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A Nonomuraea sp. strain MM565M-173N2 was isolated from deep-sea sediment off the Sanriku coast, and new antibiotics were evaluated against carbapenem-resistant Enterobacteriaceae (CRE), which is a problematic group of bacteria because of their antimicrobial resistance. From 220 l of fermented broth from strain MM565M-173N2, we isolated four new antibiotics by gel filtration and HPLC, designated as sealutomicins A (1.8 mg), B (1.5 mg), C (0.8 mg), and D (0.8 mg). Their structures were determined from MS, NMR, and CD spectra. Sealutomicin A was found to be a new enediyne antibiotic, while sealutomicins B-D were aromatized products from sealutomicin A. Sealutomicin A showed strong antibacterial activity (MIC 0.05-0.2 μg ml-1) against CRE.

Introduction Quantitative measurement of anti-A/-B antibody titers is important during ABO-incompatible living kidney transplantation (ABOi-LKT). Methods We conducted a multi-institutional study to measure the antibody titers using the automated column agglutination technique (auto-CAT) and tube test (TT) method in ABOi-LKT recipients. Statistical analysis was performed to evaluate the two methods. Results We examined 111 samples from 35 ABOi-LKT recipients at four institutions. The correlation coefficient of the two methods was >0.9; the concordance rate and clinically acceptable concordance rate for the IgG titers were 60.4% and 88.3%, respectively. Perioperative status did not influence the statistical significance. Parallel changes were observed in the IgG antibody titers measured using the auto-CAT or TT technique by desensitizing therapy in time-course monitoring. Conclusion Auto-CAT is comparable with the TT technique and is feasible for IgG anti-A/B antibody titration in ABOi-LKT recipients. This article is protected by copyright. All rights reserved.

Outcomes of allogeneic hematopoietic stem cell transplantation (HSCT) for hepatitis-associated aplastic anemia have not been fully evaluated. In the present study, the outcomes of 37 adult patients with hepatitis-associated aplastic anemia who underwent allogeneic HSCT were retrospectively analyzed using the registry database of Japan Society for Hematopoietic Cell Transplantation. The median age of the patients was 24 years (range, 16-61). The median period between diagnosis of hepatitis-associated aplastic anemia and HSCT was 6.0 months (range, 0.5-430.8). Stem cell sources were bone marrow (N = 19) or peripheral blood stem cells (N = 5) from an HLA-identical sibling or bone marrow (N = 11) and cord blood (N = 2) from an unrelated donor. The majority of conditioning regimens were fludarabine-based or high-dose cyclophosphamide-based. In all but 2 cases of early death, neutrophil engraftment was achieved. At the time of analysis, 32 patients were alive, with a median follow-up of 54.1 months. Five-year overall and failure-free survival rates were 86.0% (95% CI, 69.4-93.9%) and 75.0% (95% CI, 57.4-86.2%), respectively. Despite the heterogeneity in transplant procedures in a small number of patients, these results suggest that allogeneic HSCT is safe for use in hepatitis-associated aplastic anemia with a low rate of transplant-related mortality.

The utility of convolutional neural networks (CNNs) for sex estimation of the pelvis was evaluated using depth images generated from reconstructed three-dimensional (3D) computed tomography images. The 3D volume data were normalized by a homologous modeling technique to create polygon data with identical topology, then captured images for learning and testing. The neural networks were trained via transfer learning. As a result, a correct assignment rate >90% was obtained in most trials. The frontal view of the pelvis with 60-degree inclination achieved the best results. Selecting samples close to the average images of the sex was effective for training.

We characterized a protease of the M4 family from the cold-adapted Vibrio sp. Pr21 that was isolated from seawater at 320-m deep in Sagami Bay, Japan, and named it as PR protease based on the strain name Pr21. The PR protease had activities at 10–60 °C and 0.1–350 MPa, with the optimal temperature and pressure at 40 °C and 250 MPa. The mutant 10C9 (Q301P) obtained by error-prone PCR had higher activities than the wild-type enzyme at 10–60 °C, and the Q301P mutation contributed to the increase of the activity. The specific activity value of 10C9 was also higher than that of the wild-type enzyme at 0.1–200 MPa, but the specific activity ratios (1.28–1.59) of 10C9/wild-type enzyme at 50–200 MPa at 30 °C were smaller than those at 10–60 °C (1.73–4.39) at 0.1 MPa. The catalytic efficiency value of 10C9 was lower than that of the wild-type enzyme at 200 MPa. The homology models of PR protease suggested that the side chain of Q301 was hydrogen-bonded with the carbonyl oxygen atom of the main chain of N234 in the wild-type enzyme, and P301 had no contact with N234 in 10C9. The break of the hydrogen bond in 10C9 might strengthen the increase of the flexibility of the β-sheet near the substrate binding pocket under high-temperature conditions, whereas the flexibility of the β-sheet in 10C9 might be moderately increased compared to that in the wild-type enzyme under high-pressure conditions.

Identification of specific drug targets is very important for cancer therapy. We recently identified zinc finger protein of the cerebellum 5 (ZIC5) as a factor that promotes melanoma aggressiveness by platelet-derived growth factor D (PDGFD) expression. However, its roles in other cancer types remain largely unknown. Here we determined the roles of ZIC5 in prostate cancer (PCa) and colorectal cancer (CRC) cells. Results showed that ZIC5 was highly expressed in CRC and dedifferentiated PCa tissues, whereas little expression was observed in relevant normal tissues. Knockdown of ZIC5 decreased proliferation of several PCa and CRC cell lines with induction of cell death. ZIC5 knockdown significantly suppressed PDGFD expression transcriptionally, and PDGFD suppression also decreased proliferation of PCa and CRC cell lines. In addition, suppression of ZIC5 or PDGFD expression decreased levels of phosphorylated focal adhesion kinase (FAK) and signal transducer and activator of transcription 3 (STAT3) which are associated with PCa and CRC aggressiveness. Furthermore, knockdown of ZIC5 or PDGFD enhanced death of PCa and CRC cells induced by the anti-cancer drugs docetaxel or oxaliplatin, respectively. These results suggest that ZIC5 and PDGFD promote survival of PCa and CRC cells by enhancing FAK and STAT3 activity, and that the roles of ZIC5 are consistent across several cancer types.

