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The association of hypercholesterolemia and obesity with airway hyperresponsiveness has drawn increasing attention to the potential role of cholesterol and lipid homeostasis in lung physiology and in chronic pulmonary diseases such as asthma. We have recently shown that activation of the nuclear hormone receptor liver X receptor (LXR) stimulates cholesterol efflux in human airway smooth muscle (hASM) cells and induces expression of the ATP-binding cassette (ABC) transporters ABCA1 and ABCG1, members of a family of proteins that mediate reverse cholesterol and phospholipid transport. We show here that ABCA1 is responsible for all LXR-mediated cholesterol and phospholipid efflux to both apolipoprotein AI and high-density lipoprotein acceptors. In contrast, ABCG1 does not appear to be required for this process. Moreover, we show that hASM cells respond to increased levels of cholesterol by inducing expression of ABCA1 and ABCG1 transporters, a process that is dependent on LXR expression. These findings establish a critical role for ABCA1 in reverse cholesterol and phospholipid transport in airway smooth muscle cells and suggest that dysregulation of cholesterol homeostasis in these cells may be important in the pathogenesis of diseases such as asthma. lung; asthma; nuclear hormone receptors; ABC transporters

The nucleus pulposus is an aggrecan-rich, avascular tissue that permits the intervertebral disk to resist compressive loads. In the disk, nucleus pulposus cells express hypoxia-inducible factor (HIF)-I[alpha], a transcription factor that responds to oxygen tension and regulates glycolysis. The goal of the present study was to examine the importance of HIF-1[alpha] in rat nucleus pulposus cells and to probe the function of this transcription factor in terms of regulating aggrecan gene expression. We found that HIF-1[alpha] protein levels and mRNA stability were similar at 20 and 2% [O.sub.2]; there was a small, but significant increase in HIF-1[alpha] transactivation domain activity in hypoxia. With respect to HIF-1[alpha] target genes GAPDH, GLUT-l, and GLUT-3, mRNA and protein levels were independent of the oxygen tension. Other than a modest increase in 6-phosphofructo-2-kinase/fructose2, 6-biphosphatase reporter activity, the oxemic state did not change GAPDH, GLUT-1, and GLUT-3 promoter activities. Treatment of cells with 2-deoxyglucose (2-DG), a glycolytic inhibitor, resulted in a significant suppression in ATP synthesis in normoxia, whereas treatment with mitochondrial inhibitors did not affect ATP production and cell viability. However, measurement of the rate of fatty acid oxidation indicated that these cells contained functioning mitochondria. Finally, we showed that when HIF-1[alpha] was suppressed, irrespective of the oxemic state, there was a partial loss of aggrecan expression and promoter activity. Moreover, when cells were treated with 2-DG, there was inhibition in aggrecan promoter activity. Results of this study indicate that oxygen-independent stabilization of HIF-1[alpha] in nucleus pulposus cells is a metabolic adaptation that drives glycolysis and aggrecan expression. hypoxia; hypoxia-inducible factor-1[alpha]

The nuclear factor (NF)-[kappa]B family of transcription factors is a key participant in multiple biological processes, most notably in the immune and inflammatory response. Five proteins make up the NF-[kappa]B family, and these proteins can hetero- and homo-dimerize, giving rise to diversity. Recently, it has been shown that certain members can also interact directly with other transcription factors such as signal transducers of activated transcription, interferon regulatory factor family members and p53, providing further diversity. We propose that this promiscuity might help explain the many of roles of NF-[kappa]B in specialized cell function and fate. Furthermore, the state of a cell and its cellular background in addition to overall promoter structure and variations in the [kappa]B target sequence will all define the composition and activity of multimeric NF-[kappa]B complexes.

A report is presented on the crystal structure of inactive MtrA. It is an essential gene product for the human pathogen Mycobacterium tuberculosis.

The structural features which underlie the nucleocytoplasmic transport of FABP4 is explained. FABP4 delivers specific ligands from the cytosol to the nuclesr receptor PPARgamma in the nucleus, thereby facilitating the ligation and enhancing the transcriptional activity of the receptor.

