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

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

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: 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*

Encystation is an essential process for Acanthamoeba survival under nutrient-limiting conditions and exposure to drugs. The expression of several genes has been observed to increase or decrease during encystation. Epigenetic processes involved in regulation of gene expression have been shown to play a role in several pathogenic parasites. In the present study, we identified the protein arginine methyltransferase 5 (PRMT5), a known epigenetic regulator, in Acanthamoeba castellanii. PRMT5 of A. castellanii (AcPRMT5) contained domains found in S-adenosylmethionine-dependent methyltransferases and in PRMT5 arginine-N-methyltransferase. Expression levels of AcPRMT5 were increased during encystation of A. castellanii. The EGFP-PRMT5 fusion protein was mainly localized in the nucleus of trophozoites. A. castellanii transfected with siRNA designed against AcPRMT5 failed to form mature cysts. The findings of this study lead to a better understanding of epigenetic mechanisms behind the regulation of encystation in cyst-forming pathogenic protozoa.
Acanthamoeba castellanii ; Acanthamoeba* ; Epigenesis, Genetic ; Epigenomics ; Gene Expression Regulation ; Methyltransferases ; Parasites ; Protein-Arginine N-Methyltransferases* ; RNA, Small Interfering ; 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

We conducted both the small subunit ribosomal DNA (SSU rDNA) polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and mitochondrial (mt) DNA RFLP analyses for a genetic characterization of Acanthamoeba isolates from contact lens storage cases of students in Seoul, Korea. Twenty-three strains of Acanthamoeba from the American Type Culture Collection and twelve clinical isolates from Korean patients were used as reference strains. Thirty-nine isolates from contact lens storage cases were classified into seven types (KA/LS1, KA/LS2, KA/LS4, KA/LS5, KA/LS7, KA/LS18, KA/LS31). Four types (KA/LS1, KA/LS2, KA/LS5, KA/LS18) including 33 isolates were regarded as A. castellanii complex by riboprints. KA/LS1 type was the most predominant (51.3%) in the present survey area, followed by KA/LS2 (20.9%), and KA/LS5 (7.7%) types. Amoebae of KA/LS1 type had the same mtDNA RFLP and riboprint patterns as KA/E2 and KA/E12 strains, clinical isolates from Korean keratitis patients. Amoebae of KA/LS2 type had the identical mtDNA RFLP patterns with A. castellanii Ma strain, a corneal isolate from an American patient as amoebae of KA/LS5 type, with KA/E3 and KA/E8 strains from other Korean keratitis patients. Amoebae of KA/LS18 type had identical patterns with JAC/E1, an ocular isolate from a Japanese patient. Three types, which remain unidentified at species level, were not corresponded with any clinical isolate in their mtDNA RFLP and riboprint patterns. Out of 39 isolates analyzed in this study, mtDNA RFLP and riboprint patterns of 33 isolates (84.6%) were identical to already known clinical isolates, and therefore, they may be regarded as potentially keratopathogenic. These results suggest that contact lens wearers in Seoul should pay more attention to hygienic maintenance of contact lens storage cases for the prevention of Acanthamoeba keratitis.
Acanthamoeba/classification/*genetics/isolation & purification ; Acanthamoeba Keratitis/parasitology/prevention & control ; Animals ; Contact Lenses/*parasitology ; DNA, Mitochondrial/genetics ; DNA, Protozoan/genetics ; Human ; Korea ; Students

OBJECTIVES: We describe patterns of tumor regression based on follow-up duration after radiotherapy (RT) or chemo-RT in patients with head and neck squamous cell carcinoma. METHODS: Thirty-one patients with head and neck squamous cell carcinoma were included in this study and received definitive RT or chemo-RT. The pattern of primary tumor regression after treatment was evaluated every 1 to 2 months. Predictive factors for the length of time to full regression were also analyzed. RESULTS: Among all patients, 27 patients showed regression of the primary tumor, 24 patients showed >50% regression, and 15 patients showed total regression. The primary tumor gradually regressed during the course of follow-up. The median time to full regression was 5.2 months (range, 1.3 to 17.9 months). In the 24 patients who showed >50% regression, the rate of >50% regression increased over time as follows: 25.0% at 1 month, 62.5% at 2 months, 75.0% at 3 months, 91.7% at 4 months, and 95.8% at 5 months. Higher total RT dose and shorter RT duration were associated with longer time to full regression. CONCLUSION: A substantial number of patients showed continuous regression of the primary tumor for more than 2 months after treatment. The timing for evaluation of tumor regression must be greater than 2 months from the completion of RT or chemo-RT in patients with head and neck squamous cell carcinoma.
Carcinoma, Squamous Cell* ; Chemoradiotherapy* ; Follow-Up Studies* ; Head* ; Humans ; Neck* ; Radiotherapy*