Disease-modifying antirheumatic drug selection in Japanese patients with rheumatoid arthritis treated with biologics or JAK inhibitors without methotrexate: A retrospective hospital-based administrative claims database study.

OBJECTIVES: We evaluated the medication selection and clinical characteristics of rheumatoid arthritis patients who started treatment with/without methotrexate (MTX) (using biologic disease-modifying antirheumatic drugs or Janus kinase inhibitors instead) in Japan. METHODS: Using a Japanese hospital-based administrative claims database, rheumatoid arthritis patients who received treatment [abatacept (ABA), interleukin-6 receptor inhibitor, tumor necrosis factor inhibitor, or Janus kinase inhibitor] between 1 January 2015 and 31 December 2019 were enrolled. RESULTS: Overall, 19,301 patients were included (10,530 receiving MTX; 8771 not receiving MTX within 60 days of the first treatment). Mean ages at diagnosis were 60.7 and 65.9 years in the MTX and non-MTX groups, respectively (P < .0001). The non-MTX group had higher proportions of patients with Charlson Comorbidity Index ≥1 (P < .0001) and higher comorbidity rates. ABA was the most frequently used drug among patients with infectious/parasitic, circulatory, and respiratory diseases at baseline. Interleukin-6 receptor inhibitor had the highest use rate among patients with neoplasms; blood, gastrointestinal, and genitourinary diseases; and abnormal clinical/laboratory findings. ABA had the highest persistence probability from 6 months onward. CONCLUSIONS: MTX is used less frequently among older Japanese rheumatoid arthritis patients or those with comorbidities. In such patients, ABA is the most frequently used drug, followed by interleukin-6 receptor inhibitor, when MTX is not used at treatment start.

Factors contributing to the improvement in J-HAQ after 3 years of treatment with abatacept in biologic-naïve rheumatoid arthritis patients: Interim results of a long-term, observational, multicentre study in Japan (ORIGAMI).

OBJECTIVES: We investigated the long-term effectiveness, safety, and factors affecting Japanese Health Assessment Questionnaire (J-HAQ) improvement during abatacept treatment in Japanese rheumatoid arthritis (RA) patients. METHODS: The ORIGAMI study is an ongoing observational study of biologic-naïve RA patients with moderate disease activity treated with subcutaneous abatacept (125 mg, once-weekly). Patients treated with conventional synthetic disease-modifying antirheumatic drugs (csDMARDs) were extracted from the Institute of Rheumatology, Rheumatoid Arthritis (IORRA) registry as an historical, weighted control group. The primary endpoint for this interim analysis was the proportion of patients with J-HAQ remission (score ≤0.5) at 3 years. RESULTS: Among 279 abatacept-treated and 220 csDMARD-treated patients, J-HAQ remission was achieved at 3 years in 40.5% (95% confidence interval [CI] 34.7%-46.2%) and 28.9% (95% CI 9.9%-47.8%), respectively. Age, RA duration <1 year, baseline J-HAQ score, and Simplified Disease Activity Index remission at 6 months were associated with 3-year J-HAQ remission in the abatacept group. Overall, 24/298 patients (8.1%; safety analysis set) experienced serious adverse drug reactions with an incidence of 5.3 per 100 person-years. CONCLUSIONS: This study confirmed the 3-year effectiveness and safety, and revealed potential factors associated with J-HAQ remission in biologic-naïve RA patients treated with abatacept in real-world clinical practice.

Associations of disease duration and anti-citrullinated peptide antibody status with the effectiveness of abatacept in biologic-naïve patients with rheumatoid arthritis: Post hoc analysis of a multicentre, real-world observational study in Japan (ORIGAMI).

