No abstract

Signal transducers and activators of transcription (STAT proteins) bind to palindromic sequence elements related to interferon y (IFN-y) activation sites, which were first identified in the promoters of IFN-'yinducible genes. Although the sequences of the natural palindromic STAT-binding elements vary considerably, they conform to the general structure TT(N)5AA. We have systematically examined the effects of the spacing between the TT and AA core half sites on the binding of the STAT complexes activated by IFN-y, interleukin (IL) 6, granulocytemacrophage colony-stimulating factor, and IL-4. We show that (i) as suggested earlier, a core palindromic TT-AA motifwith a 5-bp spacing displays general STAT binding, (ii) a palindromic motif with a spacing of 4 bp selectively binds to complexes containing Stat3, and (iii) a motif with a 6-bp spacing selectively binds the STAT complexes activated by IL-4. We have examined natural elements in the promoters of cytokine-responsive genes that differ in half-site spacing and found that they display binding properties predicted from the synthetic binding sites. Furthermore, the observed differential selective binding characteristics for the most part correlate with the ability to mediate transcriptional activation of transfected test genes in response to the cytokines tested. Our results thus demonstrate that the specificity of STAT-directed transcription in response to particular cytokines or cytokine families depends in part on the spacing of half sites within the conserved response element sequence.

We have used a series of human estrogen receptor (ER) mutants to evaluate the cell- and promoter-specific transcriptional activities of the TAF1 and TAF2 transactivation regions within the human ER. We show that the manifestation of TAF1 or TAF2 function depends strongly upon promoter context; on certain promoters, both the TAF1 and TAF2 activators are required for wild-type transcriptional activity, whereas on other promoters, the TAF1 and TAF2 activators function independently. Using these constructs, we show that the antagonist activity of the triphenylethylene-derived antiestrogens, e.g. tamoxifen, arises from their intrinsic inability to activate ER TAF2 function. However, on certain promoters, these antiestrogens efficiently activate gene transcription through ER. Consistent with this observation, the TAF2 function of the ER is not required on all promoters. In these TAF2-independent promoter contexts, TAF2 function may be provided by a separate transcription factor bound to the promoter. These data suggest that 1) TAF1 may be the major transcriptional activator of the ER; and 2) TAF2 functions as a transcriptional facilitator. On promoters where TAF2 function is provided independently of the ER, the TAF1 function of the ER can function independently of TAF2 activity, allowing triphenylethylene-derived antiestrogens to demonstrate partial agonist activity. These observations provide a possible molecular explanation for the tissue-specific partial agonist properties of tamoxifen and related triphenylethylene antiestrogens observed in vivo.

CSA successfully allows most severe steroid resistant UC patients to retain their colons, and provides time for "elective" colectomy in others, especially if 6MP/aza are also given. Careful monitoring for side effects, including PCP prophylaxis, should be part of the treatment protocol.

No abstract

In an attempt to define the molecular basis of the functional diversity of K+ channels, we have isolated overlapping rat brain cDNAs that encode a neuronal delayed rectifier K+ channel, K,4, that is structurally related to the Drosophila Shaw protein. Unlike previously characterized mammalian K+ channel genes, which each contain a single protein-coding exon, K,4 arises from alternative exon usage at a locus that also encodes another mammalian Shaw homolog, NGK2. Thus, the enormous diversity of K+ channels in mammals can be generated not just through gene duplication and divergence but also through alternative splicing of RNA.The electrical excitability of cells of the nervous system is regulated, in part, by voltage-sensitive K+ channels (1). The structure of neuronal K+ channels that have been cloned has been conserved from Drosophila to humans and consists of proteins containing six hydrophobic, putative transmembrane domains, one of which, S4, has been implicated in sensing changes in the membrane potential (2-4). Although they are structurally similar, these K+ channels can be divided into four distinct classes by virtue of their sequence homology to channels encoded at the Drosophila Shaker, Shab, Shal, and Shaw loci (5-11). In Drosophila, diversity of the K+ channels within a given class arises, in part, from differential splicing of RNA encoding the channels (5-11). In contrast, mammalian K+ channel genes that have been described to date have no introns in the coding region (12)(13)(14) and the diversity of these channels appears to have resulted from gene duplication and divergence. We describe here, however, the cloning and expression of a cDNA encoding a mammalian Shaw-type K+ channel, Kv4, which arises from alternative splicing of a transcript from a gene that also encodes NGK2 (15), another mammalian channel related to Shaw. 11 MATERIALS AND METHODS K,4 cDNA Clones. An oligo(dT)-primed cDNA library, constructed from mRNA isolated from the brains of 2-weekold rats, was screened using a probe prepared from clone K41 (12), encoding part of a Shaker-type channel, labeled to a specific activity of >1 x 109 by random hexamer-primed labeling (17). The filters were hybridized at 60'C in 5 x SSPE (lx SSPE = 0.18 M NaCl/10 mM sodium phosphate, pH 7.4/1 mM EDTA), 5x Denhardt's solution, 0.5% SDS, and 1% glycerol with 200 ,ug of salmon sperm DNA per ml and probe at 1 x 106 cpm/ml and were washed to a final stringency of 0.2x SSPE/0.1% SDS at 220C. A cDNA (SA) containing a 2.6-kilobase (kb) insert was isolated and subcloned into pBS+ for sequencing. To obtain the 3' end of the coding region, another oligo(dT)-primed cDNA library, constructed from mRNA isolated from the brains of 14-day-old rats, was screened using an oligonucleotide probe (0123) that included bases 1260-1359 from 5A (Fig. 1). The probe was labeled to a specific activity of >1 x 108 cpm/pmol by filling in two overlapping oligonucleotides with the Klenow fragment of DNA polymerase I and all four [a-32P]dNTPs. Filters were hybridized at 4...

