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1.
Sci Rep ; 11(1): 14730, 2021 07 19.
Article in English | MEDLINE | ID: mdl-34282192

ABSTRACT

Although 18-fluoro-2-deoxy-glucose positron emission tomography/computed tomography (18F-FDG PET/CT) is useful for detecting synchronous colorectal cancer (CRC) in stenotic CRC, long-term outcomes of patients without synchronous FDG-avid lesions are not well reported. We investigated postoperative colonoscopy results in patients with left-sided stenosing CRC without synchronous FDG-avid lesions. In this retrospective review, 754 patients with left-sided CRC without synchronous FDG-avid lesions on preoperative 18F-FDG PET/CT were divided into two groups based on the completeness of preoperative colonoscopy. Propensity score matching was performed to balance baseline characteristics. Results of postoperative colonoscopy were compared in both the unmatched and matched cohorts. At 1 and 5 years after surgery, the cumulative risk of advanced adenoma (AA) or carcinoma (CA) in all patients, risk of CA, and additional surgical risk were 1.8% and 10.1%, 0.1% and 0.4%, and 0% and 0.5%, respectively. In both cohorts, the AA risk was significantly higher in the incomplete colonoscopy group. However, the risk of CA showed no between-group difference in the matched cohort. Additional surgical risk did not differ between the two groups. Thus, the finding of negative FDG-avid lesions in the proximal colon in addition to the target CRC ensures the absence of additional lesions warranting surgical plan changes.


Subject(s)
Adenoma/diagnosis , Colorectal Neoplasms/diagnosis , Fluorodeoxyglucose F18 , Positron Emission Tomography Computed Tomography , Adenoma/metabolism , Adenoma/pathology , Aged , Case-Control Studies , Cohort Studies , Colon/diagnostic imaging , Colon/metabolism , Colon/pathology , Colonoscopy/methods , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/pathology , Constriction, Pathologic/diagnosis , Female , Fluorodeoxyglucose F18/pharmacokinetics , Humans , Male , Middle Aged , Predictive Value of Tests , Republic of Korea , Retrospective Studies
2.
Sensors (Basel) ; 20(13)2020 Jul 07.
Article in English | MEDLINE | ID: mdl-32645894

ABSTRACT

Tactile sensors have been widely used and researched in various fields of medical and industrial applications. Gradually, they will be used as new input devices and contact sensors for interactive robots. If a tactile sensor is to be applied to various forms of human-machine interactions, it needs to be soft to ensure comfort and safety, and it should be easily customizable and inexpensive. The purpose of this study is to estimate 3D contact position of a novel image-based areal soft tactile sensor (IASTS) using printed array markers and multiple cameras. First, we introduce the hardware structure of the prototype IASTS, which consists of a soft material with printed array markers and multiple cameras with LEDs. Second, an estimation algorithm for the contact position is proposed based on the image processing of the array markers and their Gaussian fittings. A series of basic experiments was conducted and their results were analyzed to verify the effectiveness of the proposed IASTS hardware and its estimation software. To ensure the stability of the estimated contact positions a Kalman filter was developed. Finally, it was shown that the contact positions on the IASTS were estimated with a reasonable error value for soft haptic applications.


Subject(s)
Image Processing, Computer-Assisted , Touch , Humans , Software
3.
Radiat Prot Dosimetry ; 168(3): 343-9, 2016 Mar.
Article in English | MEDLINE | ID: mdl-25999332

ABSTRACT

In nuclear medicine, workers handle unsealed radioactive materials. Among the materials, (18)FDG is the most widely used in PET/CT technique. Because of the short half-life of (18)F, it is very challenging to monitor internal exposure of nuclear medicine workers using in vitro bioassay. Thus, the authors developed the new in vitro bioassay methodology for short half-life nuclides. In the methodology, spot urine sample is directly used without normalisation to 1-d urine sample and the spot urinary excretion function was newly proposed. In order to estimate the intake and committed dose for workers dealing (18)FDG, biokinetic models for FDG was also developed. Using the new methodology and biokinetic model, the in vitro bioassay for workers dealing (18)FDG was successfully performed. The authors expect that this methodology will be very useful for internal monitoring of workers who deal short-lived radionuclides in the all field as well as the nuclear medicine field.


