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The post-operative mortality and morbidity rates associated with living-donor liver transplantation (LDLT) are still relatively high. Several papers have reported the risk factors associated with post-operative infectious complications, but few have analyzed the risk factors with respect to the severity of sepsis. The aim of this study was to clarify the risk factors that affect severe sepsis after LDLT.For 63 LDLT patients at our institute, we compared peri-operative characteristics in 29 patients who developed sepsis after surgery and 34 patients who did not. The sepsis group was further divided into severe sepsis (n = 16) and sepsis (n = 13) subgroups to identify significant peri-operative risk factors.Multivariate analysis identified 3 significant risk factors for post-operative sepsis after LDLT: ABO incompatibility (P = .015), low estimated glomerular filtration rates (90 mL/min/1.73 m(2); P = .074), and low peripheral lymphocyte counts (850/μL; P = .008). Multivariate analysis showed that the only significant risk factor for severe sepsis was a low pre-operative lymphocyte count (850/μL; P = .01). In the 2 sepsis subgroups, the 5- and 10-year survival rates for the severe sepsis subgroup (37.5% and 37.5%) were significantly lower than for the sepsis subgroup (83.3% and 62.5%; P = .05). The lung was the most common site of severe sepsis (n = 8; 50.0%).Patients who developed severe sepsis after LDLT had poor long-term survival, with pre-operative lymphocyte counts 850/μL being the significant risk factor. Pre-operative nutritional intervention and rehabilitation should be considered to improve LDLT outcomes.

Microphthalmia-associated transcription factor (MITF) is a key regulator of differentiation of melanocytes and retinal pigment epithelial cells, but it also has functions in non-pigment cells. MITF consists of multiple isoforms, including widely expressed MITF-A and MITF-H. In the present study, we explored the potential role played by the Hedgehog signaling on MITF expression in two common types of primary liver cancer, using human cholangiocarcinoma cell lines, the KKU-100 and HuCCT1, along with the HepG2 human hepatocellular carcinoma cell line. Importantly, cholangiocarcinoma is characterized by the activated Hedgehog signaling. Here we show that MITF-A mRNA is predominantly expressed in all three human liver cancer cell lines examined. Moreover, cyclopamine, an inhibitor of the Hedgehog signalling, increased the expression levels of MITF proteins in HuCCT1 and HepG2 cells, but not in KKU-100 cells, suggesting that MITF expression may be down-regulated in some liver cancer cases.

Although heme oxygenase-1 (HO-1) is induced in keratinocytes after UV radiation, HO-1 expression during normal epidermal differentiation has not yet been reported. We showed by real-time PCR, western blotting, and ELISA that HO-1 mRNA and protein expression by cultured normal human keratinocytes was upregulated during epidermal differentiation induced by a high-calcium medium. Immunohistochemical staining and in situ hybridization showed the graduated expression of HO-1 in the upper epidermis, which was accompanied by suprabasal HO-1 mRNA expression, and the accumulation of bilirubin (BR) in the stratum corneum. We examined the activation of nuclear factor E2-related factor 2 (Nrf2), which is a pivotal transcription factor for HO-1 expression, by western blotting and by examining the mRNA expression of Nrf2 target genes, and excluded its role in HO-1 expression in epidermal differentiation. Next, we examined the regulation of HO-1 expression by inflammatory cytokines. IL-4 and IL-22 significantly reduced HO-1 mRNA and protein expression, whereas IL-1beta, IL-17A, and tumor necrosis factor-alpha (TNF-alpha) increased it. Finally, immunohistochemical studies on psoriatic lesional skin showed that HO-1 expression was downregulated in the parakeratotic epidermis, whereas it was retained in the orthokeratotic epidermis. These studies demonstrate that HO-1 is functionally expressed by keratinocytes in parallel with epidermal differentiation and that its expression is independently affected by several cytokines.