Allopolyploid species form through the fusion of two differentiated genomes and, in the earliest stages of their evolution, essentially all genes in the nucleus are duplicated. Because unique mutations occur in each ancestor prior to allopolyploidization, duplicate genes in these species potentially are not interchangeable, and this could influence their genetic fates. This study explores evolution and expression of a simple duplicated complex--a heterodimer between RAG1 and RAG2 proteins in clawed frogs (Xenopus). Results demonstrate that copies of RAG1 degenerated in different polyploid species in a phylogenetically biased fashion, predominately in only one lineage of closely related paralogs. Surprisingly, as a result of an early deletion of one RAG2 paralog, it appears that in many species RAG1/RAG2 heterodimers are composed of proteins that were encoded by unlinked paralogs. If the tetraploid ancestor of extant species of Xenopus arose through allopolyploidization and if recombination between paralogs was rare, then the genes that encode functional RAG1 and RAG2 proteins in many polyploid species were each ultimately inherited from different diploid progenitors. These observations are consistent with the notion that ancestry can influence the fate of duplicate genes millions of years after duplication, and they uncover a dimension of natural selection in allopolyploid genomes that is distinct from other genetic phenomena associated with polyploidization or segmental duplication.

An analysis on differential role of positional isomers of symmetric bis-2-(pyridyl)-1H-benzimidazoles as DNA binding agents was carried out. It was found that some minor chemical alterations in a set of isomeric bis-2-(n-pyridyl)-1H-benzimidazoles change their molecular structure and geometry.

A study was conducted to explore the specificity and function of interaction between the tumor suppressor protein p53 in the yeast three-hybrid system (Y3H) and RNA. The findings prove that p53 binds RNA with little sequence specificity, RNA binding has the potential to regulate DNA binding, and RNA-p53 interactions could be regulated by acetylation of the p53 C-terminus.

The research makes use of a structure model, in addition to the various energy transfer distance measurements to study the T7 RNA polymerase, which causes bending of its promoter DNA on binding by various proteins and other complexes. The analysis proves the existence of a bent and an open model for the initiation complex of T7 and also shows that in spite of the massive changes, the DNA of the active site is very much identical in initiation as well as elongation complexes.

The study employs techniques like NMR and CD calculations to describe the three-dimensional structures of the four tandem repeats of the fibroin-modular-binding protein (FMBP-1), which act as the DNA-binding domain. The analysis shows that the binding of the full-length score and three amino acid peptide repeat leads to an increase in the [alpha]-helicity of the protein regions, which then tend to form a regular secondary structure.

A study finds out how HiNF-P is regulated during the cell-cycle and examines its stability relative to [p220.sup.NPAT] using serum-stimulated cells and cells synchronized in S-phase and in mitosis. It was observed that while HiNF-P is maintained at steady-state levels in the complete cell cycle, the proteasome pathway actively degrades both HiNF-P and [p220.sup.NPAT].

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2006.08.039 Byline: Meredith Howard-Ashby, Stefan C. Materna, C. Titus Brown, Lili Chen, R. Andrew Cameron, Eric H. Davidson Keywords: Transcription factor; Sea urchin; Homeobox; Development Abstract: A set of 96 homeobox transcription factors was identified in the Strongylocentrotus purpuratus genome using permissive blast searches with a large collection of authentic homeodomain sequences from mouse, human and fly. A phylogenetic tree was constructed to compare the sea urchin homeobox gene family to those of vertebrates, with the result that with the only a few exceptions, orthologs of all vertebrate homeodomain genes were uncovered by our search. QPCR time course measurements revealed that 65% of these genes are expressed within the first 48 h of development (late gastrula). For genes displaying sufficiently high levels of transcript during the first 24 h of development (late blastula), whole mount in situ hybridization was carried out up to 48 h to determine spatial patterns of expression. The results demonstrate that homeodomain transcription factors participate in multiple and diverse developmental functions, in that they are used at a range of time points and in every territory of the developing embryo. Author Affiliation: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA Article History: Received 18 April 2006; Revised 4 August 2006; Accepted 16 August 2006

A genome-wide screen was carried out to identify GATA-3 as the most highly enriched transcription factor in the mammary ductal epithelium of pubertal mice and show that GATA-3 is localized to the body cells of terminal end buds (TEBs) and to the mature luminal cells of mammary ducts. The findings suggest that GATA-3 actively maintains luminal epithelial differentiation in the adult mammary gland, which raises important implications for the pathogenesis of breast cancer.

A methodology to quantify the binding of proteins to target DNA molecules based on single-molecule detection and real-time counting of individual free and bound fluorescently tagged molecules flowing past a detection device is presented. The methodology was used for measuring DNA binding by fluorescently tagged domains of four distinct transcription factors and these proteins represent different structural classes, quaternary states and relative affinities.