OBJECTIVES: The aim of the article is to investigate the associations of disease duration and anti-cyclic citrullinated peptide antibody (ACPA) status with the effectiveness of abatacept in biologic-naïve patients with rheumatoid arthritis (RA). METHODS: We performed post hoc analyses of the Orencia® Registry in Geographically Assembled Multicenter Investigation (ORIGAMI) study of biologic-naïve RA patients aged ≥20 years with moderate disease activity who were prescribed abatacept. Changes in the Simplified Disease Activity Index (SDAI) and Japanese Health Assessment Questionnaire (J-HAQ) at 4, 24, and 52 weeks of treatment were analysed in patients divided according to ACPA serostatus (positive/negative), disease duration (<1/≥1 year), or both. RESULTS: SDAI scores decreased from baseline in all groups. SDAI scores tended to decrease more in the ACPA-positive group and disease duration <1-year group than in the ACPA-negative group and disease duration ≥1-year group, respectively. In the disease duration <1-year group, SDAI tended to decrease more in the ACPA-positive group than in the ACPA-negative group. Disease duration was independently associated with the change in SDAI and SDAI remission at Week 52 in multivariable regression models. CONCLUSIONS: These results suggest that starting abatacept within 1 year of diagnosis was associated with greater effectiveness of abatacept in biologic-naïve patients with RA and moderate disease activity.

Contribution of the factors to EuroQol 5 Dimensions in rheumatoid arthritis patients achieving low disease activity/remission with abatacept treatment: post-hoc subgroup analyses of the Japanese real-world observational "ORIGAMI" study.

OBJECTIVES: To address improvements in quality of life (QOL), we analysed the relative contributions of factors to EuroQol 5 Dimensions (EQ-5D) in abatacept-treated rheumatoid arthritis (RA) patients in the ORIGAMI study. METHODS: Patients who were evaluable for disease activity through to Week 52 in the ORIGAMI study were divided into those achieving Simplified Disease Activity Index-remission/low disease activity (remission/LDA; n=178) and patients with moderate/high disease activity (MDA/HDA; n=99). We compared the changes in EQ-5D and other outcomes through to Week 52. Focusing on the remission/LDA group, the contribution of each factor to the variance of EQ-5D at baseline and Week 52 was examined using analysis of variance. RESULTS: The remission/LDA group showed greater improvements than the MDA/HDA group in EQ-5D, Japanese Health Assessment Questionnaire, visual analogue scale for pain (Pain VAS), and patient global assessment (PtGA). In the remission/LDA group, factors significantly contributing to EQ-5D were sex, C-reactive protein, and Pain VAS at baseline, and PtGA and age at Week 52. CONCLUSIONS: In RA patients who achieved remission/LDA during abatacept treatment, PtGA and age at Week 52 contribute to the variance of EQ-5D, suggesting that identification of factors associated with PtGA may be important to address improvements of QOL.

The importance of specific citrullinated clusterin and vimentin found in a multi-coloured bead-based citrulline-peptide array system in rheumatoid arthritis.

OBJECTIVES: The importance of citrullination in rheumatoid arthritis (RA) has been reported, but the degree to which individual citrullinated proteins affect the onset and progression of RA is still unclear. We aimed to identify citrullinated proteins that may play an important role in the onset and progression of RA using an individualised anti-citrullinated protein antibody (ACPA) evaluation system with citrullinated peptides as probes. METHODS: Serum samples from 50 normal donors and 51 RA patients were evaluated using a custom MagPlexTM bead array with 13 types of citrullinated peptide. The presence/absence of ACPAs that react to each citrullinated peptide in each subject was determined using the Z-score, which was calculated based on the fluorescence intensity distribution of a sample from a normal donor. Whether the fluorescence intensity was inhibited when free citrullinated peptides were added to a system was also evaluated. RESULTS: Median fluorescence intensities obtained from beads coupled with the 13 types of citrullinated peptide were all significantly higher in RA patients versus normal donors. With a Z-score ≥2 as the cut-off value for the presence of ACPAs, ACPAs that recognised five types of citrullinated peptides derived from fibrinogen A, fillagrin, clusterin, and vimentin were widely detected in RA patients. In addition, inhibition experiments showed that citrullinated vimentin, clusterin, and enolase 1A peptides inhibited coupling of ACPAs to other citrullinated peptides. CONCLUSIONS: ACPAs to many citrullinated proteins exhibited cross-reactivity to citrullinated clusterin and vimentin, suggesting the importance of citrullinated clusterin and vimentin in the early stages of RA pathogenesis.

Long-term safety and effectiveness of abatacept in Japanese patients with rheumatoid arthritis: 3-year follow-up of a postmarketing surveillance.