Deep-sea bacteria can produce various biotechnologically relevant enzymes due to their adaptations to high pressures and low temperatures. To identify such enzymes, we have sequenced the genome of the polycaprolactone-degrading bacterium Moritella sp. JT01, isolated from sediment samples from Japan Trench (6957 m depth), using a Illumina HiSeq2000 sequencer (12.1 million paired-end reads) and CLC Genomics Workbench (version 6.5.1) for the assembly, resulting in a 4.83-Mb genome (42 scaffolds). The genome was annotated using Rapid Annotation using Subsystem Technology (RAST), Protein Homology/analogY Recognition Engine V 2.0 (PHYRE2), and BLAST2Go, revealing 4439 protein coding sequences and 101 RNAs. Gene products with industrial relevance, such as lipases (three) and esterases (four), were identified and are related to bacterium's ability to degrade polycaprolactone. The annotation revealed proteins related to deep-sea survival, such as cold-shock proteins (six) and desaturases (three). The presence of secondary metabolite biosynthetic gene clusters suggests that this bacterium could produce nonribosomal peptides, polyunsaturated fatty acids, and bacteriocins. To demonstrate the potential of this genome, a lipase was cloned an introduced into Escherichia coli. The lipase was purified and characterized, showing activity over a wide temperature range (over 50% at 20-60 °C) and pH range (over 80% at pH 6.3 to 9). This enzyme has tolerance to the surfactant action of sodium dodecyl sulfate and shows 30% increased activity when subjected to a working pressure of 200 MPa. The genomic characterization of Moritella sp. JT01 reveals traits associated with survival in the deep-sea and their potential uses in biotechnology, as exemplified by the characterized lipase.

Earlier reports suggested that umbilical cord blood transplantation (UCBT) for aplastic anemia (AA) was feasible in alternative transplantation. To identify differences in outcomes of UCBT and HLA-matched or mismatched unrelated bone marrow transplantation (UBMT) in adults with AA, we analyzed registry data of the Japan Society for Hematopoietic Cell Transplantation and compared results of UCBT (n = 69) to 8/8-matched (n = 101), 7/8-matched (n = 65), or 6/8-matched (n = 37) UBMT. The transplantation period was from 2002 to 2012, and patients 16 years or older with AA were eligible. Median ages were 49, 35, 28, and 30 years for UCBT, 8/8-matched, 7/8-matched, and 6/8-matched UBMT, respectively. In multivariate analysis, risk of mortality was lower for 8/8-matched UBMT compared with that of UCBT (hazard ratio [HR], .55; 95% confidence interval [CI], .32 to .94; P = .029), adjusted for age and graft-versus-host disease (GVHD) prophylaxis, which were other associated factors. Mortality risks of 7/8-matched UBMT (HR, .55; 95% CI, .29 to 1.02) or 6/8-matched UBMT (HR, .67; 95% CI, .32 to 1.39) were not significantly different from those of UCBT. Risks of grade 3 or 4 acute and chronic GVHD were not different among the 4 groups. The most prevalent cause of death was graft failure in UCBT and 6/8-matched UBMT and infection in 8/8-matched and 7/8-matched UBMT. Under 40 years old,survival of UCBT was similar to that of UBMT (76%, 79%, 83%, and 83% for UCBT and 8/8-matched, 7/8-matched, and 6/8-matched UBMT, respectively, at 3 years), adjusted for transplantation period, which was another associated factor; however, for ages over 40 years, that of UCBT tended to be lower (47%, 64%, 64%, and 75% for UCBT, 8/8-matched, 7/8-matched, and 6/8-matched UBMT, respectively, at 3 years). To conclude, these data suggest that UCBT could be an alternative treatment option for younger adults when matched sibling or adequate UBMT donors are not available.

Licensing by self MHC class I ligands is required for proper natural killer (NK) cell response. NK cells with inhibitory killer cell immunoglobulin-like receptors for nonself MHC exhibit transient alloreactivity after hematopoietic stem cell transplantation (HSCT). We analyzed 3866 recipients in the Japan national registry who underwent their first allogeneic HSCT for acute myeloid leukemia (AML) or acute lymphoblastic leukemia (ALL) from HLA-A, -B, and -DRB1 allele-genomatched unrelated donors. By classifying them into 5 independent groups based on HLA-C group matching and assumed donor NK cell status, we found that for HLA-C–matched HSCT for AML in HLA-C1/C1 recipients, in whom transient alloreactivity against HLA-C2–negative leukemic cells was expected, the relapse rate was significantly lower than it was in HLA-C–matched HSCT for AML in HLA-C1/C2 recipients (hazard ratio [HR], .72; P = .011). This difference was not observed in HLA-C–matched HSCT for ALL. Compared with HLA-C–matched HSCT, significantly higher mortality was observed in HLA-C1/C1 AML patients who received transplants from HLA-C–mismatched HLA-C1/C1 donors (HR, 1.37; P = .001) and in HLA-C1/C1 ALL patients who received transplants from HLA-C2–positive donors (HR, 2.13; P = .005). In conclusion, donor selection based on leukemic subtype and donor HLA-C group matching improves transplantation outcome after HLA-C–mismatched HSCT.

Cytomegalovirus (CMV) infection is a major infectious complication after allogeneic hematopoietic cell transplantation (allo-HSCT). Recently, it was reported that CMV reactivation is associated with a decreased risk of relapse in patients with acute myeloid leukemia (AML). The aim of this study was to evaluate the impact of early CMV reactivation on the incidence of disease relapse after allo-HSCT in a large cohort of patients. The Japan Society for Hematopoietic Cell Transplantation's Transplantation-Related Complication Working Group retrospectively surveyed the database of the Transplant Registry Unified Management Program at the Japan Society for Hematopoietic Cell Transplantation. Patients with AML (n = 1836), acute lymphoblastic leukemia (ALL, n = 911), chronic myeloid leukemia (CML, n = 223), and myelodysplastic syndrome (MDS, n = 569) who underwent their first allo-HSCT from HLA-matched related or unrelated donors between 2000 and 2009 and who survived without disease relapse until day 100 after transplantation were analyzed. Patients who received umbilical cord blood transplantation were not included. Patients underwent surveillance by pp65 antigenemia from the time of engraftment, and the beginning of preemptive therapy was defined as CMV reactivation. Cox proportional hazards models were used to evaluate the risk factors of relapse, nonrelapse, and overall mortality. CMV reactivation and acute/chronic graft-versus-host disease (GVHD) were evaluated as time-dependent covariates. CMV reactivation was associated with a decreased incidence of relapse in patients with AML (20.3% versus 26.4%, P = .027), but not in patients with ALL, CML, or MDS. Among 1836 patients with AML, CMV reactivation occurred in 795 patients (43.3%) at a median of 42 days, and 436 patients (23.7%) relapsed at a median of 221 days after allo-HSCT. Acute GVHD grades II to IV developed in 630 patients (34.3%). By multivariate analysis considering competing risk factors, 3 factors were significantly associated with a decreased risk of AML relapse and 1 factor with an increased risk of AML relapse: CMV reactivation (hazard ratio [HR], .77; 95% confidence interval [CI], .59 to .99), unrelated donor compared with related donor (HR, .59; 95% CI, .42 to .84), development of chronic GVHD (HR, .77; 95% CI, .60 to .99), and pretransplantation advanced disease status compared with standard disease status (HR, 1.99; 95% CI, 1.56 to 2.52). However, CMV reactivation was associated with increased nonrelapse mortality (HR, 1.60; 95% CI, 1.18 to 2.17) and overall mortality (HR, 1.37; 95% CI, 1.11 to 1.69). A beneficial effect of CMV reactivation on subsequent risk of relapse was observed in patients with AML but not in those with other hematological malignancies. However, this benefit was nullified by the increased nonrelapse mortality. The underlying mechanism is unclear; however, immunological activation against CMV reactivation plays an essential role in this association. Thus, immune augmentation treatment options, including vaccination and adoptive T cell transfer, may be useful to take advantage of the efficacy of CMV reactivation with minimal increase in nonrelapse mortality.

