Bone dysplasia sclerosteosis results from loss of the SOST gene product, a novel cystine knot-containing protein.

Sclerosteosis is an autosomal recessive sclerosing bone dysplasia characterized by progressive skeletal overgrowth. The majority of affected individuals have been reported in the Afrikaner population of South Africa, where a high incidence of the disorder occurs as a result of a founder effect. Homozygosity mapping in Afrikaner families along with analysis of historical recombinants localized sclerosteosis to an interval of approximately 2 cM between the loci D17S1787 and D17S930 on chromosome 17q12-q21. Here we report two independent mutations in a novel gene, termed "SOST." Affected Afrikaners carry a nonsense mutation near the amino terminus of the encoded protein, whereas an unrelated affected person of Senegalese origin carries a splicing mutation within the single intron of the gene. The SOST gene encodes a protein that shares similarity with a class of cystine knot-containing factors including dan, cerberus, gremlin, prdc, and caronte. The specific and progressive effect on bone formation observed in individuals affected with sclerosteosis, along with the data presented in this study, together suggest that the SOST gene encodes an important new regulator of bone homeostasis.

Expression analysis with oligonucleotide microarrays reveals that MYC regulates genes involved in growth, cell cycle, signaling, and adhesion.

MYC affects normal and neoplastic cell proliferation by altering gene expression, but the precise pathways remain unclear. We used oligonucleotide microarray analysis of 6,416 genes and expressed sequence tags to determine changes in gene expression caused by activation of c-MYC in primary human fibroblasts. In these experiments, 27 genes were consistently induced, and 9 genes were repressed. The identity of the genes revealed that MYC may affect many aspects of cell physiology altered in transformed cells: cell growth, cell cycle, adhesion, and cytoskeletal organization. Identified targets possibly linked to MYC's effects on cell growth include the nucleolar proteins nucleolin and fibrillarin, as well as the eukaryotic initiation factor 5A. Among the cell cycle genes identified as targets, the G1 cyclin D2 and the cyclin-dependent kinase binding protein CksHs2 were induced whereas the cyclin-dependent kinase inhibitor p21(Cip1) was repressed. A role for MYC in regulating cell adhesion and structure is suggested by repression of genes encoding the extracellular matrix proteins fibronectin and collagen, and the cytoskeletal protein tropomyosin. A possible mechanism for MYC-mediated apoptosis was revealed by identification of the tumor necrosis factor receptor associated protein TRAP1 as a MYC target. Finally, two immunophilins, peptidyl-prolyl cis-trans isomerase F and FKBP52, the latter of which plays a role in cell division in Arabidopsis, were up-regulated by MYC. We also explored pattern-matching methods as an alternative approach for identifying MYC target genes. The genes that displayed an expression profile most similar to endogenous Myc in microarray-based expression profiling of myeloid differentiation models were highly enriched for MYC target genes.

Differentiating between natural progesterone and synthetic progestins: clinical implications for premenstrual syndrome and perimenopause management.

Critical differences between natural progesterone and synthetic progestins are often misunderstood. Synthetic progestins should not be used interchangeably with natural progesterone. This article describes their differences and the clinical implications for their use in managing premenstrual syndrome and perimenopause.

Architecture of protein and DNA contacts within the TFIIIB-DNA complex.

The RNA polymerase III factor TFIIIB forms a stable complex with DNA and can promote multiple rounds of initiation by polymerase. TFIIIB is composed of three subunits, the TATA binding protein (TBP), TFIIB-related factor (BRF), and B". Chemical footprinting, as well as mutagenesis of TBP, BRF, and promoter DNA, was used to probe the architecture of TFIIIB subunits bound to DNA. BRF bound to TBP-DNA through the nonconserved C-terminal region and required 15 bp downstream of the TATA box and as little as 1 bp upstream of the TATA box for stable complex formation. In contrast, formation of complete TFIIIB complexes required 15 bp both upstream and downstream of the TATA box. Hydroxyl radical footprinting of TFIIIB complexes and modeling the results to the TBP-DNA structure suggest that BRF and B" surround TBP on both faces of the TBP-DNA complex and provide an explanation for the exceptional stability of this complex. Competition for binding to TBP by BRF and either TFIIB or TFIIA suggests that BRF binds on the opposite face of the TBP-DNA complex from TFIIB and that the binding sites for TFIIA and BRF overlap. The positions of TBP mutations which are defective in binding BRF suggest that BRF binds to the top and N-terminal leg of TBP. One mutation on the N-terminal leg of TBP specifically affects the binding of the B" subunit.

TNF-alpha stimulation of fibroblast proliferation. Dependence on platelet-derived growth factor (PDGF) secretion and alteration of PDGF receptor expression.

TNF-alpha stimulates DNA synthesis and proliferation of cultured human fibroblasts. Maximal DNA synthesis in response to TNF-alpha occurs approximately 28 h after addition of TNF-alpha to quiescent fibroblasts, a delay of about 12 to 14 h as compared with DNA synthesis elicited by platelet-derived growth factor (PDGF). TNF-alpha induces PDGF A chain gene expression with a maximum at 4 h. DNA synthesis is abrogated in response to TNF-alpha by a goat anti-PDGF IgG but not by nonimmune goat IgG, suggesting induction of an autocrine PDGF-AA loop by TNF-alpha. The response to PDGF-AA requires the presence of PDGF receptor alpha-receptors. TNF-alpha does not significantly affect PDGF alpha-receptor mRNA or protein expression, nor does it alter the proliferative response to externally added PDGF-AA. In contrast, TNF-alpha reduces the levels of PDGF beta-receptor mRNA, protein expression, and cell proliferation in response to PDGF-BB. Thus, DNA synthesis in response to TNF-alpha depends upon autocrinely induced PDGF-AA. At the same time, TNF-alpha may alter the response to PDGF-BB from exogenous sources.

