Skeletal complications of rheumatoid arthritis.

Rheumatoid arthritis (RA) is associated with local and systemic inflammation that induces many changes in the skeletal health. Locally, periarticular bone loss and juxta-articular bone erosions may occur while joint ankylosis, generalized bone loss, osteoporosis, and fractures may develop secondary to inflammation. The aim of this narrative review is to summarize the clinical evidence for abnormal skeletal health in RA, the effects of disease modifying anti-rheumatic drugs (DMARDS) on bone health, and the effects of drugs for the prevention or treatment of osteoporosis in the RA population.

Meta-analysis and imputation identifies a 109 kb risk haplotype spanning TNFAIP3 associated with lupus nephritis and hematologic manifestations.

TNFAIP3 encodes the ubiquitin-modifying enzyme, A20, a key regulator of inflammatory signaling pathways. We previously reported association between TNFAIP3 variants and systemic lupus erythematosus (SLE). To further localize the risk variant(s), we performed a meta-analysis using genetic data available from two Caucasian case-control datasets (1453 total cases, 3381 total control subjects) and 713 SLE trio families. The best result was found at rs5029939 (P=1.67 x 10(-14), odds ratio=2.09, 95% confidence interval 1.68-2.60). We then imputed single nucleotide polymorphisms (SNPs) from the CEU Phase II HapMap using genotypes from 431 SLE cases and 2155 control subjects. Imputation identified 11 SNPs in addition to three observed SNPs, which together, defined a 109 kb SLE risk segment surrounding TNFAIP3. When evaluating whether the rs5029939 risk allele was associated with SLE clinical manifestations, we observed that heterozygous carriers of the TNFAIP3 risk allele at rs5029939 have a twofold increased risk of developing renal or hematologic manifestations compared to homozygous non-risk subjects. In summary, our study strengthens the genetic evidence that variants in the region of TNFAIP3 influence risk for SLE, particularly in patients with renal and hematologic manifestations, and narrows the risk effect to a 109 kb DNA segment that spans the TNFAIP3 gene.

A short sequence within two purine-rich enhancers determines 5' splice site specificity.

Purine-rich enhancers are exon sequences that promote inclusion of alternative exons, usually via activation of weak upstream 3' splice sites. A recently described purine-rich enhancer from the caldesmon gene has an additional activity by which it directs selection of competing 5' splice sites within an alternative exon. In this study, we have compared the caldesmon enhancer with another purine-rich enhancer from the chicken cardiac troponin T (cTNT) gene for the ability to regulate flanking splice sites. Although similar in sequence and length, the two enhancers demonstrated strikingly different specificities towards 5' splice site choice when placed between competing 5' splice sites in an internal exon. The 32-nucleotide caldesmon enhancer caused effective usage of the exon-internal 5' splice site, whereas the 30-nucleotide cTNT enhancer caused effective usage of the exon-terminal 5' splice site. Both enhancer-mediated splicing pathways represented modulation of the default pathway in which both 5' splice sites were utilized. Each enhancer is multipartite, consisting of two purine-rich sequences of a simple (GAR)n repeat interdigitated with two enhancer-specific sequences. The entire enhancer was necessary for maximal splice site selectivity; however, a 5- to 7-nucleotide region from the 3' end of each enhancer dictated splice site selectivity. Mutations that interchanged this short region of the two enhancers switched specificity. The portion of the cTNT enhancer determinative for 5' splice site selectivity was different than that shown to be maximally important for activation of a 3' splice site, suggesting that enhancer environment can have a major impact on activity. These results are the first indication that individual purine-rich enhancers can differentiate between flanking splice sites. Furthermore, localization of the specificity of splice site choice to a short region within both enhancers indicates that subtle differences in enhancer sequence can have profound effects on the splicing pathway.

A 32-nucleotide exon-splicing enhancer regulates usage of competing 5' splice sites in a differential internal exon.

