Bioconversion of arachidonic acid into human 14,15-hepoxilin B3 and 13,14,15-trioxilin B3 by recombinant cells expressing microbial 15-lipoxygenase without and with epoxide hydrolase.

OBJECTIVE: To produce high concentrations of 13-hydroxy-14,15-epoxy-eicosatrienoic acid (14,15-hepoxilin B3, 14,15-HXB3) and 13,14,15-trihydroxy-eicosatrienoic acid (13,14,15-trioxilin B3, 13,14,15-TrXB3) from arachidonic acid (ARA) using microbial 15-lipoxygenase (15-LOX) without and with epoxide hydrolase (EH), respectively. RESULTS: The products obtained from the bioconversion of ARA by recombinant Escherichia coli cells containing Archangium violaceum 15-LOX without and with Myxococcus xanthus EH were identified as 14,15-HXB3 and 13,14,15-TrXB3, respectively. Under the optimal conditions of 30 g cells L-1, 200 mM ARA, 25 °C, and initial pH 7.5, the cells converted 200 mM ARA into 192 mM 14,15-HXB3 and 100 mM 13,14,15-TrXB3 for 150 min, with conversion yields of 96 and 51% and productivities of 77 and 40 mM h-1, respectively. CONCLUSION: These are the highest concentrations, productivities, and yields of hepoxilin and trioxilin from ARA reported thus far.

Correction to: Bioconversion of arachidonic acid into human 14,15-hepoxilin B3 and 13,14,15-trioxilin B3 by recombinant cells expressing microbial 15-lipoxygenase without and with epoxide hydrolase.

In the original publication of the article, some chirality of Fig. 1 was published incorrectly. The corrected figure is provided below.

Short-Term Outcomes of ABO-Incompatible Living Donor Kidney Transplantation With Uniform Protocol: Significance of Baseline Anti-ABO Titer.

Antibody-mediated rejection (AMR) is one of the major causes of poor outcomes in ABO-incompatible kidney transplantation (ABOi KT). Studies investigating AMR risk factors found that anti-ABO titer is a major issue. However, the significance of antibody titer has been debated. This retrospective study analyzed AMR risk factors in 59 patients who underwent ABOi KT between August 2010 and January 2015. We also analyzed AMR risk factors in recipients with high anti-ABO baseline titers (≥1:64 on dithiothreitol at 37°C phase or ≥1:256 on antihuman globulin phase). The 2-year patient survival rate was 95.8%, and the 2-year graft survival rate was 94.9%. Nine patients (15.3%) experienced clinical (6 of 59 [10.2%]) or subclinical (3 of 59 [5.1%]) AMR. One patient experienced graft loss from hyperacute rejection. AMR risk factor analysis revealed that baseline antibody titer was associated with incidence of AMR. In patients with high baseline titers, low doses of rituximab (200-mg single-dose), an antibody against CD20, was predictive for AMR. Six patients who received pretransplant intravenous immunoglobulin did not experience AMR even when they had high baseline antibody titers. Our results indicate that a high baseline antibody titer affected the incidence of AMR. ABOi KT candidates with high baseline titers need to undergo an intensified preconditioning protocol, including high-dose rituximab (375 mg/m(2)) and intravenous immunoglobulin.

Time interval to conversion of interferon-gamma release assay after exposure to tuberculosis.

The proper interval for repeating an interferon-γ release assay (IGRA) among tuberculosis contacts with initially negative results is unknown. The interval for IGRA conversion after exposure to patients with active pulmonary tuberculosis in an outbreak setting was evaluated. In a platoon of 32 soldiers, four active pulmonary tuberculosis patients, in addition to one index patient, were diagnosed during a contact investigation. For the other 27 contacts, a tuberculin skin test (TST) and QuantiFERON® TB-Gold In-Tube (QFT-GIT) assay were performed. For soldiers with a negative result on the initial QFT-GIT assay, the test was repeated at 2, 4, 8, 14, 18 and 30 weeks until positive conversion occurred. When conversion was identified, the subject was treated for latent tuberculosis infection. Initially, 17 (63.0%) soldiers gave positive QFT-GIT results, whereas 21 (77.8%) showed positive TST results. Among 10 participants with initially negative QFT-GIT results, three showed conversion at 2 weeks, three at 4 weeks and three at 14 weeks. Conversion did not occur during the 30-week observation period in one contact. Based on the tuberculosis exposure time-points among the contacts, IGRA conversion generally occurred 4-7 weeks after exposure, although it could occur as late as 14-22 weeks after exposure.