Subject(s)
Fluorodeoxyglucose F18/urine , Occupational Exposure/analysis , Radiation Monitoring/methods , Radiopharmaceuticals/urine , Biological Assay , Humans , Nuclear Medicine , Radiation Dosage
4.
Psychiatry Investig ; 12(2): 242-8, 2015 Apr.
Article in English | MEDLINE | ID: mdl-25866526

ABSTRACT

OBJECTIVE: Atypical antipsychotic (AAP) treatment is associated with weight gain and metabolic disturbances such as dyslipidemia and dysglycemia. The metabolic disturbances are usually considered to develop secondary to weight gain. We performed the comparison of metabolic disturbances of three AAP group with different risk of metabolic side effect after adjusting for body mass to investigate whether any metabolic disturbances develop independently from body mass index (BMI). METHODS: This cross-sectional study included 174 subjects with schizophrenia who were on 1) monotherapy with clozapine (CL), olanzapine (OL), or quetiapine (QT) (n=61), 2) monotherapy with risperidone (RSP) (n=89), or 3) monotherapy with aripiprizole (ARP), or ziprasidone (ZPS) (n=24) more than 1 year. Association between the prevalence of metabolic disturbances and groups were analysed using logistic regression after adjusting confounding variables including BMI. Analysese of covariance were used to compare the AAP groups in terms of the levels of metabolic parameters. RESULTS: There were significant differences among groups in terms of the prevalence of hypertriglyceridemia (p=0.015), low HDL-cholesterol (p=0.017), and hyperglycemia (p=0.022) after adjusting for BMI. Triglyceride level (p=0.014) and the ratio of triglyceride to HDL-cholesterol (p=0.004) were significantly different among groups after adjusting for BMI. CONCLUSION: In conclusion, metabolic disturbances are significantly different in AAP groups even after adjusting BMI. AAPs may have direct effect on metabolic parameters. Blood lipid and glucose levels should be monitored regularly regardless of whether patients tend to gain weight.

6.
Psychiatr Genet ; 24(6): 249-56, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25304226

ABSTRACT

OBJECTIVES: We investigated the associations of the LEP-2548A/G and HTR2C-759C/T polymorphisms with long-term clozapine-induced weight changes and baseline BMI in chronic patients with schizophrenia. MATERIALS AND METHODS: A total of 113 patients receiving clozapine for at least 1 year were enrolled. Body weight was measured cross-sectionally and data on body weight just before starting clozapine were retrospectively extracted from medical records. RESULTS: Clozapine-induced change in BMI was correlated inversely with the baseline BMI (P<0.001, ρ=-0.347). The LEP-2548A/G polymorphism was associated significantly with the change in BMI (F=4.380, P=0.015) during clozapine use; those with the AA genotype had the highest BMI gain (1.4±3.1 kg/m), followed by those with the AG (-0.2±3.3 kg/m) and GG (-1.6±3.4 kg/m) genotypes. We also found a significant association between the leptin genotype and BMI at baseline (F=3.499, P=0.034); those with the AA genotype had the lowest baseline BMI (23.4±4.3 kg/m), followed by those with the AG (24.1±4.4 kg/m) and GG (28.8±7.3 kg/m) genotypes. In the case of the HTR2C-759C/T polymorphism, we found a trend in which T alleles were more prevalent in male patients with up to 7% increase in BMI than in those with a greater than 7% increase in BMI [12/54 (22.7%) vs. 1/27 (3.7%); Fisher's exact test: P=0.051]. CONCLUSION: This study shows an inverse correlation between the baseline BMI and change in BMI during long-term clozapine use in patients with schizophrenia, and the LEP-2548A/G polymorphism was associated significantly with both these measures.


