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Background: Tumor necrosis factor (TNF)-alpha is a cytokine that initiates liver regeneration after hepatectomy (HTx), although extensive HTx can cause liver failure with significant rise in serum TNF-alpha levels. To test our hypothesis that modulation of endogenous TNF-alpha attenuates liver failure even after extensive HTx, we used ONO-SM362, a novel TNF-alpha inhibitor, in mice subjected to 85% HTx., Methods: ICR mice were divided into 5 groups: 70% HTx, 85% HTx, 85% HTx plus ONO-SM362, 85% HTx plus monoclonal TNF-alpha antibody (mAb), and 85% HTx plus FR167653, a TNF-alpha inhibitor. We analyzed the survival rate, blood ammonia (NH(3)), serum TNF-alpha levels, TNF-alpha mRNA expression in the liver and spleen by real-time polymerase chain reaction, histologic changes, polymorphonuclear neutrophils (PMNs) infiltration, and proliferating cell nuclear antigen labeling index (PCNA LI) in the 5 groups., Results: The survival rate at 7 days after surgery was 100%, 0%, 100%, 50%, and 0%, for the 70% HTx, 85% HTx, 85% HTx + ONO-SM362, 85% HTx + mAb, and 85% HTx + FR167653, respectively. Mice that underwent 85% HTx died from liver failure associated with a significant rise in serum TNF-alpha level. ONO-SM362 and mAb improved animal survival and enhanced PCNA LI. In addition, ONO-SM362 inhibited TNF-alpha mRNA expression in the remnant liver and suppressed PMNs infiltration., Conclusions: Suppression of excessive TNF-alpha production using ONO-SM362 ameliorated liver failure after 85% HTx.

The diaphanous-related formins are actin nucleating and elongating factors. They are kept in an inactive state by an intramolecular interaction between the diaphanous inhibitory domain (DID) and the diaphanous-autoregulatory domain (DAD). It is considered that the dissociation of this autoinhibitory interaction upon binding of GTP-bound Rho to the GTPase binding domain next to DID induces exposure of the FH1-FH2 domains, which assemble actin filaments. Here, we isolated two diaphanous-related formins, mDia1 and Daam1, in platelet extracts by GTP-RhoA affinity column chromatography. We characterized them by a novel assay, where beads coated with the FH1-FH2-DAD domains of either mDia1 or Daam1 were incubated with platelet cytosol, and the assembled actin filaments were observed after staining with rhodamine-phalloidin. Both formins generated fluorescent filamentous structures on the beads. Quantification of the fluorescence intensity of the beads revealed that the initial velocity in the presence of mDia1 was more than 10 times faster than in the presence of Daam1. The actin assembly activities of both FH1-FH2-DADs were inhibited by adding cognate DID domains. GTP-RhoA, -RhoB, and -RhoC, but not GTP-Rac1 or -Cdc42, bound to both mDia1 and Daam1 and efficiently neutralized the inhibition by the DID domains. The association between RhoA and Daam1 was induced by thrombin stimulation in platelets, and RhoA-bound endogenous formins induced actin assembly, which was inhibited by the DID domains of Daam1 and mDia1. Thus, mDia1 and Daam1 are platelet actin assembly factors having distinct efficiencies, and they are directly regulated by Rho GTPases.

Background: There are few monitoring systems widely used in clinical practice for evaluating the effectiveness of aspirin therapy, so in the present study aspirin's antiplatelet effects we investigated with a whole blood aggregometer using a screen filtration pressure (SFP) method., Methods and Results: Thirty-five healthy male volunteers took 100 mg/day aspirin for 14 days. Whole-blood aggregation was analyzed at baseline and on days 7 and 14, using collagen and adenosine diphosphate as the stimuli, and compared with the platelet-rich plasma (PRP) aggregation measured by optical aggregometer. The platelet-aggregation threshold index (PATI) for both methods, which was defined as the putative agonist-concentration giving half-maximal aggregation, and the PRP-maximal aggregation rate were analyzed. The maximal aggregation rate induced by 1.6 mg/L collagen decreased from 85.5% (80.8-92.8) [median (interquartile range)] at baseline to 51.5% (39-63.8) on day 14 (p<0.0001). The PRP-PATI and whole-blood PATI for collagen increased from 0.32 (0.28-0.70) to 1.82 mg/L (1.25-2.89) (p<0.0001) and from 0.28 (0.22-0.3) to 1.06 mg/L (1.01-1.29) (p<0.0001) respectively., Conclusions: The whole-blood PATI and PRP-PATI for collagen, as well as the maximal PRP aggregation rate, clearly distinguish platelet aggregability before and after aspirin intake. However, whole-blood analysis by the SFP-method is easier to perform, and is a promising method of monitoring aspirin's effects.

Background/aims: Thermal ablation (TA) therapies such as microwave coagulation therapy (MCT) and radiofrequency interstitial thermal ablation (RFA) for colorectal metastasis to the liver cannot always achieve a complete tumor cell death, and the multiple insertions of the TA probe may lead to intrahepatic dissemination and/or distant metastasis., Methodology: The achieved local control rate, any recurrence in the residual liver, and any extrahepatic recurrence has been evaluated in 105 patients who underwent hepatectomy and/or intraoperative TA between 1994 and 2004., Results: A total of 102 unresectable liver metastatic lesions (mean size 21mm) were selectively treated with TA either as initial treatment (32 patients) and/or as re-treatment (18 patients) for recurrence in the residual liver, in combination with hepatectomy. Overall, TA achieved a high local tumor control rate of 95%. Multivariate analysis revealed that initial-TA therapy was not a significant predictive factor of hepatic recurrence or any recurrence., Conclusion: TA therapies in combination with hepatectomy may offer increased resectability without increased risk of intrahepatic dissemination or extrahepatic recurrence for certain patients who would otherwise be deemed inoperable, when relatively small tumors are indicated for TA.

Carbon nanotubes (CNTs) have been used in various fields as composites with other substances or alone to develop highly functional materials. CNTs hold great interest with respect to biomaterials, particularly those to be positioned in contact with bone such as prostheses for arthroplasty, plates or screws for fracture fixation, drug delivery systems, and scaffolding for bone regeneration. Accordingly, bone-tissue compatibility of CNTs and CNT influence on bone formation are important issues, but the effects of CNTs on bone have not been delineated. Here, it is found that multi-walled CNTs adjoining bone induce little local inflammatory reaction, show high bone-tissue compatibility, permit bone repair, become integrated into new bone, and accelerate bone formation stimulated by recombinant human bone morphogenetic protein-2 (rhBMP-2). This study provides an initial investigational basis for CNTs in biomaterials that are used adjacent to bone, including uses to promote bone regeneration. These findings should encourage development of clinical treatment modalities involving CNTs.

