Yasunari Matsuzaka

SLURP-2, a novel member of the human Ly-6 superfamily that is up-regulated in psoriasis vulgaris.

By microarray assay we identified ESTs (expressed sequence tags) whose expression was predominantly increased in the affected skin of patients with psoriasis vulgaris. Among them, a full-length cDNA sequence corresponding to one of those ESTs (AI829641) was isolated by screening of cultured human keratinocyte cDNA libraries. This cDNA encodes a novel member of the Ly-6/uPAR superfamily, designated SLURP-2 (secreted Ly-6/uPAR related protein 2). SLURP-2 has an open reading frame of 97 amino acids containing 10 conserved cysteine residues. SLURP-2 has a single functional copy within the LY6 superfamily gene cluster at chromosome 8q24.3. RT-PCR (reverse transcriptase-polymerase chain reaction) expression analysis revealed that SLURP-2 was expressed in multiple tissues, mainly in the epithelial cells including the skin and keratinocytes, but not in spleen or bone marrow. Comparison of the expression of this gene among the psoriatic lesional and nonlesional skin of patients and the normal skin of healthy individuals detected by quantitative real-time RT-PCR analysis disclosed that SLURP-2 was up-regulated threefold in psoriatic lesional skin. These findings suggest that SLURP-2 may be involved in the pathophysiology of psoriasis through its role in keratinocyte hyperproliferation and/or T cell differentiation/activation.

Identification of the hRDH-E2 gene, a novel member of the SDR family, and its increased expression in psoriatic lesion.

To identify novel psoriasis-associated genes, we focused on several ESTs (expressed sequence tags) whose expression was predominantly increased in the affected skin in patients with psoriasis vulgaris, as assessed by microarray assay. In this paper, a full-length cDNA corresponding to one of those ESTs (AI440266) was isolated by screening of cultured human keratinocyte cDNA libraries. This cDNA has an open reading frame of a 309-amino-acid protein, sharing significant homology to one of the short-chain alcohol dehydrogenase/reductase (SDR) families that can catalyze the first and rate-limiting step that generates retinaldehyde from retinol. So, this gene was designated as hRDH-E2 (human epidermal retinal dehydrogenase 2). The hRDH-E2 gene has a single functional copy on chromosome 8q12.1, spanning approximately 20kb with seven exons. The deduced amino acid sequence contains three motifs that are conserved in the SDR family. Qualitative RT-PCR demonstrated that the mRNA levels of hRDH-E2 were significantly elevated in the affected skin in psoriasis patients as compared to the unaffected skin in patients and the normal skin in healthy individual. These results suggest that hRDH-E2 may be involved in the pathogenesis of psoriasis through its critical role in retinol metabolism in keratinocyte proliferation.

Lack of an association human dioxin detoxification gene polymorphisms with endometriosis in Japanese women: results of a pilot study

ObjectivesEndometriosis is a chronic disease caused by the presence of endometrial tissue in ectopic locations outside the uterus. Chronic exposure to the environmental pollutant dioxin has been correlated with an increased incidence in the development of endometriosis in non-human primates. We have therefore examined whether there is an association between the polymorphisms of ten dioxin detoxification genes and endometriosis in Japanese women.MethodsThis was a pilot study in which 100 patients with endometriosis and 143 controls were enrolled. The prevalence of five microsatellite and 28 single nucleotide polymorphism markers within ten dioxin detoxification genes (AhR, AHRR, ARNT, CYP1A1, CYP2E1, EPHX1, GSTM1, GSTP1, GSTT1, NAT2) was examined.ResultsTaking into account that this analysis was a preliminary study due to its small sample size and genetic power, the results did not show any statistically significant difference between the cases and controls for any of the allele and genotype frequency distributions examined. In addition, no significant associations between the allele/genotype of all polymorphisms and the stage (I–II or III–IV) of endometriosis were observed.ConclusionBased on the findings of this pilot study, we conclude the polymorphisms of the ten dioxin detoxification genes analyzed did not contribute to the etiology of endometriosis among our patients.