Subject(s)
Body Mass Index , Clozapine/adverse effects , Leptin/genetics , Polymorphism, Genetic , Receptor, Serotonin, 5-HT2C/genetics , Schizophrenia/drug therapy , Weight Gain , Adult , Antipsychotic Agents/adverse effects , Antipsychotic Agents/therapeutic use , Chronic Disease , Clozapine/therapeutic use , Humans , Middle Aged
7.
Asia Pac Psychiatry ; 6(4): 386-96, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25345739

ABSTRACT

INTRODUCTION: This article aims to assess the efficacy and safety of atomoxetine in Korean adults with attention-deficit hyperactivity disorder (ADHD). METHODS: This post hoc double-blind, placebo-controlled study of atomoxetine (40-120 mg/day) over 10 weeks in adults with ADHD at 45 Japanese, Korean, and Taiwanese study sites focused on patient data from Korea (atomoxetine, n = 37; placebo, n = 37). Primary efficacy outcome was change in baseline-to-endpoint Conners' Adult ADHD Rating Scale-Investigator-rated: Screening Version (CAARS-Inv:SV) Total ADHD Symptoms score. Secondary efficacy outcomes included changes in Adult ADHD Quality of Life (AAQoL) total, Behavior Rating Inventory of Executive Function-Adult Version Self-Report (BRIEF-A:Self-Report), and Clinical Global Impression-ADHD-Severity (CGI-ADHD-S) scale scores. RESULTS: Atomoxetine-treated patients demonstrated a mean 18.9-point reduction in CAARS-Inv:SV total ADHD Symptoms score, compared with the 7.45-point reduction in placebo-treated patients (P ≤ 0.01). Significantly greater improvement was found for atomoxetine versus placebo in CGI-ADHD-S (P ≤ 0.01), BRIEF-A:Self-Report global executive composite (P ≤ 0.05), and metacognition index (P ≤ 0.01) executive function scores. Nausea, decreased appetite, and dry mouth were reported with significantly greater frequency by atomoxetine-treated patients, and only one placebo-treated patient discontinued because of adverse event. A 2.1-kg reduction in weight and a 7.5-beat/minute increase in pulse rate were observed in atomoxetine-treated patients. DISCUSSION: These data support a significant benefit of 80- to 120-mg once daily atomoxetine versus placebo for treatment of ADHD in adult Korean patients. A high placebo response rate was observed in this adult Korean sample; a higher discontinuation rate was also observed in atomoxetine-treated patients. These observations warrant further investigation.


Subject(s)
Adrenergic Uptake Inhibitors/administration & dosage , Attention Deficit Disorder with Hyperactivity/drug therapy , Propylamines/administration & dosage , Adrenergic Uptake Inhibitors/adverse effects , Adult , Atomoxetine Hydrochloride , Dose-Response Relationship, Drug , Double-Blind Method , Female , Humans , Male , Propylamines/adverse effects , Quality of Life , Republic of Korea , Treatment Outcome
8.
Clin Psychopharmacol Neurosci ; 11(2): 80-8, 2013 Aug.
Article in English | MEDLINE | ID: mdl-24023552

ABSTRACT

OBJECTIVE: To examine the prevalence of metabolic syndrome and its risk factors in a large group of schizophrenic patients. METHODS: Sociodemographic and treatment data were collected from medical records of 1,103 inpatients and outpatients treated for schizophrenia at Seoul National Hospital in Seoul, Korea. Anthropometric measurement and blood testing were conducted for collection of physical and biochemical data and diagnosis of metabolic syndrome. Data for metabolic syndrome prevalence were compared by sex, age, metabolic syndrome markers present, treatment of markers, and types of antipsychotics and individual drug agents used. RESULTS: Mean prevalence of metabolic syndrome in all subjects was 43.9% and 40.1% according to adapted Adult Treatment Panel III (ATP-IIIa) and International Diabetes Federation criteria, respectively. No significant differences were found in prevalence according to ATP-IIIa criteria between men (42.6%) and woman (45.9%). A trend toward higher prevalence with age was observed for both sexes until 50 years, followed by a continued increase for women but a decrease for men. Use of a combination of atypical antipsychotics was associated with the highest metabolic syndrome prevalence and use of aripiprazole with the lowest. High percentages of subjects with hypertension and dyslipidemia were not being treated for these conditions. CONCLUSION: Despite their higher prevalence in schizophrenic patients, metabolic syndrome and its markers are not being adequately managed in these patients. Treatment of schizophrenic patients requires attention to not only their psychiatric conditions but also associated medical conditions by individual health care practitioners and hospitals as well as the public health care sector as a whole.

