Altered acetylation of proteins in patients with rheumatoid arthritis revealed by acetyl-proteomics.

OBJECTIVES: Post-translational modifications (PTMs) are often critical for the function of proteins as well as antigenicity of proteins. We here tried to elucidate alteration of PTMs in Rheumatoid arthritis (RA), focusing on acetylation. We applied acetyl-proteomics to peripheral blood mononuclear cells (PBMCs) to elucidate PTM difference between patients with RA and healthy donors. METHODS: Proteins, extracted from peripheral blood mononuclear cells (PBMCs) of 7 RA patients and 7 healthy donors, were separated by 2-dimansional electrophoresis. Acetylation ratios of each protein spot were estimated by the combination of Sypro Ruby staining and anti-acetylated lysine antibodies. Proteins highly acetylated in the RA group were identified by mass spectrometry. Focusing on α-enolase (ENO1), one of the identified proteins, involvement of histone deacetylases (HDACs) in the high acetylation was investigated. Furthermore, the effects of acetylation on the activity of ENO1 were investigated. RESULTS: In PBMCs from the patients with RA, 29 acetylated protein spots were detected. One of highly acetylated proteins in the RA patients was identified as ENO1. The acetylation of ENO1 was found to be regulated in part by HDAC1. The enzymatic activity of ENO1 was up-regulated by acetylation. CONCLUSIONS: Highly acetylated ENO1 may play roles in the pathophysiology of RA through the maintenance of activated lymphocytes by increasing glycolysis-derived energy supply.

Establishment and characterization of a mouse neural crest derived cell line (NCCmelan5).

Stem cell factor (SCF) and endothelin 3 (EDN3) are both necessary for melanocyte development. We have established an immortal cell population of neural crest cells from C57BL/6 mice, cultivating them with SCF, EDN3 and 15% fetal calf serum without feeder cells, and have designated that line as C57NCC SE. C57NCC SE consists of a population of melanocytes in various stages of differentiation. We used a single-cell cloning method, in which only one cell is transferred to each new culture plate, and succeeded in establishing an immortal cell line named NCCmelan5. All NCCmelan5 cells were positive for KIT (SCF receptor), HMB45 (human melanosomal antigen), tyrosinase-related protein-1 (TYRP1), tyrosinase-related protein-2 (TYRP2), tyrosinase and endothelin receptor B (EDNRB) and all could oxidize 3,4-dihydroxyphenylalanine (DOPA) to form melanin. Measurement of their DNA content revealed that 88.6% of the cells were in the G0-G1 phase, suggesting that they retained normal DNA ploidy. Thus, NCCmelan5 cells have the characteristics of mature melanocytes except that they are immortal; these cells may prove useful to study factors that directly affect melanogenesis and melanocyte development without the influence of feeder cells. It is clear that our attempt to establish immortal cell lines from murine neural crest cells would have never been successful without the addition of SCF and EDN3, since C57NCC SE and NCCmelan5 cells require those factors to proliferate.

A case of black dot ringworm with a review of Japanese cases.

Black dot ringworm (BDR), caused by Trichophyton violaceum var. glabrum (T. glabrum), was observed in a 28-year-old Japanese female who had been treated with prednisolone (22.5 mg/day) for systemic lupus erythematosus. It was successfully treated with oral terbinafine (125 mg/day) for 12 weeks. The causative fungus was identified by molecular analysis as well as morphological and biochemical examination. The chitin synthase 1 (CHS1) gene cleavage pattern of the clinical isolate with restricted enzyme HinfI was identical to that of T. violaceum. We reviewed previous reports of BDR to determine the historical trend of this infection in Japan. Since 1974, 93 Japanese cases have been reported. The age distribution was bi-modal: the higher peak consisted of children (aged 0-15 years), and the lower peak was composed of the elderly (aged 60-75 years). In the elderly group, females were predominant (M:F=1:22, p<0.001). T. violaceum, including T. glabrum, was identified as the most common causative fungus of BDR (75.3%). Sixty percent of cases showed slight erythema. In 8 families, 16 cases were found to be intrafamilial infections. A history of previous steroid treatment was described in about 40%.

Stem cell factor and/or endothelin-3 dependent immortal melanoblast and melanocyte populations derived from mouse neural crest cells.

