In order to evaluate the possible roles of secretory proteases in the pathogenesis of amoebic keratitis, we purified and characterized a serine protease secreted by Acanthamoeba lugdunensis KA/E2, isolated from a Korean keratitis patient. The ammonium sulfate-precipitated culture supernatant of the isolate was purified by sequential chromatography on CM-Sepharose, Sephacryl S-200, and mono Q-anion exchange column. The purified 33 kDa protease had a pH optimum of 8.5 and a temperature optimum of 55 degrees C. Phenylmethylsulfonylfluoride and 4- (2- Aminoethyl) -benzenesulfonyl-fluoride, both serine protease specific inhibitors, inhibited almost completely the activity of the 33 kDa protease whereas other classes of inhibitors did not affect its activity. The 33 kDa enzyme degraded various extracellular matrix proteins and serum proteins. Our results strongly suggest that the 33 kDa serine protease secreted from this keratopathogenic Acanthamoeba play important roles in the pathogenesis of amoebic keratitis, such as in corneal tissue invasion, immune evasion and nutrient uptake.
Acanthamoeba/*enzymology/isolation & purification/pathogenicity ; Acanthamoeba Keratitis/*parasitology ; Animals ; Cornea/parasitology ; Humans ; Hydrogen-Ion Concentration ; Korea ; Serine Endopeptidases/chemistry/*isolation & purification/*metabolism ; Substrate Specificity ; Temperature ; Virulence Factors

To examine the expressed gene profile during encystation of Acanthamoeba castellanii Castellani, we used differentially expressed gene (DGE) screening by RT-PCR with 20 sets of random primers. From this analysis, we found that approximately 16 genes showed upregulation during encystation. We chose 6 genes, which had relatively higher expression levels, for further investigation. Based on homology search in database, DEG2 showed 55% of similarity with xylose isomerase, DEG9 showed 37% of similarity with Na P-type ATPase, and DEG14 showed 77% of similarity with subtilisin-like serine proteinase. DEG3 and DEG26 were identified as hypothetical proteins and DEG25 exhibited no significant similarity to any known protein. Encystation of Acanthamoeba has been suggested to be a process to resist adverse environmental or nutritional conditions. Further characterization studies of these genes may provide us with more information on the encystation mechanism of Acanthamoeba.
Acanthamoeba castellanii/*genetics/*growth & development ; Amino Acid Sequence ; Animals ; *Gene Expression Profiling ; Gene Expression Regulation ; *Life Cycle Stages ; Molecular Sequence Data ; Protozoan Proteins/*genetics ; Reverse Transcriptase Polymerase Chain Reaction ; Sequence Alignment ; Sequence Homology, Amino Acid ; Up-Regulation

PURPOSE: The purpose of this study was to develop an empowerment program as a nursing intervention for the patients having an acute stroke and to determine the effects of the program on their motivation, depression, and activities of daily living(ADLs). METHODS: An non-equivalent control group pretest-posttest design was used in this study. Sixty subjects were recruited from two separated institutions: 31 patients were allocated into experimental group and 29 were into control group. Six week empowerment program was provided to the experimental group. The study was conducted from November 2006 to March 2007. RESULTS: After 6 week empowerment program, rehabilitation motivation was significantly increased in the experimental group in comparison to the control group(t=-2.173, p=.036). There were no significant differences in depression and ADLs between experimental and control groups. CONCLUSION: The empowerment program effectively increased rehabilitation motivation of patients with stroke, while did not improve the levels of depression and ADLs. Future long-term intervention may benefit the patients more in terms of depression and ADLs when considering the acute stage of the patients in this study.
Activities of Daily Living ; Depression ; Humans ; Motivation ; Power (Psychology) ; Stroke

PURPOSE: To evaluate the utility of FLAIR (Fluid Attenuated Inversion Recovery) MR imaging in cerebral infarction by comparing its results with those of T2-weighted spin-echo imaging. MATERIALS AND METHODS: We retrospectively evaluated fast FLAIR images and conventional spin echo images of 82 patients (47 men and 20 women; median age 60.9 years) with cerebral infarction. MR imaging used a 1.5T MR unit with conventional T2 (TR 3900, TE 90) and fast FLAIR sequence (TR 8000, TE 105, TI 2400). We analysed the size of the main lesion and number of lesions, and discrimination between old and new lesions and between small infarction and perivascular space. RESULTS: When T2-weighted and FLAIR imaging were compared, the latter showed that the main lesion was larger in 38 cases (46%), similar in 38 (46%), and smaller in six (7%). The number of lesions was greater in 23 cases (28%), similar in 52 (63%), and fewer in seven (9%). FLAIR images discriminated between old and new lesions in 31 cases ; perivascular space and small infartion were differentiated in eight cases, and CSF inflowing artifact was observed in 66 (80%). CONCLUSION: In the diagnosis of cerebral infaretion, fast FLAIR provides images that are equal or superior to T2-weighted images. The fast FLAIR sequence may therefore be used as a part of routine MR brain study in the diagnosis of cerebral infarction.
Artifacts ; Brain ; Cerebral Infarction* ; Diagnosis ; Discrimination (Psychology) ; Female ; Humans ; Infarction ; Magnetic Resonance Imaging* ; Male ; Retrospective Studies

