of cold ischemia.

[Mediastinal emphysema after a punch on the back; report of a case].

A 19-year-old man was punched on the back, and anterior chest pain appeared about 3 hours after injury. The patient was consulted a physician complaining of anterior chest pain. On chest X-ray, mediastinal emphysema was suspected, and transferred to our hospital. Chest computed tomography (CT) revealed mediastinal emphysema. On esophageal radiography and bronchofiberscopy, no abnormal findings were detected. Conservative therapy was conducted, and symptoms had gradually improved. On the 8th hospital day, mediastinal emphysema was improved on chest CT. The patient was discharged on the 10th hospital day. The most frequent cause of mediastinal emphysema after trauma is traffic or downfall accident, and no report on this condition after the punch on the back was found.

A synergistic effect between PG490-88 and tacrolimus prolongs renal allograft survival in monkeys.

This study was undertaken to determine if PG490-88 and tacrolimus (Tac) act synergistically to prevent renal allograft rejection in monkeys and to explore possible mechanisms of synergy between these agents. MHC-mismatched renal allografts were transplanted into cynomolgus monkeys after bilateral nephrectomy. Recipients were divided into the following groups: (i) no treatment; (ii) PG490-88 (0.03 mg/kg); (iii) Tac (1 mg/kg); (iv) PG490-88 (0.01 mg/kg) + Tac (1 mg/kg) and (v) PG490-88 (0.03 mg/kg) + Tac (1 mg/kg). Through synergy PG490-88 and Tac inhibited anti-CD3/PMA-induced T-cell proliferation and IFN-gamma expression in vitro. Tac monotherapy only marginally prolonged survival (27 +/- 3.2 days), while the combination of PG490-88 and Tac significantly prolonged graft survival to a median of 99 days (PG490-88 at 0.03 mg) and 38.5 days (PG490-88 at 0.01 mg/kg). Prolonged survival correlated with inhibited IgM production as well as reduced T-cell infiltration, IL-2 protein expression and NF-AT/NF-kappaB activity. We conclude that PG490-88 and a subtherapeutic dose of Tac significantly prolong renal allograft survival in monkeys through the synergistic inhibition of T-cell activation and a decrease in IFN-gamma production and NF-AT/NF-kappaB activity.

Protective effects of PG490-88 on chronic allograft rejection by changing intragraft gene expression profiles.

Our previous study showed that PG490-88 effectively ameliorated both functional and histological changes of chronic rejection in the rat. In this experiment, we investigated the intragraft gene expression profiles of PG490-88 under successful prevention of chronic rejection in rat kidney allografts. Kidneys of F344 rats were transplanted into bilaterally nephrectomized LEW recipients. Recipients with a brief course of low-dose FK506 (1 mg/kg per day for 10 days) were dosed with PG490-88 0.5 mg/kg per day, which was predetermined and defined as the effective dose of preventing chronic allograft rejection in this model, for 90 days after grafting. Kidney grafts were harvested on day 90 after transplantation and subjected to gene expression analysis by real-time RT-PCR. Overall, the expression levels of all genes tested were upregulated in the brief course of low-dose FK506 control. PG490-88 treatment exhibited significant inhibition of intragraft m RNA levels of iNOS, IL-6, and perforin and marginal downregulation of IL-2, IFNgamma, IRF-1, TNFalpha, and TGFbeta. There was no change in IL-10, granzyme B, and PDGFalpha, when compared to the brief course of low-dose FK506 control. These results suggested that downregulation of multiple intragraft gene expression by mainly suppression of iNOS, IL-6, and perforin might be responsible for successful prevention of chronic kidney allograft nephropathy by PG490-88 in rats.

PG490-88, a new immunosuppressant, effectively prevents acute and chronic rejection in rat renal allografts.

PG490-88 is a semisynthetic derivative of the novel compound PG490 (triptolide) purified from a Chinese herb. It has been shown to prolong acute allograft survival in multiple experimental organ transplant models. However, the effect of PG490-88 on prevention of acute and chronic renal allograft rejection has not been determined. Kidneys of ACI or F344 rats were transplanted into bilaterally nephrectomized LEW recipients as the acute or chronic allograft rejection models, respectively. Treatment of LEW recipients with PG490-88 significantly prolonged ACI kidney graft survival in a dose-dependent manner when compared with the untreated allograft controls. LEW recipients of F344 kidney grafts who received PG490-88 for 90 days with a brief course of low-dose FK506 showed normal serum creatinine levels and markedly reduced histological changes of chronic rejection at day 90 after transplantation. These results suggest that PG490-88 significantly prolongs kidney allograft survival in an acute rejection model and prevents chronic allograft rejection in rats.