Little is known regarding the chimerism status after reduced-intensity conditioning transplantation when bone marrow is used as a stem cell source. We prospectively analyzed lineage-specific chimerism and retrospectively evaluated clinical outcomes in 80 adult patients who underwent unrelated donor bone marrow transplantation (URBMT) with fludarabine plus melphalan (FM) as the conditioning regimen. Mixed donor chimerism (MDC) was seen in 43 and 10 % of patients at days 14 and 28, respectively. Melphalan at ≤130 mg/m(2) was associated with an increased incidence of MDC at day 28 (P = 0.03). Patients with MDC at day 14 showed a marginally increased risk of primary graft failure and a marginally decreased risk of graft-versus-host disease. In multivariate analysis, MDC at day 14 was associated with higher overall mortality (hazard ratio (HR) = 2.1; 95 % confidence interval (CI), 1.1-4.2; P = 0.04) and relapse rate (HR = 3.0; 95 % CI, 1.2-7.5; P = 0.02), but not with non-relapse mortality (HR = 1.8; 95 % CI, 0.70-4.6; P = 0.23). Thus, the FM regimen yields prompt complete donor chimerism after URBMT, but the melphalan dose significantly impacts the kinetics of chimerism. Chimerism status evaluation at day 14 may be instrumental in predicting relapse after URBMT with the FM regimen.

Background Hematopoietic stem cell transplantation (HSCT) is one curable option for high-risk acute lymphoblastic leukemia (ALL); however, transplant-related toxicities might be severe in patients with Down syndrome and ALL (DS-ALL). Procedure HSCTs performed in patients with DS-ALL were identified in the Japan Society for Hematopoietic Cell Transplantation registry. Results In the registry data, 11 patients with DS-ALL were identified. The median age at HSCT was 9 years (range: 6–22 years). Six patients underwent HSCT at non-remission status. Allogeneic grafts were utilized in all patients, including eight patients who received HSCT from unrelated donors. Reduced intensity conditioning regimens were used in three patients. All patients achieved neutrophil engraftment by a median of day 18 (range: day 11–61). Ten patients experienced grade 3 or more infectious episodes. Six patients experienced complications of the respiratory system. The incidences of II–IV or III–IV acute GVHD were nine (81.8%) or seven patients (63.6%), respectively. Chronic GVHD was observed in five (55.6%) out of nine evaluable patients. Seven patients died at a median of 6 months (range: 0–24 months) after HSCT. Two-year relapse-free and overall survival were 33.3% (95% CI: 2.5–64.1%) or 37.5% (95% CI: 5.9–69.1%), respectively. The causes of death were relapse (n = 2), infection (n = 2), bleeding (n = 1), thrombotic microangiopathy (n = 1), and chronic GVHD (n = 1). Conclusions Therapy-related mortality accounted for five out of seven deceased patients in this case series. Attempts to reduce toxicities should be considered in HSCT for patients with DS-ALL. Pediatr Blood Cancer 2015;62:148–152. © 2014 Wiley Periodicals, Inc.

Trisomy 8 (+8) is one of the most common cytogenetic abnormalities in adult patients with acute myeloid leukemia (AML). However, the outcome of allogeneic hematopoietic stem cell transplantation (HSCT) in adult patients with AML harboring +8 remains unclear. To evaluate, the outcome and prognostic factors in patients with AML harboring +8 as the only chromosomal abnormality or in association with other abnormalities, we retrospectively analyzed the Japanese registration data of 631 adult patients with AML harboring +8 treated with allogeneic HSCT between 1990 and 2013. In total, 388 (61%) patients were not in remission at the time of HSCT. With a median follow-up of 38.5 months, the probability of overall survival and the cumulative incidence of relapse at 3 years were 40 and 34%, respectively. In the multivariate analysis, two or more additional cytogenetic abnormalities and not being in remission at the time of HSCT were significantly associated with a higher overall mortality and relapse. Nevertheless, no significant impact on the outcome was observed in cases with one cytogenetic abnormality in addition to +8. Although more than 60% of the patients received HSCT when not in remission, allogeneic HSCT offered a curative option for adult patients with AML harboring +8.

Trisomy 8 (+8) is 1 of the most common cytogenetic abnormalities in adult patients with myelodysplastic syndrome (MDS). However, the outcome of allogeneic hematopoietic stem cell transplantation (HSCT) in adult patients with MDS harboring +8 remains unclear. To evaluate the outcome and prognostic factors in patients with MDS harboring +8 as the sole cytogenetic abnormality or in association with other abnormalities, we retrospectively analyzed the Japanese registration data of 381 adult patients with MDS harboring +8 treated with allogeneic HSCT between 1990 and 2013. With a median follow-up period of 53 months, the probability of overall survival and cumulative incidence of relapse at 4 years were 51% and 22%, respectively. In the multivariate analysis, age > 50 years, 2 or more additional cytogenetic abnormalities, and a high risk at the time of HSCT according to the FAB/WHO classification were significantly associated with a higher overall mortality. Nevertheless, no significant impact of the outcome was observed in patients with 1 cytogenetic abnormality in addition to +8. Although 221 patients (58%) had advanced MDS at the time of HSCT, allogeneic HSCT offered a curative option for adult patients with MDS harboring +8.