Substrate specificity of a recombinant D-lyxose isomerase from Serratia proteamaculans that produces D-lyxose and D-mannose.

AIMS: Characterization of substrate specificity of a D-lyxose isomerase from Serratia proteamaculans and application of the enzyme in the production of D-lyxose and D-mannose. METHODS AND RESULTS: The concentrations of monosaccharides were determined using a Bio-LC system. The activity of the recombinant protein from Ser. proteamaculans was the highest for D-lyxose among aldoses, indicating that it is a D-lyxose isomerase. The native recombinant enzyme existed as a 54-kDa dimer, and the maximal activity for D-lyxose isomerization was observed at pH 7.5 and 40 degrees C in the presence of 1 mmol l(-1) Mn(2+). The K(m) values for D-lyxose, D-mannose, D-xylulose, and D-fructose were 13.3, 32.2, 3.83, and 19.4 mmol l(-1), respectively. In 2 ml of reaction volume at pH 7.5 and 35 degrees C, D-lyxose was produced at 35% (w/v) from 50% (w/v) D-xylulose by the D-lyxose isomerase in 3 h, while D-mannose were produced at 10% (w/v) from 50% (w/v) D-fructose in 5 h. CONCLUSIONS: We identified the putative sugar isomerase from Ser. proteamaculans as a D-lyxose isomerase. The enzyme exhibited isomerization activity for aldose substrates with the C2 and C3 hydroxyl groups in the left-hand configuration. High production rates of d-lyxose and d-mannose by the enzyme were obtained. SIGNIFICANCE AND IMPACT OF THE STUDY: A new D-lyxose isomerase was found, and this enzyme had higher activity for D-lyxose and D-mannose than previously reported enzymes. Thus, the enzyme can be applied in industrial production of D-lyxose and D-mannose.

Hydrolytic properties of a thermostable α-L-arabinofuranosidase from Caldicellulosiruptor saccharolyticus.

AIMS: To characterize of a thermostable recombinant α-L-arabinofuranosidase from Caldicellulosiruptor saccharolyticus for the hydrolysis of arabino-oligosaccharides to l-arabinose. METHODS AND RESULTS: A recombinant α-L-arabinofuranosidase from C. saccharolyticus was purified by heat treatment and Hi-Trap anion exchange chromatography with a specific activity of 28.2 U mg(-1). The native enzyme was a 58-kDa octamer with a molecular mass of 460 kDa, as measured by gel filtration. The catalytic residues and consensus sequences of the glycoside hydrolase 51 family of α-L-arabinofuranosidases were completely conserved in α-L-arabinofuranosidase from C. saccharolyticus. The maximum enzyme activity was observed at pH 5.5 and 80°C with a half-life of 49 h at 75°C. Among aryl-glycoside substrates, the enzyme displayed activity only for p-nitrophenyl-α-L-arabinofuranoside [maximum k(cat)/K(m) of 220 m(mol l(-1))(-1) s(-1)] and p-nitrophenyl-α-L-arabinopyranoside. This substrate specificity differs from those of other α-L-arabinofuranosidases. In a 1 mmol l(-1) solution of each sugar, arabino-oligosaccharides with 2-5 monomer units were completely hydrolysed to L-arabinose within 13 h in the presence of 30 U ml(-1) of enzyme at 75°C. CONCLUSIONS: The novel substrate specificity and hydrolytic properties for arabino-oligosaccharides of α-L-arabinofuranosidase from C. saccharolyticus demonstrate the potential in the commercial production of L-arabinose in concert with endoarabinanase and/or xylanase. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work contribute to the knowledge of hydrolytic properties for arabino-oligosaccharides performed by thermostable α-L-arabinofuranosidase.

Thiamine increases beta-glucosidase production in the newly isolated strain of Fomitopsis pinicola.