The retinal pigment epithelium (RPE) constitutes the blood-retinal barrier, whose function is impaired in various pathological conditions, including cerebral malaria, a lethal complication of Plasmodium falciparum infection. Prostaglandin (PG) D(2) is abundantly produced in the brain to regulate sleep responses. Moreover, PGD(2) is a potential factor derived from intra-erythrocyte falciparum parasites. Heme oxygenase-1 (HO-1) is important for iron homeostasis via catalysis of heme degradation to release iron, carbon monoxide and biliverdin/bilirubin, and may influence iron supply to the intra-erythrocyte falciparum parasites. Here, we showed that treatment of human RPE cell lines, ARPE-19 and D407, with PGD(2) significantly increased the expression levels of HO-1 mRNA, in a dose- and time-dependent manner. Transient expression assays showed that PGD(2) treatment increased the HO-1-gene promoter activity through the enhancer sequence, containing a Maf-recognition element. Thus, PGD(2) may contribute to the maintenance of heme homeostasis in the brain by inducing HO-1 expression.

The aim of this study was to assess the prognostic impact of the number of lymph node metastases. The medical records of 33 patients with node-positive gallbladder cancer (GBC) treated at our institution from January 1985 through December 2002 were reviewed. There were 10 cases with a single node metastasis. The sites were as follows: the cystic duct node, the pericholedochal node, the retroportal node, the hilar node, the lymph node around the common hepatic artery, and the paraaortic node. According to the International Union Against Cancer (UICC) 5th edition, 5-year survival rates for the patients with pN1, pN2, and greater than pN2 were 19.2%, 10%, and 0%, respectively (not significant). Patients with a single node metastasis had a higher 5-year survival rate (33%) than patients with two or more lymph node metastases (0%; P < 0.05). There were no lymph node recurrences in patients with a single node metastasis. Number of positive nodes and liver metastasis were factors predictive of significantly worse survival. Rather than using the topographic classification, or even simply classifying whether nodal involvement is positive or negative, classification according to the number of positive nodes will contribute to establishing a more practically useful staging system.

Microphthalmia-associated transcription factor (MITF) is responsible for differentiation of melanocytes. A recessive MITF mutant, black-eyed white Mitfmi-bw mouse, is characterized by white coat color and deafness, due to the lack of melanocytes in the skin and inner ears. By cDNA microarray analysis, we have identified lipocalin-type prostaglandin D synthase (L-PGDS), whose mRNA is undetectable in the homozygous Mitfmi-bw skin. Immunohistochemical analysis of wild-type mice identified the specific expression of L-PGDS in follicular melanocytes. L-PGDS mRNA is expressed in B16 mouse melanoma cells, but undetectable in human melanoma cell lines. RNA interference analysis against MITF suggests that L-PGDS expression is dependent on MITF in B16 melanoma cells. Furthermore, we have provided evidence that MITF is involved in the melanocyte lineage-specific transcription of the mouse L-PGDS gene. Thus, L-PGDS represents a newly identified melanocyte marker. MITF may modulate the production of prostaglandin D2 by activating the L-PGDS gene in melanocytes.

Highly oriented pyrolytic graphite (HOPG) and graphitized carbonaceous thin films prepared by plasma-assisted chemical vapor deposition (PACVD) were surface-modified by electropolymerization of thiophene. The electrochemical properties of the carbonaceous materials were studied by cyclic voltammetry and ac impedance spectroscopy. Irreversible cathodic current of the carbonaceous materials above 0.5 V (vs. Li/Li+) in the cyclic voltammograms significantly decreased by electropolymerization of thiophene, indicating that electropolymerization of thiophene suppress the decomposition of electrolytes on the carbonaceous materials. On the Nyquist plots, a semi-circle due to surface film resistance was observed, and the value significantly decreased at around 1.5 V. At potentials below 0.9 V, another semi-circle appeared in the middle to lower frequency region, which was assigned to the charge transfer resistance due to lithium-ion transfer at the surface-modified carbon electrode/electrolyte interface. The charge-transfer resistances were dependent on electrode potentials. The activation energy for lithium-ion transfer through interface between the surface-modified HOPG electrode and electrolyte was evaluated, and the value was almost identical to that obtained for an untreated HOPG electrode. Based on these results, it is concluded that electropolymerization of thiophene played an important role not in the phase transfer kinetics of lithium-ion but in reduction of the electrolyte decomposition at a graphite electrode.