9.
Clin Psychopharmacol Neurosci ; 11(2): 103-5, 2013 Aug.
Article in English | MEDLINE | ID: mdl-24023555

ABSTRACT

Clozapine use is associated with various adverse events, some of which have received little attention, including eosinophilia, pleural effusion, and hepatitis. Because of the fatality of jaundice with hepatitis, it is necessary to understand the course and management of clozapine-induced eosinophilia and hepatitis. We report on a case in which the eosinophil count began to increase shortly after clozapine use, and pleural effusion and fever then developed at the time eosinophilia was at its peak level. Jaundice with hepatitis consecutively developed when all the above symptoms subsided. The liver function recovered rapidly after clozapine was discontinued. We recommend that patients who develop rapid eosinophilia at the beginning of clozapine treatment should be monitored with LFTs, chest X-rays, and urine analysis tests.

10.
Hum Psychopharmacol ; 28(2): 107-16, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23417736

ABSTRACT

OBJECTIVE: This study assessed whether the subjective experience of patients with schizophrenia improved after switching from an oral antipsychotic to flexibly-dosed paliperidone extended-release. METHODS: We conducted a 24-week, multicenter, non-comparative, open-label trial. A total of 387 patients with schizophrenia participated the study. The primary study outcome was the change in subjective symptoms measured by the Symptom Checklist-90-Revised version (SCL-90-R) from baseline. Visual analogue scales were used for sleep and daytime somnolence as secondary subjective assessments. The clinical global impression-schizophrenia-severity scale was used to assess overall symptom severity. Social functioning was evaluated by the personal and social performance scale. Adverse events were also evaluated. RESULTS: All subjective symptoms measured by the SCL-90-R improved significantly. The early responders, who achieved >20% reduction in the SCL-90-R within 1 week, maintained significantly lower severity through the 24 weeks. The clinical global impression-schizophrenia-severity scale and personal and social performance scores also improved significantly. The visual analogue scales revealed that daytime somnolence improved significantly, whereas nocturnal sleep quality was unaltered. CONCLUSION: Our results suggest that switching to paliperidone extended-release was associated with improvements in various subjective symptoms, decreased overall symptom severity, and increased social functioning. The results also suggest that early detection and reduction of subjective symptoms are important for treatment outcome.


Subject(s)
Antipsychotic Agents/administration & dosage , Isoxazoles/administration & dosage , Pyrimidines/administration & dosage , Schizophrenia/drug therapy , Schizophrenic Psychology , Adult , Delayed-Action Preparations/administration & dosage , Female , Humans , Male , Middle Aged , Paliperidone Palmitate , Prospective Studies , Schizophrenia/diagnosis , Schizophrenia/epidemiology , Treatment Outcome
11.
Hum Psychopharmacol ; 27(4): 403-10, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22753400

ABSTRACT

INTRODUCTION: The aim of this study was to evaluate the efficacy and safety of quetiapine fumarate extended release (XR) in the treatment of Korean subjects with acute schizophrenia. METHODS: This was an 8-week, multi-center, open-label, non-comparative study to evaluate the efficacy and safety of quetiapine fumarate XR at a daily dose of 400-800 mg. Changes in total scores on the Positive and Negative Syndrome Scale (PANSS) from baseline to week 8 were analyzed to evaluate the efficacy of quetiapine XR. Additionally, the Clinical Global Impression scale and the Montgomery-Åsberg Depression Rating Scale were administered. RESULTS: The mean change in PANSS total scores was -26.8, and the mean PANSS total score at the endpoint was significantly lower than that at baseline. The mean PANSS positive score, negative score, and general score showed statistically significant reductions at the end of the study. Statistically significant changes were also observed in Clinical Global Impression-Severity and Montgomery-Åsberg Depression Rating Scale scores. The most common treatment-related adverse events in the group receiving quetiapine XR were sedation (10.6%) and constipation (9.6%). CONCLUSIONS: In this study of Korean patients with acute schizophrenia, quetiapine XR showed clinical efficacy and relatively good tolerability.


