In vitro heat effect on heterooligomeric subunit assembly of thermostable indolepyruvate ferredoxin oxidoreductase

AbstractIndolepyruvate ferredoxin oxidoreductase (IOR) from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 catalyzes the oxidative decarboxylation of arylpyruvates by forming a heterooligomeric complex (α2β2). The genes iorA and iorB which encode respective α and β subunits, were coexpressed heterologously in Escherichia coli cells under anaerobic conditions. IOR activity was detected from the cell extract containing both subunits and its activity was enhanced by in vitro heat treatment prior to the assay. The iorA and iorB were expressed individually and each subunit was examined for enzymatic activity with and without heat treatment. IOR activity was detected neither from the extract of α subunit nor β subunit. The α and β subunits were mixed and then IOR activity was examined. Weak IOR activity was detected without heat treatment, however, upon heat treatment its activity was enhanced. The mixture of individually heat treated α and β subunits did not possess any IOR activity even though the mixed sample was heat treated again. IOR α and β subunits were individually purified to homogeneity, mixed with or without heat treatment and subunit assembly was examined by determining molecular mass. Upon heat treatment, inactive α and β were converted to an active high molecular weight complex (195 kDa) which corresponds to the α2β2 structure. However, the active complex was not formed without heat treatment, suggesting that high temperature environments are important for the hetero-oligomerization of IOR subunits

Construction and high cytoplasmic expression of a tumoricidal single-chain antibody against hepatocellular carcinoma

BACKGROUND: Hep27 monoclonal (Hep27 Mab) is an antibody against hepatocellular carcinoma. Hep27 Mab itself can inhibit the growth of a hepatocellular carcinoma cell line (HCC-S102). We attempted to produce a single-chain fragment (scFv), a small fragment containing an antigen-binding site of Hep27 Mab, by using DNA-recombinant techniques. RESULTS: The sequences encoding the variable regions of heavy (V(H)) and light (V(L)) chains of a murine Hep27 Mab were linked together by a linker peptide (Gly4Ser)(3) and tagged with a hexa-histidine at the C-terminal; the resultant DNA construct was expressed in E. coli as an insoluble protein. The denatured scFv was refolded and purified by immobilized metal ion affinity chromatography (12 mg/l with a molecular weight of 27 kDa). Hep27scFv exhibited a tumoricidal activity against the HCC-S102 cell as its parental antibody (Hep27 Mab). CONCLUSION: This scFv may be a potential candidate for a targeting agent in HCC immunodiagnosis or immunotherapy

Endoscopic ultrasound-guided immunotherapy

AbstractAnti-tumoral endoscopic ultrasound-guided fine-needle injection (EUS-FNI), with its minimally invasive access for anti-tumoral agent delivery, is the most exciting field of intervention EUS. Pancreatic cancer is regarded as a systemic disease even if imaging modalities reveal no visible metastasis. From that perspective, immunological therapy is performed. To date, several reports have described immunotherapy under EUS-guidance. The first report of EUS-FNI intended for immunotherapy for advanced pancreatic cancer was published in 2000. In that study, an allogeneic mixed-lymphocyte culture was injected into tumors of eight patients with unresectable local pancreatic adenocarcinoma. The study of dendritic cells (DCs) for cancer has continued to develop in recent years. Actually, DCs are potent antigen-presenting cells for the induction of primary T-cell dependent immune response. When injected intratumorally, DCs acquire and process tumor antigens in situ, migrate to regional lymphoid organs, and initiate a strong tumor-specific immune response. To date, three reports have described EUS-FNI of DCs into pancreatic cancer: two for unresectable and one for pre-surgical operations. Every study has indicated the feasibility and safety. Furthermore, these reports showed that EUS-guided DCs injection might be an important option for treating advanced pancreatic cancer. EUS-guided immunotherapy is a very exciting field in interventional EUS for obstinate cancers

Association of the diplotype configuration at the N-acetyltransferase 2 gene with adverse events with co-trimoxazole in Japanese patients with systemic lupus erythematosus

Although co-trimoxazole (trimethoprim-sulphamethoxazole) is an effective drug for prophylaxis against and treatment of Pneumocystis pneumonia, patients often experience adverse events with this combination, even at prophylactic doses. With the aim being to achieve individual optimization of co-trimoxazole therapy in patients with systemic lupus erythematosus (SLE), we investigated genetic polymorphisms in the NAT2 gene (which encodes the metabolizing enzyme of sulphamethoxazole). Of 166 patients with SLE, 54 patients who were hospitalized and who received prophylactic doses of co-trimoxazole were included in the cohort study. Adverse events occurred in 18 patients; only two experienced severe adverse events that lead to discontinuation of the drug. These two patients and three additional ones with severe adverse events (from other institutions) were added to form a cohort sample and were analyzed in a case-control study. Genotype was determined using TaqMan methods, and haplotype was inferred using the maximum-likelihood method. In the cohort study, adverse events occurred more frequently in those without the NAT2*4 haplotype (5/7 [71.4%]) than in those with at least one NAT2*4 haplotype (13/47 [27.7%]; P = 0.034; relative risk = 2.58, 95% confidence interval = 1.34–4.99). In the case-control study the proportion of patients without NAT2*4 was significantly higher among those with severe adverse events (3/5 [60%]) than those without severe adverse events (6/52 [11.5%]; P = 0.024; odds ratio = 11.5, 95% confidence interval = 1.59–73.39). We conclude that lack of NAT2*4 haplotype is associated with adverse events with co-trimoxazole in Japanese patients with SLE