By use of electrostatic interactions of dye molecules and poly(diallyldimethylammonium chloride) (PDDA), the spin-coating technique has been successfully applied to the preparation of stable J-aggregate thin films of thiacarbocyanine dyes on a polycarbonate or quartz plate. The J-aggregate thin films were prepared by the spin-coating of PDDA aqueous solution on dye thin films prepared on a substrate by the spin-coating of 2,2,3,3-tetrafluoro-1-propanol solution of dyes. Photophysical properties of the dye thin films and J-aggregate thin films were studied by measuring the fluorescence spectra, quantum yields, and lifetimes. Coherent size of the J-aggregates was estimated to be 3-12 by means of the absorption bandwidth (full width at half maximum) or radiative lifetime. Photostability of the J-aggregate thin films was also studied in terms of photodegradation efficiency under argon and oxygen in comparison with the dye thin films, and J-aggregate thin films were found to be more stable than the corresponding dye thin films.

Non-hydrolyzable GTP analogues, such as guanosine 5'-(beta, gamma-imido)triphosphate (GppNHp), induce granule secretion from permeabilized platelets in the absence of increased intracellular Ca(2+). Here, we show that the GppNHp-induced dense granule secretion from permeabilized platelets occurred concomitantly with the activation of small GTPase Ral. This secretion was inhibited by the addition of GTP-Ral-binding domain (RBD) of Sec5, which is a component of the exocyst complex known to function as a tethering factor at the plasma membrane for vesicles. We generated an antibody against Sec5-RBD, which abolished the interaction between GTP-Ral and the exocyst complex in vitro. The addition of this antibody inhibited the GppNHp-induced secretion. These data indicate that Ral mediates the GppNHp-induced dense granule secretion from permeabilized platelets through interaction with its effector, the exocyst complex. Furthermore, GppNHp enhanced the Ca(2+) sensitivity of dense granule secretion from permeabilized platelets, and this enhancement was inhibited by Sec5-RBD. In intact platelets, the association between Ral and the exocyst complex was induced by thrombin stimulation with a time course similar to that of dense granule secretion and Ral activation. Taken together, our results suggest that the Ral-exocyst pathway participates in the regulation of platelet dense granule secretion by enhancing the Ca(2+) sensitivity of the secretion.

Genu recurvatum deformity is rare, usually resulting from injury to the anterior part of the proximal tibial growth plate. However, a small group of cases have no known cause. We describe brothers with the same type of this deformity on the same side. The degree of deformity differed between them, but deformities began at the same age in both (17 years) and progressed over a short period. There was no history of trauma, or other orthopaedic or genetic disorders. These features strongly suggested involvement of heritable risk factors.

The antibacterial activity of meropenem (MEPM) and other parenteral antibiotics against clinical isolates of 876 strains of Gram-positive bacteria, 1764 strains of Gram-negative bacteria, and 198 strains of anaerobic bacteria obtained from 30 medical institutions during 2006 was measured. The results were as follows; 1. MEPM was more active than the other carbapenem antibiotics tested against Gram-negative bacteria, especially against enterobacteriaceae and Haemophilus influenzae. MEPM was also active against most of the species tested in Gram-positive and anaerobic bacteria, except for multi-drug resistant strains including methicillin-resistant Staphylococcus aureus. 2. As for Pseudomonas aeruginosa, all of the MEPM-resistant strains were resistant to imipenem (IPM). MEPM showed low cross-resistant rate both againt IPM-resistant P. aeruginosa (41.8%) and ciprofloxacin-resistant P. aeruginosa (33.3%). 3. The proportion of extended-spectrum beta-lactamase (ESBL) strains was 4.3% (6 strains) in Escherichia coli, 1.1% (1 strain) in Citrobacter freundii, 21.7% (5 strains) in Citrobacter koseri, 3.1% (4 strains) in Klebsiella pneumoniae, 3.3% (3 strains) in Enterobacter cloacae, 0.8% (1 strain) in Serratia marcescens, and 4.9% (2 strains) in Providencia spp. The proportion of metallo-beta-lactamase strains was 3.1% (10 strains) in P. aeruginosa. 4. Of all species tested, there were no species, which MIC90 of MEPM was more than 4-fold higher than those in our previous study. Therefore, there is almost no significant decrease in susceptibility of clinical isolates to meropenem. In conclusion, the results from this surveillance study suggest that MEPM retains its potent and broad antibacterial activity and therefore is a clinically useful carbapenem at present, 11 years after available for commercial use.

A radio frequency identification (RFID) transponder covering the 13.56 MHz band was adapted to minimize its volume so that it could be placed in the pulp chamber of an endodontically treated human tooth. The minimized transponder had a maximum communication distance of 30 mm. In an animal experiment, the transponder was fixed in the cavity of a mandibular canine of a dog. An RFID reader positioned close to the dog's face could communicate with the transponder in the dog's tooth. In certain cases, the system is applicable for the personal identification procedures for hospitalized patients instead of an identification wristband.

Reorganization of the actin filament is an essential process for cell motility, cell-cell attachment and intracellular transport. Formin proteins promote nucleation and elongation of the actin filament, and thus are key regulators for this process. The formin homology 2 (FH2) domain forms a head-to-tail ring-shaped dimer, and processively moves towards the barbed end. Dishevelled-associated activator of morphogenesis (DAAM) is a Rho-regulated formin implicated in neuronal development. Here, we present the crystal structure of human DAAM1 FH2 dimer at 2.8 A resolution. This is the first dimeric structure of the mammalian formin. The core structure of human DAAM1 is similar to those of mouse mDia1 and yeast Bni1p, whereas the orientations of the FH2 dimeric rings are different between human DAAM1 and yeast Bni1p, despite their similar dimer interactions. This difference supports the previous prediction that the dimer architecture of the formin is highly flexible in the actin-free state. The results of the actin assembly assays using the DAAM1 mutants demonstrated that the length of the linker connecting the N-terminal domain and the core region is crucial for the activity.

A protein kinase C (PKC)-encoding gene (pkcA) was isolated from the filamentous fungus Aspergillus nidulans. Although we attempted to isolate pkcA deletion mutants, we obtained only heterokaryons that had both DeltapkcA and pkcA(+) nuclei. Conidia produced by the heterokaryon germinated. The germ tubes, however, lysed frequently and no colony formation was observed, indicating that the pkcA gene is essential to the viability of A. nidulans. We constructed conditional mutants (alcA(p)-pkcA mutants) that expressed pkcA under the control of the alcA promoter (alcA(p)). Under alcA(p)-repressing conditions, their colonies were smaller than those of the wild-type strains and their hyphae lysed frequently. These phenotypes were not remedied under moderate- or high-osmolarity conditions; the growth defect deteriorated further under the latter. Under alcA(p)-inducing conditions, the alcA(p)-pkcA mutants also showed growth-sensitivity to cell wall destabilizing agents. These results indicate that pkcA plays an important role in the maintenance of cell integrity.