Heme oxygenase (HO) catalyzes physiological heme degradation and consists of two structurally related isozymes, HO-1 and HO-2. Here we show that HO-2-deficient (HO-2−/−) mice exhibit hypoxemia and hypertrophy of the pulmonary venous myocardium associated with increased expression of HO-1. The hypertrophied venous myocardium may reflect adaptation to persistent hypoxemia. HO-2−/− mice also show attenuated ventilatory responses to hypoxia (10% O2) with normal responses to hypercapnia (10% CO2), suggesting the impaired oxygen sensing. Importantly, HO-2−/− mice exhibit normal breathing patterns with normal arterial CO2 tension and retain the intact alveolar architecture, thereby excluding hypoventilation and shunting as causes of hypoxemia. Instead, ventilation–perfusion mismatch is a likely cause of hypoxemia, which may be due to partial impairment of the lung chemoreception probably at pulmonary artery smooth muscle cells. We therefore propose that HO-2 is involved in oxygen sensing and responsible for the ventilation–perfusion matching that optimizes oxygenation of pulmonary blood.

Heme oxygenase 1 (HO-1) catalyzes heme breakdown, eventually releasing iron, carbon monoxide, and bilirubin IXalpha. HO-1 is induced by its substrate heme and various environmental factors, which represents a protective response against oxidative stresses. Here we show that hypoxia represses HO-1 expression in three human cell types but induces it in rat, bovine, and monkey cells, indicating the inter-species difference in the hypoxic regulation of HO-1 expression. The hypoxia-mediated repression of HO-1 expression is consistently associated with the induction of Bach1, a heme-regulated transcriptional repressor, in human cells. Bach1 is a basic leucine zipper protein, forming a heterodimer with a small Maf protein. Expression of HO-1 was also reduced in human cells when exposed to interferon-gamma or an iron chelator desferrioxamine, each of which induced Bach1 expression. In contrast, induction of HO-1 expression by CoCl(2) is associated with reduced expression of Bach1 mRNA. Thus, expression of HO-1 and Bach1 is inversely regulated. We have identified a Maf recognition element in the human HO-1 gene that is required for repression of a reporter gene by hypoxia and targeted by Bach1. Therefore, Bach1 functions as a hypoxia-inducible repressor for the HO-1 gene, thereby contributing to fine-tuning of oxygen homeostasis in human cells.

Carbonaceous thin film electrode was prepared by plasma assisted chemical vapor deposition. The films were very flat and pin-hole free with c-axis orientation. X-ray diffraction and Raman spectroscopy revealed that the bulk of the film was highly graphitized but the surface of the film was less crystallized, indicating that the surface modified graphitized thin film electrode was obtained by a single process. Electrochemical properties of this film were studied in electrolyte of propylene carbonate (PC) containing 1moldm−3LiClO4 by cyclic voltammetry. The first cycle of voltammogram showed reduction current observed at 1.0–0.5 V (vs. Li/Li+), but the reduction current at those potentials completely disappeared over the second cycle. Large reduction and oxidation currents were observed near 0 V (vs. Li/Li+), and in addition, the peak splitting was observed for oxidation peaks near 0 V. These electrochemical properties are quite similar to that of graphite electrode used in Li-ion batteries. Keywords: Carbonaceous thin film electrode, Plasma CVD, Propylene carbonate, Li-ion batteries

