Human 25-hydroxyvitamin D 3 (25(OH)D 3 ) 24-hydroxylase (CYP24) cDNA was expressed in Escherichia coli, and its enzymatic and spectral properties were revealed. The reconstituted system containing the membrane fraction prepared from recombinant E. coli cells, adrenodoxin and adrenodoxin reductase was examined for the metabolism of 25(OH)D 3 , 1a,25(OH) 2 D 3 and their related compounds. Human CYP24 demonstrated a remarkable metabolism consisting of both C-23 and C-24 hydroxylation pathways towards both 25(OH)D 3 and 1a,25(OH) 2 D 3 , whereas rat CYP24 showed almost no C-23 hydroxylation pathway [Sakaki, T. Sawada . We also succeeded in the coexpression of CYP24, adrenodoxin and NADPHadrenodoxin reductase in E. coli. Addition of 25(OH)D 3 to the recombinant E. coli cell culture yielded most of the metabolites in both the C-23 and C-24 hydroxylation pathways. Thus, the E. coli expression system for human CYP24 appears quite useful in predicting the metabolism of vitamin D analogs used as drugs. . The complicated metabolic pathways, including . 30 metabolites [7], suggested that many enzymes were related to the metabolism. However, our recent study on rat CYP24 [8] revealed that at least six-step monooxygenation toward 1a,25(OH) 2 D 3 and four-step monooxygenation toward 25(OH)D 3 could be catalyzed by rat CYP24. Although rat CYP24 showed only C-24 hydroxylation pathway, human CYP24 was reported to catalyze 23S-hydroxylation of 25(OH)D 3 [9] which is the first step in the C-23 hydroxylation pathway. In this paper, we report the further metabolism of 25(OH)D 3 to 25(OH)D 3 -26,23-lactone in C-23 hydroxylation pathway by human CYP24. Remarkable metabolism towards 25(OH)D 3 and 1a,25(OH) 2 D 3 by human CYP24 are demonstrated.Vitamin D analogs are potentially useful in the clinical treatment of type I rickets, osteoporosis, renal osteodystrophy, psoriasis, leukemia and breast cancer [7]. The metabolism of vitamin D analogs in target tissues such as kidney, small intestine and bones is pharmacologically essential as reported by Komuro et al. [10]. The major metabolic enzyme of the vitamin D analogs in these tissues is considered to be CYP24 [10,11]. Species differences observed in the metabolism of these vitamin D 3 analogs appear to originate from the specificity of CYP24-dependent reactions. Because human kidney specimens are not obtained easily, an in vitro system containing human CYP24 is required to predict drug metabolism in the human kidney. Here, we show the overexpression of human CYP24 in Escherichia coli. The expression level of CYP24 appears to be much higher than that in Sf21 cells using a baculovirus system as reported by Beckman et al. [9]. As Eur. J. Biochem. 267, 6158±6165 (2000) [25][26]24,25,26, ; tetranor 1a,23(OH) 2 , 24,25,26,27-tetranor-1a,23-dihydroxyvitamin D 3 ; tetranor 23(OH), 24,25,26,27-tetranor-23-hydroxyvitamin D 3 . Enzyme: bovine NADPH-adrenodoxin reductase (EC 1.18.1.2).

The mammalian clock genes, Period and Cryptochrome (Cry), regulate circadian rhythm. We show that circadian rhythmicity and rhythmic expression of Period in the nuclei of inflammatory synovial cells and spleen cells are disturbed in mouse models of experimental arthritis. Expressions of other clock genes, Bmal1 and Dbp, are also disturbed in spleen cells by arthritis induction. Deletion of Cry1 and Cry2 results in an increase in the number of activated CD3+ CD69+ T cells and a higher production of TNF-α from spleen cells. When arthritis is induced, Cry1−/−Cry2−/− mice develop maximal exacerbation of joint swelling, and upregulation of essential mediators of arthritis, including TNF-α, IL-1β and IL-6, and matrix metalloproteinase-3. Wee-1 kinase is solely upregulated in Cry1−/−Cry2−/− mice, in line with upregulation of c-Fos and Wee-1 kinase in human rheumatoid arthritis. The treatment with anti–TNF-α Ab significantly reduced the severity and halted the progression of the arthritis of Cry1−/−Cry2−/− mice and vice versa, ectopic expression of Cry1 in the mouse embryonic fibroblast from Cry1−/−Cry2−/− mice significantly reduced the trans activation of TNF-α gene. Thus, the biological clock and arthritis influence each other, and this interplay can influence human health and disease.

In patients with PD without autonomic failure, only cardiac MIBG uptake was severely reduced in the earliest phase of the disease (stage I). Parkinsonian syndromes other than PD did not demonstrate significant reduction in MIBG uptake in any organs except for the lower legs in MSA. In patients with PD without autonomic failure, reduction in MIBG uptake occurs selectively in the heart; this is considered to be a specific finding for PD and useful for the differential diagnosis of the parkinsonian syndromes.

A major goal of current human genome-wide studies is to identify the genetic basis of complex disorders. However, the availability of an unbiased, reliable, cost efficient and comprehensive methodology to analyze the entire genome for complex disease association is still largely lacking or problematic. Therefore, we have developed a practical and efficient strategy for whole genome association studies of complex diseases by charting the human genome at 100 kb intervals using a collection of 27,039 microsatellites and the DNA pooling method in three successive genomic screens of independent case-control populations. The final step in our methodology consists of fine mapping of the candidate susceptible DNA regions by single nucleotide polymorphisms (SNPs) analysis. This approach was validated upon application to rheumatoid arthritis, a destructive joint disease affecting up to 1% of the population. A total of 47 candidate regions were identified. The top seven loci, withstanding the most stringent statistical tests, were dissected down to individual genes and/or SNPs on four chromosomes, including the previously known 6p21.3-encoded Major Histocompatibility Complex gene, HLA-DRB1. Hence, microsatellite-based genome-wide association analysis complemented by end stage SNP typing provides a new tool for genetic dissection of multifactorial pathologies including common diseases.

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