Unlabelled: This study is for chemoradiotherapy of unresectable and recurrent cholangiocarcinoma (CCC)., Subjects and Methods: Between April 2005 and March 2007, 5 patients were evaluated for unresectable and recurrent CCC. All patients were performed percutaneous transhepatic biliary drainage (PTBD) and treated with chemoradiotherapy. The radiation method was an external beam radiotherapy (EBRT) and remote after loading system (RALS). The chemotherapy was 5-FU and gemcitabine by intravenous injection., Results: Three patients had unresectable and 2 had recurrent CCC. As for the side effects, 4 patients had a slight neutropenia and 1 had an uncontrollable gastric bleeding. No patient had cholangitis. The mean survival time for unresectable and recurrent CCC was 13.7 months and 17 months, respectively., Conclusions: Our results indicated that chemoradiotherapy had been consistent with efficacy in patients with unresectable and recurrent CCC.

We experienced successful rehabilitation of an elderly man after hip fracture (HF) who had had ipsilateral above-elbow amputation. He was a wounded soldier whose right arm had been amputated more than 60 yrs ago, and he had never used a prosthesis. He had been admitted to our hospital because of exacerbation of chronic obstructive pulmonary disease. During his admission, he had a fall-related hip fracture. We devised a special upper-limb prosthesis so that he could hold an ipsilateral crutch to enable appropriate acute-phase rehabilitation for HF. As a result, effects of disuse were prevented and he gained improvement of lower-limb function. The specialized upper-limb prosthesis also reduced his anxiety regarding walking. It can be considered that the prosthesis contributed to his quality of life with regard to activities of daily living after discharge.

Recently, we reported the application of a recombinant chicken IgY monoclonal antibody, Ab3-15, against mammalian prion protein (PrP), for the diagnosis of bovine spongiform encephalopathy in cattle. In this study, we have characterized a soluble, single-chain variable fragment (scFv) form of this antibody, sphAb3-15 using brain homogenates from mice. This sphAb3-15 antibody recognized denatured forms of both PrP(C) and PrP(Sc), and PrP(Sc) after PK-treatment, on Western blotting. In sandwich ELISAs, on dot blots and by immunoprecipitation, sphAb3-15 efficiently bound to PrP from normal brain homogenates, but weakly bound PrP from scrapie-infected brain homogenates. These results suggest that sphAb3-15 selectively recognizes PrP(C) under native conditions and that the epitope recognized by sphAb3-15 may undergo conformational changes during the conversion of PrP(C) into PrP(Sc).

We report a case of cystic echinococcosis (CE) caused by Echinococcusgranulosus, for which a modified percutaneous evacuation (PEVAC) treatment was applied. The patient had immigrated from Peru to Japan and had 2 hydatid cystic masses, 1 located in segment (S)5 of the liver and the other in S3 (5.3 and 3.5 cm in diameter, respectively), both of which were visualized as pseudotumors by ultrasound (US) examinations. Albendazole treatment showed no effects and surgical treatment was refused. After punctuation of the S5 cyst under US guidance and S3 with CT guidance, 10- and 12-French gauge catheters, respectively, with multiple side holes were inserted. About 60 ml of the cyst contents was drawn out from the S5 lesion and 2 ml from the S3 lesion. Using repetitive manual injections and aspiration of small amounts of hypertonic saline, the remaining cyst content was removed as much as possible, after which 20 and 10 ml of 98% ethanol was injected into the S5 and S3 lesions, respectively. A short-term evaluation during the 4 month-period following the procedure using US revealed nearly complete evacuation of the S5 lesion, whereas that at S3 remained as a pseudo-solid mass. We consider that percutaneous treatment is a safe therapeutic modality for hydatid cysts. This is the first case report of CE treated percutaneously in Japan.

Background: Lack of effective adjuvant therapy against advanced extrahepatic biliary tract carcinoma (BTC) requires that new therapeutic methods, such as molecular targeted therapy, be developed. The mitogen-activated protein kinase (MAPK) and Akt signaling pathways, which activate cell proliferation and suppress apoptosis, respectively, may function as important targets for such therapies. The aim of this study was to examine the expression patterns of phosphorylated MAPK (p-MAPK) and phosphorylated Akt (p-Akt) proteins in BTC cell lines and clinical specimens., Methods: Expression of p-MAPK and p-Akt proteins in four human BTC cell lines and in frozen sections of 20 advanced extrahepatic BTC specimens was analyzed by Western blotting. Thirty formalin-fixed BTC specimens were immunohistochemically stained for p-MAPK and p-Akt using labeled streptavidin-biotin conjugates., Results: Expression of p-MAPK was observed in three of four (75%) BTC cell lines, whereas no expression of p-Akt was observed. Twenty-three of 30 formalin-fixed specimens stained positive for p-MAPK (77%), whereas only 47% stained positively for p-Akt. Expression of p-MAPK relative to that of p-Akt was also seen more frequently in the frozen specimens., Conclusions: The results of this study suggest that MAPK is activated more frequently than Akt in extrahepatic biliary tract carcinoma.

Hemophagocytic lymphohistiocytosis (HLH), a disorder of the mononuclear phagocyte system, can be classified into two distinct forms: primary HLH (FHL) and secondary HLH. To clarify the epidemiology and clinical outcome for each HLH subtype, we conducted a nationwide survey of HLH in Japan. Since 799 patients were diagnosed in 292 institutions of Japan between 2001 and 2005, the annual incidence of HLH was estimated as 1 in 800,000 per year. Among them, 567 cases were actually analyzed in this study. The most frequent subtype was Epstein-Barr virus (EBV)-associated HLH, followed by other infection- or lymphoma-associated HLH. Age distribution showed a peak of autoimmune disease- and infection-associated HLH in children, while FHL and lymphoma-associated HLH occurred almost exclusively in infants and the elderly, respectively. The 5-year overall survival rate exceeded 80% for patients with EBV- or other infection-associated HLH, was intermediate for those with FHL or B-cell lymphoma-associated HLH, and poor for those with T/NK cell lymphoma-associated HLH (<15%). Although this nationwide survey establishes the heterogeneous characteristics of HLH, the results should be useful in planning prospective studies to identify the most effective therapy for each HLH subtype.

Perforin gene (PRF1) mutations cause the primary form of hemophagocytic lymphohistiocytosis (HLH). We report a genetic defect of PRF1 in a 62-year-old Japanese man with recurrent episodes of HLH. Sequencing of PRF1 from both peripheral blood mononuclear cells and nail clippings showed compound heterozygous mutation, including deletion of two base pairs at codons 1090 and 1091 (1090-1091delCT) and guanine-to-adenine conversion at nucleotide position 916 (916GAEA). Although primary HLH has been detected in infants and children, genetic mutation of PRF1 or other genes should be considered a differential diagnosis of HLH even in the elderly.