Insights into mechanisms of drug resistance could extend the efficacy of cancer therapy. To probe mechanisms in melanoma, we performed siRNA screening of genes that mediate the development of neural crest cells, from which melanocytes are derived. Here, we report the identification of ZIC5 as a mediator of melanoma drug resistance. ZIC5 is a transcriptional suppressor of E-cadherin expressed highly in human melanoma. ZIC5 enhanced melanoma cell proliferation, survival, drug resistance, in vivo growth and metastasis. Microarray analysis revealed that ZIC5 downstream signaling included PDGFD and FAK activation, which contributes to drug resistance by enhancing STAT3 activation. Silencing of ZIC5 or PDGFD enhanced the apoptotic effects of BRAF inhibition and blocked survival of melanoma cells resistant to BRAF inhibitors. Furthermore, inhibition of FAK or STAT3 suppressed expression of ZIC5, which was positively regulated by PDGFD, FAK, and STAT3 in a positive feedback loop. Taken together, our results identify ZIC5 and PDGFD as candidate therapeutic targets to overcome drug resistance in melanoma. Cancer Res; 77(2); 366–77. ©2016 AACR.

Localised brain tissue damage activates surrounding astrocytes, which significantly influences subsequent long-term pathological processes. Most existing focal brain injury models in rodents employ craniotomy to localise mechanical insults. However, the craniotomy procedure itself induces gliosis. To investigate perilesional astrocyte activation under conditions in which the skull is intact, we created focal brain injuries using light exposure through a cranial window made by thinning the skull without inducing gliosis. The lesion size was maximal at ~ 12 h and showed substantial recovery over the subsequent 30 days. Two distinct types of perilesional reactive astrocyte, identified by GFAP upregulation and hypertrophy, were found. In proximal regions the reactive astrocytes proliferated and expressed nestin, whereas in regions distal to the injury core the astrocytes showed increased GFAP expression but did not proliferate, lacked nestin expression, and displayed different morphology. Simply making the window did not induce any of these changes. There were also significant numbers of neurons in the recovering cortical tissue. In the recovery region, reactive astrocytes radially extended processes which appeared to influence the shapes of neuronal nuclei. The proximal reactive astrocytes also formed a cell layer which appeared to serve as a protective barrier, blocking the spread of IgG deposition and migration of microglia from the lesion core to surrounding tissue. The recovery was preceded by perilesional accumulation of leukocytes expressing vascular endothelial growth factor. These results suggest that, under intact skull conditions, focal brain injury is followed by perilesional reactive astrocyte activities that foster cortical tissue protection and recovery.

Structures of 3-­isopropylmalate dehydrogenase were determined at pressures ranging from 0.1 to 650 MPa. Comparison of these structures gives a detailed picture of the swelling of a cavity at the dimer interface and the generation of a new cleft on the molecular surface, which are accompanied by water penetration., Hydrostatic pressure induces structural changes in proteins, including denaturation, the mechanism of which has been attributed to water penetration into the protein interior. In this study, structures of 3-isopropylmalate dehydrogenase (IPMDH) from Shewanella oneidensis MR-1 were determined at about 2 Å resolution under pressures ranging from 0.1 to 650 MPa using a diamond anvil cell (DAC). Although most of the protein cavities are monotonically compressed as the pressure increases, the volume of one particular cavity at the dimer interface increases at pressures over 340 MPa. In parallel with this volume increase, water penetration into the cavity could be observed at pressures over 410 MPa. In addition, the generation of a new cleft on the molecular surface accompanied by water penetration could also be observed at pressures over 580 MPa. These water-penetration phenomena are considered to be initial steps in the pressure-denaturation process of IPMDH.

To understand the pressure-adaptation mechanism of deep-sea enzymes, we studied the effects of pressure on the enzyme activity and structural stability of dihydrofolate reductase (DHFR) of the deep-sea bacterium Moritella profunda (mpDHFR) in comparison with those of Escherichia call (ecDHFR). mpDHFR exhibited optimal enzyme activity at 50 MPa whereas ecDHFR was monotonically inactivated by pressure, suggesting inherent pressure-adaptation mechanisms in mpDHFR. The secondary structure of apo-mpDHFR was stable up to 80 C, as revealed by circular dichroism spectra. The free energy changes due to pressure and urea unfolding of apo-mpDHFR, determined by fluorescence spectroscopy, were smaller than those of ecDHFR, indicating the unstable structure of mpDHFR against pressure and urea despite the three-dimensional crystal structures of both DHFRs being almost the same. The respective volume changes due to pressure and urea unfolding were -45 and -53 ml/mol at 25 degrees C for mpDHFR, which were smaller (less negative) than the corresponding values of -77 and -85 ml/mol for ecDHFR. These volume changes can be ascribed to the difference in internal cavity and surface hydration of each DHFR. From these results, we assume that the native structure of mpDHFR is loosely packed and highly hydrated compared with that of ecDHFR in solution.

In this study, we investigate the potential of peritoneal macrophages to differentiate into dendritic cell (DCs) in response to preferential uptake of oligomannose-coated liposomes (OMLs). About 30% of peritoneal cells (PECs) preferentially took up OMLs that were administered into the peritoneal cavity. The OML-ingesting cells expressed CD11b and F4/80, but lacked CD11c expression, indicating that the OML-ingesting PECs with a CD11b(high)CD11c(-) phenotype are resident peritoneal macrophages. During in vitro cultivation, CD11c(+) cells arose among the PECs with ingested OMLs. CD11c(+) cells also developed among enriched peritoneal CD11b(high)CD11(-) cells from OML-treated mice, and the resulting CD11c(+) cells expressed co-stimulatory molecules and MHC class II. In addition, OML-ingesting CD11b(high)CD11c(+) cells were found in spleen after the enriched peritoneal macrophages with ingested OMLs were transplanted in the peritoneal cavity of mice. These results show that a fraction of peritoneal macrophages can differentiate into mature DCs following uptake of OMLs.

