The epidemic nature of diabetes mellitus in different regions is reviewed. The Middle East and North Africa region has the highest prevalence of diabetes in adults (10.9%) whereas, the Western Pacific region has the highest number of adults diagnosed with diabetes and has countries with the highest prevalence of diabetes (37.5%). Different classes of diabetes mellitus, type 1, type 2, gestational diabetes and other types of diabetes mellitus are compared in terms of diagnostic criteria, etiology and genetics. The molecular genetics of diabetes received extensive attention in recent years by many prominent investigators and research groups in the biomedical field. A large array of mutations and single nucleotide polymorphisms in genes that play a role in the various steps and pathways involved in glucose metabolism and the development, control and function of pancreatic cells at various levels are reviewed. The major advances in the molecular understanding of diabetes in relation to the different types of diabetes in comparison to the previous understanding in this field are briefly reviewed here. Despite the accumulation of extensive data at the molecular and cellular levels, the mechanism of diabetes development and complications are still not fully understood. Definitely, more extensive research is needed in this field that will eventually reflect on the ultimate objective to improve diagnoses, therapy and minimize the chance of chronic complications development.
The interaction between the two vitamin D response elements (DRE) located at -154 to -134 base pairs (bp) and -262 to -238 bp from the transcription initiation site has been studied using reporter gene assays and binding assays by electrophoretic gel shift measurements. 3 half-sites separated by 3 bp were found necessary for transactivation by the -154 to -125 DRE, while 2 half-sites separated by 3 bp were needed for the DRE at -262 to -238 to function. However, the two DREs together provided maximal activity. The 93-bp fragment separating the two DREs was not required and could be deleted. The most effective binding by receptor was found with the two complete DREs (dissociation constant (Kd) = 13.7 pM), although each DRE bound to the receptor and nuclear accessory factor with about 5 nM Kd. The two DREs (a total of 5 half-sites) apparently account for most if not all of the transactivation of the rat 24-hydroxylase by 1,25-dihydroxyvitamin D3. This system represents the most powerful of the DREs reported to date.
The rat calbindin D-9k gene is transcriptionally regulated by 1,25-dihydroxyvitamin D3 in the intestine. We have examined the 5'-flanking region of this gene and identified a 1,25-dihydroxyvitamin D3-responsive element (DRE) between nucleotides -489 and -445. This element confers 1,25-dihydroxyvitamin D3 responsiveness through its native promoter and the heterologous thymidine kinase promoter, and it contains the sequence GGGTGTCGGAAGCCC, which is homologous to the other previously identified DREs. Incubation of this element with the 1,25-dihydroxyvitamin D3 receptor produced a specific protein-DNA complex, which shifted to a higher molecular weight form upon the addition of a monoclonal antibody specific to the 1,25-dihydroxyvitamin D3 receptor. Therefore, the 5'-flanking region of the rat calbindin D-9k gene contains a DRE that mediates the enhanced expression of this gene by 1,25-dihydroxyvitamin D3 in the intestine.The calbindin D-9k calcium-binding protein is a member of a large family of intracellular calcium-binding proteins that act to control the transport, availability, and storage of calcium (1, 2). This protein has been detected in various tissues, including the intestine (3-5), placenta (6), uterus (7,8), yolk sac (9, 10), and fallopian tube of the rat (11). It has also been detected in small quantities in the kidney (12) and the growth cartilage (13). The primary sequences of the mouse (14), rat (15, 16), bovine (17), and porcine (18) proteins have been determined. The rat calbindin D-9k cDNA has been isolated and characterized (19). The rat gene for calbindin D-9k has also been isolated and sequenced (20,21). It is a relatively small gene, spanning about three kilobases (kb) of genomic DNA, and consists of three exons flanked by two introns (20,21).In the intestine, vitamin D appears to be the primary regulator for the calbindin D-9k gene at the transcriptional level (22-25). However, no regulation by vitamin D is exerted on the gene in the uterus, where it seems to be primarily under estrogen regulation (7,26). An active estrogen-responsive element (ERE) has been detected in the first intron of the gene at the border with the first exon (27), which is presumed to mediate the responsiveness of the gene to estrogen in the uterus. However, no vitamin D-responsive element has yet been identified in or around the calbindin D-9k gene. A number of vitamin D-responsive elements (DREs) have been identified and characterized in the 5'-flanking region of various other vitamin D-dependent genes, including the rat osteocalcin gene (28), the human osteocalcin gene (29,30), and the mouse osteopontin gene (31). All of these DREs were shown to bind the vitamin D receptor and are able to mediate vitamin D-dependent transcriptional activation.Since the isolation ofthis gene, we have tried to localize and identify a potential DRE with limited success. We describe here the identification and characterization of an active DRE in the 5' upstream region of the calbindin D-9k gene.t This element, which is homolog...
