Objective. To determine the effects of sulfasalazine (SASP) and its metabolites sulfapyridine (SP) and 5-amino salicylic acid (5ASA) on steady state mRNA levels of inflammatory cytokines [interleukin 1 beta (IL-1 beta), tumor necrosis factor alpha (TNF-alpha)], matrix metalloproteinases [collagenase (MMP1), stromelysin (MMP3), gelatinase 72 kDa (MMP2)], tissue inhibitors of metalloproteinase (TIMP 1 and TIMP 2), and the TNF-alpha receptor in rheumatoid synovial fibroblasts. Methods, Cells were dosed with each compound for 24 h in the presence or absence of PMA inducer and messenger RNA (mRNA) extracted and subjected to Northern blot analysis. Messenger RNA levels were quantitated by densitometry and normalized to GAPDH or 18S rRNA. Results. We observed some modest effects of sulfasalazine and its metabolites on steady state mRNA levels including: (1) repressed mRNA levels for TNF-alpha [approximate to 40% with 3x (drug median serum concentration) all 3 drugs], stromelysin (approximate to 24% with 3x all 3 drugs and approximate to 31% with 3x 5ASA), and collagenase (approximate to 27% with 3x 5ASA); (2) elevated mRNA levels for TIMP 2 (3.5 kb transcript) (51% with 3x SP and 44% with 3x 5ASA), gelatinase (approximate to 20% with 3x SP and 3x 5ASA), stromelysin (approximate to 40% with 3x and 1x SASP), IL-1 beta (approximate to 31% with 0.1x 5ASA); and (3) no effect on mRNA levels for TNF-alpha receptor and TIMP 1. Conclusion. (1) SASP and its metabolites showed varied effects on steady state mRNA concentrations for gene transcripts that fell into 3 categories: (a) repressed, (b) elevated, (c) no effect on mRNA levels. (2) No apparent linear dose response effect was observed for SASP or its metabolites, although a generalized suppression of mRNA levels at all doses was seen in some cases. (3) No predominant suppressive effect (greater than or equal to 50%) of mRNA levels by any of the drugs was observed for any of the genes studied; however, TIMP 2 mRNA levels increased 51% with 3x SP and 44% with 3x 5ASA.

A chimeric Adenovirus-Simian Virus 40 (AdSV40) containing the large T antigen was used to transform rheumatoid synovial fibroblasts. A rheumatoid synovial fibroblast cell line was established by infection of primary rheumatoid arthritis (RA) synovial fibroblasts at Passage 10 with AdSV40 recombinants followed by selection in semisoft agarose cultures. The transformed cells grew anchor independent, exhibited continuous proliferation (>65 passages) in monolayer culture, and formed multiple visible foci.

The chimpanzee (Pan troglodytes) alpha-fetoprotein (AFP)-encoding gene (AFP) spans 18 867 bp from the transcription start point to the polyadenylation site, and the nucleotide (nt) sequence reveals that the gene is composed of 15 exons, which are symmetrically placed within three domains of AFP. In addition, we report 3121 bp of 5'-flanking sequence and 4886 bp of 3'-flanking sequence. The entire 26 874 bp of contiguous DNA reported here was determined from three overlapping lambda phage clones. The deduced polypeptide chain is composed of a 19-amino-acid (aa) putative leader peptide, followed by 590 aa of the mature protein. The sequence of chimpanzee AFP was compared with those of the previously published human AFP [Gibbs et al., Biochemistry 26 (1987) 1332-1343] and gorilla AFP [Ryan et al., Genomics 9 (1991) 60-72]. At the aa level, the human AFP differs from the chimpanzee at 6 aa positions and from the gorilla at 4 aa positions; the chimpanzee and gorilla differ at 8 aa positions. There are four types of repetitive sequence elements (X, Alu, Xba and Kpn) in the introns and flanking regions of chimpanzee AFP, and they are located in orthologous positions in the human and gorilla AFP. However, one specific Alu and one Xba repeat in introns 4 and 7, respectively, found in human AFP, are absent from orthologous positions in chimpanzee and gorilla AFP. These two repeats represent human-specific novelties that arose from recent DNA transpositions in primate phylogeny.