Microphthalmia-associated transcription factor (MITF) regulates the differentiation and development of melanocytes and retinal pigment epithelium and is also responsible for pigment cell-specific transcription of the melanogenesis enzyme genes. Hetero zygous mutations in the MITF gene cause auditory–pigmentary syndromes. MITF consists of at least five isoforms, MITF-A, MITF-B, MITF-C, MITF-H, and MITF-M, differing at their N-termini and expression patterns. Here we show a remarkable similarity between the N-terminal domain of MITF-A and cytoplasmic retinoic acid-binding proteins. To date, four isoform-specific first exons have been identified in the MITF gene: exons 1A, 1H, 1B, and 1M in the 5′ to 3′ direction, each of which encodes the unique N-terminus of a given isoform. The 5′-flanking regions of these isoform-specific exons are termed A, H, B, and M promoters, respectively. Among these promoters, the M promoter has received particular attention, because it is functional only in melanocyte-lineage cells and is upregulated by Wnt signaling via the functional LEF-1-binding site. Moreover, the M promoter is upregulated by other transcription factors, PAX3, SOX10, and CREB. The activity and degradation of MITF-M are regulated by extracellular signals via protein phosphorylation, such as c-Kit signaling. Together, multiple signals appear to converge on the M promoter as well as on MITF proteins, leading to the proper regulation of MITF-M in melanocytes and other MITF isoforms in many cell types.

Mutations in the SOX10 gene, encoding a cell-lineage specific transcription factor, are associated with congenital deafness. We analyzed the expression of Sox10 mRNA in developing mouse inner ear by in situ hybridization. Sox10 mRNA is expressed in the entire epithelia of the otic vesicle at embryonic day 11.5 (E11.5) and in the developing cochlea and vestibule at E13.5. In postnatal day 8 and adult cochleas, Sox10 expression is restricted to the supporting cells of the organ of Corti. These expression profiles suggest that Sox10 is important for development of the cochlea.

The human tyrosinase-related protein 2 (TRP-2) gene promoter contains a cis -regulatory element (positions −447 to −416), termed DOPAchrome tautomerase distal enhancer 1 (DDE1). DDE1 functions as an enhancer in cultured melanoma cells and its core element includes a potential binding site for transcription factors containing a high-mobility-group domain. This core element is bound in vitro by multiple nuclear proteins, which are preferentially expressed in melanoma cells. DDE1 represents a composite enhancer that may be involved in melanocyte-specific transcription of the human TRP-2 gene.

Microphthalmia-associated transcription factor (Mitf) plays a critical role in the development of neural crest-derived melanocytes. Here, we show that exogenously added Wnt-3a protein, an intercellular signaling molecule, up-regulates the expression of endogenous melanocyte-specific Mitf (Mitf-M) mRNA in cultured melanocytes. The melanocyte-specific promoter of the human MITF gene (MITF-M promoter) contains a functional LEF-1-binding site, which is bound in vitro by LEF-1 and confers the preferential expression on a reporter gene in melanocytes and melanoma cells, as judged by the transient transfection assays. Moreover, the LEF-1-binding site is required for the transactivation of a reporter gene by LEF-1, beta-catenin, or their combination. Exogenously added Wnt-3a protein also transactivates the MITF-M promoter via the LEF-1-binding site; this activation was abolished when a dominant-negative form of LEF-1 was coexpressed. These results suggest that Wnt-3a signaling recruits beta-catenin and LEF-1 to the LEF-1-binding site of the MITF-M promoter. Therefore, the present study identifies Mitf-M/MITF-M as a direct target of Wnt signaling.

Microphthalmia-associated transcription factor (MITF) affects the development of many types of cells, including melanocytes and retinal pigment epithelium (RPE). MITF consists of at least three isoforms, MITF-A, MITF-H and MITF-M, differing at their amino-termini and expression patterns. Here, we characterize the structural organization of the human MITF gene. The gene contains at least four isoform-specific first exons, exons 1A, 1H, 1B and 1M in the 5' to 3' direction, each of which encodes the unique amino-terminus of a given isoform, including newly identified MITF-B. The 5'-flanking regions of these isoform-specific exons are termed promoters A, H, B and M, respectively, which showed different promoter activities, as judged by transient transfection assay. Promoter A directs the expression of a reporter gene in RPE, cervical cancer and melanoma cells, whereas promoter M is functional only in melanoma cells. Promoter H showed the significant activity in RPE and cervical cancer cells but not in melanoma cells. In contrast, the 1.7 kb 5'-flanking region of exon 1B showed no noticeable promoter activity in these cell lines. Therefore, alternative promoters provide the MITF gene with the diversity in transcriptional regulation and the capability of generating structurally different protein isoforms.