Subject(s)
Antipsychotic Agents/therapeutic use , Dibenzothiazepines/therapeutic use , Schizophrenia/drug therapy , Acute Disease , Adult , Antipsychotic Agents/administration & dosage , Antipsychotic Agents/adverse effects , Delayed-Action Preparations , Dibenzothiazepines/administration & dosage , Dibenzothiazepines/adverse effects , Female , Humans , Male , Middle Aged , Psychiatric Status Rating Scales , Quetiapine Fumarate , Republic of Korea , Schizophrenia/physiopathology , Severity of Illness Index , Treatment Outcome , Young Adult
12.
Psychiatry Investig ; 8(3): 262-8, 2011 Sep.
Article in English | MEDLINE | ID: mdl-21994515

ABSTRACT

OBJECTIVE: Genetic variation in the serotonin-2C receptor encoded by the HTR2C gene is one of the genetic determinants of antipsychotic-induced weight gain. Peroxisome proliferator-activated receptors are nuclear receptors regulating the expression of genes involved in lipid and glucose metabolism. In this cross-sectional study, we investigated whether HTR2C-759C/T, HTR2C-697G/C, PPARα V227A, and PPARγ 161C/T genotypes were associated with metabolic syndrome (MetS) in patients with schizophrenia taking clozapine. METHODS: One hundred forty-six Korean patients using clozapine for more than one year were genotyped for the HTR2C-759C/T, HTR2C-697G/C, PPARα V227A, and PPARγ 161C/T polymorphisms, and their weight, waist circumference, blood pressure, triglycerides, high-density lipoprotein-cholesterol, total cholesterol, and glucose were measured. We used the criteria for MetS proposed by the National Cholesterol Education Program-adapted Adult Treatment Panel III. RESULTS: The prevalence of MetS was 47.3% and was similar among men (49%) and women (42.9%). We found no significant differences between patients with and without MetS in terms of genotypes or allele frequencies. Logistic regression analyses also revealed no association between MetS and each genotype. CONCLUSION: We did not find significant associations between four polymorphisms (HTR2C-759C/T, HTR2C-697G/C, PPARα V227A, and PPARγ 161C/T) and MetS in patients with schizophrenia taking clozapine.

13.
Clin Neuropharmacol ; 34(5): 186-90, 2011.
Article in English | MEDLINE | ID: mdl-21725234

ABSTRACT

OBJECTIVES: The objectives of this 12-week multicenter, open-label, noncomparative study were to evaluate the overall effectiveness of paliperidone extended release (ER), the feasibility of maintaining patients on the initial dose of 6 mg, and the relationship between dose pattern and treatment response in schizophrenic patients with inadequate responses to initial treatment in a natural setting. METHODS: All patients received 6 mg of paliperidone ER during the first 2 weeks, and subsequently, the dose was adjusted at each visit based on the patient response. We examined the response rate and the effectiveness of different dose patterns of paliperidone ER such as "early increase (dose increased to 9 mg at week 2)," "late increase (dose increased to 9 mg at week 4)," and "maintenance group." RESULTS: The response rate based on the Clinical Global Impression of Improvement or Severity response criteria was 33.6% or 61.7%, respectively. The proportion of patients who stayed with the initial dose of 6 mg was 44.5% and the response rate of these patients was 79.8%. When the treatment response to the initial dose of 6 mg is inadequate (Clinical Global Impression of Improvement ≥ 4 at week 2), an early increase in the dose seems to be more effective than maintenance or late increase of the initial dose. CONCLUSIONS: This study suggests that paliperidone ER may be an effective and well-tolerated antipsychotics in the treatment of patients with schizophrenia.