OBJECTIVES: To analyse the long-term safety and effectiveness of abatacept for rheumatoid arthritis using real-world, Japanese, postmarketing surveillance data, focusing on serious infections and malignancies as priority events. METHODS: This 3-year, multicentre surveillance registered patients undergoing abatacept treatment by intravenous infusion between July 2011 and October 2012. RESULTS: The safety and effectiveness analysis sets included 647 and 596 patients, respectively. The total observation period for the safety analysis was 1280 patient-years. Over the 3-year follow-up, the incidence rates of adverse drug reactions (ADRs) and serious ADRs were 19.92 per 100 patient-years (22.87% of patients) and 4.06 per 100 patient-years (6.65% of patients), respectively. Infections and infestations were the most common ADRs (14.68%), followed by respiratory, thoracic, and mediastinal disorders (3.09%). Incidence rates of serious infections as ADRs and malignancy as adverse events were 1.95 and 1.02 per 100 patient-years, respectively. Retention rates at 1 and 3 years were 67.4% and 43.9%, respectively. Significant decreases from baseline were observed in Disease Activity Score (DAS) 28-erythrocyte sedimentation rate and DAS28-C-reactive protein, as well as Health Assessment Questionnaire-Disability Index (HAQ-DI) and modified HAQ scores. CONCLUSIONS: No new safety signals were detected during the 3-year observation period and effectiveness was maintained over time.

Japan PGx Data Science Consortium Database: SNPs and HLA genotype data from 2994 Japanese healthy individuals for pharmacogenomics studies.

Japan Pharmacogenomics Data Science Consortium (JPDSC) has assembled a database for conducting pharmacogenomics (PGx) studies in Japanese subjects. The database contains the genotypes of 2.5 million single-nucleotide polymorphisms (SNPs) and 5 human leukocyte antigen loci from 2994 Japanese healthy volunteers, as well as 121 kinds of clinical information, including self-reports, physiological data, hematological data and biochemical data. In this article, the reliability of our data was evaluated by principal component analysis (PCA) and association analysis for hematological and biochemical traits by using genome-wide SNP data. PCA of the SNPs showed that all the samples were collected from the Japanese population and that the samples were separated into two major clusters by birthplace, Okinawa and other than Okinawa, as had been previously reported. Among 87 SNPs that have been reported to be associated with 18 hematological and biochemical traits in genome-wide association studies (GWAS), the associations of 56 SNPs were replicated using our data base. Statistical power simulations showed that the sample size of the JPDSC control database is large enough to detect genetic markers having a relatively strong association even when the case sample size is small. The JPDSC database will be useful as control data for conducting PGx studies to explore genetic markers to improve the safety and efficacy of drugs either during clinical development or in post-marketing.

Treatment patterns of systemic drug use in Japanese patients with plaque psoriasis: A retrospective chart review.

Plaque psoriasis (PsO) is a chronic immune-mediated inflammatory disease with skin lesions accompanied by an inflammation-related comorbidity risk. The development of various oral drugs and biologics for PsO has provided increasing systemic treatment options for patients with PsO, and the guidance regarding the use of biologics and PsO treatment schemes are widespread in Japan. However, no comprehensive guidelines regarding systemic drug use are available, and the current treatment patterns of systemic drugs for PsO in Japan remain unclear. We conducted a retrospective chart review to clarify the current treatment patterns of systemic drugs for PsO. We enrolled 114 patients who started systemic drugs for PsO between January 2017 and December 2020 at four institutes, with a mean follow-up of 37.2 months. The mean disease duration was 7.8 (standard deviation 9.5) years at the systemic drug initiation. Of all the patients, 78.1% started with oral drugs (phosphodiesterase [PDE] 4 inhibitors 56.1%. calcineurin inhibitors 14.0%. vitamin A derivatives 7.9%), whereas 21.9% started with biologics (interleukin [IL]-17 inhibitors 9.6%. tumor necrosis factor inhibitors 7.0%. IL-23 inhibitors 3.5%. IL-12/23 inhibitors 1.8%). Oral drugs had shorter drug persistence than biologics: the 12-month persistence of the oral drugs vitamin A derivative, calcineurin inhibitor, and PDE4 inhibitor, was 35.5%, 25.8%, and 60.1%, respectively, compared with that of the biologics IL-23 and IL-17 inhibitors, which was 85.6% and 84.7%, respectively. During the study period, the incidence of treatment changes was 59.1/100 patient-years. Lack of efficacy was the most common reason for treatment changes from monotherapy (34.1%). This retrospective medical chart review allowed us to understand the real-world, long-term treatment patterns of systemic drugs for PsO and the relationships between the reasons for treatment changes and subsequent treatment selection, indicating that there is still room for improvement in the appropriate use of systemic drugs for PsO in Japan.

