Evaluation of the enzymatic properties of DNA (cytosine-5)-methyltransferase M.ApeKI from archaea in the presence of metal ions.

We previously identified M.ApeKI from Aeropyum pernix K1 as a highly thermostable DNA (cytosine-5)-methyltransferase. M.ApeKI uses the type II restriction-modification system (R-M system), among the best-studied R-M systems. Although endonucleases generally utilize Mg (II) as a cofactor, several reports have shown that MTases exhibit different reactions in the presence of metal ions. This study aim was to evaluate the enzymatic properties of DNA (cytosine-5)-methyltransferase M.ApeKI from archaea in the presence of metal ions. We evaluated the influence of metal ions on the catalytic activity and DNA binding of M.ApeKI. The catalytic activity was inhibited by Cu (II), Mg (II), Mn (II), and Zn (II), each at 5 mM. DNA binding was more strongly inhibited by 5 mM Cu (II) and 10 mM Zn (II). To our knowledge, this is the first report showing that DNA binding of typeII MTase is inhibited by metal ions.

Evaluation of the Properties of the DNA Methyltransferase from Aeropyrum pernix K1.

Little is known regarding the DNA methyltransferases (MTases) in hyperthermophilic archaea. In this study, we focus on an MTase from Aeropyrum pernix K1, a hyperthermophilic archaeon that is found in hydrothermal vents and whose optimum growth temperature is 90°C to 95°C. From genomic sequence analysis, A. pernix K1 has been predicted to have a restriction-modification system (R-M system). The restriction endonuclease from A. pernix K1 (known as ApeKI from New England BioLabs Inc. [catalog code R06435]) has been described previously, but the properties of the MTase from A. pernix K1 (M.ApeKI) have not yet been clarified. Thus, we demonstrated the properties of M.ApeKI. In this study, M.ApeKI was expressed in Escherichia coli strain JM109 and affinity purified using its His tag. The recognition sequence of M.ApeKI was determined by methylation activity and bisulfite sequencing (BS-seq). High-performance liquid chromatography (HPLC) was used to detect the position of the methyl group in methylated cytosine. As a result, it was clarified that M.ApeKI adds the methyl group at the C-5 position of the second cytosine in 5'-GCWGC-3'. Moreover, we also determined that the MTase optimum temperature was over 70°C and that it is strongly tolerant to high temperatures. M.ApeKI is the first highly thermostable DNA (cytosine-5)-methyltransferase to be evaluated by experimental evidence. IMPORTANCE In general, thermophilic bacteria with optimum growth temperatures over or equal to 60°C have been predicted to include only N4-methylcytosine or N6-methyladenine as methylated bases in their DNA, because 5-methylcytosine is susceptible to deamination by heat. However, from this study, A. pernix K1, with an optimum growth temperature at 95°C, was demonstrated to produce a DNA (cytosine-5)-methyltransferase. Thus, A. pernix K1 presumably has 5-methylcytosine in its DNA and may produce an original repair system for the expected C-to-T mutations. M.ApeKI was demonstrated to be tolerant to high temperatures; thus, we expect that M.ApeKI may be valuable for the development of a novel analysis system or epigenetic editing tool.

[Evaluation of Conventional Antifungal Agents against Recombinant Yeast Overexpressing ß-1,3-Glucanase].

　Only 4 classes of antifungal agents comprising 9 compounds are effective against deep mycosis in Japan, and it has been difficult to develop new antifungal specific agents. Micafungin, which has been used as an antifungal agent since 2002, inhibits ß-1,3-glucan synthesis in fungal cell walls, thereby killing yeast and filamentous fungi with no septum. In this study, we constructed a pYES2-BGL2 vector to overexpress ß-1,3-glucanase (BGL2) in yeast (Saccharomyces cerevisiae INVSc1) and evaluated the synergy between BGL2 overexpression and conventional antifungal agents. The recombinant yeast was incubated in SC-Ura medium, which contained galactose to induce BGL2 overexpression. The recombinant yeast with induced BGL2 overexpression was also frozen and crushed to obtain crude protein for sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), which revealed robust BGL2 overexpression compared with that in the control yeast without the expression vector. Therefore, we considered that we successfully constructed the recombinant yeast to express more BGL2. Further, 3 conventional antifungal agents (amphotericin B, micafungin, and miconazole) were more effective against the recombinant yeast than against the control yeast. From this result, it is suggested that BGL2 overexpression has an enhancing effect on conventional antifungal agents. Hence, glucanase-inducing compounds could act as novel antifungal drugs by augmenting the effectiveness of conventional antifungal agents.