To examine whether dihydrofolate reductase (DHFR) from deep-sea bacteria has undergone molecular evolution to adapt to high-pressure environments, we cloned eight DHFRs from Shewanella species living in deep-sea and ambient atmospheric-pressure environments, and subsequently purified six proteins to compare their structures, stabilities, and functions. The DHFRs showed 74–90% identity in primary structure to DHFR from S. violacea, but only 55% identity to DHFR from Escherichia coli (ecDHFR). Far-ultraviolet circular dichroism and fluorescence spectra suggested that the secondary and tertiary structures of these DHFRs were similar. In addition, no significant differences were found in structural stability as monitored by urea-induced unfolding and the kinetic parameters, K m and k cat; although the DHFRs from Shewanella species were less stable and more active (2- to 4-fold increases in k cat/K m) than ecDHFR. Interestingly, the pressure effects on enzyme activity revealed that DHFRs from ambient-atmospheric species are not necessarily incompatible with high pressure, and DHFRs from deep-sea species are not necessarily tolerant of high pressure. These results suggest that the DHFR molecule itself has not evolved to adapt to high-pressure environments, but rather, those Shewanella species with enzymes capable of retaining functional activity under high pressure migrated into the deep-sea.

手術関連死亡の最大原因は術中の大量出血であるが，その背景には外科的手技による止血が不可能な希釈性凝固障害という病態が存在する．したがって術中の大量出血を未然に防ぐには止血のための輸血治療が必要であり，その治療指針の確立が急務である． 術中の大量出血・大量輸血症例を後方視的に調査した結果，その60％強を胸部大動脈瘤手術，肝臓移植術，肝臓癌・肝門部癌切除術が占めていた．術中大量出血の背景にある止血不全の主要因は，出血量の増加にともなう凝固因子（特にフィブリノゲン）の喪失，枯渇であると考えられた．そこで上記症例の手術中に起こった低フィブリノゲン血症に対し，クリオプレシピテートおよびフィブリノゲン濃縮製剤の投与を行ったところ，速やかなフィブリノゲン値の上昇と止血の改善，および術中出血量・輸血量の顕著な減少（平均で30～40％減）を認めた． 術中の出血量増加時には，フィブリノゲン値を確認した上で速やかにフィブリノゲン濃縮製剤を投与することが，大量出血・大量輸血を未然に防ぎ，手術患者の予後改善に大きく貢献するとともに，血液製剤の使用削減・有効利用につながると考えられた．

Arabidopsis response regulators (ARRs) are involved in the His-to-Asp phosphotransfer signal transduction mechanism induced by cytokinins, a class of phytohormones in Arabidopsis thaliana. Previous studies have reported ARR mRNA accumulation in response to cytokinins. ARRs consist of 2 families (types A and B). The expression of type-A ARR genes can be induced by cytokinins, while that of type-B genes is constitutive. Recently, we have shown that plant growth induced by the endocrine disruptor bisphenol A (4,4� -isopropylidenediphenol) was similar to that induced by cytokinins. The present study is the first to show that ARR (type-A family; ARR15 and ARR16) mRNA accumulation is influenced by bisphenol A, indicating that bisphenol A is involved in inducing intracellular signal transduction.

Enzymes from organisms living in deep-sea are thought to have characteristic pressure-adaptation mechanisms in structure and function. To better understand these mechanisms in dihydrofolate reductase (DHFR), an essential enzyme in living cells, we cloned, overexpressed and purified four new DHFRs from the deep-sea bacteria Shewanella violacea (svDHFR), Photobacterium profundum (ppDHFR), Moritella yayanosii (myDHFR) and Moritella japonica (mjDHFR), and compared their structure and function with those of Escherichia coli DHFR (ecDHFR). These deep-sea DHFRs showed 33-56% primary structure identity to ecDHFR while far-ultraviolet circular dichroism and fluorescence spectra suggested that their secondary and tertiary structures were not largely different. The optimal temperature and pH for deep-sea DHFRs activity were lower than those of ecDHFR and different from each other. Deep-sea DHFRs kinetic parameters K(m) and k(cat) were larger than those of ecDHFR, resulting in 1.5-2.8-fold increase of k(cat)/K(m) except for mjDHFR which had a 28-fold decrease. The enzyme activity of ppDHFR and mjDHFR (moderate piezophilic bacteria) as well as ecDHFR decreased as pressure increased, while svDHFR and myDHFR (piezophilic bacteria) showed a significant tolerance to pressure. These results suggest that DHFRs from deep-sea bacteria possess specific enzymatic properties adapted to their life under high pressure.

Resident peritoneal macrophages (PEMs) express SIGNR1 on the cell surface as a major mannose receptor. These cells also ingest oligomannose-coated liposomes (OMLs) in an oligomannose-dependent manner following intraperitoneal administration. Therefore, the current study was conducted to investigate the possible role of SIGNR1 in capture of OMLs. Transient expression of several SIGN-related lectins potentially expressed on PEMs in CHO cells revealed that only SIGNR1 contributed to capture of OMLs. When SIGNR1 was introduced into mouse macrophage-like RAW264.7 cells, SIGNR1-expressing RAW (RAW-SIGNR1) cells recognized OMLs under serum-free conditions. OML recognition by RAW-SIGNR1 cells as well as that by PEMs was partially inhibited by an anti-SIGNR1 antibody (ER-TR9) and by mannan, and completely inhibited by EDTA. Interestingly, OML recognition by RAW-SIGNR1 cells was accelerated in the presence of serum, partially inhibited by an anti-complement receptor 3 (CR3) antibody (M1/70), and almost completely inhibited by a combination of ER-TR9 and M1/70. Complete inhibition of OML ingestion by the combination of ER-TR9 and M1/70 was also observed under serum-free conditions, suggesting that SIGNR1 and CR3 cooperate in an additive way in capture of OMLs by macrophage-like RAW cells. Administration of ER-TR9 or M1/70 into the peritoneal cavity led to a significant decrease of OML uptake by PEMs. Therefore, SIGNR1 expressed on macrophages acts as a receptor for recognition of OMLs under physiological conditions.