Treatment patterns, healthcare resource utilization, and costs in patients with moderate-to-severe psoriasis treated with systemic therapy in Japan: A retrospective claims database study.

The real-world treatment landscape for patients with moderate-to-severe psoriasis receiving systemic therapies in Japan is not well understood. This study describes the demographic and clinical characteristics, treatment patterns, healthcare resource utilization, and psoriasis-associated costs in these patients. This retrospective observational study used data from the Japan Medical Data Center database between January 2016 and December 2020. Eligible patients had a confirmed diagnosis of psoriasis, ≥1 claim for a systemic treatment of interest, medical history for ≥6 months, and follow-up data for ≥12 months. Systemic therapies comprised biologics (tumor necrosis factor and interleukin inhibitors) and oral treatments (a phosphodiesterase-4 inhibitor, immunosuppressants, and vitamin A). Patient demographics and clinical characteristics, treatment patterns, healthcare resource utilization, and costs were evaluated. The study identified 1770 patients satisfying all inclusion criteria. The mean age was 49.0 years, with 68% of patients aged 20-54 years. Overall, 90.6% and 9.4% of patients received oral medications and biologics as index treatment, respectively. Treatment patterns, healthcare resource utilization, and costs were assessed for treatments received by ≥20 patients (n = 1730). During the 12-month follow-up period, 1102/1730 patients (63.7%) discontinued index treatment, of whom 9.9% switched to alternative systemic treatments. The persistence rate was ≥70% for most biologics and <50% for oral systemic treatments. All 1730 patients had ≥1 all-cause outpatient visit (2.0 visits per person per month) and hospitalization frequency was ≤0.01 per person per month. Persistent patients incurred inflation-adjusted costs of Japanese Yen (JPY) 88 667 per person per month. Treatment switching was associated with an increase in total cost: JPY 128 039 per person per month after switching versus JPY 117 504 before switching. This study of Japanese patients with moderate-to-severe psoriasis demonstrated low persistence, high discontinuation, and low rates of treatment switching with systemic therapies. Switching was associated with increased total cost. These results indicate unmet needs for new treatments.

Cytokine receptor-like factor 1 is highly expressed in damaged human knee osteoarthritic cartilage and involved in osteoarthritis downstream of TGF-beta.

Osteoarthritis (OA) is the most prevalent joint disease and is characterized by pain and functional loss of the joint. However, the pathogenic mechanism of OA remains unclear, and no drug therapy for preventing its progress has been established. To identify genes related to the progress of OA, the gene expression profiles of paired intact and damaged cartilage obtained from OA patients undergoing joint substitution were compared using oligo microarrays. Using functional categorization combined with gene ontology and a statistical analysis, five genes were found to be highly expressed in damaged cartilage (HBEGF, ASUS, CRLF1, LOX, CDA), whereas three genes were highly expressed in intact tissues (CHST2, PTPRD, CPAN6). Among these genes, the upregulated expression of CRLF1 was reconfirmed using real-time PCR, and the in vivo expression of CRLF1 was detected in clusters of chondrocytes and fibrocartilage-like cells in damaged OA cartilages using in situ hybridization. In vitro, the transcriptional level of CRLF1 was positively regulated by TGF-beta1 in the mouse chondrogenic cell line ATDC5. Additionally, the CRLF1/CLC complex promoted the proliferation of ATDC5 cells and suppressed the expression level of aggrecan and type II collagen. Our data suggest that the CRLF1/CLC complex disrupts cartilage homeostasis and promotes the progress of OA by enhancing the proliferation of chondrocytes and suppressing the production of cartilage matrix. A component of the complex, CRLF1, may be useful as a biomarker of OA; and the corresponding receptor is a potential new drug target for OA.

Effects of kynurenine metabolites on mesangial cell proliferation and gene expression.