Enzymatic Analysis of Positional Fatty Acid Distributions in Triacylglycerols by 1(3)-Selective Transesterification with Candida antarctica Lipase B: a Collaborative Study.

The positional distributions of fatty acids (FAs) in fats and oils are principally analyzed by selectively transesterifying the target triacylglycerols (TAGs) at the 1(3) position using Pseudozyma (Candida) antarctica lipase, followed by recovering the resulting 2-monoacylglycerols (MAGs) by chromatography. FA compositions were measured by gas chromatography (GC) after methylating target TAGs and 2-MAGs. The method was collaboratively evaluated by 12 laboratories by analyzing the positional FA distributions in soybean, palm, and sardine oils. The maximum reproducibility relative standard deviations for the major FAs and those at the sn-2 positions of soybean, palm, and sardine oils were 4.41% and 3.92% (18:3n-3), 4.48% and 3.82% (18:0), and 8.93 and 8.24% (14:0), respectively. The values at the sn-2 position were always low. Therefore, these results indicated that the variations were mainly caused by the FA analysis procedure, i.e., the methylation and GC analyses, rather than the enzymatic transesterification and chromatography utilized to prepare 2-MAGs from the target oil.

Reduction of virulence factor pyocyanin production in multidrug-resistant Pseudomonas aeruginosa.

Nosocomial infections caused by metallo-ß-lactamase (MBL)-producing multidrug-resistant (MDR) Pseudomonas aeruginosa have become a worldwide problem. Pyocyanin, a representative pigment produced by P. aeruginosa, is the major virulence factor of this organismThe aim of this study was to investigate the pyocyanin-producing ability of MBL-producing MDR P. aeruginosa. A total of 50 clinical isolates of P. aeruginosa, including 20 MDR strains, were collected at 18 general hospitals in Japan. The chromaticity and luminosity produced by pyocyanin in each isolate were measured. The quantity of pyocyanin and the expression of the phzM and phzS genes coding a pyocyanin synthesis enzyme were measured. MDR strains showed a bright yellow-green, while non-MDR strains tended to show a dark blue-green. The quantities of pyocyanin in MBL-producing strains and non-producing strains were 0.015 ± 0.002 and 0.41 ± 0.10 µg, respectively. The expression of the phzM and phzS genes in the MDR strains was 11 and 14 %, respectively, of the expression in the non-MDR strains. When the MBL gene was transduced into P. aeruginosa and it acquired multidrug resistance, it was shown that the pyocyanin-producing ability decreased. The pathogenicity of MBL-producing MDR P. aeruginosa may be lower than that of non-MDR strains. These MBL-producing MDR strains may be less pathogenic than non-MDR strains. This may explain why MDR-P. aeruginosa is unlikely to cause infection but, rather, causes subclinical colonization only.

Laparoscopic radical nephrectomy under near real-time three-dimensional surgical navigation with C-arm cone beam computed tomography.

OBJECTIVE: The aim of this study was to demonstrate the advantages of C-arm cone beam computed tomography for imaging guidance of laparoscopic radical nephrectomy (LRN). METHODS: Four patients referred to the authors' institution for LRN were included in this study. To visualize the renal vascular anatomy, the Iopamiron 300 contrast agent was injected intravenously. The surgeon could adjust the viewing angle of the images and rotate the reconstructed three-dimensional (3D) image manually by using a mouse-like controller. Using the near real-time 3D navigation images, the surgeon was able to recognize the renal vascular anatomy, and more easily perform the LRN. RESULTS: All procedures were successfully performed with a satisfactory diagnostic yield or therapeutic effect without procedure-related complications. CONCLUSION: This novel technology has great potential for application in LRN because it enables accurate depiction of the renal vessels and increases surgeon confidence.

Physiological functions of iso-type short-chain fatty acid and omega 3 polyunsaturated fatty acids containing oil in obese OLETF rats.