PURPOSE: To evaluate the incidence of sacral insufficiency fracture in osteoporotic patients with compression fracture of the thoracolumbar (T-L) spine on magnetic resonance image (MRI), and to analyze the correlation of variable clinical factors and the incidence of sacral insufficiency fracture. MATERIALS AND METHODS: We retrospectively reviewed 160 patients (27 men, 133 women; age range of 50 to 89 years) who underwent spinal MRI and had compression fracture of the T-L spine. Compression fractures due to trauma or tumor were excluded. We evaluated the incidence of sacral insufficiency fracture according to the patients' age, sex, number of compression fractures, and the existence of bone marrow edema pattern of compression fracture. During the same period, we evaluated the incidence of spinal compression fracture in the patients of pelvic insufficiency fracture. RESULTS: Out of the 160 patients who had compression fracture in the T-L spine, 17 (10.6%) had insufficiency fracture of the sacrum. Compression fracture occurred almost 5 times more frequently in women (27:133), but the incidence of sacral insufficiency fracture was 2/27 for men (7.4%) and 15/133 for women (11.3%), with no statistically significant difference (p = 0.80). According to age, the ratio of insufficiency fracture to compression fracture was 0% (0/23) in the 50's, 10.6% (7/66) in the 60's, 12.5% (7/56) in the 70's, and 20.0% (3/15) in the 80's. In respect of single and multiple compression fracture, the incidence of sacral insufficiency fracture was 8/65 for men (12.3%) and 9/95 for women (9.5%), showing no significant difference (p=0.37). In the patients with and without compression fracture with bone marrow edema, insufficiency fracture occurred in 5/76 (6.6%) and 12/84 (14.3%), respectively. On the other hand, of the 67 patients who had pelvic insufficiency fracture, 27 (40.3%) also had spinal compression fracture. CONCLUSION: About 10% of the patients with osteoporotic compression fracture in the T/L spine also had pelvic sacral insufficiency fracture, which was not uncommon. These findings suggest the need to consider the possibility of pelvic sacral insufficiency fracture in cases of T/L spinal MRI for patients with osteoporotic compression fracture.
Bone Marrow ; Edema ; Female ; Fractures, Compression* ; Fractures, Stress* ; Hand ; Humans ; Incidence* ; Magnetic Resonance Imaging ; Male ; Osteoporosis ; Retrospective Studies ; Sacrum ; Spine*

The time to complete or partial (objective) response to radiotherapy in patients with hepatocellular carcinoma (HCC) is variable; thus, the reported frequency of these responses depends on the length of follow-up. However, the optimum follow-up duration is unknown. We sought to determine the optimal follow-up duration by analyzing the medical records of 25 patients with 39 HCC lesions who received definitive helical tomotherapy at a daily dose of 2 to 4 Gy at 5 fractions per week, for a total dose of 40 to 60 Gy, between January 2008 and January 2013. We determined the time to objective treatment response and local recurrence after radiotherapy and assessed several predictors of delayed treatment response. The median follow-up duration was 15.2 months (range, 7.8 to 52.1 months). Among all 39 lesions, objective responses were observed for 36 (92.3%). The median time to objective response was 3.9 months (range, 1.5 to 9.8 months). The objective response rates increased over time from 15.4% at 3 months to 71.8% at 6 months and 87.2% at 9 months. Age 60 years old or older and post-radiotherapy α-fetoprotein concentrations higher than pre-radiotherapy concentrations predicted delayed treatment response. The objective response rate continued to increase over 9 months. Therefore, to fully evaluate the treatment response of HCC, we recommend continuous observation for at least 9 months after radiotherapy.
Adult ; Aged ; Carcinoma, Hepatocellular ; blood ; radiotherapy ; Female ; Follow-Up Studies ; Humans ; Liver Neoplasms ; blood ; radiotherapy ; Male ; Middle Aged ; Retrospective Studies ; Treatment Outcome ; alpha-Fetoproteins ; analysis

Acanthamoeba keratitis has been increasing in recent years. Main risk factors are contact lens wear and their cleaning solutions. Most contact lens wearers use multipurpose disinfecting solutions (MPDS) for cleansing and disinfecting microorganisms because of its convenience. We determined amoebicidal effects of MPDS made in Korea and their cytotoxicity on human corneal epithelium cells. Fifteen commercial MPDS (A to O) were tested for their amoebicidal effects on Acanthamoeba castellanii trophozoites and cysts by using a most probable number (MPN) technique. Among them, 7 kinds of MPDS showed little or no amoebicidal effects for 24 hr exposure. Solutions A, B, G, H, L, and O showed positive amoebicidal effects, and solutions M and N killed almost all trophozoites and cysts after 24 hr exposure. However, 50%-N solution showed 56% cytotoxicity on human corneal epithelial cells within 4 hr exposure, and 50%-O solution also showed 62% cytotoxicity on human cells within 4 hr exposure. Solution A did not show any cytotoxicity on human cells. These results revealed that most MPDS made in Korea were ineffective to kill Acanthamoeba. The solutions having amoebicidal activity also showed high levels of cytotoxicity on human corneal epithelial cells. New formulations for improved MPDS that are amoebicidal but safe for host cells are needed to prevent Acanthamoeba keratitis.
Acanthamoeba castellanii* ; Acanthamoeba Keratitis ; Acanthamoeba* ; Epithelial Cells ; Epithelium, Corneal ; Humans ; Korea ; Risk Factors ; Trophozoites

