Characterization of xanthine oxidase from Cellulosimicrobium funkei possessing hypoxanthine-metabolizing activity.

AIMS: Purine-degrading enzymes are favourable as medications and diagnostic tools for hyperuricemia. This study aimed to characterize enzymes isolated from micro-organisms, which may be useful for developing a new prophylaxis for hyperuricemia. METHODS AND RESULTS: Cellulosimicrobium funkei A153 was found to be a good catalyst for hypoxanthine degradation and could oxidize hypoxanthine to xanthine and further to uric acid. The enzyme catalysing this oxidation was purified, and its partial amino acid sequences were examined. Based on this information and genome sequencing results, this xanthine dehydrogenase family protein was cloned and expressed in Rhodococcus erythropolis L88. The recombinant enzyme with a His-tag was characterized. The enzyme was a xanthine oxidase as it could utilize molecular oxygen as an electron acceptor. It was stable under 50°C and exhibited maximum activity at pH 7·0. The kcat , Km and kcat /Km values for xanthine were 1·4 s-1 , 0·22 mmol l-1 and 6·4 s-1  mmol-1  l, respectively. CONCLUSIONS: Xanthine oxidase is favourable for hyperuricemia medication because it oxidizes hypoxanthine, an easily adsorbed purine, to xanthine and further to uric acid, which are hardly adsorbed purines. SIGNIFICANCE AND IMPACT OF THE STUDY: The enzyme is useful for decreasing serum uric acid levels via conversion of easily absorbed purines to hardly absorbed purines in the intestine. Enzymes from micro-organisms may be used as a novel prophylaxis for hyperuricemia.

The anti-inflammatory effect of 10-oxo-trans-11-octadecenoic acid (KetoC) on RAW 264.7 cells stimulated with Porphyromonas gingivalis lipopolysaccharide.

BACKGROUND: There is rapidly developing interest into the role of several anti-inflammatory agents to resolve inflammation in periodontal disease. A bioactive polyunsaturated fatty acid, 10-oxo-trans-11-octadecenoic acid (KetoC), is known to have various beneficial physiological effects; however, the effect of KetoC on inflammation remains unclear. Here, we investigated the effect of KetoC on RAW 264.7 cells stimulated with Porphyromonas gingivalis lipopolysaccharide, and explored the intracellular mechanism responsible for its anti-inflammatory effects. METHODS: RAW 264.7 cells were pre-treated with or without KetoC, and then stimulated with or without P. gingivalis lipopolysaccharide. Levels of tumor necrosis factor α (TNFα), interleukin (IL)-6 and IL-1ß were determined by real-time polymerase chain reaction and enzyme-linked immunosorbent assay. Specific antagonists for G protein-coupled receptor (GPR)40 and GPR120 were used to clarify the receptor for KetoC. The intracellular mechanism was investigated using western blotting analysis to separate nuclear and cytosolic NF-κB p65 protein. RESULT: KetoC (5 µmol/L) was not toxic to RAW 264.7 cells, and significantly reduced the expression of TNFα and IL-6 mRNA and protein, and IL-1ß mRNA. No protein production of IL-1ß was observed. Additionally, when bound to GPR120, KetoC trended to downregulate nuclear NF-κB p65 protein levels. However, the antagonist for GPR40 failed to diminish the action of KetoC. CONCLUSION: KetoC suppressed the proinflammatory cytokines TNFα, IL-6 and IL-1ß via NF-κB p65, by binding to its receptor GPR120. KetoC is a promising candidate in future studies as a bioactive anti-inflammatory agent in treating periodontal disease.

Efficient enzymatic production of hydroxy fatty acids by linoleic acid Δ9 hydratase from Lactobacillus plantarum AKU 1009a.