It has been known that tissues of porpoise contain unique structured-lipids as combination of iso-valeric acid (iso-C5:0) and omega 3 polyunsaturated fatty acids (omega3 PUFAs). It is well known that omega3 PUFAs have lipid-lowering effects in animal and human studies. Although branched chain fatty acids have been interested in their unique functions, there is no data concerning the effect of iso-C5:0 on lipid metabolism. In this study we investigated the effect of structured-lipids from porpoise adipose tissue (porpoise oil) on lipid metabolism in Otsuka Long-Evans Tokushima Fatty (OLETF) rats. For 4 weeks, rats were fed semisynthetic diets containing either 10% corn oil or 5% corn oil plus 5% porpoise oil. After feeding period, the porpoise oil diet significantly alleviated hepatic triglyceride accumulation compared with the control diet in OLETF rats. Although serum triglyceride level increased, serum level of adiponectin that can protect liver function and alleviate abnormalities of lipid and glucose metabolism increased in rats fed porpoise oil diet. In conclusion, results from the present study suggest that porpoise oil feeding prevents the development of fatty liver disease through the enhancement of lipoprotein secretion and increase of adiponectin production in obese rats.

Separation and quantification of sn-1 and sn-2 fatty acid positional isomers in phosphatidylcholine by RPLC-ESIMS/MS.

Endogenous phosphatidylcholine in biological membranes exists as isomers with acyl moieties at the sn-1 or sn-2 positions of the glycerol backbone. However, detailed biochemical information on these positional isomers is not generally available. This study is the first report on the separation and identification of positional isomers of endogenous phosphatidylcholine using reversed-phase LC-ESIMS/MS. The separation of positional isomers in PC was achieved by using ultra performance LC, which uses a high-resolution HPLC system. To identify positional isomers in individual PC species, their lyso-PC-related fragments and fatty acids, which were obtained by MS/MS analysis in the negative ion mode, were used. From the application results of biological samples, the lipid extracts of mouse brain were found to be abundant in PC containing 22:6 at the sn-1 position of the glycerol backbone. However, the lipid extracts from mouse heart and liver were not abundant in positional isomers. This achievement demonstrates that the relative amounts of positional isomers in various tissues or molecular species differ. These results will be useful for the clarification of the biological mechanisms of remodelling enzymes such as phospholipase and acyltransferase. Thus, our report provides a novel and critical milestone in understanding how molecular composition of phospholipids is established and their biological roles.

Evaluation of dentin bonding performance and acid-base resistance of the interface of two-step self-etching adhesive systems.

The purpose of this study was to evaluate dentin bond strengths and to observe the adhesive-dentin interface after acid-base challenge using fluoride-free and fluoride-releasing self-etching adhesive systems; Clearfil SE Bond (SE), FL-Bond (FL) and FL-Bond II(FL II). Fifteen dentin surfaces from human molars were ground and bonded with one of three adhesive systems. The microtensile bond strength (muTBS) test was performed at a crosshead speed of 1 mm/min. The interface of the bonded specimens after acid-base challenge were also examined by a SEM. The muTBS of SE were significantly higher than those of FL and FL II (p<0.05), however, there were no significant differences between FL and FL II (p>0.05). An acid-base resistant zone (ABRZ) was observed in all the groups, however, formation of the ABRZ was material dependent. Fluoride-release from the adhesive is a key factor to create thick ABRZ.

Analysis of oxidized phosphatidylcholines as markers for oxidative stress, using multiple reaction monitoring with theoretically expanded data sets with reversed-phase liquid chromatography/tandem mass spectrometry.

Recently, it was reported that oxidized phosphatidylcholine shows biological activities via scavenger receptor CD36 or Toll-like receptor 4 (TLR4)-TRIF. Thus, the analysis of oxidized phospholipids is essential in understanding these biological roles. Here, we report an analytical method for oxidized phosphatidylcholines using multiple reaction monitoring (MRM) with theoretically expanded data sets. This analytical method was performed by a quadrupole linear ion trap mass spectrometer with ultra performance LC (UPLC). To investigate whether this established analytical method was applicable to biological samples, we performed variation analysis of oxidized PCs using a myocardial ischemia-reperfusion model. Most oxidized PCs were detected in higher amounts in the ischemic myocardium than in the non-ischemic myocardium. From these application results, this established method is a valuable tool for the global analysis of oxidized PCs. In the future, our study can provide further understanding of how oxidized phospholipids are produced and are correlated to various diseases.