Two Cases of Retroperitoneal Liposarcoma Diagnosed Using Endoscopic Ultrasound-Guided Fine-Needle Aspiration (EUS-FNA)

This report describes our experience with two cases that were ultimately diagnosed as retroperitoneal liposarcoma using endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA). Case 1 is that of a 54-year-old woman with chief complaints of nausea and abdominal distention. Computed tomography (CT) and magnetic resonance imaging (MRI) revealed a large (15 cm diameter) tumor, which was significantly compressing the stomach and apparently occupied the entire left abdominal cavity. Although advanced primary gastrointestinal stromal tumor (GIST) or retroperitoneal tumor was inferred as the differential diagnosis, a definitive diagnosis was difficult using imaging alone. After EUS-FNA was done, the tumor was diagnosed histopathologically as high-grade liposarcoma. Case 2 is that of a 73-year-old man. Abdominal ultrasonography and CT showed a 6 cm diameter tumor within the pelvic cavity. The tumor had high MRI signal-intensity on both T1 and T2 images. Endorectal EUS showed a hyperechoic mass. The images suggested lipoma or liposarcoma containing lipoma-like components. Myxoid liposarcoma was revealed by subsequent EUS-FNA. Performing EUS-FNA was clinically useful for determining the subsequent therapeutic strategy in these cases where a tumor of unknown origin existed in the retroperitoneum

Nitro-fatty acids and cyclopentenone prostaglandins share strategies to activate the Keap1-Nrf2 system: a study using green fluorescent protein transgenic zebrafish

Nitro-fatty acids are electrophilic fatty acids produced in vivo from nitrogen peroxide that have many physiological activities. We recently demonstrated that nitro-fatty acids activate the Keap1-Nrf2 system, which protects cells from damage owing to electrophilic or oxidative stresses via transactivating an array of cytoprotective genes, although the molecular mechanism how they activate Nrf2 is unclear. A number of chemical compounds with different structures have been reported to activate the Keap1-Nrf2 system, which can be categorized into at least six classes based on their sensing pathways. In this study, we showed that nitro-oleic acid (OA-NO2), one of major nitro-fatty acids, activates Nrf2 in the same manner that of a cyclopentenone prostaglandin 15-deoxy-Δ12,14-prostaglandin J2 (15d-PGJ2) using transgenic zebrafish that expresses green fluorescent protein (GFP) in response to Nrf2 activators. In transgenic embryos, GFP was induced in the whole body by treatment with OA-NO2, 15d-PGJ2 or diethylmaleate (DEM), but not with hydrogen peroxide (H2O2), when exogenous Nrf2 and Keap1 were co-overexpressed. Induction by OA-NO2 or 15d-PGJ2 but not DEM was observed, even when a C151S mutation was introduced in Keap1. Our results support the contention that OA-NO2 and 15d-PGJ2 share an analogous cysteine code as electrophiles and also have similar anti-inflammatory roles

Mapping dusty star formation in and around a cluster at z=0.81 by wide-field imaging with AKARI

We present environmental dependence of dusty star forming activity in and around the cluster RXJ1716.4+6708 at z=0.81 based on wide-field and multi-wavelength observations with Suprime-Cam on the Subaru telescope and IRC onboard the AKARI satellite. Our optical data shows that the optical colour distribution of galaxies starts to dramatically change from blue to red at the medium-density environment such as cluster outskirts, groups and filaments. By combining with infrared data, we find that 15 micron galaxies tend to have optical colours between the red sequence and the blue cloud with a tail into the red sequence. The spatial distribution of the 15 micron galaxies over ~200 arcmin^2 around the cluster reveals that few 15 micron galaxies are detected in the cluster central region. This is probably due to the low star forming activity in the cluster core. However, interestingly, the fraction of 15 micron galaxies in the medium-density environments is as high as in the low-density field, despite the fact that the optical colours start to change in the medium-density environments. Furthermore, we find that 15 micron galaxies which have optically red colours (candidates for dusty red galaxies) and galaxies with high specific star formation rates are also concentrated in the medium-density environment. These results imply that the star forming activity in galaxies in groups and filaments is enhanced due to some environmental effects specific to the medium-density environment, and such a phenomenon is probably directly connected to the truncation of star forming activity in galaxies seen as the dramatic change in optical colours in such environments.Comment: 15 pages, 14 figures, accepted for publication in MNRA

Successful Endoscopic Closure Using Polyglycolic Acid Sheets with Fibrin Glue for Nonhealing Duodenal Ulcer with Perforation after Proton Beam Therapy of Liver Tumor

We describe the first case of a nonhealing duodenal ulcer with perforation after proton beam therapy (PBT) of a liver tumor that was successfully treated endoscopically using polyglycolic acid (PGA) sheets with fibrin glue. A 69-year-old man received PBT for a liver tumor. Esophagogastroduodenoscopy (EGD) 3 months after PBT revealed a duodenal ulcer. A proton pump inhibitor was administered for 7 weeks, and the ulcer healed. Six months after the EGD, recurrence of the duodenal ulcer with perforation occurred. An emergency open surgery with placement of the omental patch was performed. However, 5 days after the surgery, because the EGD revealed a perforation site in the duodenal ulcer that was not closed, the conservative treatment was continued. Twenty-eight days after the surgery, EGD revealed that the perforation size had increased. Therefore, we conducted endoscopic closure therapy using PGA sheets with fibrin glue. Eleven days after the closure procedure, the EGD showed that the perforation site was filled with granulation tissue and was closed. Forty-nine days after the procedure, EGD revealed that the ulcer had healed. This endoscopic closure treatment was effective for a nonhealing duodenal ulcer with perforation after PBT of a liver tumor