Oncocytic biliary cystadenocarcinoma is a form of intraductal oncocytic papillary neoplasm of the liver.

Biliary cystadenocarcinoma with oncocytic differentiation was first reported in 1992. This is a report of a second case. The patient (a 71-year-old man) was admitted to our hospital complaining of abdominal fullness. Multicystic lesions were identified in the left hepatic lobe radiologically. The patient died of peritoneal dissemination of carcinoma 20 months later. At autopsy, the tumor of the left hepatic lobe was found to be composed of adjoining multiple cystic lesions and a solid lesion with infiltration of the hepatic hilus and peritoneal dissemination. Histologically, the multicystic lesions were covered by papillary neoplastic epithelial cells with an eosinophilic granular cytoplasm resembling that of oncocytes and a fine fibrovascular core. The cyst wall was fibrous, but there was no mesenchymal stroma. In the solid lesion and infiltrated areas, acidophilic and granular carcinoma cells formed small glandular or solid cord patterns with much mucin secretion (mucinous carcinoma). Immunohistochemically, carcinoma cells of both components were found to contain many mitochondria and showed the phenotypes of hepatocytes and cholangiocytes. Interestingly, the intrahepatic biliary tree also was invaded by carcinoma cells. This may be a case of intraductal oncocytic papillary neoplasm of the left hepatic lobe followed by secondary cystic dilatation of the affected bile duct.

Photochemistry and photoinduced proton-transfer by pharaonis phoborhodopsin.

Phoborhodopsin (pR or sensory rhodopsin II, sRII) is a photoreceptor of the negative phototaxis of Halobacterium salinarum, and pharaonis phoborhodopsin (ppR or pharaonis sensory rhodopsin II, psRII) is a corresponding protein of Natronobacterium pharaonis. The photocycle of ppR is essentially as follows: ppR(498) --> ppRK(approximately 540) --> ppRKL(512) --> ppRL(488) --> ppRM(390) --> ppRO(560) --> ppR (numbers in parenthesis denote the maximum absorbance). The photocycle is very similar to that of bacteriorhodopsin, but the rate of initial pigment recovery is about two-orders of magnitude slower. By low-temperature spectroscopy, two K-intermediates were found but the L intermediate was not detected. The lack of L indicates extraordinary stability of K at low temperature. ppRM is photoactive similar to M of bR. The ground state ppR contains only all-trans retinal whereas ppRM and ppRO contain 13-cis and all-trans, respectively. ppR has the ability of light-induced proton transport from the inside to the outside. Proton uptake occurs at the formation of ppRO and the release at its decay. ppR associates with its transducer and this complex transmits a signal to the cytoplasm. The proton transport ability is lost when the complex forms, but the proton uptake and release still occur, suggesting that the proton movement is non-electrogenic (release and uptake occur from the same side). The stoichiometry of the complex between ppR and the transducer is 1 : 1. ppR or pR has absorption maximum at approximately 500 nm, which is blue-shifted from those of other archaeal rhodopsins. The molecular mechanism of this color regulation is not yet solved.

Effect of Sotalol in the prevention of atrial fibrillation following coronary artery bypass grafting.

OBJECTIVE: We assessed the efficacy of postoperatively administered oral Sotalol in preventing the occurrence of postoperative atrial fibrillation. METHODS: Subjects were 80 consecutive patients undergoing coronary artery bypass grafting (CABG) randomized alternately into a Sotalol group (40 patients) administered 80 mg of oral Sotalol daily starting on the postoperative day 1 and continued for 14 days, and a control group (40 patients) matched for age and gender. RESULTS: The incidence of postoperative atrial fibrillation (21 patients) was significantly lower in the Sotalol group (6/40 patients; 15%) than in controls (15/40; 37.5%) (p < 0.05). Significant bradycardia or hypotension, necessitating drug withdrawal, occurred in 3 of 40 (7.5%) patients in the Sotalol group. None in the Sotalol group developed Torsardes de Pointes or sustained ventricular arrhythmias or other severe side effects. The sinus heart rate increased in both groups but less in the Sotalol group. QT, QRS, and QTc durations did not differ between groups. Postoperative hospital stay did not differ between groups. CONCLUSIONS: Oral Sotalol administration of 80 mg daily was associated with a significant decrease in postoperative atrial fibrillation in patients undergoing CABG without appreciable side effects. Sotalol should thus be considered in preventing postoperative atrial fibrillation in patients undergoing CABG in the absence of heart failure and significant left ventricular dysfunction.