Background. In the present study we attempted to evaluate the role of the graft as an alloantigen in induction and maintenance of donor-specific prolongation of allograft survival by short course(s) of FK506 treatment in rat heart transplantation. Materials and methods. In the WKA/Qdj-to-LEW combination group, FK506 (3 mg/kg/day, 7 days, sc) was administered from Day 0 after cardiac transplantation. A second WKA/Qdj cardiac graft was transplanted on Day 14 into the LEW recipient after removal of the first graft on Day 3 or Day 7. In other groups an additional course of FK506 (3 mg/kg/day, 7 days, sc) was given from Day 35. A second WKA/Qdj cardiac graft was transplanted on Day 49 into the LEW recipient after removal of the first graft on Day 7 or Day 28. Results. A short course of FK506 treatment allowed for survival prolongation of a cardiac allograft [mean survival time (MST) = 42.8 days]. Removal of the first graft on Day 7 (MST = 16.4 days) but not on Day 3 (MST = 10.2 days) caused donor-specific prolongation of the second allograft survival, which was significantly shorter than that in the recipient without the graftectomy (MST = 32.8 days). When graftectomy was performed on Day 3, there were immunohistochemically detectable levels of donor class II expressing cells in the recipient spleen on Day 7, indicating that the presence of the graft more effectively induced unresponsiveness than microchimerism alone. The additional course of FK506 treatment on Day 35 maintained graft survival (MST > 90.6 days). Removal of the first graft on Day 7 (MST = 13.5 days) or Day 28 (MST = 24.7 days) did not show significant prolongation of the second allograft survival, whereas significant survival prolongation of the second graft was observed in the recipient without the graftectomy (MST = 38.8 days). However, marked survival prolongation of the second donor-specific allograft in half of the recipients that had received the second course of FK506 treatment after graftectomy on Day 28 indicates that residual donor antigen can function as tolerizing antigen with an FK506 supplement. Conclusions. We concluded that the persistence of a graft more effectively induces and maintains donor-specific unresponsiveness than does the chimeric state of graft-derived cells alone under immunosuppression by FK506 in the rat heart transplantation model.

Aim This study was conducted to clarify the management of perioperative infectious complications after adult living donor liver transplantation (LDLT). Patients and methods Fourteen adult LDLT patients were enrolled in this study. We examined the occurrence of infectious complications in these cases and the relationships of infectious complications to UNOS status and MELD score. Surveillance culture and immunoserologic analyses were performed. From the results of these analyses, we made a diagram of infection surveillance using a matrix of time and sampling site. Using the diagram, we chose sensitive antibiotics as soon as possible. Results The infection site and its pathogen were able to be detected in four (28.5%) patients, all of whom had MRSA infections, together with lung aspergillosis in one case, pseudomonas pneumonia in another, and both in another. Two patients died of lung aspergillosis. Bacteria detected in the airway tended to spread to other sites during the postoperative period. In all four patients in whom infectious diseases were detected, and in a fifth patient in whom the site of infection was not known, the UNOS status was 1. The MELD score was calculated in eight patients, six of whom had high MELD scores (>20). Conclusion Most cases were manageable by choosing and changing antibiotics and antifungal drugs according to the results of surveillance cultures twice a week. However, aspergillosis had an extremely poor prognosis. Patients with a high MELD score or low UNOS status, or both, showed poor prognosis; and in them, multiple drug resistance bacteria caused severe perioperative infectious complications.