AIMS: This study aims to produce hydroxy fatty acids efficiently. METHODS AND RESULTS: Escherichia coli overexpressing linoleic acid Δ9 hydratase from Lactobacillus plantarum AKU 1009a was employed to produce hydroxy fatty acids with industrial potential. We found that 280 g l(-1) of linoleic acid (1 mol l(-1)) was converted into (S)-10-hydoxy-cis-12-octadecenoic acid (HYA) with a high conversion rate of 98% (mol/mol) and more than 99·9% enantiomeric excess (e.e.) by recombinant E. coli cells in the presence of FAD and NADH. In the same way, many kinds of C18 unsaturated fatty acids with Δ9 carbon double bond (280 g l(-1)) were converted into corresponding 10-hydroxy fatty acids with the conversion rates over 95% (mol/mol). We also produced HYA at a high rate of accumulation (289 g l(-1) ) with a high yield (97 mol%) in a reaction mixture that contained glucose instead of NADH. CONCLUSIONS: We developed a process for producing several types of hydroxy fatty acids with high accumulation rates and high yields. SIGNIFICANCE AND IMPACT OF THE STUDY: Hydroxy fatty acids are important materials for the chemical, food, cosmetic and pharmaceutical industries, and thus they have recently attracted much interest in a variety of research fields. However, the mass production of hydroxy fatty acids has been limited. This method of hydroxy fatty acids production will facilitate the widespread application of hydroxy fatty acids in various industries.

Production of cis-11-eicosenoic acid by Mortierella fungi.

AIM: To find cis-11-eicosenoic acid (20:1ω9, EA)-producing micro-organisms. METHODS AND RESULTS: We found EA-producing fungi by screening about 300 fungal strains and identified a major fatty acid accumulated in the Mortierella fungi as EA by means of GC-MS analysis. In particular, Mortierella chlamydospora CBS 529.75 produced a high amount of EA (36.3 mg g(-1) of dried cells) on cultivation at 28°C for 4 days and then at 12°C for 3 days. In the result of lipid analysis, most of the EA was a component of triacylglycerols, not phospholipids. CONCLUSION: We found that M. chlamydospora CBS 529.75 was the best producer for the microbial production of EA. SIGNIFICANCE AND IMPACT OF THE STUDY: EA is beneficial as a raw material for medical supplies and a moisturizing component of cosmetic creams. This is the first report of microbial production of EA.

Identification of insulin-like growth factor 2 mRNA-binding protein 3 as a radioresistance factor in squamous esophageal cancer cells.

Identification of reliable markers of radiosensitivity and the key molecules that donate susceptibility to anticancer treatments to esophageal cancer cells would be highly desirable. We found that the mRNA expression of insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) was higher in radioresistant TE-5 and TE-9 cells than in radiosensitive TE-12 cloneA1 cells. Conversely, knocking down expression of IGF2BP3 mRNA in TE-5 and TE-9 cells using small interfering RNA significantly enhanced their radiosensitivity. Furthermore, patients with squamous cell esophageal cancers strongly expressing IGF2BP3 tended to respond poorly to chemoradiation. These data suggest that IGF2BP3 may be a key marker of radiosensitivity that diminishes the susceptibility of squamous cell esophageal cancer cells to radiotherapy. IGF2BP3 may, thus, be a useful target for improving radiotherapy for patients with esophageal squamous cell carcinoma.

A novel Fe(II)/α-ketoglutarate-dependent dioxygenase from Burkholderia ambifaria has ß-hydroxylating activity of N-succinyl l-leucine.

An Fe(II)/α-ketoglutarate-dependent dioxygenase, SadA, was obtained from Burkholderia ambifaria AMMD and heterologously expressed in Escherichia coli. Purified recombinant SadA had catalytic activity towards several N-substituted l-amino acids, which was especially strong with N-succinyl l-leucine. With the NMR and LC-MS analysis, SadA converted N-succinyl l-leucine into N-succinyl l-threo-ß-hydroxyleucine with >99% diastereoselectivity. SadA is the first enzyme catalysing ß-hydroxylation of aliphatic amino acid-related substances and a potent biocatalyst for the preparation of optically active ß-hydroxy amino acids.

Outcomes of endoscopic and surgical resection for a second primary cancer in the residual cervical esophagus after thoracic esophagectomy.

