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At least 1,153 records · Page 64Linked to original sources
PubMed2005

Identification of novel steroid target genes through the combination of bioinformatics and functional analysis of hormone response elements.

Steroid hormone receptors including androgen receptor (AR), glucocorticoid receptor (GR), progesterone receptor (PR), and mineralocorticoid receptor (MR) recognize and bind to identical consensus hormone response elements (HREs), which consist of two hexameric half-sites (5'-AGAACA-3') arranged as inverted repeats with a 3-bp spacer. Although only a few near-consensus HRE sequences have been identified in the transcriptional regulatory regions of known steroid target genes, it has been unclear whether the exact consensus sequences function as bona fide HREs in vivo. A genome-wide in silico screening of palindromic HREs identified 565 exact consensus sequences in human genome (NCBI 35 assembly). In this study, of 565 exact consensus elements, functional in vivo receptor binding was evaluated regarding 26 sequences located within 10 kb upstream to the 5' end of annotated genes through chromatin immunoprecipitation (ChIP) assay using cells endogenously expressing steroid hormone receptors. Hormone responsiveness of proximal gene expression was examined through quantitative RT-PCR. As far as performing ChIP assay for AR, GR, and PR, 14 of 26 elements significantly recruited at least one of the receptors by hormone treatment (>2-fold enrichment versus vehicle). In terms of gene expression in the vicinity of the above 14 functional perfect HREs, four genes were upregulated by >2-fold with hormone treatment. The present data suggest that the combination of bioinformatics analysis and quantitative experimental evaluation is useful to identify novel functional HREs that may contribute to the transcriptional regulation of steroid target genes.

Cell Line, Tumor↗
PubMed1998

Identification and functional analysis of the mouse lens filensin gene promoter.

Filensin (also called CP94; CP95; CP97; 115kDa protein) is a component of the lens-specific beaded filament which is believed to be functionally important in lens fiber cell differentiation and in maintaining lens fiber cell conformation and transparency. A 17.2kb fragment containing the 5'-upstream sequence of the filensin gene was isolated. S1-mapping analysis determined the transcription start point (tsp; +1) which locates at 94base pairs upstream from the initiating ATG on the filensin gene. In addition to a major tsp, a minor tsp (-136) was observed. DNA sequence of the fragment around the tsp (-2144 to +155) was identified. Analysis of the DNA sequence of the promoter region around tsp revealed two motifs with sequence homology to Sox2 and Maf recognition sequences in addition to one GATA-1 site, two Sp1 binding sites, and three AP-2 binding motifs. No TATA-box or CCAAT-motif was found around the tsp region. A series of sequentially deleted fragments of (-2144 to +40) were fused to firefly luciferase reporter plasmid pGL2 and tested for activity in chicken embryonic lens explants. A minimal promoter region for mouse filensin of (-70 to +40) was identified. The lens-specific promoter activity was detected using lens explants cultured within 12h after dissection. The activity was remarkably enhanced by culture in the presence of 5ng/ml of basic fibroblast growth factor. Each one of the Sp1 and AP-2 binding motifs was localized to the fragment of (-27 to +40) using electrophoretic mobility shift assays. These are the first data to identify the basic elements to the 5'-upstream sequences of the filensin gene, namely the tsp and the minimal filensin promoter.

Animals↗
PubMed1999

A soluble form of the avian hepatitis B virus receptor. Biochemical characterization and functional analysis of the receptor ligand complex.

Avian hepatitis B virus infection is initiated by the specific interaction of the extracellular preS part of the large viral envelope protein with carboxypeptidase D (gp180), the primary cellular receptor. To functionally and biochemically characterize this interaction, we purified a soluble form of duck carboxypeptidase D from a baculovirus expression system, confirmed its receptor function, and investigated the contribution of different preS sequence elements to receptor binding by surface plasmon resonance analysis. We found that preS binds duck carboxypeptidase D with a 1:1 stoichiometry, thereby inducing conformational changes but not oligomerization. The association constant of the complex was determined to be 2.2 x 10(7) M-1 at 37 degreesC, pH 7.4, with an association rate of 4.0 x 10(4) M-1 s-1 and a dissociation rate of 1.9 x 10(-3) s-1, substantiating high affinity interaction of avihepadnaviruses with their receptor carboxypeptidase D. The separately expressed receptor-binding domain, comprising about 50% of preS as defined by mutational analysis, exhibits similar constants. The domain consists of an essential element, probably responsible for the initial receptor contact and a part that contributes to complex stabilization in a conformation sensitive manner. Together with previous results from cell biological studies these data provide new insights into the initial step of hepadnaviral infection.

Amino Acid Sequence↗
PubMed1999

Functional analysis of the proteasome regulatory particle.