This study sought to determine whether a prognostic score is a useful indicator of donor safety using 13 consecutive donors enrolled for liver transplantation. The donor operations were right hepatic lobectomies (n = 10) and left hepatic lobectomies (n = 3). The postoperative maximal level of serum total bilirubin was used to assess the magnitude of surgical stress. Variables such as donor age, percentage of liver resection (PLR), indocyanine green 15-minute retention rate (ICGR15), operative blood loss, operation time, prognostic score and graft weight were evaluated as predictors of the magnitude of surgical stress. The PLR and prognostic score (PS) were calculated according to the following formulae: PLR (%) = 100*Graft weight (g)/standard liver volume of the donor (mL); PS = -84.6 + 0.933*PLR (%) +1.11*ICGR15 (%) +0.999*age (years); Standard liver volume (mL) = 706.2*body surface area (m2) + 2.39. No serious complications occurred after the donor operations. Maximal bilirubin ranged from 1.9 to 10.9 mg/dL. There were no mortalities, although there were two morbidities, bile leakage and prolonged liver dysfunction. Postoperative hyperbilirubinemia was observed in two donors and in one Gilbert's syndrome donor. Linear regression analysis of each variable indicated poor correlations between those variables and maximal bilirubin. However, close correlations were seen between maximal bilirubin and both donor age and PS except for the three patients who showed postoperative hyperbilirubinemia. In these uneventful donors, statistical formulae were obtained as follows: maximal bilirubin (PMB) = 0.271 + 0.056*donor age (correlation coefficient 0.612, P.008), PMB = 1.541 + 0.059*PS (correlation coefficient 0.597, P.009). In conclusion, PS is useful to predict maximal bilirubin and to ensure donor safety.

Heme oxygenase-1, an essential enzyme in heme catabolism, and metallothionein IIA, a small metal-binding protein with clusters of cysteins, are remarkably induced in HeLa cells following the treatment with cadmium or zinc. Both proteins are considered to be involved in the defense system against metal toxicity. Here we showed by transient expression assays that the cadmium-responsive element (CdRE) of the human heme oxygenase-1 gene is not responsive to zinc, whereas a metal-regulatory element (MRE) of the human metallothionein IIA gene is able to respond to either cadmium or zinc. The CdRE is recognized by a certain nuclear protein(s) which is however unable to bind to an MRE of the metallothionein IIA gene. These results suggest that the metal-selective activation of each gene promoter is mediated by a separate mechanism.

Heme oxygenase is an essential enzyme in heme catabolism and is inducible by various environmental derangements such as cadmium. The activity and mRNA levels of heme oxygenase were remarkably increased in HeLa cells by the treatment with cadmium. As a first step in studying the molecular mechanisms of this induction, we performed transient expression assays in four human cell lines including HeLa to analyze the cadmium-mediated inducibility of the fusion genes, containing the firefly luciferase gene as a reporter under the human heme oxygenase gene promoter. By determining the luciferase activity expressed in the transfected cells, we found the region between about 4.5 and 4 kilobase pairs upstream from the transcriptional initiation site of the heme oxygenase gene which confers cadmium-mediated inducibility on the fusion gene. The region was then subjected to further functional analysis in HeLa cells, which allowed us to localize the cadmium-responsive element to 20 base pairs. Gel mobility shift assays demonstrated that this 20-base pair element is specifically bound by nuclear protein(s) of HeLa cells, the binding activities of which were however unchanged by the treatment with cadmium. Using the synthetic cadmium-responsive elements containing various base changes, we have identified a 10-base pair sequence, TGCTAGATTT, required for the cadmium-mediated inducibility and in vitro protein binding. We thus suggest that this binding protein(s) is involved in the cadmium-mediated activation of the heme oxygenase gene. Incidentally, the consensus sequence of AP-1 binding site, TGAGTCA, is present downstream of this cadmium-responsive element. However, we provide evidence that AP-1 is not directly involved in the cadmium-mediated induction of the human heme oxygenase gene.