In the present study, we examined the effects of kynurenine metabolites on cultured mesangial cells (MCs) and demonstrated for the first time that they affect MC proliferation and gene expression. Anthranilic acid and 3-hydroxy-DL-kynurenine suppressed MC proliferation by 32% and 43%, respectively at 10(-6) M compared to the control. In contrast, quinolinic acid and l-kynurenine promoted MC proliferation by 49% and 35% at 10(-8) M respectively, although promoting activities declined at higher concentrations. In addition, quinolinic acid upregulated the gene expression of platelet-derived growth factor-B, collagen type-Ialpha1, and collagen type-IValpha1. However, the gene expression of hepatocyte growth factor (HGF) was downregulated. We further examined the gene expressions in the glomeruli of high serum IgA (HIGA) mice with IgA nephropathy using microarray technology and found that the gene expression of insulin-like growth factor-1 was higher, but that of HGF was lower at 40 weeks of age compared to 8 weeks of age. In Balb/c mice, the gene expression of three kynurenine pathway enzymes (kynurenine aminotransferase I, kynurenine aminotransferase II, and quinolinate phospho-ribosyltransferase) increased 2- to 3.5-fold, whereas those in HIGA mice did not change significantly. These results suggest that abnormalities in the kynurenine pathway are associated with the dysfunction of MCs and progression of chronic kidney diseases.

Social isolation rearing-induced impairment of the hippocampal neurogenesis is associated with deficits in spatial memory and emotion-related behaviors in juvenile mice.

Experiences during brain development may influence the pathogenesis of developmental disorders. Thus, social isolation (SI) rearing after weaning is a useful animal model for studying the pathological mechanisms of such psychiatric diseases. In this study, we examined the effect of SI on neurogenesis in the hippocampal dentate gyrus (DG) relating to memory and emotion-related behaviors. When newly divided cells were labeled with 5-bromo-2'-deoxyuridine (BrdU) before SI, the number of BrdU-positive cells and the rate of differentiation into neurons were significantly decreased after 4-week SI compared with those in group-housed mice. Repeated treatment of fluoxetine prevented the SI-induced impairment of survival of newly divided cells and ameliorated spatial memory impairment and part of aggression in SI mice. Furthermore, we investigated the changes in gene expression in the DG of SI mice by using DNA microarray and real-time PCR. We finally found that SI reduced the expression of development-related genes Nurr1 and Npas4. These findings suggest that communication in juvenile is important in the survival and differentiation of newly divided cells, which may be associated with memory and aggression, and raise the possibility that the reduced expression of Nurr1 and/or Npas4 may contribute to the impairment of neurogenesis and memory and aggression induced by SI.

Downregulated expression in high IgA (HIGA) mice and the renal protective role of meprinbeta.

This study discusses the critical role of the metalloproteinase meprinbeta in the progression of glomerulonephritis. Using a microarray technique, the gene expression profiles in glomeruli isolated from high serum IgA (HIGA) mice with a purity of 97% or greater were examined. HIGA mice are a valid model of human IgA nephropathy (IgAN), with the typical pathological features of this condition, including a consistently high serum IgA level as well as dominant mesangial IgA deposition and mesangial enlargement. Among the many upregulated/downregulated genes after the development of IgAN, the downregulation of meprinbeta was intriguing. The expression level of the meprinbeta gene at 40 weeks of age was 52% of that observed at 8 weeks of age (prior to the development of IgAN), although in the control BALB/c mice, a 2.19-fold elevation was seen. These results were also confirmed by semi-quantitative RT-PCR and immunostaining analyses. As meprinbeta is a subunit of metalloproteinase meprins (meprin A, meprin B) and meprins are capable of proteolytically degrading extracellular matrix (ECM) components and proteolytically processing bioactive peptides, the downregulation of meprinbeta may contribute to the progression of glomerulonephritis and the eventual glomerular scarring. This working hypothesis was examined using an in vivo meprinbeta inhibition study. The inhibition of meprins by actinonin exacerbated some parameters of renal injury in mice afflicted with anti-glomerular basement membrane (anti-GBM) antibody-associated nephritis. These in vitro and in vivo results suggest that meprinbeta may play a protective role against the progression of renal injury through the degradation of ECM and bioactive peptides.

DBTSS: DataBase of Human Transcription Start Sites, progress report 2006.