Antibacterial activity of ZnO powder with crystallographic orientation.

ZnO powder with crystallographic orientation was prepared from the mixed aqueous solution of zinc chloride, tri-ethanol amine and thio-urea. From X-ray diffraction measurement, as-prepared powder was found to have the orientation along a-b axes of hexagonal structure, and a needle-like shape with the aspect ratio of 5 was observed by scanning electron microscope, indicating that as-prepared powder had crystallographic orientation. In the tests of antibacterial activity by colony count method, ZnO powders with and without crystallographic orientation were used in present work. Survival ratio of bacteria decreased with increasing powder concentration, i.e., increase in antibacterial activity. The antibacterial activity in ZnO powder with crystallographic orientation was weaker than that in commercial ZnO powder without orientation at same powder concentration. Regarding specific surface area of the powders used in antibacterial tests, however, antibacterial activity in powder with orientation was found to be similar to that without orientation; that is, the crystallographic orientation of ZnO did not affect antibacterial activity. The activity toward Staphylococcus aureus was stronger than that toward Escherichia coli, irrespective of the kind of powders.

Novel determination system for urea in alcoholic beverages by using an FIA system with an acid urease column.

A novel determination method for urea using an acid urease column-FIA system was developed, and the system was applied to the determination of urea in rice wine. This novel FIA system was characterized by CO2 detection due to the property of acid urease and by a microfluidic gas-diffusion device with the use of an ultra-thin hollow fiber membrane. A biosensing system fabricated in this study was assembled with a double-plunger pump, a sample-injection valve, an immobilized acid urease column as a recognition element for the assay of urea, a gas-diffusion unit, and a flow-type spectrophotometer. The gas-diffusion unit consisted of a double-tubing structure in which the outer tubing was made of PTFE (i.d. 1.0 mm; o.d. 1.5 mm) and the inner tubing was of porous PTFE (i.d. 0.19 mm; o.d. 0.25 mm). Standard urea solutions (20 microl) were measured through monitoring variations in the absorbance of a coloring agent solution resulting from a pH shift due to carbon dioxide molecules being enzymatically generated. A wide and linear relationship was obtained between the concentration of urea (16 microM - 1.0 mM) and the change in absorbance. This FIA system has great advantages that the system did not suffer from ammonia and ethanol in samples. This system, armed with a microfluidic gas-diffusion device, was applicable to the determination of various substrates of many kinds of decarboxylase, amino-acid oxidase, and amino-acid oxygenase, producing CO2 and NH3 molecules.

Comparison of whole-body 18F-FDG PET, 99mTc-MIBI SPET, and post-therapeutic 131I-Na scintigraphy in the detection of metastatic thyroid cancer.