Patients who have received subtotal esophagectomy for thoracic esophageal cancer must be closely monitored for second primary malignancies. The purpose of this study is to review and assess patients who developed a second primary esophageal cancer in the residual cervical esophagus. Between 1996 and 2010, 10 patients were diagnosed in our hospital with esophageal squamous cell cancer in the residual cervical esophagus after undergoing thoracic esophagectomy and were treated with endoscopic or surgical resection. Data from these patients were reviewed retrospectively. Seven of the 10 patients (70%) had multiple primary carcinoma lesions at the time of their esophagectomy. A second primary cancer in the residual cervical esophagus was detected in eight patients during follow-up endoscopic examinations while the patients were still asymptomatic. Seven of the patients underwent endoscopic resection for a superficial cancer. None of those patients experienced any complications, and all are currently alive and cancer-free. The remaining three patients underwent resection of the cervical esophagus with regional lymph node dissection. Two of those patients experienced severe complications; one subsequently died (hospital death) from pneumonia, 12 months after surgery, while the other died from recurrence of his cancer. The third patient is alive and cancer-free. Early detection of a second primary malignancy in the residual cervical esophagus followed by endoscopic resection is the best treatment strategy for patients who previously received subtotal esophagectomy for thoracic esophageal cancer. Surgical resection puts patients at high risk of mortality or morbidity.

[Thoracic surgery when the patients have cerebrovascular disease].

In cancer patients, cerebrovascular disease is recognized as the 2nd most common complication involving the central nervous system. Once cancer occurs in stroke patients, or vice versa, neurological outcomes significantly worsen and prognosis tends to be poor. Perioperative stroke after noncardiac, nonneurosurgical procedures is more common than generally acknowledged. It is reported to have an incidence of 0.05~7.4% of patients. "The analysis of lung cancer registry for resected cases in 1994", jointly established by the Japan Lung Cancer Society and the Japanese Association for Chest Surgery, reported a 2.4% frequency of cerebrovascular disease. Cerebrovascular disease is an unavoidable comorbidity for thoracic surgeons. Most are thrombotic in origin and are noted after discharge from the postanesthetic care unit. The mortality is more than 2 times greater than in strokes occurring outside the hospital. Delayed diagnosis and a synergistic interaction between the inflammatory changes normally associated with stroke, and those normally occurring after surgery, may explain this increase. Emergency non-contrast scanning of the brain is the primary diagnostic brain imaging study for evaluation of patients with suspected stroke, and the goal is to complete the computer tomography examination within 25 min of the provisional diagnosis being made.

Therapeutic strategy for the treatment of postoperative recurrence of esophageal squamous cell carcinoma: clinical efficacy of radiotherapy.

We investigated the effectiveness of chemoradiotherapy for the treatment of lymph node recurrence and hematogenous metastasis after esophagectomy for esophageal squamous cell carcinoma. Between 2001 and 2006, 216 patients with thoracic esophageal squamous cell carcinoma had curative esophagectomy. Of those, 23 with lymph node recurrence received chemoradiotherapy (50.0-68.8 Gy). In addition, five patients had isolated recurrences in a distant organ and received chemoradiotherapy (50.0-60.0 Gy). We analyzed outcomes from the radiotherapy for recurrent esophageal cancer. The 1-, 2-, and 5-year survival rates after recurrence for the 23 patients whose lymph node recurrence was treated with chemoradiotherapy were 52, 31, and 24%, respectively, and the median survival time was 13 months. Among the five patients with recurrent tumors in a distant organ, chemoradiotherapy produced a complete response in two patients, a partial response in one patient, and stable disease in two patients, giving an effectiveness rate of 60% (complete response + partial response). Chemoradiotherapy has a beneficial prognostic effect in patients with lymph node recurrence of esophageal squamous cell carcinoma. Chemoradiotherapy for a metastatic tumor in a distant organ may be the treatment of choice in cases where systemic chemotherapy has proven ineffective.