In the long-chain dicarboxylic acids (DCA)-hyperproducing mutant Candida maltosa strains, methyl-ends of n-alkanes and fatty acids are hydroxylated by n-alkane inducible cytochromes P450 (P450alk), presumably as an essential step in DCA production. A significantly higher production of P450alks was observed in response to n-alkane in the DCA-hyperproducing mutant strain M2030 than in the wild-type strain 1098. Northern analysis demonstrated that n-tetradecane induction levels of mRNAs of all four ALK genes encoding major P450alk isoforms involved in n-alkane assimilation were significantly higher in the DCA-hyperproducing mutant than in the wild-type strain. Among these four ALK genes, enhancement of the transcriptional induction level of ALK5, which prefers fatty acids as substrates, was prominent in the mutant. In agreement with Northern analysis, promoters of ALK genes, especially that of ALK5, more strongly responded to n-alkanes in the DCA-hyperproducing mutant than in the wild-type strain. These results suggest that the transcriptional control of ALK genes in the DCA-hyperproducing mutant strains was altered preferably to accelerate DCA production.

Background: Congestive heart failure (CHF) is associated with altered energy homeostasis and myocardial inflammation, hypertrophy, and fibrosis. Adiponectin, an insulin-sensitizing adipocytokine, may affect these pathogenic factors, and the circulating adiponectin level may serve as a biological marker of CHF. This study aimed to assess the significance of serum adiponectin as a prognostic marker for Japanese CHF patients., Methods and Results: The serum adiponectin levels were compared between 54 (24 ischemic and 30 non-ischemic) CHF patients with left ventricular systolic dysfunction and 55 age- and gender-matched control subjects. The CHF patients also underwent simultaneous clinical assessment and measurements for brain natriuretic peptide (BNP) and parameters of lipid or glucose metabolism. Compared with the controls, the CHF patients showed significantly increased serum adiponectin levels [6.7 (4.9-12.6) vs 14.6 (9.7-25.4) microg/ml, p<0.0001]. In the CHF patients, the log-transformed values of the serum adiponectin levels positively correlated with the log-transformed values of the plasma BNP levels (p=0.0003, r=0.48) and inversely correlated with the body mass index (p=0.0006, r=-0.46). Furthermore, an increase in the serum adiponectin level was associated with higher mortality (p<0.05), particularly in the ischemic CHF patients (p<0.005)., Conclusions: An increase in the circulating adiponectin level was associated with higher mortality in the ischemic CHF patients. Adiponectin may be an informative risk marker for Japanese CHF patients.

Photobrightening and photobluing effects of photoluminescence (PL) from CdSe/ZnS/TOPO nanocrystal ensembles, i.e. ca. 1000 nanocrystals, have been investigated in several environments. The PL intensity increases more than twice with blue shift under continuous light irradiation in a wet nitrogen (H(2)O/N(2)) atmosphere, while in a pure nitrogen atmosphere and vacuum their behaviors are nothing particular. In addition, increasing rates of both the brightening and bluing effects in the H(2)O/N(2) atmosphere depend on irradiation intensities. These results suggest that both effects should be associated with photo-adsorption of water molecules onto nanocrystal surfaces. As for the brightening effect, we propose that the photo-adsorption of water molecules provides some restoring functions to PL-quenching defects or trap-sites on or near nanocrystal surfaces. On the other hand, the origin of the bluing effect is not clear from the results of the ensemble measurements. To get further insight into the bluing effect, PL spectra from single nanocrystals are measured under continuous light irradiation in cyclic environments in which the H(2)O/N(2) atmosphere and vacuum are alternating. As a result, blue-shifted PL induced by the light irradiation in the H(2)O/N(2) atmosphere recovers almost to its initial one reversibly under evacuation. The mechanism of the photobluing effect will be discussed also on the bases of the photobrightening model introduced above.

Background: The prognosis of gallbladder carcinoma is poor; therefore, investigating the efficacy of new chemotherapy agents is essential for the treatments for this tumor. Recently, several studies have reported clinical trials using gemcitabine as treatment for advanced gallbladder cancers. However, the antitumor effects of gemcitabine on gallbladder carcinoma have not been examined in in vitro and in vivo model systems., Methods: We examined the cytotoxicity of gemcitabine in four biliary tract cancer cell lines using the WST-1 assay. In addition, we examined the effect of gemcitabine on gallbladder cancers resulting from orthotopic inoculation of NOZ gallbladder tumor cells into nude mice. One week after transplantation, the mice were randomized into two groups: In Group A, the mice were treated by an intra-peritoneal injection of 0.9% sodium chloride for three weeks after inoculation (control). In Group B, the mice were treated by an intra-peritoneal injection of gemcitabine (125 mg / kg) for three weeks. All mice were sacrificed one week after the end of treatment, and macroscopic and histological findings were evaluated. The expression levels of proliferating-cell nuclear antigen (PCNA) were examined to investigate cellular proliferation activity, and Tunnel assays were performed to determine apoptotic status. Survival duration of the mice after gemcitabine treatment was compared to that of untreated mice., Results: The gemcitabine sensitivity of the four biliary tract cancer cell lines was similar in a dose dependent manner. In the in vivo models, the Group A mice showed huge tumors of the gallbladder, with liver invasion and lymph node metastases. However, there were no abdominal tumors in the Group B mice, and microscopic gallbladder cancer could only be detected from histological findings. The mean percent of PCNA-positive tumor cells was significantly higher in tumors from mice in Group A (71.9%) compared to those of Group B (34.7%). The mean percent of Tunnel-positive tumor cells was significantly lower in mice from Group A (2.0%) than those from Group B (5.7%). Survival duration was prolonged significantly in the gemcitabine-treated mice relative to untreated mice., Conclusions: Gemcitabine treatment may inhibit tumor progression and prolong survival in gallbladder cancer by inhibiting cell proliferation and inducing apoptosis.

We performed random sequencing of cDNAs from nine biologically or industrially important cultures of the industrially valuable fungus Aspergillus oryzae to obtain expressed sequence tags (ESTs). Consequently, 21 446 raw ESTs were accumulated and subsequently assembled to 7589 non-redundant consensus sequences (contigs). Among all contigs, 5491 (72.4%) were derived from only a particular culture. These included 4735 (62.4%) singletons, i.e. lone ESTs overlapping with no others. These data showed that consideration of culture grown under various conditions as cDNA sources enabled efficient collection of ESTs. BLAST searches against the public databases showed that 2953 (38.9%) of the EST contigs showed significant similarities to deposited sequences with known functions, 793 (10.5%) were similar to hypothetical proteins, and the remaining 3843 (50.6%) showed no significant similarity to sequences in the databases. Culture-specific contigs were extracted on the basis of the EST frequency normalized by the total number for each culture condition. In addition, contig sequences were compared with sequence sets in eukaryotic orthologous groups (KOGs), and classified into the KOG functional categories.

