Isolation of Acanthamoeba with Sabouroud's glucose agar plate

Isolation of Acanthamoeba or finding out the cysts from corneal leasions is necessary to confirm the diagnosis of Acanthamoeba keratitis. However, we cannot always find out the cyst from corneal scrapings of the lesions. In these cases the isolation of Acanthamoeba is very important to confirm the diagnosis. Non-nutrient agar plates are generally used to isolate Acanthamoeba, but it is difficult to get them easily at many hospitals. To find out another agar that can be used to isolate Acanthamoeba and got easily, Sabouroud's glucose agar plates were compared with non-nutrient agar plates for the isolation of Acanthamoeba. Both agar plates seeded with Escherichia coli showed a nice growth of trophozoites first and cysts later. A clinical material, stock solution of contact lens used by a suspected patient of Acanthamoeba keratitis, was put on Sabouroud's glucose agar plate seeded with Escherichia coli. After three days of incubation at 25°C Acanthamoeba was found on the plate. Sabouroud's glucose agar plate was a very useful agar plate for the isolation of Acanthamoeba

Electron microscopic studies of Acanthamoeba : 2 Morphological changes of cultured Acanthamoeba due to anti-amoebic agents

Morphological changes of Acanthamoeba in Peptone-Yeast-Glucose medium containing anti-amoebic agents were studied with transmission electron microscope. In fradiomycin-treated amoeba, decrease of acanthopodia, vacuolar formation in the ectoplasm and cell collapse were observed. In polyhexamethylene biguanide (PHMB)-treated amoeba, rupture of plasma membrane and degeneration of mitochondria were observed at the early stage. These results indicate that PHMB is more effective than fradiomycin for the treatment of Acanthaoeba keratitis

Methylation of the KEAP1 gene promoter region in human colorectal cancer

<p>Abstract</p> <p>Background</p> <p>The Keap1-Nrf2 pathway has been reported to be impaired in several cancers. However, the status of Keap1-Nrf2 system in human colorectal cancer (CRC) has not been elucidated.</p> <p>Methods</p> <p>We used colorectal cancer (CRC) cell lines and surgical specimens to investigate the methylation status of the <it>KEAP1 </it>promoter region as well as expression of Nrf2 and its downstream antioxidative stress genes, <it>NQO-1 </it>and <it>AKR1C1</it>.</p> <p>Results</p> <p>DNA sequencing analysis indicated that all mutations detected were synonymous, with no amino acid substitutions. We showed by bisulfite genomic sequencing and methylation-specific PCR that eight of 10 CRC cell lines had hypermethylated CpG islands in the <it>KEAP1 </it>promoter region. HT29 cells with a hypermethylated <it>KEAP1 </it>promoter resulted in decreased mRNA and protein expression but unmethylated Colo320DM cells showed higher expression levels. In addition, treatment with the DNA methyltransferase inhibitor 5-Aza-dC combined with the histone deacetylase inhibitor trichostatin A (TSA) increased <it>KEAP1 </it>mRNA expression. These result suggested that methylation of the <it>KEAP1 </it>promoter regulates its mRNA level. Time course analysis with the Nrf2-antioxidant response element (ARE) pathway activator t-BHQ treatment showed a rapid response within 24 h. HT29 cells had higher basal expression levels of <it>NQO-1 </it>and <it>AKR1C1 </it>mRNA than Colo320DM cells. Aberrant promoter methylation of <it>KEAP1 </it>was detected in 53% of tumor tissues and 25% of normal mucosae from 40 surgical CRC specimens, indicating that cancerous tissue showed increased methylation of the <it>KEAP1 </it>promoter region, conferring a protective effect against cytotoxic anticancer drugs.</p> <p>Conclusion</p> <p>Hypermethylation of the <it>KEAP1 </it>promoter region suppressed its mRNA expression and increased nuclear Nrf2 and downstream ARE gene expression in CRC cells and tissues.</p

Sphere therapy for corneal endothelium deficiency in a rabbit model. Invest Ophthalmol Vis Sci.

PURPOSE. To isolate precursor cells derived from rabbit corneal endothelium (CE) and to use them for the treatment of CE deficiency in a rabbit model. METHODS. A sphere-forming assay was performed to isolate precursor cells from rabbit CE. Immunocytochemistry was used to examine marker expressions of neural and mesenchymal cells in the sphere colonies and their progenies. The pump function of the CE sheet was evaluated by measurement of the potential difference and short circuit current. Precursors obtained from rabbit CE by a sphere-forming assay were injected into the anterior chamber of the eye, after which an eye-down (i.e., CE up) position was maintained for 24 hours to allow attachment by gravitation (sphere eye-down group). The sphere eye-down and control groups, observed for 28 days after surgery, underwent histologic and fluorescence microscopic examinations. RESULTS. Rabbit CE formed primary and secondary sphere colonies. The progeny expressed ␣-smooth muscle actin, nestin, and neural markers and showed a CE-like hexagonal shape and adequate transport activity. Mean corneal thickness in the sphere eye-down group was significantly less than in the other control groups 3, 7, 14, 21, and 28 days (P Ͻ 0.05) after surgery. CE-like hexagonal cells were detected on Descemet&apos;s membrane, and corneal edema was substantially suppressed. DiI-labeled cells were spread over the rear corneal surface in the sphere eye-down group only. CONCLUSIONS. Precursors from rabbit CE were isolated by a sphere-forming assay. Rabbit CE-derived sphere therapy is an effective treatment in a rabbit CE deficiency model. (Invest Ophthalmol Vis Sci. 2005;46:3128 -3135