＜背景·目的＞肝臓移植術を受けるレシピエントには肝機能が極度に低下した例が多く，血小板減少に加え凝固障害を合併しており，しばしば大量出血をきたす．当院では肝臓移植術中に血液凝固能を評価するとともに，凝固能低下が進行した際の輸血治療について検討した．＜対象·方法＞対象は2003年から2005年にかけて行った肝臓移植術31例．術前および肝臓摘出時，門脈血流再開時，門脈血流再開4時間後，10時間後，16時間後および24時間後の4ポイントで血液凝固検査を行い，凝固能低下の程度と出血量の相関について検討した．著明な凝固能低下を認めた場合にはフィブリノゲン製剤を投与し，出血量および輸血量の変化について検討した．＜結果＞術中もっとも減少幅の大きかったのはフィブリノゲン値で，門脈血流再開時に最低値（82±49mg/dl）を示した．術中出血量が5l以上の群と5l未満の群では，肝摘出∼門脈血流再開4時間後のフィブリノゲン値に有意差を認め，前者では最低フィブリノゲン値が75mg/dl未満であった．術中の最低フィブリノゲン値が75mg/dl未満の群と75mg/dl以上の群で門脈血流再開2時間後までの出血量を比較すると，後者において有意に出血量が少なかった．術中にフィブリノゲン値100mg/dlを維持するようフィブリノゲン製剤を投与したところ，出血量は約30%減少し，輸血量も大幅に減らすことができた（平均で赤血球製剤20%減，新鮮凍結血漿50%減，血小板製剤60%減）．＜考察＞肝臓移植術中にはフィブリノゲン値100mg/dlを維持するようフィブリノゲン製剤を投与することにより，出血量および輸血量を大幅に減少させうると考えられた．

＜背景·目的＞胸部大動脈瘤に対する大血管置換術では，人工心肺使用時の血液のヘパリン化，および体外循環にともなう血小板数の減少や血小板機能の低下などによって大量出血をきたし，しばしば多量の輸血を必要とする．しかし大動脈瘤患者における出血傾向の原因のひとつとして重要なのは，瘤局所における凝固·線溶系の活性化により凝固異常が起こっているという点である．当院では大動脈瘤の手術において速やかに止血を図るため，手術室で全身麻酔導入後から人工心肺装置作動までの間に血小板アフェレーシスを実施して患者の自己血小板を採取し，人工心肺離脱後に患者に輸血するという治療を行ってきた．今回，その止血効果についてレトロスペクティブな検討を行った．＜対象·方法＞胸部大動脈瘤の手術を実施する26名の患者に対して，全身麻酔導入後から人工心肺装置作動までの間に，血液成分分離装置を使用して血小板アフェレーシスを実施した．採取した自己血小板15∼20単位は人工心肺離脱後に患者に輸血した．血小板アフェレーシスを実施しなかった過去の34症例と出血量·同種血液製剤の輸血量を比較，検討した．＜成績＞自己血小板の採取·輸血を実施しなかった症例での出血量は1,322±1,498ml（平均±標準偏差; 以下同様）であり，同種血液製剤の使用量は赤血球製剤12.8±14.2単位，新鮮凍結血漿17.1±20.8単位，濃厚血小板13.5±12.2単位であった．それに対して自己血小板を採取した症例では，出血量が688±493ml，同種血液製剤の使用量は赤血球製剤5.7±7.3単位，新鮮凍結血漿8.5±10.8単位，濃厚血小板2.7±6.5単位と，いずれも劇的に減少していた．＜結論＞大動脈瘤手術において患者から自己血小板を採取し，人工心肺離脱直後に輸血して止血を図ることは，出血量·輸血量の大幅な減少に寄与すると考えられた．

Shewanella violacea DSS12 is facultative piezophile isolated from the deep-sea. The expression of cydDC genes (required for d-type cytochrome maturation) of the organism is regulated by hydrostatic pressure. In this study, we analyzed the nucleotide sequence upstream of cydDC in detail and found that there are putative binding sites for the NarL protein which is part of a two-component regulatory system also containing the sensor protein NarX. Furthermore, we identified the narQP genes (homologues of narXL) from S. violacea DSS12 and demonstrated the heterologous expression of narP in Escherichia coli. These results will be helpful in examining pressure regulation of gene expression in S. violacea at the molecular level.

Purpose: A positive response to gefitinib in non–small cell lung cancer (NSCLC) has been correlated to mutations in epidermal growth factor receptor (EGFR) gene. Previous reports have been based mainly on diagnostic screening by sequencing. However, sequencing is a time-consuming and complicated procedure, not suitable for routine clinical use. Experimental Design: We have developed rapid, simple, and sensitive mutation detection assays based on the SMart Amplification Process (SMAP) and applied it for analyzing EGFR gene mutations in clinical samples. By using SMAP, we can detect mutations within 30 min including sample preparation. To validate the assay system for potential use in clinical diagnostics, we examined 45 NSCLC patients for EGFR mutations using sequencing and SMAP. Results: The outcomes of the SMAP assay perfectly matched the sequencing results, except in one case where SMAP was able to identify a mutation that was not detected by sequencing. We also evaluated the sensitivity and specificity of SMAP in mutation detection for EGFR. In a serial dilution study, SMAP was able to find a mutation in a sample containing only 0.1% of the mutant allele in a mixture of wild-type genomic DNA. We also could show amplification of mutated DNA with only 30 copies per reaction. Conclusions: The SMAP method offers higher sensitivity and specificity than alternative technologies, while eliminating the need for sequencing to identify mutations in the EGFR gene of NSCLC. It provides a robust and point-of-care accessible approach for a rapid identification of most patients likely to respond to gefitinib.

Clinical characteristics of Japanese patients with t(8;21) acute myeloid leukemia (AML) have not been well described. From January 2000 to December 2005, a total of 147 Japanese adult de novo AML (FAB: M2) patients were newly diagnosed with t(8;21) AML (n=46) or without t(8;21) AML (n=101) in collaborating hospitals. Patients with t(8;21) (median age, 49.5 years; range, 18–86 years) were significantly younger than AML(M2) patients without t(8;21) (median age, 60 years; range 17–90 years) (p Figure Figure

The standard conditioning regimen in allogeneic hematopoietic stem cell transplantation (HSCT) for aplastic anemia from a human leukocyte antigen (HLA)-identical sibling has been high-dose cyclophosphamide (CY 200 mg/kg). In the present study, results for 203 patients with aplastic anemia aged 16 years or older who underwent allogeneic HSCT from HLA-identical siblings were retrospectively analyzed using the registry database of Japan Society for Hematopoietic Cell Transplantation. Conditioning regimens were defined as a (1) high-dose CY (200 mg/kg or greater)-based (n = 117); (2) reduced-dose CY (100 mg/kg or greater, but less than 200 mg/kg)-based (n = 38); and (3) low-dose CY (less than 100 mg/kg)-based (n = 48) regimen. Patient age and the proportion of patients receiving fludarabine were significantly higher in the reduced- and low-dose CY groups than the high-dose CY group. Engraftment was comparable among the groups. Five-year overall survival (OS) tended to be higher in the low-dose CY group [93.0 % (95 % CI 85.1-100.0 %)] than the high-dose CY [84.2 % (95 % CI 77.1-91.3 %)] or reduced-dose CY groups [83.8 % (95 % CI 71.8-95.8 %); P = 0.214]. Age-adjusted OS was higher in the low-dose CY group than the high- and reduced-dose CY groups with borderline significance (P = 0.067). These results suggest that CY dose can safely be reduced without increasing graft rejection by adding fludarabine in allogeneic HSCT for aplastic anemia from an HLA-identical sibling.