We developed a microfabricated electrochemical DNA chip for detection of polymerase chain reaction (PCR) products from 16S rRNA sequences of Clostridium piliforme (Cp), Helicobacter bilis (Hb) and Helicobacter hepaticus (Hh), and the nucleocapsid protein gene of mouse hepatitis virus (MHV). This chip does not require DNA labeling, and the hybridization signal can be detected as an anodic current. The average anodic currents of 9 (Cp), 5 (Hb), 8 (Hh) and 7 (MHV) PCR positive samples derived from feces of spontaneously infected mice (Cp, Hb and Hh) and MHV-contaminated tumor cells were 27.9+/-7.2, 31.9+/-8.1, 29.3+/-10.1, and 27.6+/-3.0 nA, respectively. On the other hand, the average anodic currents of 19 (Cp), 27 (Hb), 18 (Hh), and 13 (MHV) PCR negative samples were 0.3+/-2.9, 3.7+/-2.4, -1.0+/-1.7, and -2.3+/-2.7 nA, respectively. The anodic current increased with increasing concentrations of pathogens. For experimentally infected samples, the results of PCR/electrophoresis were in complete accord with those of this system when anodic currents of 6.1 (Cp), 8.5 (Hb), 2.4 (Hh), and 3.1 nA (MHV) were taken as the cut-off value. The results suggested that the electrochemical DNA chip system is useful for specific and quantitative detection of PCR products.

We generated two recombinant chicken IgYs, designated Ab3-15 and Ab4-19, against mammalian prion protein (PrP) from the single chain fragment of variable region (scFv) antibodies. These two antibodies recognized PrP(Sc) from bovine spongiform encephalopathy (BSE) in cattle and were more sensitive than the corresponding scFv antibodies. These antibodies also recognized PrP(Sc) from other scrapie-infected mammals. These results indicate that Ab3-15 and Ab4-19 are useful for diagnosis of BSE as well as other prion diseases.

Vesicular traffic during exocytosis is regulated by Rab GTPase, Sec4p in yeast, which is activated by a guanine nucleotide exchange factor (GEF) called Sec2p. The GEF activity is localized in the N-terminal 160 residues of Sec2p, which lacks sequence similarity with any other GEFs with known structures, and thereby the guanine nucleotide exchange mechanism by Sec2p remains unknown. Here, we report the crystal structure of the Sec2p GEF domain at 3.0 A resolution. The structure unexpectedly consists of a homodimeric, parallel coiled coil that extends over 180 A. Pull-down and guanine nucleotide exchange analyses on a series of deletion and point mutants of Sec2p unveiled the catalytic residues for its GEF activity as well as the Sec4p binding site, thus presenting a nucleotide exchange mechanism by a simple coiled coil. The present functional analyses allow us to build the Sec2p:Sec4p complex model, which explains the specificity for Rab GTPases by their respective GEF proteins.

Chitinases are thought to be involved in the morphogenesis and autolysis of filamentous fungi. We cloned a gene (chiB) encoding a class V chitinase from Aspergillus nidulans. ChiB expressed in Escherichia coli had chitin-hydrolyzing activity, indicating that chiB encoded a chitinase. Deletion of chiB affected neither germination efficiency nor hyphal growth rate, but considerably reduced the intracellular and extracellular chitinase activities. The decrease in hyphal dry weight during autolytic phase was slower in the mutant than in the wild-type strain. Western blot analysis indicated that the quantity of ChiB significantly increased when the wild-type mycelia were starved for carbon sources, a condition that induced hyphal autolysis. These results suggest that chiB plays an important role in the autolytic process in A. nidulans.

Mammalian interleukin-13 (IL-13) is an important regulatory T2 cytokine secreted by activated T lymphocytes. The IL-13 receptor (IL-13R) has two different chains, IL-13Ralpha1 and IL-13Ralpha2. Although the chicken IL-13 gene is well characterized, little is known about IL-13Rs. We cloned a cDNA encoding the 380 amino acid pro-peptide of chicken IL-13Ralpha2 (chIL-13Ralpha2) and developed a monoclonal antibody (mAb), HU13-1, against it. The chIL-13Ralpha2 amino acid sequence showed 37-39% sequence identity with mammalian homologs. High levels of chIL-13Ralpha2 mRNA were expressed in liver, testis, ovary, brain, and lipopolysaccharide (LPS)-stimulated IN24 cells. HU13-1 specifically recognized recombinant chIL-13Ralpha2 in ELISAs, and western blots identified a 45-kDa glycoprotein or a 41-kDa non-glycosylated protein in LPS-stimulated IN24 cell lysates. LPS induced a gradual increase in HU13-1-positive IN24 cells over 20 h. These results indicate that mAb HU13-1 recognizes native chIL-13Ralpha2 and will be valuable for further studies of chIL-13Rs.

Anoxomat Mart II (Mart Microbiology BV, Lichtenvooorde, Netherlands, Central Scientific Commerce Inc., Tokyo, Japan) is an anaerobic jar apparatus which uses a vacuum pump in combination with catalyst as gas replacement procedure to remove all traces of oxygen. As we had a chance to use Anoxomat Mart II, we compared it with other two anaerobic culture methods; namely AnaeroPack anaero (Mitsubishi Gas Chemical Co., Tokyo, Japan) which employs anaerobic jar method, and Concept400 (RUSKINN TECHNOLOGY LTD, England; Central Scientific Commerce INc., Tokyo, Japan) which uses anaerobic chamber method. We used 10 different species of anaerobic bacteria obtained from ATCC. One strain each of 10 species was cultured and examined for measurement of the sensitibity of an anaerobic indicator, th number of bacteria after 48 hour culture, the diameter of colonies, and MIC value. As a result, the time to reach the anaerobic condition was around 30 minutes by the Mart II against around 60 minutes by the AnaeroPack anaero. There was no difference concerning the number of bacteria after 48 hour culture among three methods. But anaerobic bacteria cultured by Mart II tended to make bigger colonies compared to other two methods in the 5 strains out of 9, except for one strain in which the diameter of colonies could not be measured. On the other hand, the comparison of MIC value showed good correlation in 11 antibiotics out of 12 among three methods. The MIC value of 11 antibiotics fitted within 1-fold difference, and 2-fold difference was observed in only one antibiotic. Mart II is so small that it does cheep consumables. From these reasons, we concluded that Mart II can be one of the useful anerobic culture methods.