UNLABELLED: The usefulness of fluorine-18 fluorodeoxyglucose (FDG) positron emission tomography (PET) in differentiated thyroid cancer (DTC) has been demonstrated by many investigators, but in only a small number of studies have FDG-PET images been compared with those obtained using other non-iodine tumour-seeking radiopharmaceuticals. In most of the studies, planar imaging was performed for comparison using thallium-201 chloride or technetium-99m 2-methoxyisobutylisonitrile ((99m)Tc-MIBI). Furthermore, FDG-PET studies were not always performed in the hypothyroid state with increased levels of thyroid stimulating hormone (TSH), which are known to increase FDG uptake by DTC. The aim of this study was to compare the ability of FDG-PET to detect metastatic DTC with that of (99m)Tc-MIBI whole-body single-photon emission tomography (SPET) and post-therapeutic iodine-131 scintigraphy, evaluated under TSH stimulation. Nineteen patients (8 men, 11 women; age range, 38-72 years, mean 60 years; 17 thyroidectomised and 2 inoperable patients following (131)I ablation of the remaining thyroid tissue; 16 papillary and 3 follicular carcinomas) with metastatic DTC underwent FDG-PET whole-body scan (WBS) and (99m)Tc-MIBI SPET WBS at an interval of less than 1 week, followed by (131)I therapy. The SPET images were reconstructed using the maximum likelihood expectation maximisation (ML-EM) method. All patients were hypothyroid at the time of each scan. (131)I WBS was performed 3-5 days after oral administration of the therapeutic dose. A total of 32 lesions [10 lymph node (LN), 15 lung, 6 bone, 1 muscle] were diagnosed as metastases, as confirmed by histopathology and/or other imaging modalities (X-ray, US, CT, MRI, bone, (201)Tl and (131)I scans). FDG-PET, (99m)Tc-MIBI SPET and post-therapeutic (131)I scintigraphy respectively revealed a total of 26 (81.3%), 20 (62.5%) and 22 (68.8%) lesions. These techniques respectively demonstrated nine (90.0%), eight (80.0%) and six (60.0%) LN metastases, and eleven (73.3%), seven (46.7%) and ten (66.7%) lung metastases. They each demonstrated five of the six bone metastases (83.3%). FDG-PET and (99m)Tc-MIBI SPET were positive in 17 (78.3%) and 14 (63.6%) of the 22 (131)I-positive lesions, respectively, and also in nine (90.0%) and six (60.0%) of the ten (131)I-negative lesions, respectively. Three of the five (131)I-positive and FDG-PET-negative lesions were miliary type lung metastases with a maximal nodular diameter of less than 10 mm. Comparison of FDG-PET with (99m)Tc-MIBI SPET revealed concordant results in 24 lesions, and discordant results in eight lesions (seven with positive FDG-PET alone and one with positive (99m)Tc-MIBI SPET alone). IN CONCLUSION: (a) even using whole-body SPET, FDG PET is superior to (99m)Tc-MIBI in terms of ability to detect metastases of DTC; (b) the higher sensitivity of FDG-PET compared with the previous studies could partly be due to increased serum TSH.

Seasonal variation in relapse rate of graves' disease after thionamide drug treatment.

OBJECTIVE: Controversy abounds on the issue of seasonal variation in new onset of Graves' disease, partly due to the difficulty of precisely dating the exact start of symptoms. To address the possible relationship between climatic changes and disease activity from a different perspective, we reviewed time of relapse during regular follow-up after successful drug treatment with thionamides. DESIGN: Retrospective analysis of a case series in a university clinic. PATIENTS AND MEASUREMENTS: We consecutively registered patients who experienced re-emergence of hyperthyroidism between 1992 and 2001 after successful antithyroid drug therapy. Excluded were subjects with superimposing painless thyroiditis, in postpartum, on immunomodulatory drugs, or off thionamides prematurely on their own volition. RESULTS: Fifty-two patients recurred 2 to 36 months after drug cessation. The frequency was higher in spring and summer (March to August) than in autumn and winter (September to February). With a new coated-tube radioreceptor assay, TSH binding inhibitor immunoglobulin activity was detected in sera from 87.5% of the reworsened patients. CONCLUSIONS: Graves' disease tends to relapse more frequently in spring and summer. Further clinical studies are warranted to clarify underlying mechanism (s) for this seasonal variation.

Chemo-radionuclide therapy for thyroid cancer: initial experimental study with cultured cells.

UNLABELLED: Radioiodine therapy has long been used for distant metastases of thyroid cancer. Although partially effective in most cases, it can render a complete cure only in a limited number of patients. One way to enhance its efficacy would be to combine it with antineoplastic agents. Here we describe an initial in vitro evaluation with 4 thyroid cancer cell lines. METHODS: Cells were sparsely seeded in microtiter plates and allowed to grow for 2 days; then they were exposed to sublethal concentrations of cisplatin (CDDP), doxorubicin (Dox), or 5-fluorouracil (5-FU), followed by treatment with I-131 for 48 hr. Cell survival was measured with a commercial kit based on the colorimetry of succinate dehydrogenase activity. RESULTS: Chemotherapeutic drugs exerted similar concentration-dependent cytotoxic effects in all 4 cell lines. The doses necessary to reduce the surviving fraction to half of the control were about 3 microg/ml for CDDP, 0.3 microg/ml for Dox, and 3 microg/ml for 5-FU (when used continuously for 48 hours). On the other hand, sensitivity to I-131 irradiation differed among the lines; same doses (7.4-14.8 MBq/ml) caused the greatest damage in FRO cells, a modest effect in NPA and WRO, and only minimal change in B-CPAP. The combined effect was most demonstrable in wells treated with Dox and radioiodine, whereas the addition of CDDP or 5-FU had marginal or insignificant merit, respectively. In FRO cells, half-lethal doses of the above mentioned CDDP, Dox, and 5-FU, when used together with 14.8 MBq/ml I-131, reduced cell survival to 54.5%, 29.4% and 33.4%, respectively, vs. 60.2% with radioiodine alone. CONCLUSION: In vitro, clinical concentrations of Dox can accelerate the killing of thyroid cancer cells by radioiodine. These favorable experimental results warrant future studies to evaluate whether this new bidisciplinary approach is clinically relevant and feasible.