Environment around the chromophore in pharaonis phoborhodopsin: mutation analysis of the retinal binding site.

Phoborhodopsin (pR or sensory rhodopsin II, sRII) and pharaonis phoborhodopsin (ppR or pharaonis sRII, psRII) have a unique absorption maximum (lambda(max)) compared with three other archaeal rhodopsins: lambda(max) of pR and ppR is approx. 500 nm and of others (e.g. bacteriorhodopsin, bR) is 560-590 nm. To determine the residue contributing to the opsin shift from ppR to bR, we constructed various ppR mutants, in which a single residue was substituted for a residue corresponding to that of bR. The residues mutated were those which differ from that of bR and locate within 5 A from the conjugated polyene chain of the chromophore or any methyl group of the polyene chain. The shifts of lambda(max) of all mutants were small, however. We constructed a mutant in which all residues which differ from those of bR in the retinal binding site were simultaneously substituted for those of bR, but the shift was only from 499 to 509 nm. Next, we constructed a mutant in which 10 residues located within 5 A from the polyene as described above were simultaneously substituted. Only 44% of the opsin shift (lambda(max) of 524 nm) from ppR to bR was obtained even when all amino acids around the chromophore were replaced by the same residues as bR. We therefore conclude that the structural factor is more important in accounting for the difference of lambda(max) between ppR and bR rather than amino acid substitutions. The possible structural factors are discussed.

Clinical characteristics of B-cell lymphoma-associated hemophagocytic syndrome (B-LAHS): comparison of CD5+ with CD5- B-LAHS.

OBJECTIVE: B-cell lymphoma-associated hemophagocytic syndrome (B-LAHS) has been increasingly reported in Asia and is regarded as a variant of intravascular lymphomatosis (IVL). Recently CD5 was reported to be expressed in some cases of diffuse large cell lymphoma and IVL. We therefore examined the expression of CD5 on lymphoma cells in B-LAHS and compared the clinical and laboratory data between CD5+ and CD5- B-LAHS. METHODS: The expression of CD5 on lymphoma cells was examined using flow cytometry and immunohistochemical analysis of paraffin sections. The clinical records were reviewed to characterize clinical features. PATIENTS: Twelve patients with B-LAHS; ten men and two women, age ranging from 41 to 82 years (median, 63.5 years) were included in this study. RESULTS: B-LAHS is characterized by fever and hepatosplenomegaly without lymphadenopathy at the initial presentation. Histological examination showed hemophagocytosis and infiltration of lymphoma cells in the bone marrow, and in some cases intravascular proliferation of lymphoid cells characteristic of IVL. All patients showed increased levels of lactate dehydrogenase, C-reactive protein, ferritin and soluble interleukin-2 receptor. In eight of the twelve patients, lymphoma cells were positive for CD5. But no differences were observed in the clinical or laboratory findings between CD5+ B-LAHS and CD5- B-LAHS. CONCLUSION: No clinical differences were observed between CD5+ B-LAHS and CD5- B-LAHS. Further studies are required to elucidate the differences in pathogenesis between these two subgroups of B-LAHS.

Scavenger cells with gram-positive bacterial lipoteichoic acid infiltrate around the damaged interlobular bile ducts of primary biliary cirrhosis.

BACKGROUND/AIMS: Gram-positive bacterial DNA is frequently detectable in gallbladder bile of primary biliary cirrhosis (PBC) patients. To advance these findings, lipoteichoic acid (LTA) of gram-positive bacteria with high antigenicity was examined in liver specimens and bile from PBC patients and controls. METHODS: LTA was examined by Western blotting in the gallbladder bile from 15 PBC, 11 cholecystolithiasis and six normal subjects, and by immunohistochemistry in liver specimens from 16 PBC, six primary sclerosing cholangitis (PSC), eight chronic viral hepatitis C (CVH-C) and five normal subjects. RESULTS: In the gallbladder bile, there was no significant difference in the positive rate of LTA between PBC and controls. LTA-containing mononuclear cells were frequently detected in the portal tracts, particularly around the bile ducts and in hepatic sinusoids in PBC, while they were infrequent or occasional in control livers. These LTA-containing cells were sinusoidal endothelial cells and Kupffer cells, and portal monocytes, which frequently expressed scavenger receptor class B type 1. CONCLUSIONS: LTA derived from bacterial fragments may reach the bile, not only in the diseased state but also under normal conditions. Such LTA may be involved in the development and progression of portal tract lesions, particularly bile duct lesions, in PBC.

Pharaonis phoborhodopsin binds to its cognate truncated transducer even in the presence of a detergent with a 1:1 stoichiometry.