We have developed S. cerevisiae as a model system for mechanistic studies of the 26S proteasome. The subunits of the yeast 19S complex, or regulatory particle (RP), have been defined, and are closely related to those of mammalian proteasomes. The multiubiquitin chain binding subunit (S5a/Mcb1/Rpn10) was found, surprisingly, to be nonessential for the degradation of a variety of ubiquitin-protein conjugates in vivo. Biochemical studies of proteasomes from deltarpn10 mutants revealed the existence of two structural subassemblies within the RP, the lid and the base. The lid and the base are both composed of 8 subunits. By electron microscopy, the base and the lid correspond to the proximal and distal masses of the RP, respectively. The base is sufficient to activate the 20S core particle for degradation of peptides, but the lid is required for ubiquitin-dependent degradation. The lid subunits share sequence motifs with components of the COP9/signalosome complex, suggesting that these functionally diverse particles have a common evolutionary ancestry. Analysis of equivalent point mutations in the six ATPases of the base indicate that they have well-differentiated functions. In particular, mutations in one ATPase gene, RPT2, result in an unexpected defect in peptide hydrolysis by the core particle. One interpretation of this result is that Rpt2 participates in gating of the channel through which substrates enter the core particle.

Adenosine Triphosphatases↗
PubMed2001

Rapid in vivo functional analysis of transgenes in mice using whole body imaging of luciferase expression.

The use of transgenic animals in biomedical research is increasing rapidly and may be the best means of determining gene function. Generating transgenic animals typically requires time-consuming screening processes, and gene function is assessed by an array of difficult phenotypic and biochemical assays performed ex vivo. To address the unmet need in transgenic research for functional assays performed with ease in living animals, we demonstrate here that in vivo detection of luciferase enzyme as a transcriptional reporter facilitates rapid screening for both the presence and function of transgenes in intact living mice. Using this approach we identified three bioluminescent transgenic founders where the transgene consisted of the heme oxygenase promoter fused to the modified coding sequence of the luciferase gene. These founders were identified from 183 pups and confirmed by PCR analysis. Identification of HO-1-luc homozygotes from back- crossed F2 littermates was then accelerated by in vivo imaging. In another transgenic mouse line, where the transgene was comprised of the bone morphogenic-4 (BMP4) promoter fused to the modified luciferase gene, we were able to identify transgenic animals and in each line we were able to visualize patterns of expression in living animals over time. The light production from these transgenic mice indicated that the desired DNA fragment was functional and different expression profiles apparent at different ages and after gene induction.

Animals↗
PubMed1996

Functional analysis of simian immunodeficiency virus SIVAGM long terminal repeat.

We have previously shown that long terminal repeats (LTRs) derived from various isolates of SIVAGM share a unique functional property. In the absence of viral Tat, all SIVAGM LTRs act as much more efficient promoters than any of the other LTRs derived from representative primate immunodeficiency viruses. In the presence of Tat, however, SIVAGM LTRs are activated relatively inefficiently. To map the elements that confer these features on the SIVAGM LTR, a number of deletion mutants were constructed, and their promoter activities were determined using a bacterial CAT gene as a marker. The results obtained indicated that various elements located in the U3 region may contribute to the high basal promoter activity and that no negative elements are present in the region. The Tat-responsive sequence TAR was localized to the R region as observed for the other LTRs. A mutant carrying a single nucleotide deletion in this region completely lost responsiveness to Tat protein.

Base Sequence↗
PubMed2004

Functional analysis of the classical, alternative, and MBL pathways of the complement system: standardization and validation of a simple ELISA.

Primary defence against invading microorganisms depends on a functional innate immune system and the complement system plays a major role in such immunity. Deficiencies in one of the components of the complement system can cause severe and recurrent infections, systemic diseases, such as systemic lupus erythematosus (SLE) and renal disease. Screening for complement deficiencies in the classical or alternative complement pathways has mainly been performed by haemolytic assays. Here, we describe a simple ELISA-based format for the evaluation of three pathways of complement activation. The assays are based on specific coatings for each pathway in combination with specific buffer systems. We have standardized these assays and defined cut off values to detect complement deficiencies at the different levels of the complement system. The results demonstrate the value of these ELISA-based procedures for the functional assessment of complement deficiencies in clinical practice. The assay is now available commercially in kit form.

Complement Activation↗
PubMed2002

Functional analysis of initial cell divisions defines the subsequent fate of individual human CD34(+)CD38(-) cells.