A new transcription factor of the CCAAT-binding family, MtHAP2-1, is identified in the model legume Medicago truncatula, for which RNA interference (RNAi) and in situ hybridization experiments have indicated a key role during nodule development. The complementary expression pattern of miR169 and MtHAP2-1 and the phenotype of miR-resistant MtHAP2-1 nodules have suggested that the miR169-mediated restriction of MtHAP2-1 expression to the nodule meristematic zone is necessary for the differentiation of nodule cells.

The effects of the turn, the hydrophobic cluster, and a disulfide cross-linker on the folding kinetics of a designed alpha-helical hairpin, Z34C, are examined using an infrared temperature-jump method in conjunction with site-specific mutagenesis. Results reveal that Z34C folds with an ultrafast rate and support a folding mechanism in which the rate-limiting step corresponds to the formation of the reverse turn.

The C-terminal fragment of [Ca.sub.v]1.2, an L-type voltage-gated calcium channel (LTC), associated transcription regulator (CCAT) binds to a nuclear protein, associates with an endogenous promoter and regulates the expression of a wide variety of endogeneous genes important for neuronal signaling and excitability. The results have provided evidence that voltage-gated calcium channels can directly activate transcription and have described a mechanism linking voltage-gated channels to the function and differentiation of excitable cells.

The functions and mechanism of action of the myocardin family of coactivators and the physiological significance of transcriptional coactivation in the context of signal-dependent and cell-type-specific gene regulation are reviewed. Members of the myocardin family of coactivators activate genes involved in cell proliferation, migration, and myogenesis by associating with serum response factor (SRF).

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2006.02.020 Byline: Lorenza Penengo (1), Marina Mapelli (1), Andrea G. Murachelli (1), Stefano Confalonieri (1), Laura Magri (1), Andrea Musacchio (2), Pier Paolo Di Fiore (1)(2)(3), Simona Polo (1)(2), Thomas R. Schneider (1)(2) Abstract: The interaction between ubiquitinated proteins and intracellular proteins harboring ubiquitin binding domains (UBDs) is critical to a multitude of cellular processes. Here, we report that Rabex-5, a guanine nucleotide exchange factor for Rab5, binds to Ub through two independent UBDs. These UBDs determine a number of properties of Rabex-5, including its coupled monoubiquitination and interaction in vivo with ubiquitinated EGFRs. Structural and biochemical characterization of the UBDs of Rabex-5 revealed that one of them (MIU, motif interacting with ubiquitin) binds to Ub with modes superimposable to those of the UIM (ubiquitin-interacting motif):Ub interaction, although in the opposite orientation. The other UBD, RUZ (Rabex-5 ubiquitin binding zinc finger) binds to a surface of Ub centered on Asp58.sub.Ub and distinct from the 'canonical' Ile44.sub.Ub-based surface. The two binding surfaces allow Ub to interact simultaneously with different UBDs, thus opening new perspectives in Ub-mediated signaling. Author Affiliation: (1) IFOM, the FIRC Institute for Molecular Oncology Foundation, Via Adamello 16, 20139 Milan, Italy (2) European Institute of Oncology, Via Ripamonti 435, 20141 Milan, Italy (3) University of Milan, 20122 Milan, Italy Article History: Received 7 December 2005; Revised 6 February 2006; Accepted 14 February 2006 Article Note: (miscellaneous) Published online: February 16, 2006

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2005.10.042 Byline: Outi Hallikas (1), Kimmo Palin (2), Natalia Sinjushina (3), Reetta Rautiainen (1), Juha Partanen (3), Esko Ukkonen (2), Jussi Taipale (1) Abstract: Understanding the regulation of human gene expression requires knowledge of the 'second genetic code,' which consists of the binding specificities of transcription factors (TFs) and the combinatorial code by which TF binding sites are assembled to form tissue-specific enhancer elements. Using a novel high-throughput method, we determined the DNA binding specificities of GLIs 1-3, Tcf4, and c-Ets1, which mediate transcriptional responses to the Hedgehog (Hh), Wnt, and Ras/MAPK signaling pathways. To identify mammalian enhancer elements regulated by these pathways on a genomic scale, we developed a computational tool, enhancer element locator (EEL). We show that EEL can be used to identify Hh and Wnt target genes and to predict activated TFs based on changes in gene expression. Predictions validated in transgenic mouse embryos revealed the presence of multiple tissue-specific enhancers in mouse c-Myc and N-Myc genes, which has implications for organ-specific growth control and tumor-type specificity of oncogenes. Author Affiliation: (1) Molecular and Cancer Biology Program, Biomedicum Helsinki, PO Box 63, FIN-00014 University of Helsinki, Finland (2) Department of Computer Science, PO Box 68, FIN-00014 University of Helsinki, Finland (3) Developmental Biology Program, Institute of Biotechnology, PO Box 56, FIN-00014 University of Helsinki, Finland Article History: Received 14 June 2005; Revised 21 September 2005; Accepted 21 October 2005 Article Note: (miscellaneous) Published: January 12, 2006

