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1.  Absence of Autophagy-Related Proteins Expression Is Associated with Poor Prognosis in Patients with Colorectal Adenocarcinoma 
Background/Aim. Autophagy, a cellular degradation process, has paradoxical roles in tumorigenesis and the progression of human cancers. The aim of this study was to investigate the expression levels of autophagy-related proteins in colorectal cancer (CRC) and to evaluate their prognostic significance. Methods. This study is a retrospective review of immunohistochemical and clinicopathological data. All specimens evaluated were obtained from 263 patients with colorectal cancer who had undergone surgery between November 1996 and August 2007. The primary outcomes measured were the expression levels of three autophagy-related proteins (ATG5, BECN1/Beclin 1, and Microtubule-associated protein 1 light chain 3B (LC3B)) by immunohistochemistry and its association in clinicopathological parameters and patient survival. Results. The autophagy-related protein expression frequencies were 65.1% (151/232) for ATG5, 71.3% (174/244) for BECN1, and 74.7% (186/249) for LC3B for the 263 patients. Correlation between the expression of autophagy-related proteins was significant for all protein pairs. Multivariate analysis showed that negative LC3B expression and absence of autophagy-related proteins expression were independently associated with poor prognosis. Conclusion. Absence of autophagy-related proteins expression is associated with poor clinical outcome in CRC, suggesting that these proteins have potential uses as novel prognostic markers.
PMCID: PMC3960741  PMID: 24723943
2.  CDH13 and HCRTR2 May Be Associated with Hypersomnia Symptom of Bipolar Depression: A Genome-Wide Functional Enrichment Pathway Analysis 
Psychiatry Investigation  2015;12(3):402-407.
Although bipolar disorder is highly heritable, the identification of specific genetic variations is limited because of the complex traits underlying the disorder. We performed a genome-wide association study of bipolar disorder using a subphenotype that shows hypersomnia symptom during a major depressive episode. We investigated a total of 2,191 cases, 1,434 controls, and 703,012 single nucleotide polymorphisms (SNPs) in the merged samples obtained from the Translational Genomics Institute and the Genetic Association Information Network. The gene emerging as the most significant by statistical analysis was rs1553441 (odds ratio=0.4093; p=1.20×10-5; Permuted p=6.0×10-6). However, the 5×0-8 threshold for statistical significance required in a genome-wide association study was not achieved. The functional enrichment pathway analysis showed significant enrichments in the adhesion, development-related, synaptic transmission-related, and cell recognition-related pathways. For further evaluation, each gene of the enriched pathways was reviewed and matched with genes that were suggested to be associated with psychiatric disorders by previous genetic studies. We found that the cadherin 13 and hypocretin (orexin) receptor 2 genes may be involved in the hypersomnia symptom during a major depressive episode of bipolar disorder.
PMCID: PMC4504925  PMID: 26207136
Genome-wide association study; Bipolar disorder; Hypersomnia; Functional enrichment pathway analysis; Bipolar depression
3.  DC-Based Immunotherapy Combined with Low-Dose Methotrexate Effective in the Treatment of Advanced CIA in Mice 
Journal of Immunology Research  2015;2015:834085.
We have previously demonstrated that semimature dendritic cell- (smDC-) based immunotherapy is effective for the treatment of collagen-induced arthritis (CIA) prior to disease onset. In the present study, we examined the efficacy of combination therapy with smDCs and methotrexate (MTX) in advanced CIA with a score of 2-3. Combination therapy with low-dose MTX and type II collagen- (CII-) pulsed smDCs (CII-smDCs) was more effective in inhibiting disease progression than high or low-dose MTX alone or a combination of high dose MTX and CII-smDCs. The effect of CII-smDCs alone was also comparable to the combination therapy. CD4+Foxp3+ Treg populations and IL-10 secretion markedly increased, and CII-specific autoreactive T cells decreased in mice treated with CII-smDCs alone or in combination with MTX. Combination therapy reduced the secretion of interferon-γ (IFN-γ) and IL-17 with little influence on the IL-4 secretion in the mixed leukocyte reaction. These results imply that the combination therapy with low-dose MTX and smDCs is effective in controlling advanced CIA by enhancing Treg population and suppresses antigen-specific Th1/Th17 immunity, rather than initiating Th1 to Th2 immune deviation. Our findings provide a better understanding of the DC therapy in combination with MTX for the treatment of patients with rheumatoid arthritis (RA).