In order to elucidate the molecular adaptation mechanisms of enzymes to the high hydrostatic pressure of the deep sea, we cloned, purified, and characterized more than ten dihydrofolate reductases (DHFRs) from bacteria living in deep-sea and ambient atmospheric pressure environments. The nucleotide and amino acid sequences of these DHFRs indicate the deep-sea bacteria are adapted to their environments after the differentiation of their genus from ancestors inhabiting atmospheric pressure environments. In particular, the backbone structure of the deep-sea DHFR from Moritella profunda (mpDHFR) almost overlapped with the normal homolog from Escherichia coli (ecDHFR). Thus, those of other DHFRs would also overlap on the basis of their sequence similarities. However, the structural stability of both DHFRs was quite different: compared to ecDHFR, mpDHFR was more thermally stable but less stable against urea and pressure unfolding. The smaller volume changes due to unfolding suggest that the native structure of mpDHFR has a smaller cavity and/or enhanced hydration compared to ecDHFR. High hydrostatic pressure reduced the enzymatic activity of many DHFRs, but three deep-sea DHFRs and the D27E mutant of ecDHFR exhibited pressure-dependent activation. The inverted activation volumes from positive to negative values indicate the modification of their structural dynamics, conversion of the rate-determining step of the enzymatic reaction, and different contributions of the cavity and hydration to the transition-state structure. Since the cavity and hydration depend on amino acid side chains, DHFRs would adapt to the deep-sea environment by regulating the cavity and hydration by substituting their amino acid side chains without altering their backbone structure. The results of this study clearly indicate that the cavity and hydration play important roles in the adaptation of enzymes to the deep-sea environment.

We studied two cases with leukemia that relapsed in the central nervous system (CNS) after allogeneic stem cell transplantation. One patient underwent peripheral blood stem cell transplantation (SCT) from a related, yet haplotype-mismatched, donor for chronic myelomonocytic leukemia. She was kept in complete remission (CR) in the bone marrow (BM) for 7 months, until relapse in the cerebrospinal fluid (CSF) was evident. In the other patient, with acute lymphoblastic leukemia, systemic relapse occurred when he was still on immunosuppression 6 months after SCT from an unrelated donor. After induction chemotherapy following cessation of immunosuppression, the BM examination proved CR. During consolidation chemotherapy, however, he developed leukemic dissemination in the CSF, despite the fact that the BM was in CR. Chimerism status in the BM mononuclear cells and fractionated peripheral blood (PB) cells (granulocytes, T-lymphocytes, and the others) was assessed by short tandem repeat analysis. In both patients, the BM cells and all the fractions of the PB cells proved donor-type chimeras. These results seem to suggest that the graft-versus-leukemia effects might not be as effective in the CNS as in the BM, even when complete T-lymphoid chimerism is achieved.

Upon a certain DNA damage including cisplatin treatment, p73 is stabilized and exerts its growth-suppressive and/or proapoptotic function. However, the precise molecular basis by which the intracellular levels of p73 are regulated remains unclear. In the present study, we have identified RanBPM as a novel binding partner of p73alpha by yeast-based two-hybrid screening, and also found that RanBPM has an ability to stabilize p73alpha. GST pull-down assays and co-immunoprecipitation experiments revealed that RanBPM directly bound to the extreme COOH-terminal region of p73alpha, whereas it failed to interact with p53. Co-expression of RanBPM with p73alpha resulted in the nuclear translocation of RanBPM, and both proteins co-localized in cell nucleus as examined by indirect immunofluorescent staining. It is worth noting that the expression of RanBPM inhibited the ubiquitination of p73alpha, and thereby prolonged its half-life. Subsequent studies demonstrated that the proapoptotic activity of p73alpha was significantly enhanced in the presence of RanBPM. Taken together, our present findings implicate a novel role for RanBPM in the regulation of p73 stability and function.

Some rod-shaped bacteria, including Escherichia coli, exhibit cell filamentation without septum formation under high-hydrostatic-pressure conditions, indicating that the cell-division process is affected by hydrostatic pressure. The effects of elevated pressure on FtsZ-ring formation in E. coli cells were examined using indirect immunofluorescence microscopy. Elevated pressure of 40 MPa completely inhibited colony formation of E. coli cells under the cultivation conditions used, and the cells exhibited obviously filamentous shapes. In the elongated cells, normal cell-division processes appeared to be inhibited, because no FtsZ rings were observed by indirect immunofluorescent staining. In addition, it was observed that hydrostatic pressure dissociated the E. coli FtsZ polymers in vitro. These results suggest that high hydrostatic pressure directly affects cell survival and morphology through the dissociation of the cytoskeletal frameworks.

We have cloned the rpoZ gene, encoding RNA polymerase omega protein, by PCR approach from the deep-sea piezophilic and psychrophilic bacterium, Shewanella violacea strain DSS12. The cloned gene, 285bp in length, was found to encode a protein consisting of 94 amino acid residues with a molecular mass of 10,327 Da. Significant homology was evident comparing the RpoZ protein of S. violacea with that of Shewanella oneidensis (69% identity), Vibrio cholerae (65% identity), Escherichia coli K-12 (64% identity) and Haemophilus influenzae (61% identity). From the Northern blot analysis, S. violacea rpoZ gene was expressed constitutively under pressure conditions of 0.1, 30 and 50MPa. We constructed expression plasmid to overproduce the RpoZ protein and transformed into E. coli JM109 as a host of overproduction. Upon induction, the recombinant protein encoded by plasmid pQrpoZ was overexpressed and purified using Ni2+ affinity column.

Approximately 20% of familial amyotrophic lateral sclerosis (FALS) arises from germ-line mutations in the superoxide dismutase-1 (SOD1) gene. However, the molecular mechanisms underlying the process have been elusive. Here, we show that a neuronal homologous to E6AP carboxyl terminus (HECT)-type ubiquitin-protein isopeptide ligase (NEDL1) physically binds translocon-associated protein-delta and also binds and ubiquitinates mutant (but not wild-type) SOD1 proportionately to the disease severity caused by that particular mutant. Immunohistochemically, NEDL1 is present in the central region of the Lewy body-like hyaline inclusions in the spinal cord ventral horn motor neurons of both FALS patients and mutant SOD1 transgenic mice. Two-hybrid screening for the physiological targets of NEDL1 has identified Dishevelled-1, one of the key transducers in the Wnt signaling pathway. Mutant SOD1 also interacted with Dishevelled-1 in the presence of NEDL1 and caused its dysfunction. Thus, our results suggest that an adverse interaction among misfolded SOD1, NEDL1, translocon-associated protein-delta, and Dishevelled-1 forms a ubiquitinated protein complex that is included in potentially cytotoxic protein aggregates and that mutually affects their functions, leading to motor neuron death in FALS.