Large alternatively spliced internal exons are uncommon in vertebrate genes, and the mechanisms governing their usage are unknown. In this report, we examined alternative splicing of a 1-kb internal exon from the human caldesmon gene containing two regulated 5' splice sites that are 687 nucleotides apart. In cell lines normally splicing caldesmon RNA via utilization of the exon-internal 5' splice site, inclusion of the differential exon required a long purine-rich sequence located between the two competing 5' splice sites. This element consisted of four identical 32-nucleotide purine-rich repeats that resemble exon-splicing enhancers (ESE) identified in other genes. One 32-nucleotide repeat supported exon inclusion, repressed usage of the terminal 5' splice site, and functioned in a heterologous exon dependent on exon enhancers for inclusion, indicating that the caldesmon purine-rich sequence can be classified as an ESE. The ESE was required for utilization of the internal 5' splice site only in the presence of the competing 5' splice site and had no effect when placed downstream of the terminal 5' splice site. In the absence of the internal 5' splice site, the ESE activated a normally silent cryptic 5' splice site near the natural internal 5' splice site, indicating that the ESE stimulates upstream 5' splice site selection. We propose that the caldesmon ESE functions to regulate competition between two 5' splice sites within a differential internal exon.

Cloning of cDNAs encoding human caldesmons.

Caldesmon (CDM) is a potential actomyosin regulatory protein found in smooth muscle and nonmuscle cells. Domain mapping and physical studies suggest that CDM is an elongated molecule with an N-terminal myosin/calmodulin-binding domain and a C-terminal tropomyosin/actin/calmodulin-binding domain separated by a 40-nm-long central helix. An 1100-nucleotide (nt) cDNA probe encoding the C terminus of avian caldesmon (aCDM) was used to screen a human aorta library and clone smooth-muscle and non-muscle CDM-encoding cDNAs (CDM). The human (h) smooth-muscle hCDM is 3050-3630 nt long, having variation in length in the 3'-untranslated region. The predicted hCDM protein has a high degree of identity, greater than 90%, to aCDM in the N- and C-terminal-binding domains. The central helical domain is more variable, but retains characteristic repeated peptides and an 'i, i + 4' acidic/basic amino acid (aa) motif found in aCDM which can form intra-helical salt bridges to stabilize the central helix. The predicted smooth-muscle protein is 793 aa long (93,262 Da) with a calculated pI of 5.75. As is the case for the chicken, nonmuscle hCDM is missing the central helical domain, 256 aa overall. Our nonmuscle clone is not full length, but the C-terminal end is identical to the smooth-muscle form. If the N-terminal domain is identical, as it is in the chicken, the predicted protein is 537 aa (62,558 Da). Examination of the 'junctions' at either end of the deleted central domain gives a clear indication of the splice sites and suggests that the nonmuscle form is generated by exon skipping.(ABSTRACT TRUNCATED AT 250 WORDS)

Photoaffinity labeling and partial purification of the beta cell sulfonylurea receptor using a novel, biologically active glyburide analog.

An iodinated analog of the sulfonylurea, glyburide, has been synthesized which can be labeled to high specific activity and used to photolabel the sulfonylurea receptor. 5-Iodo-2-hydroxy-"glyburide", has an iodo group replacing the chlorine at position 5 and a methoxy residue replacing the hydroxy group at position 2 on the benzamido ring. This analog retains biologic activity stimulating insulin secretion from a hamster beta cell line (HIT cells) at the same ED50 (0.4 nM) as glyburide. Scatchard analysis demonstrated high and low affinity binding sites on HIT cell membranes (Kd values of 0.36 nM and 277 nM and Bmax values of 1.6 and 100 pmol/mg of membrane protein, respectively). Competitive binding assays with unlabeled glyburide or 5-iodo-2-hydroxyglyburide yield Ki values of 0.5 and 1.0 nM, respectively. The analog can be covalently linked by ultraviolet irradiation to a membrane protein of Mr = 140,000. The photolabeling is completely blocked by unlabeled glyburide or the analog. Two other species of Mr = 65,000 and 43,000 are also photolabeled; these may be the low affinity sites. After photolabeling, the receptor has been purified partially by chromatographic procedures and is suitable for obtaining peptide sequence. The 140,000 molecular weight protein is identified as the sulfonylurea receptor since its binding constant, 0.36 nM, is closely correlated with its ability to stimulate insulin secretion (ED50 congruent to 0.4 nM).

Argonne intense pulsed neutron source used to solve the molecular structure of a novel organometallic complex.

The single-crystal structure of Mn(CO)(3)(C(7)H(11)) is the first to be solved by direct methods based on time-of-flight neutron diffraction data obtained at the Argonne Intense Pulsed Neutron Source. The molecule contains an unusual three-center, two-electron manganese-hydrogen-carbon interaction.