We have previously demonstrated that Rab27 regulates dense granule secretion in platelets. Here, we analyzed the activation status of Rab27 using the thin layer chromatography method analyzing nucleotides bound to immunoprecipitated Rab27 and the pull-down method quantifying Rab27 bound to the GTP-Rab27-binding domain (synaptotagmin-like protein (Slp)-homology domain) of its specific effector, Slac2-b. We found that Rab27 was predominantly present in the GTP-bound form in unstimulated platelets due to constitutive GDP/GTP exchange activity. The GTP-bound Rab27 level drastically decreased due to enhanced GTP hydrolysis activity upon granule secretion. In permeabilized platelets, increase of Ca(2+) concentration induced dense granule secretion with concomitant decrease of GTP-Rab27, whereas in non-hydrolyzable GTP analogue GppNHp (beta-gamma-imidoguanosine 5'-triphosphate)-loaded permeabilized platelets, the GTP (GppNHp)-Rab27 level did not decrease upon the Ca(2+)-induced secretion. These data suggested that GTP hydrolysis of Rab27 was not necessary for inducing the secretion. Taken together, Rab27 is maintained in the active status in unstimulated platelets, which could function to keep dense granules in a preparative status for secretion.

Concentrations above the MIC of caspofungin allow growth of some Candida isolates. A strain demonstrating paradoxical growth was grown in the presence and absence of caspofungin, and the cell wall content was analyzed. Beta-1,3-glucan declined 81% in the presence of caspofungin, as expected. Beta-1,6-glucan declined 73%. Chitin increased 898%, demonstrating a mechanism for paradoxical growth-a rapid shift in the key polymer.

The extracellular polysaccharides (EPS) produced by lactic acid bacteria (LAB) are associated with the rheology, texture, and mouthfeel of fermented milk products, including yogurt. This study investigated the immunomodulatory effects of EPS purified from the culture supernatant of Lactobacillus delbrueckii ssp. bulgaricus (L. bulgaricus) OLL1073R-1. The crude EPS were prepared from the culture supernatant of L. bulgaricus OLL1073R-1 by standard chromatographic methods, and were fractionated into neutral EPS and acidic EPS (APS). Acidic EPS were further fractionated into high molecular weight APS (H-APS) and low molecular weight APS (L-APS). High molecular weight APS were shown to be phosphopolysaccharides containing D-glucose, D-galactose, and phosphorus. Stimulation of mouse splenocytes by H-APS significantly increased interferon-gamma production, and, moreover, orally administered H-APS augmented natural killer cell activity. Oral administration of yogurt fermented with L. bulgaricus OLL1073R-1 and Streptococcus thermophilus OLS3059 to mice showed a similar level of immunomodulation as H-APS. However, these effects were not detected following administration of yogurt fermented with the starter combination of L. bulgaricus OLL1256 and S. thermophilus OLS3295. We conclude from these findings that yogurt fermented with L. bulgaricus OLL1073R-1, containing immunostimulative EPS, would have an immunomodulatory effect on the human body.

Deficiency of osteoprotegerin (OPG), a soluble decoy receptor for receptor activator of nuclear factor-kappaB ligand (RANKL), in mice induces osteoporosis caused by enhanced bone resorption. Serum concentrations of RANKL are extremely high in OPG-deficient (OPG(-/-)) mice, suggesting that circulating RANKL is involved in osteoclastogenesis. RANKL(-/-) mice exhibit osteopetrosis, with the absence of osteoclasts. We examined the requirements for osteoclastogenesis using OPG(-/-) mice, RANKL(-/-) mice, and a system involving bone morphogenetic protein 2 (BMP-2)-induced ectopic bone formation. When collagen disks containing BMP-2 (BMP-2-disks) or vehicle were implanted into OPG(-/-) mice, osteoclast-like cells (OCLs) and alkaline phosphatase-positive OCLs appeared in BMP-2-disks but not the control disks. F4/80-positive osteoclast precursors were similarly distributed in both BMP-2- and control disks. Cells expressing RANKL were detected in the BMP-2-disks, and the addition of OPG to the disk inhibited OCL formation. Muscle cells in culture differentiated into alkaline phosphatase-positive cells in the presence of BMP-2 and accordingly expressed RANKL mRNA in response to PTH. This suggests that RANKL expressed by osteoblasts is a requirement for osteoclastogenesis. We then examined how osteoblasts are involved in osteoclastogenesis other than RANKL expression, using RANKL(-/-) mice. BMP-2- and control disks were implanted into RANKL(-/-) mice, which were injected with RANKL for 7 d. Many OCLs were observed in the BMP-2-disks and bone tissues but not the control disks. These results suggest that osteoblasts also play important roles in osteoclastogenesis through offering the critical microenvironment for the action of RANKL.

Liddle's syndrome (excessive absorption of sodium ions) and PHA-1 (pseudohypoaldosteronism type 1) with decreased sodium absorption are caused by the mutations in the amiloride-sensitive epithelial sodium channel ENaC. Rab proteins are small GTPases involved in vesicle transport, docking, and fusion. Earlier, we reported that Rab27a inhibits ENaC-mediated currents through protein-protein interaction in HT-29 cells. We hereby report that Rab27a-dependent inhibition is associated with the GTP/GDP status as constitutively active or GTPase-deficient mutant Q78L inhibits amiloride-sensitive currents whereas GDP-locked inactive mutant T23N showed no effect. In order to further explore the molecular mechanism of this regulation, we performed competitive assays with two Rab27a-binding proteins: synaptotagmin-like protein (SLP-5) and Munc13-4 (a putative priming factor for exocytosis). Both proteins eliminate negative modulation of Rab27a on ENaC function. The SLP-5 reversal of Rab27a effect was restricted to C-terminal C2A/C2B domains assigned for putative phospholipids-binding function while the Rab27a-binding SHD motif imparted higher inhibition. The ENaC-mediated currents remain unaffected by Rab27a though SLP-5 appears to strongly bind it. The immunoprecipitation experiments suggest that in the presence of excessive Munc13-4 and SLP-5 proteins, Rab27a interaction with ENaC is diminished. Munc13-4 and SLP-5 limit the Rab27a availability to ENaC, thus minimizing its effect on channel function. These observations decisively prove that Rab27a inhibits ENaC function through a complex mechanism that involves GTP/GDP status, and protein-protein interactions involving Munc13-4 and SLP-5 effector proteins.