Stem cell factor (SCF) and endothelin-3 (ET3) are both necessary for melanocyte development. In order to obtain immortal cell populations of melanoblasts that can survive without feeder cells, we first obtained an immortal cell population of neural crest cells (NCCs) from Sl/+ and +/+ mice of strain WB by incubating with a culture medium supplemented with SCF and ET3, and then we designated them as NCC-SE3 cells. NCC-SE3 cells were bipolar, polygonal, or round in shape and possessed melanosomes of stages I-III (mainly stage I). They were positive to dihydroxyphenylalanine (DOPA) reaction and expressed KIT (a receptor tyrosine kinase), tyrosinase, tyrosinase-related protein-1 (TRP1), tyrosinase-related protein-2 (TRP2), and endothelin-B receptor (ETRB) as determined by immunostaining. We next cultured NCC-SE3 cells by changing culture medium from the one supplemented with SCF + ET3 to the one supplemented with SCF or ET3. NCC-SE3 cells cultured with ET3 alone, designated as NCC-E3 cells, were bipolar in shape and had mainly stage II melanosomes and expressed the same proteins as did NCC-SE3 cells. However, NCC-SE3 cells cultured with SCF alone, designated as NCC-S4.1 cells, were polygonal in shape and had mainly stage I melanosomes. They are thought to be more immature because they were positive to KIT, TRP1, and TRP2, but not to ETR(B), tyrosinase, and DOPA reaction. When 12-O-tetradecanoylphorbol 13-acetate and cholera toxin were added to the culture medium, NCC-S4.1 cells changed shape from polygonal to bipolar and became DOPA-positive. This suggests that NCC-S4.1 cells are melanoblasts that have the potential to differentiate into melanocytes. These cell populations will be extremely useful to study factors that affect melanocyte development and melanogenesis.

Differences among chitin synthase I gene sequences in Trichophyton rubrum and T. violaceum.

Nucleotide sequences of chitin synthase 1 (CHS1) gene were analysed for the phylogenetic relation between Trichophyton violaceum and T. rubrum, including two isolates of T. raubitschekii and one isolate of T. rubrum var. nigricans. About 620-bp genomic DNA fragments of the CHS1 gene were amplified from these dermatophytes by polymerase chain reaction (PCR) and sequenced. The CHS1 nucleotide sequences of these dermatophytes showed more than 95% similarity between the species. The phylogenetic analysis of their sequences revealed that T. rubrum was genetically distinct from T. violaceum. The specific restriction endonuclease site for HinfI was present in the CHS1 gene sequence of T. rubrum but not in that of T. violaceum. A molecular analysis of CHS1 genes will provide useful information for the identification of these Trichophyton species and the understanding of their evolution.

Molecular confirmation of a Trichophyton violaceum isolate from human black-dot ringworm.

A clinical isolate from a black-dot ringworm lesion of a 28-year-old female Japanese was investigated by morphological and biochemical analyses as well as molecular analyses. The isolate grew well on thiamine enriched agar and did not produce violet pigment, macro-conidia or micro-conidia on Sabouraud's dextrose agar. Approximately 620-bp genomic DNA fragments of the CHS1 gene were amplified from Trichophyton mentagrophytes, T. rubrum, T. tonsurans and T. violaceum by polymerase chain reaction (PCR) and sequenced. The chitin synthase 1 (CHS1) nucleotide sequences of the clinical isolate showed more than 97% similarity to that of T. violaceum and less than 96% similarity to that of T. mentagrophytes, T. rubrum and T. tonsurans. The phylogenetic analysis of their sequences revealed that the clinical isolate was genetically close to T. violaceum and distinct from T. mentagrophytes, T. rubrum and T. tonsurans. Therefore, the isolate was confirmed as T. violaceum by mycological examination and molecular analyses.

Evaluation of the effect of glucan on hepatic, renal and mammary tumorigenesis in rats.

Glucan was evaluated for its ability to modify the hepatic and renal tumorigenesis induced in partially hepatectomized Sprague-Dawley female rats by diethylnitrosamine (DEN) and phenobarbital (PB), and the mammary tumorigenesis induced in intact Sprague-Dawley female rats by N-nitrosomethylurea (NMU). In both models the rats received every two weeks i.v. glucan (10 mg/kg) or equivalent amounts of dextrose. The results indicated that glucan did not significantly modify the incidence of the chemically-induced hepatic, renal and mammary tumors.