TFIIIC determines RNA polymerase III specificity at the TATA-containing yeast U6 promoter.

The gene encoding yeast U6 snRNA that is transcribed by RNA polymerase III (Pol III) contains both a TATA box upstream of the transcription start site and a downstream binding site for the factor TFIIIC. This juxtaposition of elements typical of both Pol II- and Pol III-transcribed genes raises the question of how polymerase specificity is determined. The upstream U6 promoter containing the TATA box and transcription start site was shown previously to be transcribed by Pol III in vitro. We therefore tested whether the upstream promoter of yeast U6 encodes Pol III specificity. One model is that polymerase specificity is conferred by the homologous Pol II and Pol III transcription factors TFIIB and BRF1. However, we found no specificity in the binding of BRF1 or TFIIB to TATA-containing promoters of genes specifically transcribed by Pol III or Pol II. Yeast strains deficient for Pol II or Pol III transcription were employed to examine U6 polymerase specificity in vivo. We find that the U6 upstream promoter is Pol II-specific in vivo and is converted to Pol III specificity by TFIIIC. Thus, preferential recruitment of TFIIIB by TFIIIC probably excludes the Pol II general factors and promotes Pol III transcription, thereby determining polymerase specificity.

The role of the TATA-binding protein in the assembly and function of the multisubunit yeast RNA polymerase III transcription factor, TFIIIB.

The Saccharomyces cerevisiae RNA polymerase III transcription factor (TF)IIIB has been assembled from three components. An assembly pathway of these polypeptides, which specifies their interactions, has been determined. The TATA-binding protein, TBP, and the TFIIB-related BRF1 gene product BRF, together reconstitute the transcription factor activity and TFIIC-dependent DNA-binding activity of the B' component of TFIIIB. BRF alone weakly binds to a TFIIIC-tRNA gene complex; TBP greatly stabilizes this interaction. B" transcription factor activity is recovered with its previously identified 90 kd polypeptide from SDS-polyacrylamide gels. Incorporation of the 90 kd B" protein into the transcription complex requires TBP. The heparin-resistant TFIIIB-DNA complex retains all three of its constituent proteins, TBP, BRF, and B".

A yeast TFIIB-related factor involved in RNA polymerase III transcription.

A suppressor gene was identified, which in high copy number rescues a temperature-sensitive mutation in yeast TATA-binding protein (TBP). Suppression was allele specific because the suppressor did not rescue the temperature-sensitive phenotype of another TBP mutant. This suppressor gene encodes a 596-amino-acid protein of which the amino-terminal half is homologous to the Pol II-specific factor TFIIB. Disruption of this gene, termed BRF1, showed it to be essential for growth of yeast. Deletion of sequences at either the amino or carboxyl terminus of BRF1 gave both temperature- and cold-sensitive phenotypes. These temperature- and cold-sensitive strains were used to prepare extracts deficient in BRF1 activity and were tested for transcriptional activity by RNA polymerases I, II, and III in vitro. BRF1-deficient extracts are defective in Pol III transcription and can be reconstituted for Pol III transcription by the addition of recombinant BRF1. Western analysis shows that BRF1 is present in TFIIIB but not the TFIIIC fraction, suggesting that it is a component of TFIIIB. We propose that BRF1 plays a role in Pol III initiation analogous to the role played by TFIIB for Pol II in its interaction with TBP and polymerase. The identification of a Pol III-specific TFIIB-like factor extends the previously noted similarity of transcriptional initiation by the three nuclear polymerases.

The major 85-kDa surface antigen of the mammalian-stage forms of Trypanosoma cruzi is a family of sialidases.

Trypanosoma cruzi, an intracellular protozoan parasite infecting a wide variety of vertebrates, is the agent responsible for Chagas disease in humans. An estimated 15-20 million people in South and Central America are infected with the parasite. Chagas disease often results in severe autoimmune and inflammatory pathology and is the major cause of heart failure in endemic areas. Nevertheless, little is known about the host-parasite interactions that lead to this pathology. We have previously cloned several members of a large gene family (SA85-1) and shown that these genes encode 85-kDa T. cruzi, mammalian-stage-specific, surface antigens. Here we report that members of the SA85-1 family possess sialidase activity and are shed by the parasite. We suggest that the sialidases may contribute to the pathology during T. cruzi infection by cleaving sialic acid from cells of the immune system.

Public input into health care policy: controversy and contribution in California.

KIE: This is one of a set of six short articles, grouped under the umbrella title "Grassroots bioethics revisited: health care priorities and community values," with a very brief introduction by Bruce Jennings. The articles focus on the involvement of community health decisions projects with public policy issues of access to health care, allocation of resources, setting health care priorities, cost containment, and rationing.^ieng