Diagnostic approach for patients with unidentified fever according to the classical criteria of fever of unknown origin in the field of autoimmune disorders

ArticleImmunological medicine. 42(4): 176-184 (2019)journal articl

Long-term maintenance of the mucosal healing induced by azacitidine therapy in a patient with intestinal Behçet's-like disease accompanied with myelodysplastic syndrome involving trisomy 8

ArticleImmunological medicine. 42(3): 135-141 (2019)journal articl

Gene Organization in Rice Revealed by Full-Length cDNA Mapping and Gene Expression Analysis through Microarray

Rice (Oryza sativa L.) is a model organism for the functional genomics of monocotyledonous plants since the genome size is considerably smaller than those of other monocotyledonous plants. Although highly accurate genome sequences of indica and japonica rice are available, additional resources such as full-length complementary DNA (FL-cDNA) sequences are also indispensable for comprehensive analyses of gene structure and function. We cross-referenced 28.5K individual loci in the rice genome defined by mapping of 578K FL-cDNA clones with the 56K loci predicted in the TIGR genome assembly. Based on the annotation status and the presence of corresponding cDNA clones, genes were classified into 23K annotated expressed (AE) genes, 33K annotated non-expressed (ANE) genes, and 5.5K non-annotated expressed (NAE) genes. We developed a 60mer oligo-array for analysis of gene expression from each locus. Analysis of gene structures and expression levels revealed that the general features of gene structure and expression of NAE and ANE genes were considerably different from those of AE genes. The results also suggested that the cloning efficiency of rice FL-cDNA is associated with the transcription activity of the corresponding genetic locus, although other factors may also have an effect. Comparison of the coverage of FL-cDNA among gene families suggested that FL-cDNA from genes encoding rice- or eukaryote-specific domains, and those involved in regulatory functions were difficult to produce in bacterial cells. Collectively, these results indicate that rice genes can be divided into distinct groups based on transcription activity and gene structure, and that the coverage bias of FL-cDNA clones exists due to the incompatibility of certain eukaryotic genes in bacteria

血清アルカリホファターゼ: その少年期における活性値の性・年令変化及びアイソェンザイム種について

金沢大学医療技術短期大学

Identification of a Novel C-Terminal Truncated WT1 Isoform with Antagonistic Effects against Major WT1 Isoforms.

The Wilms' tumor gene WT1 consists of 10 exons and encodes a zinc finger transcription factor. There are four major WT1 isoforms resulting from alternative splicing at two sites, exon 5 (17AA) and exon 9 (KTS). All major WT1 isoforms are overexpressed in leukemia and solid tumors and play oncogenic roles such as inhibition of apoptosis, and promotion of cell proliferation, migration and invasion. In the present study, a novel alternatively spliced WT1 isoform that had an extended exon 4 (designated as exon 4a) with an additional 153 bp (designated as 4a sequence) at the 3' end was identified and designated as an Ex4a(+)WT1 isoform. The insertion of exon 4a resulted in the introduction of premature translational stop codons in the reading frame in exon 4a and production of C-terminal truncated WT1 proteins lacking zinc finger DNA-binding domain. Overexpression of the truncated Ex4a(+)WT1 isoform inhibited the major WT1-mediated transcriptional activation of anti-apoptotic Bcl-xL gene promoter and induced mitochondrial damage and apoptosis. Conversely, suppression of the Ex4a(+)WT1 isoform by Ex4a-specific siRNA attenuated apoptosis. These results indicated that the Ex4a(+)WT1 isoform exerted dominant negative effects on anti-apoptotic function of major WT1 isoforms. Ex4a(+)WT1 isoform was endogenously expressed as a minor isoform in myeloid leukemia and solid tumor cells and increased regardless of decrease in major WT1 isoforms during apoptosis, suggesting the dominant negative effects on anti-apoptotic function of major WT1 isoforms. These results indicated that Ex4a(+)WT1 isoform had an important physiological function that regulated oncogenic function of major WT1 isoforms

Apoptotic function of an Ex4a(+)WT1 isoform.

<p>(<b>A</b>) The role of Ex4a(+)WT1 in apoptosis. Ex4a(+) or Mock vector was transfected into WT1-expressing HT-1080 cells. Frequencies (%) of Annexin V-positive apoptotic cells and cells with loss of MMP were determined by flowcytometry after 24 h. Left, Frequencies (%) of Annexin V-positive apoptotic cells are shown. Right, Frequencies (%) of cells with mitochondrial membrane potential (MMP) loss are shown. Results are means and S.D. of three independent experiments. *, p<0.05. (<b>B</b>) Expression of Ex4a(+) and major WT1 isoforms during apoptosis. K562 cells were treated with the indicated concentrations of Dox for 12 h and analyzed for Annexin-V positive apoptotic cells and expression of Ex4a(+) and major WT1 isoforms by flowcytometry and RT-PCR, respectively. Upper, Frequencies (%) of Annexin V-positive apoptotic cells. Lower, RT-PCR using Ex4-F and Ex6-R primer pair that amplifies both Ex4a(+) and major WT1 isoforms. GAPDH is used as an internal control. Results are representative of three independent experiments. (<b>C</b>) Change of Ex4a(+)WT1 and major WT1 isoforms during apoptosis. K562 cells were treated with the indicated concentrations of Dox for 12 h and expression of Ex4a(+)WT1 and total WT1 isoforms including both Ex4a(+) and major WT1 isoforms were determined by quantitative real-time RT-PCR using Ex4a-F and Ex6-R primer pair and Ex6-F and Ex7-R primer pair, respectively. Actin is used as an internal control for normalization. Expression levels of Ex4a(+)WT1 and total WT1 in Dox-untreated cells are defined as 1.0. (<b>D</b>) Suppression of Ex4a(+)WT1 inhibits Dox-induced apoptosis. K562 cells were transfected with one μg of either of two WT1 Ex4a-specific siRNAs (si-4a-1 and si-4a-2) or a control siRNA (si-control) together with 2.0 μg of Ex4a(+)WT1 vector or 2.0 μg of empty vector (Mock), cultured for 24 h, treated with 4.0 μM Dox for 12 h, and then analyzed for Annexin-V positive apoptotic cells by flowcytometry. Frequencies (%) of Annexin V-positive apoptotic cells are shown. Results are mean and S.D. of three independent experiments. *, p<0.05.</p