PMCID: PMC4499408  PMID: 26221616
4.  Use of laparoscopy for diagnosing experimentally induced acute pancreatitis in dogs 
Journal of Veterinary Science  2014;15(4):551-556.
Diagnosis of acute pancreatitis in dogs remains a significant challenge despite the development of advanced diagnostic methodologies. Visual inspection and pancreas biopsy using laparoscopy are generally considered to be procedures free of complications when conducted on healthy animals. However, the usefulness of laparoscopy for diagnosing acute pancreatitis has not been assessed. In the present study, the efficacy of laparoscopy for diagnosing acute pancreatitis in dogs was evaluated in animals with experimentally induced acute pancreatitis. Gross appearance of the pancreatic area was examined by laparoscopy to survey for the presence of edema, adhesions, effusion, pseudocysts, hemorrhage, and fat necrosis. Laparoscopic biopsy was performed and the histopathologic results were compared to those of pancreatic samples obtained during necropsy. The correlation between laparoscopy and histopathologic findings of the pancreas was evaluated. The presence of adhesions, effusion, and hemorrhage in the pancreatic area observed by laparoscopy significantly correlated with the histopathologic results (p < 0.05). There was no significant relationship between the histopathologic and laparoscopic biopsy findings. Results of this study suggested that laparoscopic evaluation of gross lesions has clinical significance although the laparoscopic biopsy technique has some limitations. This method combined with additional diagnostic tools can be effective for diagnosing acute pancreatitis in dogs.
PMCID: PMC4269598  PMID: 24962411
acute pancreatitis; biopsy; dog; laparoscopy
5.  The Difference in Comorbidities and Behavioral Aspects between Internet Abuse and Internet Dependence in Korean Male Adolescents 
Psychiatry Investigation  2014;11(4):387-393.
This study examined the differences in psychiatric comorbidities and behavioral aspects in accordance with the severity of Internet addiction in male adolescents.
One hundred and twenty-five adolescents from four middle and high schools in Seoul were enrolled in this study. The subjects were divided into non-addict, abuse, and dependence groups according to a diagnostic interview by psychiatrists. The psychiatric comorbidities and behavioral aspects of subjects were evaluated through psychiatric clinical interviews based on the Diagnostic and Statistical Manual of Mental Disorders (4th edition), the Children's Depression Inventory, the State-Trait Anxiety Inventory, the Internet Addiction Test, and a self-reported questionnaire about behavioral aspects.
The psychiatric comorbidity distributions were significantly different in the abuse and dependence groups, particularly in terms of attention-deficit hyperactivity disorder and mood disorder items. The Children's Depression Inventory, the State-Trait Anxiety Inventory, and the Internet Addiction Test scores were also significantly different among the three groups. There were significant differences in 10 of the 20 items of the Internet Addiction Test between the non-addict, abuse, and dependence groups. There were significant differences in seven items between the non-addict and abuse groups, but no differences between subjects in the abuse and dependence groups. Significant differences were observed in three items between the abuse and dependence groups, but there were no significant differences between the non-addict and abuse groups. In terms of behavioral aspects, scores for abusive, sexual, and decreased social interest behaviors were highest in the dependence group, and lowest in the non-addict group. However, the behavioral aspects of decreased interpersonal relationships did not show this difference between groups.
This study suggests that there are differences in psychiatric comorbidities and behavioral aspects between adolescent males with characteristics of Internet abuse and Internet dependence.