Encystation mediating cyst specific cysteine proteinase (CSCP) of Acanthamoeba castellanii is expressed remarkably during encystation. However, the molecular mechanism involved in the regulation of CSCP gene expression remains unclear. In this study, we focused on epigenetic regulation of gene expression during encystation of Acanthamoeba. To evaluate methylation as a potential mechanism involved in the regulation of CSCP expression, we first investigated the correlation between promoter methylation status of CSCP gene and its expression. A 2,878 bp of promoter sequence of CSCP gene was amplified by PCR. Three CpG islands (island 1–3) were detected in this sequence using bioinformatics tools. Methylation of CpG island in trophozoites and cysts was measured by bisulfite sequence PCR. CSCP promoter methylation of CpG island 1 (1,633 bp) was found in 8.2% of trophozoites and 7.3% of cysts. Methylation of CpG island 2 (625 bp) was observed in 4.2% of trophozoites and 5.8% of cysts. Methylation of CpG island 3 (367 bp) in trophozoites and cysts was both 3.6%. These results suggest that DNA methylation system is present in CSCP gene expression of Acanthamoeba. In addition, the expression of encystation mediating CSCP is correlated with promoter CpG island 1 hypomethylation.
Acanthamoeba castellanii* ; Acanthamoeba* ; Computational Biology ; CpG Islands ; Cysteine Proteases ; DNA Methylation* ; DNA* ; Epigenomics ; Gene Expression Regulation ; Gene Expression* ; Methylation ; Negotiating ; Polymerase Chain Reaction ; Trophozoites

Protein arginine methyltransferase (PRMT) is an important epigenetic regulator in eukaryotic cells. During encystation, an essential process for Acanthamoeba survival, the expression of a lot of genes involved in the encystation process has to be regulated in order to be induced or inhibited. However, the regulation mechanism of these genes is yet unknown. In this study, the full-length 1,059 bp cDNA sequence of Acanthamoeba castellanii PRMT1 (AcPRMT1) was cloned for the first time. The AcPRMT1 protein comprised of 352 amino acids with a SAM-dependent methyltransferase PRMT-type domain. The expression level of AcPRMT1 was highly increased during encystation of A. castellanii. The EGFP-AcPRMT1 fusion protein was distributed over the cytoplasm, but it was mainly localized in the nucleus of Acanthamoeba. Knock down of AcPRMT1 by synthetic siRNA with a complementary sequence failed to form mature cysts. These findings suggested that AcPRMT1 plays a critical role in the regulation of encystation of A. castellanii. The target gene of AcPRMT1 regulation and the detailed mechanisms need to be investigated by further studies.
Acanthamoeba castellanii* ; Acanthamoeba* ; Amino Acids ; Clone Cells ; Cytoplasm ; DNA, Complementary ; Epigenomics ; Eukaryotic Cells ; Protein-Arginine N-Methyltransferases* ; RNA, Small Interfering

PURPOSE: Predictive factors for radiation pneumonitis (RP) after helical tomotherapy (HT) may differ from those after linac-based radiotherapy. In this study, we identified predictive factors for RP in patients with lung cancer treated with HT. MATERIALS AND METHODS: We retrospectively analyzed clinical, treatment-related and dosimetric factors from 31 patients with lung cancer treated with HT. RP was graded according to Common Terminology Criteria for Adverse Events version 4.0 and grade > or =2 RP was defined as a RP event. We used Kaplan-Meier methods to compute the actuarial incidence of RP. For univariate and multivariate analysis, the log-rank test and the Cox proportional regression hazard model were used. We generated receiver-operating characteristics (ROC) curves to define the cutoff values for significant parameters. RESULTS: The median follow-up duration was 6.6 months (range, 1.6 to 38.5 months). The 2-, 4-, and 6-month actuarial RP event rates were 13.2%, 58.5%, and 67.0%, respectively. There was no grade 4 or more RP. Ipsilateral V5, V10, V15, and contralateral V5 were related with RP event on univariate analysis. By multivariate analysis, ipsilateral V10 was factor most strongly associated with RP event. On the ROC curve, the cutoff values of ipsilateral V5, V10, V15, and contralateral V5 were 67.5%, 58.5%, 50.0%, and 55.5%, respectively. CONCLUSION: In our study, ipsilateral V5, V10, V15, and contralateral V5 were significant predictive factors for RP after HT.
Follow-Up Studies ; Humans ; Incidence ; Lung Neoplasms* ; Lung* ; Methods ; Multivariate Analysis ; Proportional Hazards Models ; Radiation Pneumonitis* ; Radiotherapy ; Radiotherapy, Intensity-Modulated* ; Retrospective Studies ; Risk Factors ; ROC Curve