DBTSS was first constructed in 2002 based on precise, experimentally determined 5' end clones. Several major updates and additions have been made since the last report. First, the number of human clones has drastically increased, going from 190,964 to 1,359,000. Second, information about potential alternative promoters is presented because the number of 5' end clones is now sufficient to determine several promoters for one gene. Namely, we defined putative promoter groups by clustering transcription start sites (TSSs) separated by <500 bases. A total of 8308 human genes and 4276 mouse genes were found to have putative multiple promoters. Third, DBTSS provides detailed sequence comparisons of user-specified TSSs. Finally, we have added TSS information for zebrafish, malaria and schyzon (a red algae model organism). DBTSS is accessible at http://dbtss.hgc.jp.

Gene expression analysis in the lung of the rasH2 transgenic mouse at week 4 prior to induction of malignant tumor formation by urethane and N-methylolacrylamide.

The rasH2 transgenic (Tg) mice are susceptible to genotoxic and some non-genotoxic carcinogens. In carcinogenicity studies carried out using rasH2 Tg mice, the carcinogenic potential of chemicals are evaluated over a 26-week experimental period. In the present study, we examined the comprehensive gene expressions in the lungs of Tg and non-Tg mice prior to the induction of malignant tumors. Urethane (UR), a mutagenic carcinogen, was administered for 4 weeks, and thereafter withdrawn for 22 weeks. N-methylolacrylamide (NMA), a non-mutagenic carcinogen, was administered for 26 weeks. At week 4, gene expression analysis of non-neoplastic part of the lungs demonstrated changes in the expressions of the cell-cycle and inflammation related genes following UR and NMA treatment, respectively, in both the Tg and non-Tg mice. The gene expressions of epireguline, aurora kinase B, and cyclin B1 increased in the UR-treated Tg mice. We also found an increase in the plasma carcinoembryonic antigen level in the UR-treated Tg mice. Although UR treatment induced the formation of adenomas or adenocarcinomas in the lungs in all mice, earlier induction was apparent in the Tg mice. NMA treatment was found to induce the formation of adenomas and adenocarcinomas at week 26 in the Tg mice, but not in the non-Tg mice, and no expressions of specific genes were apparent in either genotype of mice. Our results indicate that analysis of cancer-related gene expressions in the lungs and plasma biomarkers at week 4 in rasH2 Tg mice could be a screening tool for carcinogenicity, especially of mutagenic carcinogens.

Transcriptional induction and translational inhibition of Arc and Cugbp2 in mice hippocampus after transient global ischemia under normothermic condition.

Mild hypothermia protects against neuronal damage after transient global ischemia in experimental animals. The exact mechanism of this protective effect remains to be elucidated. The purpose of the present study was to investigate the molecular mechanisms relevant to different neurologic responses to hypothermia and normothermia. Transient global ischemia was induced in C57BL/6 mice by bilateral common carotid artery occlusion for 10 min. Hypothermia provided robust neuroprotection in the hippocampus region and dramatically reduced the mortality rate. Using adaptor-tagged competitive polymerase chain reaction, we obtained the relative transcription levels of 1210 genes in the hippocampal region and compared the expression patterns of these genes. Two genes, Activity-regulated cytoskeleton-associated protein (Arc) and CUG-binding protein-2 (Cugbp2), showed remarkable and persistent increases in their expression levels in normothermic mice, compared with in both sham and hypothermic mice. Despite the increased transcription of Arc and Cugbp2, an immunohistochemistry analysis did not show comparable increases in the translations of both genes. Only a transient increase in Arc protein was observed in the granule cells of the dentate gyrus at 6 h after reperfusion. A remarkable decrease in Cugbp2 protein was observed in the pyramidal cells of the hippocampal CA1-CA3, in accordance with the progress of neuronal degeneration. A decrease in Cugbp2 protein was not observed in hypothermic mice. These results suggest that transient global ischemia induces the translational inhibition of genes with increased expression not in hypothermic, but in normothermic mice. Thus, translational inhibition might play an important role in the progress of neuronal injury after transient global ischemia.

Distinct class of putative "non-conserved" promoters in humans: comparative studies of alternative promoters of human and mouse genes.