OBJECTIVE: We assessed the relationship of individual cell divisional behavior with the functional fate of stem cell candidates at the single cell level. MATERIALS AND METHODS: Individual CD34(+)CD38(-) cells derived from cord blood (88-352 cells in each of 25 experiments) were cultured in early-acting conditioned medium (EACM) or late-acting proliferation medium (LAPM). The initial cell divisions were microscopically monitored every 12 to 24 hours and then assessed for primitive function in the myeloid lymphoid-initiating cell assay and committed function in the colony-forming cell (CFC) assay. RESULTS: Despite a higher proliferative capacity in LAPM, significantly more quiescent cells (11.1 +/- 1.7%) were found in LAPM than in EACM cultures (1.1 +/- 0.4%; p < 0.001). No differences were observed in the initially plated CD34(+)Cd38(-) cells that produced asymmetrically dividing progeny. The majority of cells with subsequent ML-IC function divided in EACM but were found among quiescent cells in LAPM conditions. All cycling cells with subsequent ML-IC capacity initially remained quiescent for at least 96 hours. All ML-IC had been recruited exclusively (100%) from either cytokine nonresponsive (quiescent) or slow and asymmetrically dividing cells (1-2 divisions). In contrast, the majority of CFCs entered the cell cycle immediately after plating, have divided more than two times, and only 20.2 +/- 5.5% of the cycled CFC divided asymmetrically. CONCLUSIONS: Asymmetric divisional behavior of CD34(+)CD38(-)cells cannot be influenced by culture conditions. Primitive ML-IC can be distinguished from committed CFC by initial quiescence or asymmetric divisions. Committed CFC cycle rapidly and symmetrically.

ADP-ribosyl Cyclase↗
PubMed1996

Localization and functional analysis of human cortical area V5 using magneto-encephalography.

Using a multi-channel SQUID-based neuromagnetometer, we have determined the location, temporal dynamics and functional response properties of the human homologue of the primate cortical area V5 (MT). We provide evidence that area V5 in humans is located near the occipito-temporal border in a minor sulcus immediately below the superior temporal sulcus. this area is selective for low spatial frequencies ( < or = 4.0 c/deg), responds to a wide range of temporal frequencies ( < or = 35 Hz) and shows response saturation for stimulus contrasts greater than 10%. In addition, we find that this area is not responsive to purely chromatic patterns but is responsive to motion-contrast stimuli. Our results are consistent with the hypothesis that area V5 in humans represents a stage of processing within the magnocellular pathway. We discuss our results in relation to the widespread belief that area V5 in humans is specifically concerned with motion perception.

Adult↗
PubMed2004

Structure-function analysis of the presumptive Arabidopsis auxin permease AUX1.

We have investigated the subcellular localization, the domain topology, and the amino acid residues that are critical for the function of the presumptive Arabidopsis thaliana auxin influx carrier AUX1. Biochemical fractionation experiments and confocal studies using an N-terminal yellow fluorescent protein (YFP) fusion observed that AUX1 colocalized with plasma membrane (PM) markers. Because of its PM localization, we were able to take advantage of the steep pH gradient that exists across the plant cell PM to investigate AUX1 topology using YFP as a pH-sensitive probe. The YFP-coding sequence was inserted in selected AUX1 hydrophilic loops to orient surface domains on either apoplastic or cytoplasmic faces of the PM based on the absence or presence of YFP fluorescence, respectively. We were able to demonstrate in conjunction with helix prediction programs that AUX1 represents a polytopic membrane protein composed of 11 transmembrane spanning domains. In parallel, a large aux1 allelic series containing null, partial-loss-of-function, and conditional mutations was characterized to identify the functionally important domains and amino acid residues within the AUX1 polypeptide. Whereas almost all partial-loss-of-function and null alleles cluster in the core permease region, the sole conditional allele aux1-7 modifies the function of the external C-terminal domain.

Alleles↗
PubMed1985

In vivo treatment of rats with monoclonal anti-T-cell antibodies. Immunohistochemical and functional analysis in normal rats and in experimental allergic neuritis.

The effects of intraperitoneal injection of monoclonal anti-rat T-lymphocyte antibodies were evaluated immunohistochemically and functionally in normal rats and in rats with experimental allergic neuritis. In the normal animals a single injection of OX8 antibodies, reactive with suppressor/cytotoxic T cells, completely eliminated OX8-reactive cells from peripheral lymphoid organs and from circulation, whereas the 'pan' T-cell-reactive W3/13 antibodies and the helper T-cell-reactive W3/25 antibodies only caused a partial elimination of their respective target cells. Injection of the W3/13 and W3/25 antibodies but not of OX8 antibodies led to a diminished responsiveness to allogeneic stimulation in vitro for spleen cells obtained from the treated rats, whereas the OX8 injection caused a complete elimination of the in vitro cytotoxic response to allogeneic cells in the mixed lymphocyte reaction-activated spleen cell population. When Lewis rats were injected with peripheral nerve myelin and Freund's adjuvant for the induction of EAN, treatment with W3/13 antibodies completely prevented the onset of disease, whereas treatment with the OX8 antibodies exaggerated the disease symptoms.