Microbial production of conjugated gamma-linolenic acid from gamma-linolenic acid by Lactobacillus plantarum AKU 1009a.

AIMS: Optimal production conditions of conjugated gamma-linolenic acid (CGLA) from gamma-linolenic acid using washed cells of Lactobacillus plantarum AKU 1009a as catalysts were investigated. METHODS AND RESULTS: Washed cells of Lact. plantarum AKU 1009a exhibiting a high level of CGLA productivity were obtained by cultivation in a nutrient medium supplemented with 0.03% (w/v) alpha-linolenic acid as an inducer. Under the optimal reaction conditions with 13 mg ml(-1)gamma-linolenic acid as a substrate in 5 -ml reaction volume, the washed cells [32% (wet cells, w/v) corresponding to 46 mg ml(-1) dry cells] as the catalysts produced 8.8 mg CGLA per millilitre reaction mixture (68% molar yield) in 27 h. The produced CGLA was a mixture of two isomers, i.e., cis-6,cis-9,trans-11-octadecatrienoic acid (CGLA1, 40% of total CGLA) and cis-6,trans-9,trans-11-octadecatrienoic acid (CGLA2, 60% of total CGLA), and accounted for 66% of total fatty acid obtained. The CGLA produced was obtained as free fatty acids adsorbed mostly on the surface of the cells of Lact. plantarum AKU1009a. CONCLUSION: The practical process of CGLA production from gamma-linolenic acid using washed cells of Lact. plantarum AKU 1009a was successfully established. SIGNIFICANCE AND IMPACT OF THE STUDY: We presented the first example of microbial production of CGLA. CGLA produced by the process is valuable for evaluating their physiological and nutritional effects, and chemical characteristics.

Small cell carcinoma of the esophagus treated with esophagectomy and following chemotherapy: case report with review of the literature.

We report 2 cases of small cell carcinoma of the esophagus treated with esophagectomy as a primary treatment and following chemotherapy. One patient (pT1N1M0) achieved long-term survival, while the other patient (pT1N1M1-lym) died 18 months after surgery. We used reports on 47 Japanese patients receiving esophagectomy as a primary treatment to determine when esophagectomy for small cell carcinoma of the esophagus is indicated. We conclude that esophagectomy as a local treatment provides relatively good long-term survival only in patients without lymph node involvement.

Selective production of cis-9,trans-11 isomer of conjugated linoleic acid from trans-vaccenic acid methyl ester by Delacroixia coronata.

AIMS: Bio-process development for isomer selective and efficient production of cis-9,trans-11-octadecadienoic acid (CLA) from trans-vaccenic acid (t-VA, trans-11-octadecenoic acid) through microbial fatty acid Delta9-desaturation reaction. METHODS AND RESULTS: A total of 550 strains of fungi and yeasts were screened for CLA production from t-VA through Delta9 desaturation. Delacroixia coronata IFO 8586 was selected as a potent producer of CLA from t-VA. Efficient CLA production was observed during cultivation in medium supplemented with the methyl ester of t-VA (t-VAME). Under the optimal conditions with 33.3 mg ml(-1) of t-VAME as substrate, 10.5 mg ml(-1) CLA was produced by D. coronata IFO 8586 after 7 days of cultivation in the medium containing dextrin (5.0%), tryptone (2.0%) and thiourea (0.83 micromol ml(-1)). The strain produced the cis-9,trans-11 isomer of CLA selectively (98% of total CLA), with a small amount of the trans-9,trans-11 isomer (2% of total CLA), mainly in the form of triacylglycerols (69% of total CLA). CONCLUSIONS: A practical bio-process for selective production of cis-9,trans-11 isomer of CLA using filamentous fungus D. coronata IFO 8586 was successfully established. SIGNIFICANCE AND IMPACT OF THE STUDY: Isomer selective bio-process for the practical production of cis-9,trans-11-CLA was first established. The process is benefitable for expanding the application of CLA for medicinal and nutraceutical purposes.

[Sentinel lymph node mapping in non-small cell lung cancer].