A 44-day-old male infant with familial hemophagocytic lymphohistiocytosis (FHL) associated with the MUNC13-4 mutation is reported. He presented with fever and poor feeding, lymphocytosis with thrombocytopenia and CSF pleocytosis without virological explanation. On the basis of progressive hyperferritinemia (1323 ng/ml), anemia (hemoglobin: 5.2 g/dl), hypertriglyceridemia (547 mg/dl) and increased LDH (1063 IU/l) with hemophagocytosis in the bone marrow, hemophagocytic lymphohistiocytosis was diagnosed. He showed a good response to corticosteroid therapy and the disease was stable for more than 5 months. Thereafter, he suffered from central nervous system complications, and successfully underwent unrelated cord blood stem cell transplantation. A remission was observed for more than 2 years, with mild mental retardation. Genetic analysis revealed that he had a compound heterozygous mutation of MUNC13-4; namely a novel 2163G>A mutation resulting in W721X, and 754-1G>C resulting in a premature stop codon in this gene. Western blot analysis showed the complete loss of the MUNC13-4 protein, whereas other molecules associated with the SNARE systems were detected at normal levels. Conclusion. FHL may have a broad clinical spectrum, and further analysis on its phenotype-genotype association is required to establish an appropriate treatment strategy, including immunochemotherapy and stem cell transplantation in the future.

Molecular orientation of poly(di-n-hexylsilane) adsorbed on poly(vinyl alcohol) film has been studied by making use of the stretching technique. Dichroic ratio, Rd, strongly depended on the thickness of poly(di-n-hexylsilane) thin film and the highest value ca. 19 was observed at the film thickness of 110 +/- 30 nm. The thermal hysteresis of the molecular orientation was observed in the heating-cooling cycles. By studying the fluorescence spectrum it was confirmed that a portion of the poly(di-n-hexylsilane) molecules were in transoid conformation even at 320 K, although most of poly(di-n-hexylsilane) molecules were in disordered conformation (conformation D). This poly(di-n-hexylsilane) in transoid conformation is formed in the stretching process and may play a role of crystallization nucleus to induce the whole orientation of the poly(di-n-hexylsilane) in the film state.

The International Committee on Taxonomy of Viruses (ICTV) recently accepted Endornavirus as a new genus of plant dsRNA virus. We have determined the partial nucleotide sequences of the RNA-dependent RNA polymerase regions from the large dsRNAs (about 14 kbp) isolated from barley (Hordeum vulgare), kidney bean (Phaseolus vulgaris), melon (Cucumis melo), bottle gourd (Lagenaria siceraria), Malabar spinach (Basella alba), seagrass (Zostera marina), and the fungus Helicobasidium mompa. Phylogenetic analyses of these seven dsRNAs indicate that these dsRNAs are new members of the genus Endornavirus that are widely distributed over the plant and fungal kingdoms.

Photophysical and photochemical processes of 9-methyl- and 9-phenyl-9,10-dihydro-9-silaphenanthrene derivatives have been studied at room temperature and 77 K in comparison with the carbon analogue, 9,10-dihydrophenanthrene. These 9,10-dihydro-9-silaphenanthrene derivatives show smaller fluorescence quantum yield and remarkably larger Stokes shifts than those of the carbon analogue. In contrast, their phosphorescence quantum yields are two times larger than those of the carbon analogue, although the absolute value is not so large (approximately 0.1). Reaction products and intermediates produced by the 266 nm light photolysis have been studied, and it has been confirmed that 9-methyl- and 9-phenyl-9-silaphenanthrenes have been photochemically formed in methylcyclohexane at 77 K, in addition to the formation of radical cations of 9,10-dihydro-9-silaphenanthrene derivatives and the carbon-centered radical: 9-hydro-9-silaphenanthrenyl radical.

The polarized synthesis of cell wall components such as chitin is essential for the hyphal tip growth of filamentous fungi. The actin cytoskeleton is known to play important roles in the determination of hyphal polarity in Aspergillus nidulans. Previously, we suggested that CsmA, a chitin synthase with a myosin motor-like domain (MMD), was involved in polarized chitin synthesis in a manner dependent on the interaction between the MMD and the actin cytoskeleton. The genome database indicates that A. nidulans possesses another gene encoding another chitin synthase with an MMD. In this study, we characterized this gene, which we designated csmB. The csmB null mutants examined were viable, although they exhibited defective phenotypes, including the formation of balloons and intrahyphal hyphae and the lysis of subapical regions, which were similar to those obtained with csmA null mutants. Moreover, csmA csmB double null mutants were not viable. Mutants in which csmB was deleted and the expression of csmA was under the control of the alcA promoter were viable but severely impaired in terms of hyphal growth under alcA-repressing conditions. We revealed that CsmB with three copies of a FLAG epitope tag localized at the hyphal tips and forming septa, and that the MMD of CsmB was able to bind to actin filaments in vitro. These results suggest that CsmA and CsmB perform compensatory functions that are essential for hyphal tip growth.

Background: Although 5-Fluorouracil (5-FU) is widely used in chemotherapeutic treatment for gallbladder cancer, there is little evidence of its effectiveness. Thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) are the major determinants of individual sensitivity to 5-FU, and the impact of TS and DPD expression on prognosis and 5-FU efficacy has been studied in gastrointestinal carcinomas., Materials and Methods: TS and DPD enzymatic activities in frozen samples of 7 gallbladder cancer tissues and immunohistochemical TS and DPD protein expressions in 53 resected gallbladder cancers were evaluated., Results: The DPD activity of gallbladder cancers was significantly higher than that of gastric and colon cancers (p=0.041). TS and DPD protein expressions increased in pT2 and pT3 cases compared to those in pT1 cases, and cases both with high TS and DPD expressions had a worse prognosis than both low TS and DPD expressions. However, TS and DPD expressions were not independent prognostic factors., Conclusion: TS and DPD protein expressions in gallbladder cancers were frequently high in pT2 and pT3 gallbladder cancers and control of high TS or DPD levels may be important for advanced gallbladder cancer therapy.

We describe a simple method for humanizing chicken monoclonal antibody (mAb). Humanization of mAbs by simple CDR-grafting often results in loss of affinity because certain framework residues of the antibody variable regions can participate in antigen-antibody interaction. In this study, humanization of chicken mAbs was achieved by CDR-grafting, followed by framework fine-tuning using a chicken phage-displayed mAb, phAb4-31, as a model antibody. In order to fine-tune the framework, we used the phage-displayed combinatorial library with permutation of important framework residues. After panning the humanized library, the "most humanized" variants were selected and analyzed for antigen-binding activity. All of these clones retained affinity comparable to the parental chicken mAb. These results suggest that chicken mAbs can easily be humanized, and thus humanized chicken mAbs may be practically applied as therapeutic agents.

We tested the effects of several combinations of bait and fish components of the yeast two-hybrid detection system for estrogenic activity. A combination of the full-length human estrogen receptor alpha with the nuclear receptor-binding domain of co-activator steroid receptor co-activator-1 (SRC-1) or transcriptional intermediate factor-2 (TIF-2) was most effective for estrogen-dependent induction of the chromosome-integrated UAS(GAL)-CYC1(p)-lacZ reporter construct among the two-hybrid systems so far tested.