AIMS: To isolate a high beta-glucosidase (BGL)-producing strain and to optimize BGL production in the isolated strain. METHODS AND RESULTS: A high BGL-producing strain was isolated and identified as Fomitopsis pinicola KMJ812 based on its morphology and a comparison of sequence of its internal transcribed spacer rDNA gene. To increase BGL production, F. pinicola was supplemented with various vitamins. Supplementation with thiamine (20 mg l(-1)) improved BGL production in F. pinicola cultures by 3.7-fold to give a specific activity of 114.4 micromol min(-1) mg(-1) protein, one of the highest among BGL-producing micro-organisms. The increased production of BGL in the thiamine-supplemented culture was confirmed by 2D electrophoresis followed by MS/MS sequencing. The BGL purified from F. pinicola culture showed the highest catalytic efficiency ever reported. CONCLUSION: Supplemental thiamine remarkably increased BGL production by a novel BGL-producing strain, F. pinicola KMJ812. SIGNIFICANCE AND IMPACT OF THE STUDY: Our results provide a high BGL-producing strain and the production media for BGL production, and should contribute to better industrial production of glucose via biological processes.

Intraductal papillary mucinous tumour of the pancreas: differentiation of malignancy and benignancy by CT.

AIM: To retrospectively identify signs predictive of malignant intraductal papillary mucinous tumour (IPMT) of the pancreas on computed tomography (CT) images. MATERIALS AND METHODS: Thirty-four benign and 21 malignant pancreatic IPMTs were evaluated. Preoperative helical CT images in these 55 cases of pathologically proven pancreatic IPMT were reviewed by two radiologists unaware of the histological grading. Tumour morphological types, locations, numbers and sizes of cystic lesions, maximum main pancreatic duct diameters, the presence of septa, mural nodule, wall thickening, and calcification in cysts, communication with the main pancreatic duct, peripancreatic haziness, protrusion of duodenal papilla, pancreatic atrophy, lymphadenopathy and distant metastasis were analysed using univariate and multivariate analysis. RESULTS: Main duct IPMTs were more likely to be malignant (71%) than branch duct (23%) or combined type IPMTs (28%; p=0.002). Among the branch duct type and combined types, large cystic lesion (p=0.018), the presence of a mural nodule (p=0.018), a thickened wall (p=0.009), and peripancreatic haziness (p=0.039) were found to predict malignancy. CONCLUSION: CT is helpful in the preoperative differentiation of malignant and benign pancreatic IPMT. The presence of a dilated main pancreatic duct, mural nodules, thickened wall and peripancreatic haziness may be used as independent predictive signs of malignancy.

Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase that increases the production rate of D-tagatose.

AIMS: Characterization of a mutated Geobacillus stearothermophilus L-arabinose isomerase used to increase the production rate of D-tagatose. METHODS AND RESULTS: A mutated gene was obtained by an error-prone polymerase chain reaction using L-arabinose isomerase gene from G. stearothermophilus as a template and the gene was expressed in Escherichia coli. The expressed mutated L-arabinose isomerase exhibited the change of three amino acids (Met322-->Val, Ser393-->Thr, and Val408-->Ala), compared with the wild-type enzyme and was then purified to homogeneity. The mutated enzyme had a maximum galactose isomerization activity at pH 8.0, 65 degrees C, and 1.0 mM Co2+, while the wild-type enzyme had a maximum activity at pH 8.0, 60 degrees C, and 1.0-mM Mn2+. The mutated L-arabinose isomerase exhibited increases in D-galactose isomerization activity, optimum temperature, catalytic efficiency (kcat/Km) for D-galactose, and the production rate of D-tagatose from D-galactose. CONCLUSIONS: The mutated L-arabinose isomerase from G. stearothermophilus is valuable for the commercial production of D-tagatose. SIGNIFICANCE AND IMPACT OF THE STUDY: This work contributes knowledge on the characterization of a mutated L-arabinose isomerase, and allows an increased production rate for D-tagatose from D-galactose using the mutated enzyme.

Characterization of a thermostable recombinant beta-galactosidase from Thermotoga maritima.