PMCID: PMC4225202  PMID: 25395969
Internet abuse; Dependence; Comorbidity; Behavioral aspects
6.  Rectourethral Fistula: Systemic Review of and Experiences With Various Surgical Treatment Methods 
Annals of Coloproctology  2014;30(1):35-41.
A rectourethral fistula (RUF) is an uncommon complication resulting from surgery, radiation or trauma. Although various surgical procedures for the treatment of an RUF have been described, none has gained acceptance as the procedure of choice. The aim of this study was to review our experience with surgical management of RUF.
The outcomes of 6 male patients (mean age, 51 years) with an RUF who were operated on by a single surgeon between May 2005 and July 2012 were assessed.
The causes of the RUF were iatrogenic in four cases (two after radiation therapy for rectal cancer, one after brachytherapy for prostate cancer, and one after surgery for a bladder stone) and traumatic in two cases. Fecal diversion was the initial treatment in five patients. In one patient, fecal diversion was performed simultaneously with definitive repair. Four patients underwent staged repair after a mean of 12 months. Rectal advancement flaps were done for simple, small fistula (n = 2), and flap interpositions (gracilis muscle flap, n = 2; omental flap, n = 1) were done for complex or recurrent fistulae. Urinary strictures and incontinence were observed in patients after gracilis muscle flap interposition, but they were resolved with simple treatments. The mean follow-up period was 28 months, and closure of the fistula was achieved in all five patients (100%) who underwent definitive repairs. The fistula persisted in one patient who refused further definitive surgery after receiving only a fecal diversion.
Depending on the severity and the recurrence status of RUF, a relatively simple rectal advancement flap repair or a more complex gracilis muscle or omental flap interposition can be used to achieve closure of the fistula.
PMCID: PMC3953168  PMID: 24639969
Rectal fistula; Urinary fistula; Surgical flap; Complication
7.  Effect of Gaseous Ozone Exposure on the Bacteria Counts and Oxidative Properties of Ground Hanwoo Beef at Refrigeration Temperature 
This study was designed to elucidate the effect of ozone exposure on the bacteria counts and oxidative properties of ground Hanwoo beef contaminated with Escherichia coli O157:H7 at refrigeration temperature. Ground beef was inoculated with 7 Log CFU/g of E. coli O157:H7 isolated from domestic pigs and was then subjected to ozone exposure (10×10−6 kg O3 h−1) at 4℃ for 3 d. E. coli O157:H7, total aerobic and anaerobic bacterial growth and oxidative properties including instrumental color changes, TBARS, catalase (CAT) and glutathione peroxidase (GPx) activity were evaluated. Ozone exposure significantly prohibited (p<0.05) the growths of E. coli O157:H7, total aerobic and anaerobic bacteria in ground beef samples during storage. Ozone exposure reduced (p<0.05) the CIE a* value of samples over storage time. The CIE L* and CIE b* values of the samples fluctuated over storage time, and ozone had no clear effect. Ozone exposure increased the TBARS values during 1 to 3 d of storage (p<0.05). The CAT and GPx enzyme activities were not affected by ozone exposure until 2 and 3 d of storage, respectively. This study provides information about the use of ozone exposure as an antimicrobial agent for meat under refrigerated storage. The results of this study provide a foundation for the further application of ozone exposure by integrating an ozone generator inside a refrigerator. Further studies regarding the ozone concentrations and exposure times are needed.
PMCID: PMC4662157  PMID: 26761291
ozone; ground Hanwoo beef; Escherichia coli O157:H7; bacteria counts; oxidative properties
8.  Active Escape of Orientia tsutsugamushi from Cellular Autophagy 
Infection and Immunity  2013;81(2):552-559.