The sentinel node (SN) concept is that lymphatic flux from a primary tumor initially flows into the SN. If this concept is correct, and metastasis is not found in a SN, it almost certainly that metastasis is not present in more distal lymph nodes. SN mapping and biopsy were developed as techniques for staging the lymphatic basin without the potential morbidity of lymph edema and nerve injury in cases of melanoma, or lymph edema of the arm in cases of breast cancer. Although there is also evidence of the existence of SN in non-small cell lung cancer (NSCLC), SN mapping is not widely used in the treatment of NSCLC. The potential benefit of SN mapping in NSCLC is enabling surgeons to know more precise staging of cancer. More sensitive techniques can be employed with a limited amount of tissue to detect micrometastasis. In addition, SN mapping can be applied to appropriate segmentectomy for NSCLC.

Preventive wrapping of the fragile tracheobronchial wall using a flap of latissimus dorsi muscle during esophagectomy after chemoradiotherapy.

BACKGROUND: Thanks to significant recent progress in the application of chemoradiotherapy (CRT) in the treatment of esophageal cancer, the numbers of operations on patients who have undergone preoperative CRT are increasing. In these patients, preoperative CRT often leads to formation of an unstable, scleroid layer comprised of what appears to be scar tissue around the tracheobronchial wall. This scleroid layer must be removed with the tumor, thereby weakening the wall. METHOD: We performed preventive intrathoracic transposition of a pedicled latissimus dorsi muscle flap on 6 patients with clinically T4 esophageal cancer (6 males; age: 63-75 years). All 6 patients had undergone preoperative CRT (median, 43.0 Gy; range, 32.0-60.0 Gy; with cisplatin + 5-fluorouracil infusion) to relieve direct invasion of the airway. RESULTS: In 6 patients, preventive use of a pedicled latissimus dorsi muscle flap resulted in uneventful recovery from the tracheobronchial wall weakness. Anastomotic leakage, recurrent nerve paralysis and pneumonia occurred in 2 patients each (33.3%); there were no life-threatening complications or operation-related deaths, however. CONCLUSIONS: When performing an esophagectomy for thoracic esophageal squamous-cell cancer in patients who have received preoperative CRT, the weakened tracheobronchial wall can potentially be reinforced using a pedicled latissimus dorsi muscle flap.

[Experience of twenty-one cases of tracheo-bronchoplasty].

Twenty-one cases of tracheo-bronchoplasty were performed in Akita University Hospital from 1997 to 2007. There are 14 cases of squamous cell carcinoma, 3 cases of adenocarcinoma, 2 cases of adenoid cystic carcinoma, 1 case of inflammatory tracheal stenosis, and 1 case of inflammatory bronchial stenosis. We performed 12 cases of right upper sleeve lobectomy, 4 cases of left upper sleeve lobectomy, 2 cases of left lower sleeve lobectomy, 1 case of right sleeve pneumonectomy, and 2 cases of tracheoplasty. Of 3 cases, we added sleeve resection of pulmonary artery. The ends of the bronchus are anastomosed end-to-end. The bronchial anastomotic suture was carried out peri-cartilaginously through all layers using an interrupted suture technique except for membranous portion. Membranous portion was sutured a continuous anastomotic technique. We use monofilament, absorbable suture material.

Expression of alpha-1,3-fucosyltransferase type IV and VII genes is related to poor prognosis in lung cancer.

To date, five alpha-1,3-fucosyltransferase genes (Fuc-TIII, IV, V, VI, and VII) have been cloned. To examine the role of alpha-1,3-fucosyltransferase in the synthesis of sialyl Lewis x and the prognosis of lung cancer, PCR amplification of five fucosyltransferase genes and immunohistochemical staining of sialyl Lewis x were performed in 333 patients with lung cancer who underwent surgical resection from 1980 to 1993. The frequencies of Fuc-TIII/V, Fuc-TVI, Fuc-TIV, and Fuc-TVII expression were 9%, 26%, 75%, and 66%, respectively. The frequency of sialyl Lewis x expression (75%) was comparable to Fuc-TIV and Fuc-TVII expression. However, the grading of sialyl Lewis x staining correlated only with the grading of Fuc-TVII gene amplification. Survival of the patients whose tumors showed strong expression of Fuc-TIV and/or FucT-VII was significantly shorter than that of the patients whose tumors did not express either Fuc-TIV or Fuc-TVII. These results suggest that Fuc-TIV and Fuc-TVII expression may be of prognostic value among patients with lung cancer by participating in the biosynthesis of sialyl Lewis x.