Subject(s)
Antipsychotic Agents/therapeutic use , Isoxazoles/therapeutic use , Pyrimidines/therapeutic use , Schizophrenia/drug therapy , Adult , Delayed-Action Preparations/administration & dosage , Dose-Response Relationship, Drug , Female , Humans , Male , Paliperidone Palmitate , Patient Dropouts/statistics & numerical data , Psychological Tests , Schizophrenia/diagnosis , Schizophrenia/physiopathology , Tablets , Treatment Outcome
15.
Appl Biochem Biotechnol ; 160(6): 1585-99, 2010 Mar.
Article in English | MEDLINE | ID: mdl-19440663

ABSTRACT

The Thermococcus peptonophilus (Tpe) DNA polymerase gene was expressed under the control of the T7lac promoter on pET-22b(+) in Escherichia coli BL21-CodonPlus(DE3)-RIL in order to fully elucidate its biochemical properties and evaluate its feasibility in polymerase chain reaction (PCR) application. The expressed enzyme was then purified by heat treatment followed by two steps of column chromatography after which optimum pH and temperature of the enzyme were evaluated to be 7.0 and 75 degrees C, respectively. The optimal buffer for PCR with Tpe DNA polymerase consisted of 50 mM Tris-HCl (pH 8.0), 2 mM MgCl(2), 80 mM KCl, and 0.02% Triton X-100. Tpe DNA polymerase revealed a 3.6-fold higher fidelity (3.37 x 10(-6)) than Taq DNA polymerase (12.13 x 10(-6)) and performed significantly more efficiently in PCR amplification than both Taq and Pfu DNA polymerases. Ratios of 31:1 of Taq to Tpe DNA polymerases allowed PCR amplification of targets up to 15 kb in length with a 2.2-fold higher fidelity than Taq DNA polymerase. The results of the PCR experiments indicate that Tpe DNA polymerase may provide a higher fidelity DNA amplification in a shorter reaction time.


Subject(s)
DNA-Directed DNA Polymerase/metabolism , Hot Temperature , Polymerase Chain Reaction/methods , Thermococcus/enzymology , DNA-Directed DNA Polymerase/isolation & purification , Electrophoresis, Polyacrylamide Gel , Enzyme Stability , Exonucleases/metabolism , Hydrogen-Ion Concentration , Plasmids/genetics
16.
J Artif Organs ; 12(4): 274-7, 2009.
Article in English | MEDLINE | ID: mdl-20035402

ABSTRACT

A new tailor-made bone implant (TI) with six horizontal cylindrical holes fabricated from alpha-tricalcium phosphate powder, as described in our previous report, was modified to include five additional vertical holes (TI-v) in an attempt to accelerate the bone regeneration through the holes. This TI-v implant and hydroxyapatite implants (HI) as controls were transplanted into experimental skull defects in dogs. Computed tomography (CT) was performed immediately after the surgery and then every 4 weeks. The dogs were killed for histological analysis at 24 weeks of implantation. On CT, bone bridging between the implant and the skull was observed in the TI-v group from 8 weeks of implantation, whereas a clear bone bridge was not formed in the HI group after 24 weeks of implantation. Histological analysis revealed collagen tissues and new bone formation in the horizontal cylindrical holes in most of the TI-v group, whereas mainly connective tissues invaded the porous structures in the HI group. In the Ti-v group, at the middle of the horizontal holes where they crossed the vertical holes, fibrous collagen tissues and muscular tissue filled up the hole and new bone formation seemed to be blocked. However, in the TI-v group more collagen and bone tissues were formed than in the HI group; when compared with the data in our previous report, however, the total volume of regenerated bone in the horizontal cylindrical holes in the TI-v seemed to be less than that in the TI. Thus, the addition of vertical cylindrical holes in the TI-v was not effective in promoting the faster stabilization of the TI-v in the skull of the dog.