Orientia tsutsugamushi, the causative agent of scrub typhus, is an obligate intracellular pathogen. After entry into host cells, the bacterium rapidly escapes from the endosomal pathway and replicates in the cytosol of eukaryotic host cells. Here we show that O. tsutsugamushi infection efficiently promotes cellular autophagy, a cell-autonomous defense mechanism of innate immunity. However, most of the internalized bacteria barely colocalized with the induced autophagosomes, even when stimulated with rapamycin, a chemical inducer of autophagy. Treatment of infected cells with tetracycline suppressed bacterial evasion from autophagy and facilitated O. tsutsugamushi targeting to autophagosomes, suggesting that the intracellular pathogen may be equipped with a bacterial factor or factors that block autophagic recognition. Finally, we also found that chemical modulators of cellular autophagy or genetic knockout of the atg3 gene does not significantly affect the intracellular growth of O. tsutsugamushi in vitro. These results suggest that O. tsutsugamushi has evolved to block autophagic microbicidal defense by evading autophagic recognition even though it activates the autophagy pathway during the early phase of infection.
PMCID: PMC3553808  PMID: 23230293
9.  Amelioration of Behavioral Abnormalities in BH4-deficient Mice by Dietary Supplementation of Tyrosine 
PLoS ONE  2013;8(4):e60803.
This study reports an amelioration of abnormal motor behaviors in tetrahydrobiopterin (BH4)-deficient Spr−/− mice by the dietary supplementation of tyrosine. Since BH4 is an essential cofactor for the conversion of phenylalanine into tyrosine as well as the synthesis of dopamine neurotransmitter within the central nervous system, the levels of tyrosine and dopamine were severely reduced in brains of BH4-deficient Spr−/− mice. We found that Spr−/− mice display variable ‘open-field’ behaviors, impaired motor functions on the ‘rotating rod’, and dystonic ‘hind-limb clasping’. In this study, we report that these aberrant motor deficits displayed by Spr−/− mice were ameliorated by the therapeutic tyrosine diet for 10 days. This study also suggests that dopamine deficiency in brains of Spr−/− mice may not be the biological feature of aberrant motor behaviors associated with BH4 deficiency. Brain levels of dopamine (DA) and its metabolites in Spr−/− mice were not substantially increased by the dietary tyrosine therapy. However, we found that mTORC1 activity severely suppressed in brains of Spr−/− mice fed a normal diet was restored 10 days after feeding the mice the tyrosine diet. The present study proposes that brain mTORC1 signaling pathway is one of the potential targets in understanding abnormal motor behaviors associated with BH4-deficiency.
PMCID: PMC3618182  PMID: 23577163
10.  Disseminated Herpes Zoster in an Immunocompetent Elderly Patient 
The Korean Journal of Pain  2013;26(2):195-198.
Herpes zoster is a cutaneous infection that is characterized by an acute vesicobullous rash with ipsilateral one or two dermatomal distribution and painful allodynia, while predominantly being found in the elderly. Extensive cutaneous dissemination has been reported in immune-compromised patients, such as those who suffer from HIV infections, cancer, chemotherapy, and corticosteroid therapy patients. However, we report a case of disseminated herpes zoster infection in an immuno-competent elderly individual.
PMCID: PMC3629351  PMID: 23614086
cell mediated immunity; disseminated herpes; Ramsay Hunt syndrome
11.  Detection of Antibodies against Orientia tsutsugamushi Sca Proteins in Scrub Typhus Patients and Genetic Variation of sca Genes of Different Strains 
Scrub typhus, caused by Orientia tsutsugamushi infection, is one of the main causes of acute febrile illness in the Asian-Pacific region. Although early diagnosis and immediate antibiotic treatment are critical for reducing disease severity and mortality, current diagnostic methods using serological and molecular approaches have some limitations in sensitivity and applicability in clinical laboratories. In this study, we identified and characterized O. tsutsugamushi surface cell antigen (sca) family genes encoding autotransporter proteins in order to test them as novel diagnostic targets. We evaluated antibody responses against the Sca proteins in scrub typhus patient sera and examined the genetic diversity of these genes in different strains after PCR amplification. Specific antibody responses against ScaA and ScaC were observed in patients with high indirect immunofluorescence assay titers (≥1:640), whereas specific responses against ScaB and ScaE were relatively low. Genetic analysis using genomic DNAs revealed the sca genes to be quite variable among the different strains. In contrast to scaA, scaC, and scaD, which were detected in all of the tested strains, scaB and scaE were amplified differentially from the different strains, suggesting a differential presence of the genes in the genomes. Among the members of the gene family, the sequence of scaC is the most highly conserved between the different strains, and the size of scaD is the most variable due to the presence of different numbers of internal repeat sequences. These results suggest that the sca genes of O. tsutsugamushi may be valuable targets for use in combination with classical assay methods for scrub typhus diagnosis.