Leukemia-inhibitory factor (LIF) is indispensable for maintaining the undifferentiated state when propagating mouse embryonic stem (ES) cells. We previously cloned chicken LIF (chLIF) cDNA and demonstrated that it maintained chicken ES cell cultures in an undifferentiated state. Here, we developed two monoclonal antibodies, HUL-1 and HUL-2, against chLIF, which specifically recognized recombinant chLIF (rchLIF) produced by Escherichia coli and Chinese hamster ovary K1 cells, in enzyme-linked immunosorbent assays and Western blot analysis. In addition, HUL-2 inhibited the phosphorylation of signal transducer and activator of transcription 3 by rchLIF in chicken blastodermal cells (CBCs), but not that of mitogen-activated protein kinase kinase. Furthermore, the addition of HUL-2 to CBC cultures resulted in embryoid bodies forming earlier than in normal cultures. These results indicated that HUL-2 recognized not only rchLIF but also native chLIF, and suggested that CBCs in culture produce LIF, which functions in autocrine signaling.

Objective: Our aim is to assess the clinicopathological significance of E-cadherin and beta-catenin expression, as well as their association with apoptosis in gallbladder cancers., Methods: The expression of E-cadherin and beta-catenin proteins was examined in 4 biliary tract cancer cell lines and 49 gallbladder cancer specimens by immunofluorescent or immunohistochemical methods and Western blotting. The apoptotic status was evaluated in the cell lines by poly(ADP-ribose) polymerase Western blotting and in the tumors by the TdT-mediated dUTP nick end labeling assay., Results: Expression of poly(ADP-ribose) polymerase (apoptosis) was only seen in cell lines that expressed both E-cadherin and beta-catenin. Reduced expression of E-cadherin and beta-catenin was frequently seen in advanced gallbladder cancer cases (61 and 83%, respectively) relative to pT1 cases (25 and 63%, respectively). The 5-year survival rate in cases with reduced E-cadherin expression was 26%, significantly lower than in cases with preserved E-cadherin expression (70%; p = 0.017). Cases with reduced expression of both had lower apoptotic indices and showed a worse prognosis compared with cases with reduced expression of either E-cadherin or beta-catenin (p = 0.04 and 0.049, respectively)., Conclusions: The expression of E-cadherin or beta-catenin frequently diminishes as the tumor progresses, and abnormalities of E-cadherin and beta-catenin expression were associated with decreased apoptosis in gallbladder cancers. E-cadherin expression might be a useful prognostic marker in this tumor.

Interleukin-6 (IL-6) is a multifunctional cytokine that plays roles in regulating immune responses, acute phase reactions and hematopoiesis. IL-6 signaling is regulated by two receptors, a specific alpha chain (IL-6Ralpha) and a signal transducer, gp130. In this study, cDNA encoding the 445 amino acid propeptide of chicken IL-6Ralpha (chIL-6Ralpha) was identified. The predicted 445 amino acids showed approximately 40% sequence identity with mammalian homologues. In a domain search, chIL-6Ralpha had a signal peptide of 20 residues, an immunoglobulin-like (IG) domain of 71 residues and a fibronectin-type III (FN III) domain of 85 residues. On comparison with mammalian homologues, four conserved cysteine residues and the WSXWS motif were observed in the N- and C-terminal regions of the FN III domain, respectively. Expression analysis revealed that chIL-6Ralpha is strongly expressed in liver and the chicken hepatoma cell line LMH. These findings indicate that the identified chicken cDNA sequence encodes a chIL-6Ralpha homologue.

Antiplatelet therapy has been established as a preventive medicine for ischemic cardiovascular diseases both at acute and chronic phases. This therapy is also crucial for the prevention of thrombotic events after coronary stent implantation. So far, many lines of clinical evidence have demonstrated the beneficial effects of aspirin (an irreversible cyclooxygenase inhibitor) and thienopyridine derivatives (adenosine diphosphate (ADP)-receptor P2Y12 inhibitors). Recently, it has been reported that the cardiovascular risk is elevated in patients with platelets resistant to these drugs, compared to the good responders. One of the current problems to be solved in antiplatelet therapy is to find out patients resistant to the antiplatelet therapy and improve its preventive effects. In addition to aspirin and thienopyridines, several types of drugs with antiplatelet effects are currently available in clinical practice. Clinical evidence has recently been accumulating for these drugs that can be potential alternatives in patients with aspirin or thienopyridine resistance. In this review, the mechanisms, evidence and approach to the present problems of drugs with antiplatelet effects are discussed.

In this chapter, we describe the methods we have used to show that chicken leukemia inhibitory factor (LIF) maintains chicken embryonic stem (ES) cells in an undifferentiated state in culture. Recombinant chicken LIF (rchLIF) was expressed as a fusion protein linked to glutathione S-transferase (GST) and purified to greater than 90% purity in two chromatography stages, the first an affinity step using the GST tail, which was cleaved before further purification by gel chromatography. Chicken ES cells were obtained by culturing chicken blastodermal cells isolated from stage X embryos of freshly laid chicken eggs. These cells can be maintained in media containing rchLIF for at least 9 d without any other cytokines or feeder cells. Chicken ES cells were characterized by the expression of alkaline phosphatase activity, stage-specific embryonic antigen (SSEA)-1 and embryonal carcinoma cell monoclonal antibody-1. In addition, the phosphorylation of signal transducers and activators of transcription-3 by LIF, which is sufficient to maintain the undifferentiated state of ES cells, was detected by Western blotting analysis.

The genome of Aspergillus oryzae, a fungus important for the production of traditional fermented foods and beverages in Japan, has been sequenced. The ability to secrete large amounts of proteins and the development of a transformation system have facilitated the use of A. oryzae in modern biotechnology. Although both A. oryzae and Aspergillus flavus belong to the section Flavi of the subgenus Circumdati of Aspergillus, A. oryzae, unlike A. flavus, does not produce aflatoxin, and its long history of use in the food industry has proved its safety. Here we show that the 37-megabase (Mb) genome of A. oryzae contains 12,074 genes and is expanded by 7-9 Mb in comparison with the genomes of Aspergillus nidulans and Aspergillus fumigatus. Comparison of the three aspergilli species revealed the presence of syntenic blocks and A. oryzae-specific blocks (lacking synteny with A. nidulans and A. fumigatus) in a mosaic manner throughout the genome of A. oryzae. The blocks of A. oryzae-specific sequence are enriched for genes involved in metabolism, particularly those for the synthesis of secondary metabolites. Specific expansion of genes for secretory hydrolytic enzymes, amino acid metabolism and amino acid/sugar uptake transporters supports the idea that A. oryzae is an ideal microorganism for fermentation.