Subject(s)
Bone Regeneration , Bone Substitutes , Calcium Phosphates , Prostheses and Implants , Skull Fractures/surgery , Animals , Dogs , Female , Male , Skull Fractures/diagnostic imaging , Skull Fractures/pathology , Tomography, X-Ray Computed
17.
Extremophiles ; 13(4): 657-67, 2009 Jul.
Article in English | MEDLINE | ID: mdl-19412677

ABSTRACT

The family B DNA polymerase gene from the archaeon Thermococcus marinus (Tma) contains a long open reading frame of 3,939 bp that encodes 1,312 amino acid residues. The gene is split by one intervening sequence that forms a continuous open reading frame with the two polymerase exteins. In this study, the Tma DNA polymerase gene both with (precursor form) and without (mature form) its intein was expressed in Escherichia coli, purified by heat treatment and HiTrap Heparin HP column chromatography and characterized. Primary sequence analysis of the mature Tma polymerase showed high sequence identity with DNA polymerases in the genus Thermococcus. The expressed precursor form was easily spliced during purification steps. The molecular mass of the purified Tma DNA polymerases is about 90 kDa, as estimated by SDS-PAGE. Both Tma DNA polymerases showed the same properties. PCR performed with this enzyme was found to be optimal in the presence of 50 mM Tris-HCl (pH 8.4), 40 mM KCl, 12.5 mM (NH(4))(2)SO(4,) 2 mM MgCl(2,) 0.05% Triton X-100 and 0.0075% BSA. Furthermore, long-range PCR and time-saving PCR were performed using various specific ratios of Taq and Tma DNA polymerases (Tma plus DNA polymerase).


Subject(s)
DNA-Directed DNA Polymerase/chemistry , Polymerase Chain Reaction/instrumentation , Polymerase Chain Reaction/methods , Thermococcus/enzymology , Amino Acid Sequence , Cloning, Molecular , DNA Primers/chemistry , Exonucleases/metabolism , Gene Expression Profiling , Genes, Archaeal , Hydrogen-Ion Concentration , Models, Genetic , Molecular Sequence Data , Open Reading Frames , Phylogeny , Sequence Analysis, DNA
18.
Appl Environ Microbiol ; 74(21): 6563-9, 2008 Nov.
Article in English | MEDLINE | ID: mdl-18791030

ABSTRACT

The known archaeal family B DNA polymerases are unable to participate in the PCR in the presence of uracil. Here, we report on a novel archaeal family B DNA polymerase from Nanoarchaeum equitans that can successfully utilize deaminated bases such as uracil and hypoxanthine and on its application to PCR. N. equitans family B DNA polymerase (Neq DNA polymerase) produced lambda DNA fragments up to 10 kb with an approximately 2.2-fold-lower error rate (5.53 x 10(-6)) than Taq DNA polymerase (11.98 x 10(-6)). Uniquely, Neq DNA polymerase also amplified lambda DNA fragments using dUTP (in place of dTTP) or dITP (partially replaced with dGTP). To increase PCR efficiency, Taq and Neq DNA polymerases were mixed in different ratios; a ratio of 10:1 efficiently facilitated long PCR (20 kb). In the presence of dUTP, the PCR efficiency of the enzyme mixture was two- to threefold higher than that of either Taq and Neq DNA polymerase alone. These results suggest that Neq DNA polymerase and Neq plus DNA polymerase (a mixture of Taq and Neq DNA polymerases) are useful in DNA amplification and PCR-based applications, particularly in clinical diagnoses using uracil-DNA glycosylase.


Subject(s)
DNA-Directed DNA Polymerase/metabolism , Nanoarchaeota/enzymology , Polymerase Chain Reaction/methods , Bacteriophage lambda/genetics , DNA, Viral/genetics , DNA-Directed DNA Polymerase/isolation & purification , Hypoxanthine/metabolism , Molecular Weight , Substrate Specificity , Uracil/metabolism
19.
Environ Microbiol ; 10(12): 3212-24, 2008 Dec.
Article in English | MEDLINE | ID: mdl-18647334