PMCID: PMC3428396  PMID: 22787193
12.  Development and Validation of a Smartphone Addiction Scale (SAS) 
PLoS ONE  2013;8(2):e56936.
The aim of this study was to develop a self-diagnostic scale that could distinguish smartphone addicts based on the Korean self-diagnostic program for Internet addiction (K-scale) and the smartphone's own features. In addition, the reliability and validity of the smartphone addiction scale (SAS) was demonstrated.
A total of 197 participants were selected from Nov. 2011 to Jan. 2012 to accomplish a set of questionnaires, including SAS, K-scale, modified Kimberly Young Internet addiction test (Y-scale), visual analogue scale (VAS), and substance dependence and abuse diagnosis of DSM-IV. There were 64 males and 133 females, with ages ranging from 18 to 53 years (M = 26.06; SD = 5.96). Factor analysis, internal-consistency test, t-test, ANOVA, and correlation analysis were conducted to verify the reliability and validity of SAS.
Based on the factor analysis results, the subscale “disturbance of reality testing” was removed, and six factors were left. The internal consistency and concurrent validity of SAS were verified (Cronbach's alpha = 0.967). SAS and its subscales were significantly correlated with K-scale and Y-scale. The VAS of each factor also showed a significant correlation with each subscale. In addition, differences were found in the job (p<0.05), education (p<0.05), and self-reported smartphone addiction scores (p<0.001) in SAS.
This study developed the first scale of the smartphone addiction aspect of the diagnostic manual. This scale was proven to be relatively reliable and valid.
PMCID: PMC3584150  PMID: 23468893
13.  Orientia tsutsugamushi Subverts Dendritic Cell Functions by Escaping from Autophagy and Impairing Their Migration 
Dendritic cells (DCs) are the most potent antigen-presenting cells that link innate and adaptive immune responses, playing a pivotal role in triggering antigen-specific immunity. Antigen uptake by DCs induces maturational changes that include increased surface expression of major histocompatibility complex (MHC) and costimulatory molecules. In addition, DCs actively migrate to regional lymph nodes and activate antigen-specific naive T cells after capturing antigens. We characterize the functional changes of DCs infected with Orientia tsutsugamushi, the causative agent of scrub typhus, since there is limited knowledge of the role played by DCs in O. tsutsugamushi infection.
Methodology/Principal Finding
O. tsutsugamushi efficiently infected bone marrow-derived DCs and induced surface expression of MHC II and costimulatory molecules. In addition, O. tsutsugamushi induced autophagy activation, but actively escaped from this innate defense system. Infected DCs also secreted cytokines and chemokines such as IL-6, IL-12, MCP5, MIP-1α, and RANTES. Furthermore, in vitro migration of DCs in the presence of a CCL19 gradient within a 3D collagen matrix was drastically impaired when infected with O. tsutsugamushi. The infected cells migrated much less efficiently into lymphatic vessels of ear dermis ex vivo when compared to LPS-stimulated DCs. In vivo migration of O. tsutsugamushi-infected DCs to regional lymph nodes was significantly impaired and similar to that of immature DCs. Finally, we found that MAP kinases involved in chemotactic signaling were differentially activated in O. tsutsugamushi-infected DCs.