Neutrophils contain on their surface a receptor for the Fc portion of IgA. Cross-linking of this receptor in the fluid phase induces superoxide production and release of granule constituents, but the response to surface associated IgA has not been previously studied. Neutrophils incubated with surface-associated IgA (SAIgA) release significant amounts of activated collagenase in addition to the granule proteins myeloperoxidase and lactoferrin. This activation is associated with release of superoxide as well as hydrogen peroxide and hypochlorous acid. Although neutrophils incubated with soluble aggregates of IgA also release granule proteins and produce superoxide, soluble aggregates of IgA do not trigger the release of activated collagenase and do not generate hydrogen peroxide or hypochlorous acid. In summary, neutrophils activated by surface associated IgA respond differently than when cells are activated by soluble aggregates of IgA. These differences may be important in understanding the mechanisms of tissue injury in patients with inflammatory disorders.

We have identified four genetic novelties that are fixed in specific primate lineages and hence can serve as phylogenetic time markers. One Alu DNA repeat is present in the human lineage but is absent from the great apes. Another Alu DNA repeat is present in the gorilla lineage but is absent from the human, chimpanzee, and orangutan. A progenitor Xba1 element is present in the human, chimpanzee, gorilla, and orangutan, but only in the human lineage did it give rise to a transposed progeny, Xba2. The saltatory appearance of Xba2 is an example of a one-time event in the evolutionary history of a species. The enolase pseudogene, known to be present as a single copy in the human, was found to be present in four other primates, including the baboon, an Old World monkey. Using the accepted value of 5 x 10(-9) nucleotide substitutions per site per year as the evolutionary rate for pseudogenes, we calculated that the enolase pseudogene arose approximately 14 million years ago. The calculated age for this pseudogene and its presence in the baboon are incongruent with each other, since Old World monkeys are considered to have diverged from the hominid lineage some 30 million years ago. Thus the rate of evolution in the enolase pseudogene is only about 2.5 x 10(-9) substitutions per site per year, or half the rate in other pseudogenes. It is concluded that rates of substitution vary between species, even for similar DNA elements such as pseudogenes. We submit that new DNA repeats arise in the genomes of species in irreversible and punctuated events and hence can be used as molecular time markers to decipher phylogenies.

Primary cell cultures derived from 14-day-old embryonic quail intestine were transformed with Rous sarcoma virus. An intestinal epithelial cell line was established and grown for 4 mo, and survived 8 passages. Mock infected control cells died after 3 wk of culture. Our data represent the first reported successful transformation of avian intestinal epithelial cells.

Expression of the vitamin D induced calbindin-D28K Protein is transcriptionally controlled by the steroid hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) in a tissue-specific manner in the intestine and kidney. In order to examine the cis-acting elements of the calbindin-D28K promoter and its modulation by 1,25-dihydroxyvitamin D3, chimeric plasmids containing 2.1 kb of 5' flanking region linked to the reporter gene chloramphenicol acetyl transferase (CAT) were transfected by lipofection into primary cultures of chick kidney cells. Transfected chick kidney cells exhibited a high basal expression of the chloramphenicol acetyl transferase gene, reflecting the strong activity of the calbindin-D28K promoter. Expression of the pCaBP2.1 reporter gene was increased 2-fold in the presence of the hormone 1,25(OH)2D3 in the primary kidney cells. Deletion of a 1.42 kb fragment ending -679 base pairs upstream from the transcription start site led to a 2-fold repression in the reporter gene activity by the hormone 1,25(OH)2D3 in primary chick kidney cultures. These preliminary results suggest that both positive and negative elements normally act to regulate the expression of the calbindin-D28K gene in primary chick kidney cells.

1,25-Dihydroxyvitamin D3, the hormonally active form of vitamin D (1,25(OH)2D3), plays a major role in the transcriptional regulation of the vitamin D-induced calcium binding protein calbindin-D28k in the chick intestine. Sequence-specific protein-DNA interactions within the promoter of the calbindin-D28k gene were studied by DNAse I footprinting analysis to obtain information on the mechanism by which the 1,25(OH)2D3 receptor and other transcription factors regulate its expression. Restriction fragments spanning nucleotides -679 to +44 of the calbindin-D28k gene were used as probes Intestinal nuclear extracts prepared from vitamin D-deficient chicks generated several protected regions. Two prominent areas of protection against DNase I digestion were located at nucleotides -595 to -572 (21 bp) and -372 to -337 (36 bp). The -372 to -337 protected segment includes a CACCC sequence motif. Additional protection regions (-333/-328, -319/-315 and -308/-304) were observed within and near the candidate chicken calbindin-D28k 1,25(OH)2D3-response element (-329/-313) and the CCAAT box (-326/-322). DNase I digestion patterns obtained with liver nuclear extracts, containing low levels of 1,25(OH)2D3 receptor, revealed weaker protein-DNA interactions in these regions.