Proline 61 A (P61A) mutation related to the factor for inversion stimulation (FIS) where a homodimeric DNA-binding protein found in enteric bacteria is presented. It confirmed the existence of thermostable dimeric intermediate of P61A FIS by glutaraldehyde cross-linking experiments at 95 degree Celsius.

Microcalorimatric and optical methods are used to study the thermodynamic properties and DNA binding ability of the N-terminal DNA binding domains of interferon regulatory factors IRF-1 (DBD1) and IRF-3 (DBD3). It is found that DBD1 has more sequence specificity than DBD3 and the strength of the DBDs' specific association with DNA is inversely related to the stability of the free DBDs.

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.ydbio.2005.07.023 Byline: Patrick Allard (a)(b), Qin Yang (b), William F. Marzluff (c), Hugh J. Clarke (a)(b) Keywords: Oocyte; Histones; Translational control; SLBP; Meiotic maturation Abstract: Although messenger RNAs encoding the histone proteins are among the most abundant in mammalian oocytes, the mechanism regulating their translation has not been identified. The stem-loop binding protein (SLBP) binds to a highly conserved sequence in the 3'-untranslated region (utr) of the non-polyadenylated histone mRNAs in somatic cells and mediates their stabilization and translation. We previously showed that SLBP, which is expressed only during S-phase of proliferating cells, is expressed in growing oocytes at G2 of the cell cycle and accumulates substantially during meiotic maturation. We report here that elevating the amount of SLBP in immature (G2) oocytes is sufficient to increase translation of a reporter mRNA bearing the histone 3'-utr and endogenous histone synthesis and that this effect is not mediated through increased stability of the encoding mRNAs. We further report that translation of the reporter mRNA increases dramatically during meiotic maturation coincident with the accumulation of SLBP. Conversely, when SLBP accumulation during maturation is prevented using RNA interference, both translation of the reporter mRNA and synthesis of endogenous histones are significantly reduced. This effect is not mediated by a loss of the encoding mRNAs. Moreover, following fertilization, SLBP-depleted oocytes also show a significant decrease in pronuclear size and in the amount of acetylated histone detectable on the chromatin. These results demonstrate that histone synthesis in immature and maturing oocytes is governed by a translational control mechanism that is directly regulated by changes in the amount of SLBP. Author Affiliation: (a) Department of Biology, McGill University, Montreal, QC, Canada (b) Department of Obstetrics and Gynecology, McGill University, Montreal, QC, Canada (c) Program in Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27514, USA Article History: Received 21 April 2005; Revised 22 June 2005; Accepted 19 July 2005

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.cell.2005.08.012 Byline: Rashu B. Seth, Lijun Sun, Chee-Kwee Ea, Zhijian J. Chen Abstract: Viral infection triggers host innate immune responses through activation of the transcription factors NF-[kappa]B and IRF3, which coordinately regulate the expression of type-I interferons such as interferon-[beta] (IFN-[beta]). Herein, we report the identification of a novel protein termed MAVS (mitochondrial antiviral signaling), which mediates the activation of NF-[kappa]B and IRF3 in response to viral infection. Silencing of MAVS expression through RNA interference abolishes the activation of NF-[kappa]B and IRF3 by viruses, thereby permitting viral replication. Conversely, overexpression of MAVS induces the expression of IFN-[beta] through activation of NF-[kappa]B and IRF3, thus boosting antiviral immunity. Epistasis experiments show that MAVS is required for the phosphorylation of IRF3 and I[kappa]B and functions downstream of RIG-I, an intracellular receptor for viral RNA. MAVS contains an N-terminal CARD-like domain and a C-terminal transmembrane domain, both of which are essential for MAVS signaling. The transmembrane domain targets MAVS to the mitochondria, implicating a new role of mitochondria in innate immunity. Author Affiliation: Howard Hughes Medical Institute, Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390 Article History: Received 8 June 2005; Revised 8 August 2005; Accepted 11 August 2005 Article Note: (miscellaneous) Published online: August 25, 2005