These results suggest that O. tsutsugamushi can target DCs to exploit these sentinel cells as replication reservoirs and delay or impair the functional maturation of DCs during the bacterial infection in mammals.
Author Summary
Scrub typhus is an acute febrile illness caused by Orientia tsutsugamushi infection and is one of the main causes of febrile illness in the Asia-Pacific region. If not properly treated with antibiotics, patients often develop severe vasculitis that affects multiple organs, and the mortality rate of untreated patients reaches up to 30%. To understand the pathogenic mechanisms of the infectious disease, we characterized the functional changes of O. tsutsugamushi–infected dendritic cells (DCs), which play a pivotal role in orchestrating innate and adaptive immune responses. The obligate intracellular bacteria efficiently infected bone marrow-derived DCs and activated the cells as measured by induced surface expression of MHC II and costimulatory molecules, secretion of cytokines and chemokines, and autophagy induction. However, the live bacteria actively escaped from host autophagosomes and the migration of infected cells was severely impaired in vitro, ex vivo, and in vivo infection models. Finally, we found that MAP kinases involved in chemotactic signaling were differentially activated in O. tsutsugamushi-infected DCs. These results suggest that O. tsutsugamushi can target DCs to exploit these sentinel cells as replication reservoirs and delay or impair the functional maturation of DCs during the bacterial infection in mammals.
PMCID: PMC3536799  PMID: 23301113
14.  Autophagy induction by tetrahydrobiopterin deficiency 
Autophagy  2011;7(11):1323-1334.
Tetrahydrobiopterin (BH4) deficiency is a genetic disorder associated with a variety of metabolic syndromes such as phenylketonuria (PKU). In this article, the signaling pathway by which BH4 deficiency inactivates mTORC1 leading to the activation of the autophagic pathway was studied utilizing BH4-deficient Spr-/- mice generated by the knockout of the gene encoding sepiapterin reductase (SR) catalyzing BH4 synthesis. We found that mTORC1 signaling was inactivated and autophagic pathway was activated in tissues from Spr-/- mice. This study demonstrates that tyrosine deficiency causes mTORC1 inactivation and subsequent activation of autophagic pathway in Spr-/- mice. Therapeutic tyrosine diet completely rescued dwarfism and mTORC1 inhibition but inactivated autophagic pathway in Spr-/- mice. Tyrosine-dependent inactivation of mTORC1 was further supported by mTORC1 inactivation in Pahenu2 mouse model lacking phenylalanine hydroxylase (Pah). NIH3T3 cells grown under the condition of tyrosine restriction exhibited autophagy induction. However, mTORC1 activation by RhebQ64L, a positive regulator of mTORC1, inactivated autophagic pathway in NIH3T3 cells under tyrosine-deficient conditions. In addition, this study first documents mTORC1 inactivation and autophagy induction in PKU patients with BH4 deficiency.
PMCID: PMC3242797  PMID: 21795851
tetrahydrobiopterin; autophagy; mTORC1; tyrosine; phenylalanine; phenylketonuria; Akt; AMPK
15.  Perspectives of Integrative Cancer Genomics in Next Generation Sequencing Era 
Genomics & Informatics  2012;10(2):69-73.
The explosive development of genomics technologies including microarrays and next generation sequencing (NGS) has provided comprehensive maps of cancer genomes, including the expression of mRNAs and microRNAs, DNA copy numbers, sequence variations, and epigenetic changes. These genome-wide profiles of the genetic aberrations could reveal the candidates for diagnostic and/or prognostic biomarkers as well as mechanistic insights into tumor development and progression. Recent efforts to establish the huge cancer genome compendium and integrative omics analyses, so-called "integromics", have extended our understanding on the cancer genome, showing its daunting complexity and heterogeneity. However, the challenges of the structured integration, sharing, and interpretation of the big omics data still remain to be resolved. Here, we review several issues raised in cancer omics data analysis, including NGS, focusing particularly on the study design and analysis strategies. This might be helpful to understand the current trends and strategies of the rapidly evolving cancer genomics research.