AIMS: Characterization of a thermostable recombinant beta-galactosidase from Thermotoga maritima for the hydrolysis of lactose and the production of galacto-oligosaccharides. METHODS AND RESULTS: A putative beta-galactosidase gene of Thermotoga maritima was expressed in Escherichia coli as a carboxyl terminal His-tagged recombinant enzyme. The gene encoded a 1100-amino acid protein with a calculated molecular weight of 129,501. The expressed enzyme was purified by heat treatment, His-tag affinity chromatography, and gel filtration. The optimum temperatures for beta-galactosidase activity were 85 and 80 degrees C with oNPG and lactose, respectively. The optimum pH value was 6.5 for both oNPG and lactose. In thermostability experiments, the enzyme followed first-order kinetics of thermal inactivation and its half-life times at 80 and 90 degrees C were 16 h and 16 min, respectively. Mn2+ was the most effective divalent cation for beta-galactosidase activity on both oNPG and lactose. The Km and Vmax values of the thermostable enzyme for oNPG at 80 degrees C were 0.33 mm and 79.6 micromol oNP min(-1) mg(-1). For lactose, the Km and Vmax values were dependent on substrate concentrations; 1.6 and 63.3 at lower concentrations up to 10 mm of lactose and 27.8 mm and 139 micromol glucose min(-1) mg(-1) at higher concentrations, respectively. The enzyme displayed non-Michaelis-Menten reaction kinetics with substrate activation, which was explained by simultaneous reactions of hydrolysis and transgalactosylation. CONCLUSIONS: The results suggest that the thermostable enzyme may be suitable for both the hydrolysis of lactose and the production of galacto-oligosaccharides. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work contribute to the knowledge of hydrolysis and transgalactosylation performed by beta-galactosidase of hyperthermophilic bacteria.

Increased expression of caveolin-1 and microvessel density correlates with metastasis and poor prognosis in clear cell renal cell carcinoma.

OBJECTIVE: To investigate the relationship of caveolin-1 expression and microvessel density (MVD), a reflection of angiogenesis, with metastasis and prognosis in patients with clear cell renal cell carcinoma (RCC). PATIENTS AND METHODS: Formalin-fixed, paraffin-embedded tissue sections of clear cell RCC from 67 patients who had undergone radical nephrectomy were stained immunohistochemically with specific antibodies against caveolin-1 and CD34. Caveolin-1 immunostaining was semi-quantitatively estimated based on the proportion (percentage of positive cells) and intensity. MVD was determined with CD34-stained slides. The expression pattern of caveolin-1 and MVD was compared with the clinicopathological variables. RESULTS: Eighteen patients had either synchronous or metachronous metastases and 11 died during the follow-up. Caveolin-1 intensity was significantly correlated with tumour size (P = 0.005), TNM stage (P = 0.028), M stage (P = 0.012), grade (P = 0.015), and metastasis (synchronous or metachronous; P < 0.001). The caveolin-1 proportion (P = 0.037) and MVD (P = 0.011) were significantly correlated with metastasis. MVD was correlated with caveolin-1 intensity (r = 0.385, P = 0.001) and caveolin-1 proportion (r = 0.388, P = 0.001). There was no difference in the expression of caveolin-1 and MVD between primary and metastatic sites. The survival of patients with higher caveolin-1 intensity was significantly worse than that of patients with lower caveolin-1 intensity. Multivariate analyses indicated that only M-stage was an independent prognostic factor for cancer-specific survival and caveolin-1 expression was not an independent factor. CONCLUSIONS: Increased expression of caveolin-1 and MVD is associated with metastasis and a worse prognosis in clear cell RCC. Caveolin-1 expression is correlated with MVD. These results suggest that caveolin-1 may be important in the progression of clear cell RCC and angiogenesis may be affected by caveolin-1 during the progression of RCC.

Lactulose production by beta-galactosidase in permeabilized cells of Kluyveromyces lactis.