NFKBIL1 Confers Resistance to Experimental Autoimmune Arthritis Through the Regulation of Dendritic Cell Functions

We and others have reported that human NF‐κB inhibitor‐like‐1 (NFKBIL1) was a putative susceptible gene for autoimmune diseases such as rheumatoid arthritis (RA). However, its precise role in the pathogenesis of RA is still largely unknown. In this study, we generated transgenic mice expressing human NFKBIL1 (NFKBIL1‐Tg) and examined whether NFKBIL1 plays some role(s) in the development of autoimmune arthritis. In both a collagen‐induced arthritis model and a collagen antibody‐induced arthritis model, NFKBIL1‐Tg mice showed resistance to arthritis compared to control mice, indicating that the gene product of NFKBIL1 was involved in the control of thusly induced arthritis. Total spleen cells of NFKBIL1‐Tg mouse showed decreased proliferation to mitogenic stimuli, consistent with its resistance to arthritis. Unexpectedly, purified T cells of NFKBIL1‐Tg mouse showed increased proliferation and cytokine production. This apparent discrepancy was accounted for by the impaired functions of antigen‐presenting cells of NFKBIL1‐Tg mouse; both T/B cell‐depleted spleen cells and bone marrow‐derived dendritic cells of the Tg mouse induced less prominent proliferation and IL‐2 production of T cells. Furthermore, dendritic cells (DCs) derived from NFKBIL1‐Tg mouse showed lower expression of co‐stimulatory molecules and decreased production of inflammatory cytokines when they were activated by lipopolysaccharide. Taken together, these results indicated that NFKBIL1 affected the pathogenesis of RA at least in part through the regulation of DC functions.

Identification and characterization of novel variants of the thioredoxin reductase 3 new transcript 1 TXNRD3NT1

We have identified and characterized a new gene sequence, TXNRD3NT1, whose transcripts, corresponding to the EST AA430236, were found by Affymetrix DNA chip analysis to be significantly down regulated in affected psoriatic tissue. The full-length cDNA of TXNRD3NT1 was isolated and characterized by combining 5′- and 3′-RACE (rapid amplication of cDNA ends) with screening a keratinocyte cDNA library, designing appropriate PCR primers, cloning amplified products, sequencing, and sequence analysis. Because part of this gene overlaps the previously described thioredoxin reductase 3 (TXNRD3) gene, we have named it TXNRD3NT1 (TXNRD3 new transcript 1). The full-length TXNRD3NT1 cDNA has 1133 nucleotides with a 251-bp 3-UTRand 2 poly(A)signal variants and 2 poly (A) sites. The TXNRD3NT1 cDNA ORF encodes for 133 amino acids, with the first four residues coding for a tubulin-β mRNA autoregulation signal. Mapping the cDNA nucleotide sequence to the human genome sequence revealed that the TXNRD3NT1 gene has 4 exons located on Chromosome 3, at position 3q21. Exons 1 and 2 of the TXNRD3NT1 gene overlap with exons 15 and 16 of the thioredoxin reductase 2 gene which has different ORFs to that of TXNRD3NT1. The translation initiation codon ATG was found in exon 3 of the TXNRD3NT1 gene. RT-PCR showed that the full-length variant of the TXNRD3NT1 gene was expressed in only four issues (pancreas, esophagus, bone marrow, and keratinocytes) of the 30 different tissues tested. In most other tissues, an aberrant and truncated form of the transcript (i.e., missing exon 3 and part of exon 4) was detected. The result of a preliminary association study between psoriasis and single microsatellite marker of the TXNRD3NT1 gene suggests that it may not be a significant genetic determinant of psoriasis. However, we cannot exclude the possibility that other sequence variants may still exist within the TXNRD3NT1 gene. Sequence analysis of the TXNRD3NT1 gene from 8 psoriasis patients and 8 healthy controls revealed a number of synonymous SNPs that may be useful markers for future disease association studies.

Failure to detect significant association between estrogen receptor-alpha gene polymorphisms and endometriosis in Japanese women

ObjectivesThe aim of the study was to test whether estrogen receptor 1 (ESR1) gene polymorphisms are correlated with the risk of the development of endometriosis in Japanese women, as a preliminary study.MethodsTo compare allelic frequencies and genotype distributions, a case-control study of 100 affected women and 143 women with no evidence of disease was performed using 10 microsatellite repeat markers and 66 single-nucleotide polymorphisms (SNPs) in the ESR1 gene region.ResultsAlthough our results might be insufficient to detect genetic susceptibility, owing to the small sample size and low genetic power, statistical analysis of the differences in allelic frequency between the cases and controls at each microsatellite locus demonstrated that no microsatellite locus in the ESR1 gene displayed a significant association with the disease when multiple testing was taken into account. Also, there were no statistically significant differences in the SNP allele frequencies and genotypes between the cases and controls when multiple testing was taken into account.ConclusionThe findings in our pilot study suggest that ESR1 polymorphisms do not contribute to endometriosis susceptibility.