[Survival after surgical resection for stage IV non-small cell lung cancer].

We analyzed 20 patients with stage IV non-small cell lung cancer operated from 1988 to 2003. Fourteen out of 20 were cases with pulmonary metastasis (pm2). The prognosis of patients with pm2 was better than that of those with distant organ metastasis. In pm2 patients, the survival rate of cases without lymph node metastases was higher than those with lymph node metastases. It is suggested that in cases of pm2 without lymph node metastases, surgical operation is possibly effective treatment of choice.

Novel bacterial peroxidase without catalase activity from Flavobacterium meningosepticum: purification and characterization.

A novel bacterial peroxidase co-produced intracellularly with H(2)O(2)-forming nucleoside oxidase, was purified from the cell-free extract of Flavobacterium meningosepticum to homogeneity with 10.3% overall recovery through simple purification procedures including successive DEAE-Sephacel, phenyl-Sepharose CL-4B and Sephacryl S-300 chromatography. The relative molecular mass of the native enzyme was 220¿ omitted¿000 Da, and that of its subunit was 54¿ omitted¿000 Da. In contrast to other major intercellular peroxidases of bacterial origin, the enzyme did not show any catalase activity. The amino acid sequences of the 92 NH(2)-terminal amino acids and three internal peptides showed no significant homology with known peroxidases. The enzyme was not sensitive to the typical peroxidase inhibitors NaCN, NaF and NaN(3), while mercuric ion strongly inhibited the enzyme activity, and some carbonyl reagents were also found to have inhibitory effects. The enzyme showed a small K(m) value for H(2)O(2) (9.5 microM) compared to other peroxidases. On the basis of its visible absorption spectrum, the enzyme contained about 1.3 mol of heme per molecule.

Extended Region of Nodulation Genes in Rhizobium meliloti 1021. I. Phenotypes of Tn5 Insertion Mutants.

Rhizobium meliloti Nod(-) mutant WL131, a derivative of wild-type strain 102F51, was complemented by a clone bank of wild-type R. meliloti 1021 DNA, and clone pRmJT5 was recovered. Transfer of pRmJT5 conferred alfalfa nodulation on other Rhizobium species, indicating a role in host range determination for pRmJT5. Mutagenesis of pRmJT5 revealed several segments in which transposon insertion causes delay in nodulation, and/or marked reduction of the number of nodules formed on host alfalfa plants. The set of mutants indicated five regions in which nod genes are located; one mutant, nod-216, is located in a region not previously reported to encode a nodulation gene. Other mutant phenotypes correlated with the positions of open reading frames for nodH, nodF and nodE , and with a 2.2-kb EcoRI fragment. A mutant in nodG had no altered phenotype in this strain. One nodulation mutant was shown to be a large deletion of the common nod gene region. We present a discussion comparing the various studies made on this extended nod gene region.

[Thymolipoma in association with myasthenia gravis; report of a case].

An 18-year-old man was admitted with easy fatigue and muscle weakness. A diagnosis of myasthenia gravis was made. Chest X-ray showed no mediastinal abnormality, however, chest computed tomography (CT) showed a soft tissue mass in the thymus. The patient underwent extended thymectomy and small tumor (2 x 2 x 2 cm) was resected. On histological examination the tumor proved to be a thymolipoma composed of mature adipose elements containing cords and nests of thymic tissue. Symptoms of myasthenia gravis were dramatically improved after the operation. This case is the 12th in the world literature in which a thymolipoma is associated with symptoms of myasthenia gravis.