Lactulose production from lactose and fructose was investigated with several commercial beta-galactosidases. The enzyme from Kluyveromyces lactis exhibited the highest lactulose productivity among the beta-galactosidases tested. The reaction conditions for lactulose production were optimized using cells that had been permeabilized by treatment with 50% (v/v) ethanol: cell concentration, 10.4 g l(-1); concentration of substrates, 40% (w/v) lactose and 20% (w/v) fructose; temperature, 60( degrees )C; pH 7.0. Under these conditions, the permeabilized cells produced approximately 20 g l(-1) lactulose in 3 h with a lactulose productivity of 6.8 g l(-1) h(-1). These results represent 1.3- and 2.1-fold increases in lactulose concentration and productivity compared with untreated washed cells. This is the first reported trial of enzymatic synthesis of lactulose using permeabilized yeast cells.

Low-frequency collective chain dynamics in a model biomembrane.

Proton NMR was employed as a probe for the collective hydrocarbon chain dynamics in decylammonium chloride (C10H21NH3Cl), a model biomembrane undergoing an irreversible structural phase transition sequence. Our rotating frame spin-lattice relaxation measurements revealed a low-frequency critical collective chain dynamics in the kHz regime, which is associated with the interdigitated to noninterdigitated chain configurational phase transition.

Improvement of tagatose conversion rate by genetic evolution of thermostable galactose isomerase.

To enhance the isomerization rate of galactose into tagatose, a thermostable galactose isomerase, which was isolated from bacteria growing in a hot spring, was genetically improved using the error-prone PCR method. From 500 mutant clones, a clone showing improved conversion activity was selected. The sequence of the selected clone had five amino acid changes: His(228)-->Asp, Gly(384)-->Asp, Ser(393)-->Thr, Lys(428)-->Asn and Asp(475)-->Lys. The improved galactose isomerase had an 11-fold higher reaction rate than the original.

Increased erythritol production in fed-batch cultures of Torula sp. by controlling glucose concentration.

The effect of glucose concentration on erythritol production by Torula sp. was investigated. The maximum volumetric productivity of erythritol was obtained at an initial glucose concentration of 300 g l(-1) in batch culture. The volumetric productivity was maximal at a controlled glucose concentration of 225 g l(-1), reducing the lag time of the erythritol production. A fed-batch culture was established with an initial glucose concentration of 300 g l(-1) and with a controlled glucose concentration of 225 g l(-1) in medium containing phytic acid as a phosphate source. In this fed-batch culture, a final erythritol production of 192 g l(-1) was obtained from 400 g l(-1) glucose in 88 h. This corresponded to a volumetric productivity of 2.26 g l(-1) h(-1) and a 48% yield.

Fluorescein dermofluorometry for the assessment of diabetic microvascular disease.

BACKGROUND/AIMS: Fluorescein dermofluorometry can be used to relate the uptake of fluorescein in the skin to blood flow. We have characterized the uptake of the dye by a wash-in time constant that is inversely proportional to the local blood flow. The purpose of this study was to explore the use of dermofluorometry in the assessment of patients with diabetic microvascular disease. METHODS: Fluorescein dermofluorometry was performed in four groups of patients: non-diabetic control patients, diabetic control patients, diabetic patients with chronic foot ulcers, and diabetic patients with acute foot ulcers. The outcomes of the patients with foot ulcers were documented 4-14 months after participation. Following an intravenous injection of sodium fluorescein, the change in the fluorescein signal with time was continuously measured at the plantar surface of the foot. Both the initial slope of the signal and the wash-in time constant were calculated in each subject. RESULTS: Significant differences in the wash-in time constant were found between diabetic and non-diabetic subjects and between diabetic subjects with and without foot ulcers. Of the eight patients with foot ulcers, two of them did not display an early wash-out in the dermofluorometer signal and later both required amputations. CONCLUSION: The fluorescein wash-in time constant demonstrated better correlation with the presence of diabetic microvascular disease than did the initial slope of the signal. Differences in the wash-in time constants of non-diabetic and diabetic subjects support the hemodynamic hypothesis for the development of microvascular disease. The indication of early wash-out of the fluorescein signal may also be useful in the prediction of ulcer healing.

Biological modification of the fatty acid group in an emulsan by supplementing fatty acids under conditions inhibiting fatty acid biosynthesis.