ABSTRACT

DNA ligases are divided into two groups according to their cofactor requirement to form ligase-adenylate, ATP-dependent DNA ligases and NAD(+)-dependent DNA ligases. The conventional view that archaeal DNA ligases only utilize ATP has recently been disputed with discoveries of dual-specificity DNA ligases (ATP/ADP or ATP/NAD(+)) from the orders Desulfurococcales and Thermococcales. Here, we studied DNA ligase encoded by the hyperthermophilic crenarchaeon Sulfophobococcus zilligii. The ligase exhibited multiple cofactor specificity utilizing ADP and GTP in addition to ATP. The unusual cofactor specificity was confirmed via a DNA ligase nick-closing activity assay using a fluorescein/biotin-labelled oligonucleotide and a radiolabelled oligonucleotide. The exploitation of GTP as a catalytic energy source has not to date been reported in any known DNA ligase. This phenomenon may provide evolutionary evidence of the nucleotide cofactor utilization by DNA ligases. To bolster this hypothesis, we summarize and evaluate previous assertions. We contend that DNA ligase evolution likely started from crenarchaeotal DNA ligases and diverged to eukaryal DNA ligases and euryarchaeotal DNA ligases. Subsequently, the NAD(+)-utilizing property of some euryarchaeotal DNA ligases may have successfully differentiated to bacterial NAD(+)-dependent DNA ligases.


Subject(s)
Coenzymes/pharmacology , DNA Ligases/genetics , DNA Ligases/metabolism , Desulfurococcaceae/enzymology , Nucleotides/pharmacology , Adenosine Diphosphate/metabolism , Adenosine Triphosphate/metabolism , Amino Acid Sequence , DNA/metabolism , DNA Breaks, Single-Stranded , DNA, Archaeal/chemistry , DNA, Archaeal/genetics , Desulfurococcaceae/genetics , Desulfurococcaceae/metabolism , Evolution, Molecular , Guanosine Triphosphate/metabolism , Molecular Sequence Data , Sequence Alignment , Sequence Analysis, DNA
20.
Appl Microbiol Biotechnol ; 80(5): 785-94, 2008 Oct.
Article in English | MEDLINE | ID: mdl-18626641

ABSTRACT

In this study, the gene encoding Bacillus sp. HJ171 uracil-DNA glycosylase (Bsp HJ171 UDG) was cloned and sequenced. The Bsp HJ171 UDG gene consists of a 738-bp DNA sequence, which encodes for a protein that is 245-amino-acid residues in length. The deduced amino acid sequence of the Bsp HJ171 UDG had a high sequence similarity with other bacterial UDGs. The molecular mass of the protein derived from this amino acid sequence was 27.218 kDa. The Bsp HJ171 UDG gene was expressed under the control of a T7lac promoter in the pTYB1 plasmid in Escherichia coli BL21 (DE3). The expressed enzyme was purified in one step using the Intein Mediated Purification with an Affinity Chitin-binding Tag purification system. The optimal temperature range, pH, NaCl concentration, and KCl concentration of the purified enzyme was 20-25 degrees C, 8.0, 25 and 25 mM, respectively. The half-life of the enzyme at 40 degrees C and 50 degrees C were approximately 131 and 45 s, respectively. These heat-labile characteristics enabled Bsp HJ171 UDG to control carry-over contamination in the polymerase chain reaction product (PCR) without losing the PCR product.


Subject(s)
Bacillus/enzymology , Bacterial Proteins/chemistry , Polymerase Chain Reaction/standards , Uracil-DNA Glycosidase/chemistry , Amino Acid Sequence , Bacillus/classification , Bacillus/genetics , Bacterial Proteins/genetics , Bacterial Proteins/isolation & purification , Bacterial Proteins/metabolism , Cloning, Molecular , Enzyme Stability , Escherichia coli/genetics , Escherichia coli/metabolism , Molecular Sequence Data , Phylogeny , Polymerase Chain Reaction/methods , Sequence Alignment , Uracil-DNA Glycosidase/genetics , Uracil-DNA Glycosidase/isolation & purification , Uracil-DNA Glycosidase/metabolism
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