Despite limited understanding of therapeutic aetiopathogenesis of ulcerative colitis and Crohn's disease, there is a strong evidence base for the efficacy of pharmacological and biological therapies. It is equally important to recognise toxicity of the medical armamentarium for inflammatory bowel disease (IBD). Sulfasalazine consists of sulfapyridine linked to 5-aminosalicylic acid (5-ASA) via an azo bond. Common adverse effects related to sulfapyridine 'intolerance' include headache, nausea, anorexia, and malaise. Other allergic or toxic adverse effects include fever, rash, haemolytic anaemia, hepatitis, pancreatitis, paradoxical worsening of colitis, and reversible sperm abnormalities. The newer 5-ASA agents were developed to deliver the active ingredient of sulfasalazine while minimising adverse effects. Adverse effects are infrequent but may include nausea, dyspepsia and headache. Olsalazine may cause a secretory diarrhoea. Uncommon hypersensitivity reactions, including worsening of colitis, pancreatitis, pericarditis and nephritis, have also been reported. Corticosteroids are commonly prescribed for treatment of moderate to severe IBD. Despite short term efficacy, corticosteroids have numerous adverse effects that preclude their long term use. Adverse effects include acne, fluid retention, fat redistribution, hypertension, hyperglycaemia, psycho-neurological disturbances, cataracts, adrenal suppression, growth failure in children, and osteonecrosis. Newer corticosteroid preparations offer potential for targeted therapy and less corticosteroid-related adverse effects. Azathioprine and mercaptopurine are associated with pancreatitis in 3 to 15% of patients that resolves upon drug cessation. Bone marrow suppression is dose related and may be delayed. The adverse effects of methotrexate include nausea, leucopenia and, rarely, hypersensitivity pneumonia or hepatic fibrosis. Common adverse effects of cyclosporin include nephrotoxicity, hypertension, headache, gingival hyperplasia, hyperkalaemia, paresthesias, and tremors. These adverse effects usually abate with dose reduction or cessation of therapy. Seizures and opportunistic infections have also been reported. Antibacterials are commonly employed as primary therapy for Crohn's disease. Common adverse effects of metronidazole include nausea and a metallic taste. Peripheral neuropathy can occur with prolonged administration. Ciprofloxacin and other antibacterials may be beneficial in those intolerant to metronidazole. Newer immunosuppressive agents previously reserved for transplant recipients are under investigation for IBD. Tacrolimus has an adverse effect profile similar to cyclosporin, and may cause renal insufficiency. Mycophenolate mofetil, a purine synthesis inhibitor, has primarily gastrointestinal adverse effects. Biological agents targeting specific sites in the immunoinflammatory cascade are now available to treat IBD. Infliximab, a chimeric antibody targeting tumour necrosis factor-or has been well tolerated in clinical trials and early postmarketing ...

The BAH genomic locus encodes three distinct proteins: junctin, humbug, and BAH. All three proteins share common exons, but differ significantly based upon the use of alternative terminal exons. The biological roles of BAH and humbug and their functional relationship to junctin remain unclear. To evaluate the role of BAH in vivo, the catalytic domain of BAH was specifically targeted such that the coding regions of junctin and humbug remained undisturbed. BAH null mice lack measurable BAH protein in several tissues, lack aspartyl ␤-hydroxylase activity in liver preparations, and exhibit no hydroxylation of the epidermal growth factor (EGF) domain of clotting Factor X. In addition to reduced fertility in females, BAH null mice display several developmental defects including syndactyly, facial dysmorphology, and a mild defect in hard palate formation. The developmental defects present in BAH null mice are similar to defects observed in knock-outs and hypomorphs of the Notch ligand Serrate-2. In this work, ␤-hydroxylation of Asp residues in EGF domains is demonstrated for a soluble form of a Notch ligand, human Jagged-1. These results along with recent reports that another post-translational modification of EGF domains in Notch gene family members (glycosylation by Fringe) alters Notch pathway signaling, lends credence to the suggestion that aspartyl ␤-hydroxylation may represent another post-translational modification of EGF domains that can modulate Notch pathway signaling. Previous work has demonstrated increased levels of BAH in certain tumor tissues and a role for BAH in tumorigenesis has been proposed. The role of hydroxylase in tumor formation was tested directly by crossing BAH KO mice with an intestinal tumor model, APCmin mice. Surprisingly, BAH null/APCmin mice show a statistically significant increase in both intestinal polyp size and number when compared with BAH wild-type/APCmin controls. These results suggest that, in contrast to expectations, loss of BAH catalytic activity may promote tumor formation.