When the concentration of the antibiotic cerulenin was increased up to 3.0 mg/l in medium containing ethanol as a carbon source, the specific growth rate of Acinetobacter calcoaceticus and the fatty acid content of the emulsan decreased from 0.179 h(-1) and 13.9% to 0.015 h(-1) and 3.4%, respectively. The emulsifying activity in medium containing cerulenin decreased with increasing cerulenin concentration. In the culture containing 3.0 mg/l cerulenin, fatty acid biosynthesis was inhibited. Various fatty acids were added to this inhibitory culture as a second carbon source to modify the fatty acid group in the emulsan. When an odd-numbered fatty acid was added, the resulting emulsan was found to have other odd-numbered fatty acids that were not present originally. Among the emulsan produced from even-numbered fatty acids, the emulsan produced from myristic acid (C14) contained the greatest amount of the same-numbered fatty acids. When the amount of supplemental myristic acid was increased, the myristic acid content in the emulsan increased, but its emulsifying activity decreased.

Increase of xylitol yield by feeding xylose and glucose in Candida tropicalis.

Candida tropicalis, a strain isolated from the sludge of a factory manufacturing xylose, produced a high xylitol concentration of 131 g/l from 150 g/l xylose at 45 h in a flask. Above 150 g/l xylose, however, volumetric xylitol production rates decreased because of a lag period in cell growth. In fed-batch culture, the volumetric production rate and xylitol yield from xylose varied substantially with the controlled xylose concentration and were maximum at a controlled xylose concentration of 60 g/l. To increase the xylitol yield from xylose, feeding experiments using different ratios of xylose and glucose were carried out in a fermentor. The maximum xylitol yield from 300 g/l xylose was 91% at a glucose/xylose feeding ratio of 15%, while the maximum volumetric production rate of xylitol was 3.98 g l-1 h-1 at a glucose/xylose feeding ratio of 20%. Xylitol production was found to decrease markedly as its concentration rose above 250 g/l. In order to accumulate xylitol to 250 g/l, 270 g/l xylose was added in total, at a glucose/xylose feeding ratio of 15%. Under these conditions, a final xylitol production of 251 g/l, which corresponded to a yield of 93%, was obtained from 270 g/l xylose in 55 h.

Increase of xylitol production rate by controlling redox potential in Candida parapsilosis.

The effect of redox potential on xylitol production by Candida parapsilosis was investigated. The redox potential was found to be useful for monitoring the dissolved oxygen (DO) level in culture media, especially when the DO level was low. An increase in the agitation speed in a 5 L fermentor resulted in an increased culture redox potential as well as enhanced cell growth. Production of xylitol was maximized at a redox potential of 100 mV. As the initial cell concentration increased from 8 g/L to 30 g/L, the volumetric productivity of xylitol increased from 1.38 g/L. h to 4.62 g/L. h. A two-stage xylitol production strategy was devised, with stage 1 involving rapid production of cells under well-aerated conditions, and stage 2 involving cultivation with reduced aeration such that the culture redox potential was 100 mV. Using this technique, a final xylitol concentration of 180 g/L was obtained from a culture medium totally containing 254.5 g/L xylose in a 3,000 L pilot scale fermentor after 77 h fermentation. The volumetric productivity of xylitol during the fermentation was 2.34 g/L. h.

Effect of soybean oil and glucose on sophorose lipid fermentation by Torulopsis bombicola in continuous culture.

The effect of soybean oil and glucose on the growth of Torulopsis bombicola and sophorose lipid production in continuous culture was investigated. As the dilution rate in 100 g/l glucose and 100 g/l soybean oil medium was increased, the dry cell weight and sophorose lipid concentration decreased. Sophorose lipid productivity, however, was maximum at a dilution rate of 0.03 h-1. The cell yield from glucose and the sophorose lipid production from soybean oil were approximately constant regardless of the dilution rate. The specific consumption rate of soybean oil was closely related to the specific production rate of sophorose lipid. These results suggest that soybean oil was used only for sophorose lipid production whereas glucose was used only for cell mass and maintenance. When the soybean oil concentration was varied at fixed dilution rate in 100 g/l glucose medium, a high concentration of soybean oil was found to inhibit sophorose lipid production.