Comparison of dry- and wet-based fine bead homogenizations to extract DNA from fungal spores

The present study explored DNA extraction kinetics from fungal spores, i.e., Aspergillus niger, Penicillium chrysogenum and Cladosporium sphaerospermum, by fine bead mill homogenization. In particular, the study aimed to investigate basic differences between the dry- and wet-based methods. The results showed higher initial rates of the DNA extractions by the dry-based method than by the wet-based method, due to higher collision efficiency among fine beads and fungal spores. Based on the experimental results, we constructed kinetic models. While the results by the wet-based method were fitted well with an existing first-order release-degradation model, the results by the dry-based method were not fitted well. Meanwhile, a newly constructed first-order release-degradation model, assuming a proportion of the DNA remained inside the disrupted spore cells and protected from further sheer stress, showed good correlations. The real-time PCR assays showed the PCR efficiencies of the DNA obtained by the dry-based method were higher than those by the wet-based method likely due to increased moderate fragmentation of the DNA by the dry-based method. Thus, although wet-based methods have been commonly used, dry-based methods might also be applicable to achieve efficient extraction and PCR amplification of fungal DNA.

Mapping of susceptibility locus for endometriosis within the HLA region using microsatellite markers in Japanese women

Endometriosis is a female disorder characterized by the presence of uterine endometrial tissue in ectopic loci. Previous studies reported a higher prevalence of particular human leukocyte antigen (HLA) in endometriosis. In order to confirm the association between endometriosis and the HLA region, 15 polymorphic microsatellite markers distributed in the HLA class II to class III region were subjected to association analysis by polymerase chain reaction (PCR)-based DNA typing of 89 patients and 136 healthy controls. Statistical analysis of the allelic frequency at each microsatellite locus showed that there were no statistically significant differences in the allele frequency distributions between the cases and controls. This finding suggests that the etiology of endometriosis does not involve the HLA class II genomic region and a portion of class III genomic region in the Japanese population.

Association of sick building syndrome with neuropathy target esterase (NTE) activity in Japanese

Sick building syndrome (SBS) is a set of several clinically recognizable symptoms reported by occupants of a building without a clear cause. Neuropathy target esterase (NTE) is a membrane bound serine esterase and its reaction with organophosphates (OPs) can lead to OP‐induced delayed neuropathy (OPIDN) and nerve axon degeneration. The aim of our study was to determine whether there was a difference in NTE activity in the peripheral blood mononuclear cells (PBMCs) of Japanese patients with SBS and healthy controls and whether PNPLA6 (alias NTE) gene polymorphisms were associated with SBS. We found that the enzymatic activity of NTE was significantly higher (P < 0.0005) in SBS patients compared with controls. Moreover, population with an AA genotype of a single nucleotide polymorphism (SNP), rs480208, in intron 21 of the PNPLA6 gene strongly reduced the activity of NTE. Fifty‐eight SNP markers within the PNPLA6 gene were tested for association in a case–control study of 188 affected individuals and 401 age‐matched controls. Only one SNP, rs480208, was statistically different in genotype distribution (P = 0.005) and allele frequency (P = 0.006) between the cases and controls (uncorrected for testing multiple SNP sites), but these were not significant by multiple corrections. The findings of the association between the enzymatic activity of NTE and SBS in Japanese show for the first time that NTE activity might be involved with SBS.

Association study between sick building syndrome and polymorphisms of seven human detoxification genes in the Japanese

Sick building syndrome (SBS) is a chronic disorder caused by exposure to diverse indoor environmental or chemical pollutants. This study examined the association between seven detoxification genes (CYP1A1, CYP2E1, EPHX1, GSTM1, GSTT1, GSTP1, and NAT2) and SBS in the Japanese population. One hundred eighty patients with SBS and 401 healthy controls were enrolled in this study. We examined the prevalence for total of eleven genetic polymorphisms of detoxification genes. However, no statistically significant differences in allele and genotype frequency distributions of eleven genetic polymorphisms of these detoxification genes were found between patients and controls. On this basis, we conclude that the polymorphisms that we assessed for the detoxification genes do not contribute to the etiology of SBS.