PMCID: PMC3480879  PMID: 23105932
cancer genomics; integromics; next generation sequencing; research design
16.  A New Reporter Vector System Based on Flow-Cytometry to Detect Promoter Activity 
Immune Network  2009;9(6):243-247.
In this study, we report the development of a new dual reporter vector system for the analysis of promoter activity. This system employs green fluorescence emitting protein, EGFP, as a reporter, and uses red fluorescence emitting protein, DsRed, as a transfection control in a single vector. The expression of those two proteins can be readily detected via flow cytometry in a single analysis, with no need for any further manipulation after transfection. As this system allows for the simultaneous detection of both the control and reporter proteins in the same cells, only transfected cells which express the control protein, DsRed, can be subjected to promoter activity analysis, via the gating out of all un-transfected cells. This results in a dramatic increase in the promoter activity detection sensitivity. This novel reporter vector system should prove to be a simple and efficient method for the analysis of promoter activity.
PMCID: PMC2816957  PMID: 20157611
Dual reporter vector system; Transcription activity; Promoter assay; FACS
17.  A Gartner duct cyst of the vagina causing dysuria and dyschezia in a Yorkshire Terrier 
Journal of Veterinary Science  2007;8(4):427-429.
A 5 year-old, intact female Yorkshire terrier was referred for dysuria and dyschezia. The radiographic and ultrasound examination showed a round shaped mass caudal to the urinary bladder that contained anechoic fluid within the thin walls. During surgery, the cyst was noted to be attached to the outer wall of the vagina, not connected to the vaginal lumen. Cystic fluid was removed and the cystic wall was resected. Then the remaining cystic wall was omentalized to prevent a recurrence.
Histological examination confirmed that the cyst was of Wolffian duct origin. In this case, a large Gartner duct cyst causing urological problems was diagnosed and removed by surgical resection.
PMCID: PMC2868161  PMID: 17993759
dog; dyschezia; dysuria; Gartner duct cyst; vaginal cyst
18.  Directed Evolution of Thermus Maltogenic Amylase toward Enhanced Thermal Resistance 
The thermostability of maltogenic amylase from Thermus sp. strain IM6501 (ThMA) was improved greatly by random mutagenesis using DNA shuffling. Four rounds of DNA shuffling and subsequent recombination of the mutations produced the highly thermostable mutant enzyme ThMA-DM, which had a total of seven individual mutations. The seven amino acid substitutions in ThMA-DM were identified as R26Q, S169N, I333V, M375T, A398V, Q411L, and P453L. The optimal reaction temperature of the recombinant enzyme was 75°C, which was 15°C higher than that of wild-type ThMA, and the melting temperature, as determined by differential scanning calorimetry, was increased by 10.9°C. The half-life of ThMA-DM was 172 min at 80°C, a temperature at which wild-type ThMA was completely inactivated in less than 1 min. Six mutations that were generated during the evolutionary process did not significantly affect the specific activity of the enzyme, while the M375T mutation decreased activity to 23% of the wild-type level. The molecular interactions of the seven mutant residues that contributed to the increased thermostability of the mutant enzyme with other adjacent residues were examined by comparing the modeled tertiary structure of ThMA-DM with those of wild-type ThMA and related enzymes. The A398V and Q411L substitutions appeared to stabilize the enzyme by enhancing the interdomain hydrophobic interactions. The R26Q and P453L substitutions led potentially to the formation of genuine hydrogen bonds. M375T, which was located near the active site of ThMA, probably caused a conformational or dynamic change that enhanced thermostability but reduced the specific activity of the enzyme.
PMCID: PMC169122  PMID: 12902281

Results 1-18 (18)