The caiddll (25-hydroxyvitamin D3) 24-hydroxylase is one of the key enzymes in the metabolism of vitm D. This enzyme acts on both calcidlol and calcitriol (1,25-dhydroxyvitamin D3) Cell Transfection and Chloramphenicol Acetltranferase (CAT) Assay. NRK rat kidney cells (American Type Culture Collection) were cultured to =50%o confluency 24 hr before transfection. The cells were transfected with 2-10 pg of test plasmid by using the standard lipofectin method (GIBCO/ BRL). The DNA/lipofectin solution was left on the cells for 24 hr after which the medium was changed to DMEM (GIBCO/BRL)/10%o fetal calfserum (HyClone), and the cells were dosed with 40 nM of 1,25-(OH)%D3 or vehicle and incubated for an additional 24-36 hr at 370C. The cells were harvested by scraping them into 0.25 M Tris'HCI, pH 8, and lysed by three cycles of freezing and thawing; the CAT enzyme was assayed as described (19) by using 100 mg of lysate protein, as measured by the Bradford protein assay (20). The acetylated chloramphenicol was quantitated by using the Betascope model 603 blot analyzer (Betagen, Waltham, MA). Fig. 1 shows the sequence of the 5' upstream region of the 24-hydroxylase gene from -1749 to +76. Bases +76 to -516 coincide with the sequence published by Ohyama et al. (6). The position ofthe identified DRE is marked by asterisks and lies between nt-262 and nt-238 from the transcription start site, and sequences of possible cAMP-response elements lie between nt-368 and nt-126. The position of the DRE is closer to the transcription start site than previously identified positive DREs (8)(9)(10)(11)13). RESULTSBecause all known DREs lie within the first 500 bases upstream of the transcription start site, the search for the DRE in the rat 24-hydroxylase gene was performed by a Abbreviations: 1,25-(OH)2D3, calcitriol (la,25-dihydroxyvitamin D3); VDR, vitamin D receptor; DRE, vitamin D response element; CAT, chloramphenicol acetyltransferase.
Uptake and efflux of 64Cu were examined to determine whether hepatic parenchymal cells exhibit the kinetic criteria of a specific transport system for copper and related trace metals. Saturation kinetics were clearly indicated by both v versus [Cu] and 1/v versus 1/[Cu] plots (Km = 11 +/- 0.6 microM and Vmax = 2.7 nmol Cu X min-1 X mg prot-1). Identical results were obtained by cold-copper analyses, and contributions from simple diffusion or nonspecific binding were not detected. Virtually all of the accumulated 64Cu was intracellular by 0.5 min (the initial velocity period), with approximately 40% in the cytosolic fraction. Several related trace metals inhibited 64Cu uptake, but Ni(II) at a 10:1 molar excess did not. Zn(II) acted as a simple competitive inhibitor of 64Cu uptake (Ki = 16 microM). Efflux from preloaded cells was biphasic, with an initial rapid phase of approximately 5 min. Approximately 35% of preloaded 64Cu was transported out of the cells by 40 min, and little efflux occurred thereafter. Thus, hepatocytes exhibit saturation kinetics, competition by related substrates, and countertransport criteria of specific facilitated transport. A wide variety of metabolic inhibitors have no effect on 64Cu uptake under the same conditions that inhibit the active transport of bile acids. Specific inhibitor tests for electrogenic coupling were also negative. Because the identical kinetic parameters were obtained for free 64Cu and the 1:1 64Cu-histidine complex, it is inferred that copper is probably transported as the free ion. Cells incubated with greater than or equal to 10 microM 64Cu showed a net loss of copper after 40- to 60-min incubation, which may involve specific hepatic mechanisms in copper homeostasis.
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