The receptor for the steroid hormone 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] belongs to a superfamily of steroid and triidothyronine (T3) receptors. As yet no 1,25(OH)2D3 response element (DRE) has been identified. Since the T3 and 1,25(OH)2D3 receptors are structurally homologous, we have used the nucleotide sequence of the T3 response element to carry out a computer search of the promoter region of several 1,25(OH)2D3 regulated genes. We report here one candidate DRE from the chick calbindin-D28K gene (AGCCCAATGGCTGAACA) and two candidate DRE's from the rat osteocalcin gene (TCCCCACTGGATGAGCG and CCTGCACTGGGTGAATGA).

The seco-steroid hormone 1,25-dihydroxyvitamin D3 is known to induce the expression of a calcium binding protein termed calbindin-D28K in a variety of target tissues. In order to comprehend the mechanism of induction we have cloned and sequenced the chicken calbindin-D28K gene. The gene spans some 18.5 kilobases (kb) of chromosomal DNA from the putative Cap site to the polyadenylation site of the 2.8 kb mRNA. It is split into 11 coding exons by 10 intervening sequences. The promoter region of this gene is markedly G + C-rich (60-80%) extending from -225 to +400. Within this region we find 70 CpG dinucleotides, four G-C boxes, and numerous known promoter regulatory signals. These putative regulatory signals include a TATA box (ATAAATA) at -30 and a CAT box (CCAAT) at -326. Ten additional variant CAT boxes are found in the upstream promoter region (-218 to -770) of this gene. Furthermore we have identified a glucocorticoid-like responsive element at -410 (TCTACACACTGTTCC) and this element overlaps a metal responsive element (TGCACTC) and a variant CAT box (CCAAAT) and juxtaposes an enhancer-like core element (AAATGGT) on its 3'-side. In addition, the calbindin-D28K promoter is composed of a variety of simple repeated sequences, some of which are components of putative regulatory signals. All splice junctions were found to conform to the GT-AG rule. A consensus sequence of the 5'-splice junction reads AG/GTAAG-TTATA. A consensus sequence of the 3'-splice site consists of two elements: a pyrimidine track (mainly T) followed by ACAG/G-T. A two-dimensional model of calbindin-D28K was constructed which projects the existence of 6 alpha-helix-loop-alpha-helix regions characteristic of calcium binding domains. The 3'-end of the gene consists of a single large (2039 base pair) uninterrupted exon, an organizational feature common to other members of the calcium binding protein gene family which include calmodulin, parvalbumin, Spec I, myosin light chains, etc. Another feature common to the gene family is the presence of the repeated sequence ATTT or TTTA located in the 3'-untranslated exons. These simple repeat sequences could be involved in regulating mRNA degradation by serving as a ribonuclease recognition signal.

Our understanding of how vitamin D mediates biological responses has entered a new era. It is now clear that the bulk of the biological responses supported by vitamin D occur as a consequence of its metabolism to its daughter metabolite 1 alpha,25-dihydroxyvitamin D3 (a steroid hormone). The fact that 1,25(OH)2D3 receptors are ubiquitous in tissue distribution opens the possibility for unforeseen biological functions of the vitamin D endocrine system. For example, 1,25(OH)2D3 serves as an immunoregulatory hormone and a differentiation hormone besides its classical role in mineral homeostasis. The avian 1,25)OH)2D3 receptor has recently been cloned and shown to be a member of the nuclear transacting receptor family that includes estrogen, progesterone, glucocorticoid, thyroxine (T3), aldosterone, and retinoic acid receptors. We have compiled an extensive number of RNA polymerase II-transcribed genes that are regulated by 1,25(OH)2D3. Classification of these genes on functional grounds identifies and formulates the several genetic circuits or biochemical systems in which 1,25(OH)2D3 plays an essential regulatory role. These systems include genes that govern oncogene and lymphokine expression as well as those involved in mineral homeostasis, vitamin D metabolism, and regulation of a set of replication-linked genes (c-myc, c-myb, and histone H4), which are critical for rapid cellular proliferation. An integrated analysis of the combinations of genetic circuits regulated by 1,25(OH)2D3 suggests that they may be collectively tied to a DNA replication-differentiation switch.