Pharaonis phoborhodopsin (ppR) (also pharaonis sensory rhodopsin II) is a receptor of the negative phototaxis of Natronobacterium pharaonis. ppR forms a complex with its pharaonis halobacterial transducer (pHtrII), and this complex transmits the light signal to the sensory system in the cytoplasm. The expressed C-terminal-His tagged ppR and C-terminal-His tagged truncated pHtrII (t-Htr) in Escherichia coli (His means the 6x histidine tag) form a complex even in the presence of 0.1% of n-dodecyl-beta-D-maltoside, and the M-decay of the complex became about twice slower than that of ppR alone. The photocycling rates under varying concentration ratios of ppR to t-Htr in the presence of detergent were measured. The data were analyzed on the following assumptions: (1) the M-decay of both ppR alone and the complex followed a single exponential decay with different time constants; and (2) the M-decay under varying concentration ratios of ppR to t-Htr, therefore, followed a biexponential decay function which combined the decay of the free ppR and that of the complex as photoreactive species. From these analyses we estimated the dissociation constant (15.2 +/- 1.8 microM) and the number of binding sites (1.2 +/- 0.08).

Reproducibility of [11 C]FLB 457 binding in extrastriatal regions.

Extrastriatal D2 dopamine receptors represent an important target of research into the pathophysiology and pharmacotherapy of psychiatric disorders. The high affinity radioligand [11C]FLB 457 makes possible the measurement of low concentrations of D2 receptors in extrastriatal regions using positron emission tomography (PET). The aim of this study was to assess the test/retest variability and reliability of [11C]FLB 457 binding using a reference tissue model. Eight healthy male subjects (aged 20-33 years) underwent two [11C]FLB 457 PET examinations. Radioactivity in the cerebellum was used as the reference. The binding potentials (BPs) for five cortical regions of interest (ROIs) were calculated using the reference tissue model. The BP was also calculated for each pixel in the form of parametric images. Reproducibility was assessed both for the ROI method and for the parametric images. The test/retest reproducibility for [11C]FLB 457 binding was good, with a mean variability ranging from 4.5% for the thalamus to 15.5% for the hippocampus. The parametric images also demonstrated good reproducibility. These results support the suitability of using [11C]FLB 457 for the quantitative evaluation of extrastriatal D2 receptors and for protocols requiring repeated measurements in the same individual.

Studies on transcriptional regulation of Cyp3a16 gene in mouse livers by application of direct DNA injection method.

CYP3A16 is expressed in the mouse liver specifically during a fetal and puberty life. Functional regions responsible for the transcriptional regulation of the Cyp3a16 gene were identified by an in vivo direct DNA injection into mouse livers followed by a luciferase assay. The results of the deletion analysis of a 5'-flanking sequence suggested the existence of an adult-specific repressor(s) interacting with the Cyp3a16 gene. In addition, a positive regulatory element was assumed to be present in a region from -146 to -56 of the Cyp3a16 gene. A hepatocyte nuclear factor 4 (HNF-4)-binding consensus sequence was found in this region. In fact, HNF-4alpha bound to this sequence as evidenced by a gel mobility shift assay. The role of the HNF-4-binding sequence was further examined by an introduction of mutations in this sequence. The introduction of the mutations resulted in a reduced activity of a luciferase in the assay. These results obtained by an application of the direct DNA injection method suggest that the HNF-4alpha activates the transcription of the Cyp3a16 gene in vivo.

Age-related reduction of extrastriatal dopamine D2 receptor measured by PET.

Although the aging effect of dopamine D2 receptor in the striatum is well-documented, the effect of age on the extrastriatal dopamine D2 receptor has not been fully examined. Since the density of extrastriatal dopamine D2 receptor is very low, suitable ligands are limited. In this study, we used [11C]FLB 457 to quantify the extrastriatal dopamine D2 receptor in the living human brain. Twenty-seven healthy male subjects aged from 21 to 82 years participated in the positron emission tomography study. Extrastriatal [11C]FLB 457 binding was quantified with a reference tissue model using cerebellum as a reference region. Binding potentials corresponding to Bmax/Kd were used to evaluate age-related change. We found age-related decreases of D2 receptor binding in all measured extrastriatal regions. The decrease of D2 receptor binding was 13.8% per decade in frontal cortex, 12.0% in temporal cortex, 13.4% in parietal cortex, 12.4% in occipital cortex, 12.2% in hippocampus, and 4.8% in thalamus. These findings suggest that the amounts of D2 receptor declines in all brain regions as part of the normal aging process.