Although imatinib mesylate has shown encouraging activity in chronic myelogenous leukemia (CML), disease progression during therapy has been observed, manifested by clonal expansion of imatinib mesylate-resistant leukemia cells. On the other hand, myelosuppression related to treatment of imatinib mesylate is often managed with temporary interruption of treatment or dose reduction. We here report two CML patients who had imatinib mesylate-sensitive blast crisis (BC) immediately after discontinuation of imatinib mesylate therapy. The patients discontinued therapy because of neutropenia. Although there was no evidence of blastic phase during therapy, BC occurred 2 weeks after the withdrawal of treatment in both cases. Interestingly, additional chromosomal abnormalities were detected following the withdrawal of imatinib mesylate and disappeared by re-introduction of this agent. The same doses of imatinib mesylate was still effective and remission was sustained with imatinib mesylate alone again. Our report suggests the possibility that withdrawal of imatinib mesylate may lead to proliferation of blast clones even in patients showing good responses to imatinib mesylate without signs of disease progression.

Neuroblastoma (NBL), one of the most common childhood solid tumors, has a distinct nature in different prognostic subgroups. However, the precise mechanism underlying this phenomenon remains largely unknown. To understand the molecular and genetic bases of neuroblastoma, we have generated its cDNA libraries and identified a human ortholog of tubulin tyrosine ligase gene (hTTL/Nbla0660) as a differentially expressed gene at high levels in a favorable subset of the tumor. Tubulin is subjected to several types of evolutionarily conserved posttranslational modification, including tyrosination and detyrosination. Tubulin tyrosine ligase catalyzes ligation of the tyrosine residue to the COOH terminus of the detyrosinated form of alpha-tubulin. The measurement of hTTL mRNA expression in 74 primary neuroblastomas by quantitative real-time reverse transcription-PCR revealed that its high expression was significantly associated with favorable stages (1, 2 and 4s; p = 0.0069), high TrkA expression (p = 0.002), a single copy of MYCN (p < 0.00005), tumors found by mass screening (p = 0.0042), nonadrenal origin (p = 0.0042) and good prognosis (p = 0.023). The log-rank test showed that high expression of hTTL was an indicator of favorable prognosis (p = 0.026). Immunohistochemical analysis using specific antibodies generated by us demonstrated that tyrosinated tubulin (Tyr-tubulin), detyrosinated tubulin (Glu-tubulin) and hTTL as well as Delta2-tubulin were positive in favorable tumors, whereas only Delta2-tubulin was positive in the tumors with MYCN amplification. In an RTBM1 neuroblastoma cell line, hTTL was increased after treating the cells with bone morphogenetic protein 2 (BMP2) or all-trans retinoic acid (RA), which induced neuronal differentiation. These results suggest that the deregulated tubulin tyrosination/detyrosination cycle caused by decreased expression of hTTL is associated with inhibition of neuronal differentiation and enhancement of cell growth in the primary neuroblastomas with poor outcome.

We report a 28-year-old male suffering from convergence insufficiency due to a unilateral lesion in the rostral part of the superior colliculus (SC). The patient visited the emergency clinic complaining of vertigo. Cerebral CT revealed a high-density area in the left SC. Head MRI showed swelling in the rostal part of the left SC. No abnormalities were detected elsewhere. The present case supports data from animal experiments suggesting that the rostral SC participates in convergence.

When non-extremophiles encounter extreme environmental conditions, which are natural for the extremophiles, stress reactions, e.g., expression of heat shock proteins (HSPs), are thought to be induced for survival. To understand how the extremophiles live in such extreme environments, we studied the effects of high hydrostatic pressure on cellular contents of HSPs and their mRNAs during growth in a piezophilic bacterium, Shewanella violacea. HSPs increased at high hydrostatic pressures even when optimal for growth. The mRNAs and proteins of these HSPs significantly increased at higher hydrostatic pressure in S. violacea. In the non-piezophilic Escherichia coli, however, their mRNAs decreased, while their proteins did not change. Several transcriptional start sites (TSSs) for HSP genes were determined by the primer extension method and some of them showed hydrostatic pressure-dependent increase of the mRNAs. A major refolding target of one of the HSPs, chaperonin, at high hydrostatic pressure was shown to be RplB, a subunit of the 50S ribosome. These results suggested that in S. violacea, HSPs play essential roles, e.g., maintaining protein complex machinery including ribosomes, in the growth and viability at high hydrostatic pressure, and that, in their expression, the transcription is under the control of σ(32).

We investigated whether vitamin E (VE) supplementation alleviates collagen-induced arthritis in its stages by altering immune functions in DBA/1J mice. Mice were divided into 4 groups; control (VE 30 IU/kg in diet), A-VE0, A-VE30 and A-VE500 (animals with induced arthritis given VE 0, VE 30, VE 500 IU/kg in diet respectively). Arthritis was induced with Type II Collagen (TIIC). The extent of arthritis in the joint did not decline in neither A-VE0 nor A-VE500 groups compared with A-VE30. The proliferation of splenocytes stimulated with ConA was suppressed in both A-VE0 and A-VE500 groups. The production of Th1 cytokine stimulated with TIIC was increased in both A-VE0 and A-VE500 groups, but production of Th2 cytokine was not changed. Our observations suggest that VE supplementation does not always improve the progress of arthritis. The influence of VE on cytokine balance in autoimmune (Th1 predominant) arthritis should be further investigated.

The gene encoding the principal a factor (rpoD) of the piezophilic bacterium Shewanella violacea was cloned and sequenced. The rpoD gene was found to encode a polypeptide consisting of 614 amino acid residues, showing 75.6 and 64.3% identity to those of Escherichia coli and Pseudomonas putida, respectively. Comparison with E. coli sigma70 and P. putida sigma70 showed that significant similarity exists in four conserved regions known to be required for promoter recognition and core binding. Using an expression plasmid harboring the rpoD gene, the S. violacea sigma70 factor was overexpressed in E. coli and successfully purified to near homogeneity.