Publisher Summary This chapter elaborates why vitamin D is essential for the maintenance of calcium homeostasis, its primary function being to maintain normal serum calcium and phosphate levels through a direct stimulation of intestinal transport and mobilization of mineral from bone. The genomic action of 1,25(OH)2D3-receptor complex involves binding to specific DNA sequences of calbindin-D28K gene, but this remains to be experimentally determined. Vitamin D plays a physiological role in pancreatic function and particularly in insulin secretion. In the model for calcium transport in intestinal epithelial ceils, calcium is internalized at the brush border membrane in endocytic vesicles that subsequently fuse with lysosomes. Calcium-bearing lysosomes are then transported to the basal lateral membrane along microtubules, where exocytosis of the vesicular contents completes the transport process. 1,25(OH)2D3 from Bone Marrow Macrophages (BMM) and Pulmonary Alveolar Macrophages (PAM) may be effective locally in a paracrine sense because both bone marrow cells and activated lymphocytes have been recognized as target cells for 1,25(OH)2D3. The 1,25(OH)2D3 produced in the bone marrow may influence myeloid stem cell differentiation toward mature macrophages.

The human albumin-alpha-fetoprotein genomic domain contains 13 repetitive DNA elements randomly distributed throughout the symmetrical structures of these genes. These repeated sequences are located at different sites within the two genes. The human albumin gene contains five Alu elements within four of its 14 intervening sequences. Two of these repeats are located in intron 2, and the remaining three are located in introns 7, 8, and 11. The human alpha-fetoprotein gene contains three of these Alu elements, one in intron 4 and the remaining two in the 3'-untranslated region. In addition, the human alpha-fetoprotein gene contains a Kpn repeat and two classes of novel repeats that are absent from the human albumin gene. Six of the Alu elements within the two genes are bound by short direct repeats that harbor five base substitutions in 120 possible positions (60 bp times 2 termini). The absence of Alu repeats from analogous positions in rodents indicates that these repeats invaded the albumin-alpha-fetoprotein domain less than 85 Myr ago (the time of mammalian radiation). Furthermore, considering the conservation of terminal repeats flanking the Alu sequences of the albumin-alpha-fetoprotein domain (0.042 changes per site), we submit that the average time of Alu insertion into this gene family could have been as recently as 15-30 Myr ago.

The human albumin gene spans 16,961 nucleotides from the putative "Cap" site to the first poly(A) addition site. It is split into 15 exons by 14 intervening sequences which are symmetrically placed within the three domains of albumin. The 5' region is highly conserved up to position -250 and contains the putative TATA (-32) and CAT (-88) boxes. A consensus 5' splice sequence reads /GTAGAGT while the 3' splice sequence is pyrimidine rich and contains CTAG/ at the splice junction. The gene contains three polyadenylation signals, and this 3' region presumably arose by triplication of a shorter fragment prior to mammalian radiation. The albumin gene exhibits a high degree of DNA polymorphism and appears to have been recently invaded by Alu repetitive sequences.

We conducted the present study in an attempt to correlate function with the rate of molecular evolution for serum albumin and alpha-fetoprotein. We found a high rate of silent substitution (between 5 X 10(-9) and 7 X 10(-9)/site/year) for both the albumin and alpha-fetoprotein genes, perhaps the highest so far reported for an expressed nuclear gene. The rates of effective substitution and amino acid changes were also very high, but in contrast to silent substitutions, they are higher for alpha-fetoprotein than for albumin by approximately 70%. For alpha-fetoprotein, the rate of effective substitution (1.5 X 10(-9)/site/year) may be approaching that for nonfunctional pseudogenes (about 3 X 10(-9)/site/year). Evolutionary divergence was also estimated at the amino acid level. It was found that the rate of change of alpha-fetoprotein (55% amino acids replaced in 100 Myr) approaches that of the fastest-evolving fibrinopeptides (92% amino acids replaced in 100 Myr). This high rate may indicate that alpha-fetoprotein can tolerate a great deal of molecular variation without its function being impaired in the process. Albumin evolves at a slower rate (39% amino acids replaced in 100 Myr), although still faster than either hemoglobin (17% amino acids replaced in 100 Myr) or cytochrome c (5% amino acids replaced in 100 Myr). The slower evolutionary rate may indicate that albumin has more refined functional specifications and hence can tolerate fewer mutational changes. The latter conclusion remains, however, to be reconciled with the condition of inherited analbuminemia, where a virtually complete absence of albumin produces surprisingly few symptoms.