A patient with two thyroid papillary carcinomas demonstrating hot and cold lesions on 113I thyroid scintigraphy.

A 44-year-old euthyroid woman had two palpable nodules in the thyroid gland. 123I thyroid scintigraphy showed a hot nodule in the right lobe and a cold one in the left lobe. Total thyroidectomy was performed, and histopathologic examination revealed that both tumors contained papillary carcinoma. Thus, hot nodules on a thyroid scintigram with 123I do not necessarily preclude malignancy.

Preparation of chiral 4-benzyloxymethyldihydrofuran-2-one using lipase-catalyzed kinetic resolution: synthesis of (-)-virginiae butanolide C (VB C).

Lipase-catalyzed kinetic resolution of the N,N-dialkyl-3-benzyloxymethyl-4-hydroxybutanamide 10a,b afforded the acetate 11a,b with (R) configuration, whereas the N-monoalkyl-3-benzyloxymethyl-4-hydroxybutanamide 10c-e gave the acetate 11c-e with (S) configuration. The butanamide 10 smoothly cyclized to give chiral 4-benzyloxymethyldihydrofuran-2-one 9 without racemization, which was effectively transformed into highly stereocontrolled virginiae butanolide C (VB C).

Causes of appearance of scintigraphic hot areas on thyroid scintigraphy analyzed with clinical features and comparative ultrasonographic findings.

This study was done retrospectively to analyze the ultrasonographic (US) findings in thyroid scintigraphic hot areas (HA). Three-thousand, eight-hundred and thirty-nine consecutive patients who underwent 99mTc-pertechnetate (n = 3435) or 123I (n = 457) scintigraphy were analyzed. HA were regarded as present when the tracer concentration was greater than the remaining thyroid tissue, or when hemilobar uptake was observed. High-resolution US examinations were performed with a real-time electronic linear scanner with a 7.5 or 10 MHz transducer. One hundred and four (2.7%) were found to be scintigraphic HA (n = 120). US revealed a nodular lesion or well-demarcated thyroid tissue corresponding to the HA in 94 areas (78.4%, Category 1), an ill-defined region with different echogenicity in 13 areas (10.8%, Category 2), and no correlating lesion in 13 areas (10.8%, Category 3). These 104 patients included 43 with adenomatous goiter (59 areas), 33 with adenoma, 11 with Hashimoto's thyroiditis, 5 with primary thyroid cancer, 4 with euthyroid ophthalmic Graves' disease (EOG), 3 with hemilobar atrophy or hypogenesis, 2 with hemilobar agenesis, 2 with hypothyroidism with blocking-type TSH-receptor antibodies (TSHRAb), I with acute suppurative thyroiditis. Among the 59 adenomatous nodules and 33 adenomas, 51 (86.4%) and 32 (97.0%), respectively, belonged to Category 1. A solitary toxic nodule was significantly larger and occurs more often in older patients than in younger patients. On the other hand, all 17 patients with known autoimmune thyroid diseases including Hashimoto's thyroiditis, EOG and hypothyroidism with blocking TSHRAb belonged to Category 2 or 3. Possible underlying mechanisms are 1) hyperfunctioning tumors or nodules, 2) localized functioning thyroid tissue freed from autoimmune destruction, inflammation or tumor invasion, 3) congenital abnormality, 4) clusters of hyperactive follicular cells caused by long-term TSH and/or TSHRAb stimulation, 5) asymmetry, etc. Scintigraphic HA are observed in patients with various thyroid diseases and high-resolution US appears to be helpful clinically for the differential diagnosis of the above mentioned disorders.