Although recent studies have revealed that the majority of human genes are subject to regulation of alternative promoters, the biological relevance of this phenomenon remains unclear. We have also demonstrated that roughly half of the human RefSeq genes examined contain putative alternative promoters (PAPs). Here we report large-scale comparative studies of PAPs between human and mouse counterpart genes. Detailed sequence comparison of the 17,245 putative promoter regions (PPRs) in 5463 PAP-containing human genes revealed that PPRs in only a minor fraction of genes (807 genes) showed clear evolutionary conservation as one or more pairs. Also, we found that there were substantial qualitative differences between conserved and non-conserved PPRs, with the latter class being AT-rich PPRs of relative minor usage, enriched in repetitive elements and sometimes producing transcripts that encode small or no proteins. Systematic luciferase assays of these PPRs revealed that both classes of PPRs did have promoter activity, but that their strength ranges were significantly different. Furthermore, we demonstrate that these characteristic features of the non-conserved PPRs are shared with the PPRs of previously discovered putative non-protein coding transcripts. Taken together, our data suggest that there are two distinct classes of promoters in humans, with the latter class of promoters emerging frequently during evolution.

Diversification of transcriptional modulation: large-scale identification and characterization of putative alternative promoters of human genes.

By analyzing 1,780,295 5'-end sequences of human full-length cDNAs derived from 164 kinds of oligo-cap cDNA libraries, we identified 269,774 independent positions of transcriptional start sites (TSSs) for 14,628 human RefSeq genes. These TSSs were clustered into 30,964 clusters that were separated from each other by more than 500 bp and thus are very likely to constitute mutually distinct alternative promoters. To our surprise, at least 7674 (52%) human RefSeq genes were subject to regulation by putative alternative promoters (PAPs). On average, there were 3.1 PAPs per gene, with the composition of one CpG-island-containing promoter per 2.6 CpG-less promoters. In 17% of the PAP-containing loci, tissue-specific use of the PAPs was observed. The richest tissue sources of the tissue-specific PAPs were testis and brain. It was also intriguing that the PAP-containing promoters were enriched in the genes encoding signal transduction-related proteins and were rarer in the genes encoding extracellular proteins, possibly reflecting the varied functional requirement for and the restricted expression of those categories of genes, respectively. The patterns of the first exons were highly diverse as well. On average, there were 7.7 different splicing types of first exons per locus partly produced by the PAPs, suggesting that a wide variety of transcripts can be achieved by this mechanism. Our findings suggest that use of alternate promoters and consequent alternative use of first exons should play a pivotal role in generating the complexity required for the highly elaborated molecular systems in humans.

Molecular characterization of mammalian dicarbonyl/L-xylulose reductase and its localization in kidney.

In this report, we first cloned a cDNA for a protein that is highly expressed in mouse kidney and then isolated its counterparts in human, rat hamster, and guinea pig by polymerase chain reaction-based cloning. The cDNAs of the five species encoded polypeptides of 244 amino acids, which shared more than 85% identity with each other and showed high identity with a human sperm 34-kDa protein, P34H, as well as a murine lung-specific carbonyl reductase of the short-chain dehydrogenase/reductase superfamily. In particular, the human protein is identical to P34H, except for one amino acid substitution. The purified recombinant proteins of the five species were about 100-kDa homotetramers with NADPH-linked reductase activity for alpha-dicarbonyl compounds, catalyzed the oxidoreduction between xylitol and l-xylulose, and were inhibited competitively by n-butyric acid. Therefore, the proteins are designated as dicarbonyl/l-xylulose reductases (DCXRs). The substrate specificity and kinetic constants of DCXRs for dicarbonyl compounds and sugars are similar to those of mammalian diacetyl reductase and l-xylulose reductase, respectively, and the identity of the DCXRs with these two enzymes was demonstrated by their co-purification from hamster and guinea pig livers and by protein sequencing of the hepatic enzymes. Both DCXR and its mRNA are highly expressed in kidney and liver of human and rodent tissues, and the protein was localized primarily to the inner membranes of the proximal renal tubules in murine kidneys. The results imply that P34H and diacetyl reductase (EC ) are identical to l-xylulose reductase (EC ), which is involved in the uronate cycle of glucose metabolism, and the unique localization of the enzyme in kidney suggests that it has a role other than in general carbohydrate metabolism.