A series of oligonucleotides containing modified purines and pyrimidines at the recognition site were prepared to probe their mechanism of selective interaction to bind to the methyl-binding domain (MBD) of the methyl-CpG-binding protein 2 (MeCP2). DNA-protein interface mediated by structured water as opposed to hydrophobic interactions elucidated the improved binding of the MBD to oligonucleotides containing several cytosine analogues

The self-assembly properties of highly purified, recombinant L4-22K protein is characterized to understand the thermodynamics of assembly of two human adenoviral (Ad) proteins, IVa2 and L4-22K. L4-22K which reversibly assembles into higher-order structures according to an indefinite, isodesmic assembly scheme could provide significant insight into the thermodynamics of assembly of L4-22K and IVa2 on the PS.

A pair of furan-based cyclic homooligopeptides is prepared to specifically target G-quadruplexes and UV melting has shown that these ligands have effectively induced G-quadruplex structures in the G-rich 22-mer c-MYC DNA sequence and have stabilized the structure. The studies have shown that the present molecules are effective in specifically binding and stabilizing quadruplexes and have provided a suitable scaffold to develop into a quadruplex-targeting therapeutic agent.

The equilibrium binding Deinococcus radiodurans single-stranded (ss) DNA binding protein (DrSSB) to ssDNA is examined for comparison with that of Escherichia coli SSB (EcoSSB). The binding enthalpy has shown a large sensitivity to NaCl concentration, similar to that seen for EcoSSB and these common features for SSB proteins having multiple DNA binding domains enable versatility of SSB function in vivo.

Two X-ray structures of chicken smooth muscle tropomyosin overlap complex are described. The interactions between the N- and C-terminal coiled coils of smooth muscle tropomyosin have displayed curvature, which has differed between the two structures and has implied flexibility in the overlap complex, at least in solution.

The properties of six individually expressed human methylated DNA binding protein 2 (MeCP2) domains spanning the entire protein were analyzed in detail to better understand their interactions with each other, with DNA, and with nucleosomal arrays. The results offer new mechanistic and biochemical insights related to the conformational modulations of intrinsically disordered MeCP2 protein, and its context-dependent in vivo roles.

Isothermal titration calorimetry was used to probe the specificity and examine the thermodynamics of single-stranded DNA binding protein (SSB) binding to two Escherichia coli proteins important for DNA replication, the x subunit of DNA polymerase III holoenzyme and the PriA helicase. The results suggested that DNA binding proteins could use the interaction of SSB with both x and PriA to gain access to ssDNA by first interacting with the SSB C-termini.

Coiled-coil arrays were used to study the interaction specificity of four viral basic-region leucine-zipper transcription factors (bZIPs) and assess their self-associations as well as heterointeractions with 33 representative human bZIPs. The new interactions detected for HBZ with MAFB, MAFG, ATF2, CEBPG, and CREBZF and MEQ with NFIL3 were confirmed in solution by circular dichroism.

The fluorescent single-stranded DNA binding protein (SSB) is used to develop the kinetic mechanism by which the protein and ssDNA associate and dissociate and understand the interaction between single-stranded DNA (ssDNA) and SSB. The complex dissociation kinetics of the fluorescent SSB*ssDNA complex is found to be greatly accelerated by the presence of free wild-type SSB.

A high-resolution NMR solution structure of recombinant ribosomal protein L14e, a component of the large ribosomal subunit in both archaea and eukaryotes is characterized to understand the classic SH3 fold structure adopted by the N-terminal 57 residues. Great similarities observed between the N-terminal sequence of L14e of any Sulfolobus protein and N-terminal sequence of Sac8b, a DNA-binding protein indicated the potential use of structure of L14e to serve as model to study other members of the L14e family, including COG2163 group, L6e and L27e.

Nuclear magnetic resonance spectroscopy and proton exchange were used to characterize two RNA-DNA hybrids from the tR2 intrinsic transcription terminator site of phage [lambda]. The results suggested for the first time that the RNA-DNA hybrid structures with the same base sequence, the opening dynamics and the stability of individual base pairs are strongly influenced by the chemical nature of each strand.