Animals↗
PubMed2006

Normothermic ex vivo lung perfusion of non-heart-beating donor lungs in pigs: from pretransplant function analysis towards a 6-h machine preservation.

Donor shortage urges optimal use of all lungs available. Ex vivo lung perfusion (EVLP) is a method to evaluate lung function before implantation. EVLP was performed in pigs to evaluate lung function, using two different clinical non-heart-beating (NHS) donor protocols: flush perfusion and topical cooling after 1-h warm ischaemia (n = 5 each). Secondly, we investigated whether EVLP can be used for 6 h ex vivo machine preservation (n = 4). In comparison with topical cooling, flush perfusion preserved lung function better during EVLP. During 6 h normothermic EVLP, gas exchange remained stable; however, the pulmonary artery pressure and ventilation pressure showed a significant increase. EVLP is a reliable method for evaluation of lung graft function. Flush perfusion with Perfadex is preferred above topical cooling in NHB lung donation. Six-hour normothermic EVLP is feasible but should be further improved to make ex vivo machine preservation or treatment of lung grafts successful.

Animals↗
PubMed2000

Structural and functional analysis of the riboflavin synthesis genes encoding GTP cyclohydrolase II (ribA), DHBP synthase (ribBA), riboflavin synthase (ribC), and riboflavin deaminase/reductase (ribD) from Helicobacter pylori strain P1.

The functions of the riboflavin synthesis gene homologues ribA, ribBA, ribC, and ribD from Helicobacter pylori strain P1 were confirmed by complementation of defined Escherichia coli mutant strains. The H. pylori ribBA gene, which is similar to bifunctional ribBA genes of Gram-positive bacteria, fully complemented the ribB mutation and partially restored growth in a ribC mutant. However, ribBA did not complement the ribA mutation in E. coli, thus explaining the presence of the additional separate copy of the ribA gene in the H. pylori chromosome. In E. coli exclusively ribA conferred hemolytic activity and gave rise to production of molecules with fluorescence characteristics similar to flavins, as observed earlier. The E. coli hemolysin ClyA was not involved in causing the hemolytic phenotype. No riboflavin synthesis genes on plasmids conferred iron uptake functions to a siderophore-deficient mutant of E. coli. Marker exchange mutagenesis of the genes in H. pylori was not successful indicating that riboflavin synthesis is essential for basic metabolic functions of the gastric pathogen.

Aminohydrolases↗
PubMed2003

Functional analysis of the TCR binding domain of toxic shock syndrome toxin-1 predicts further diversity in MHC class II/superantigen/TCR ternary complexes.

Superantigens (SAGs) aberrantly alter immune system function through simultaneous interaction with lateral surfaces of MHC class II molecules on APCs and with particular variable regions of the TCR beta-chain (Vbeta). To further define the interface between the bacterial SAG toxic shock syndrome toxin-1 (TSST-1) and the TCR, we performed alanine scanning mutagenesis within the putative TCR binding region of TSST-1 along the central alpha helix adjacent to the N-terminal alpha helix and the beta7-beta9 loop as well as with two universally conserved SAG residues (Leu(137) and Tyr(144) in TSST-1). Mutants were analyzed for multiple functional activities, and various residues appeared to play minor or insignificant roles in the TCR interaction. The locations of six residues (Gly(16), Trp(116), Glu(132), His(135), Gln(136), and Gln(139)), each individually critical for functional activity as well as direct interaction with the human TCR Vbeta2.1-chain, indicate that the interface occurs in a novel region of the SAG molecule. Based on these data, a model of the MHC/TSST-1/TCR ternary complex predicts similarities seen with other characterized SAGs, although the CDR3 loop of Vbeta2.1 is probably involved in direct SAG-TCR molecular interactions, possibly contributing to the TCR Vbeta specificity of TSST-1.

Amino Acid Substitution↗
PubMed1997

Expression and functional analysis of voltage-activated Na+ channels in human prostate cancer cell lines and their contribution to invasion in vitro.

Ion channels are important for many cellular functions and disease states including cystic fibrosis and multidrug resistance. Previous work in the Dunning rat model of prostate cancer has suggested a relationship between voltage-activated Na+ channels (VASCs) and the invasive phenotype in vitro. The objectives of this study were to 1) evaluate the expression of VASCs in the LNCaP and PC-3 human prostate cancer cell lines by Western blotting, flow cytometry, and whole-cell patch clamping, 2) determine their role in invasion in vitro using modified Boyden chambers with and without a specific blocker of VASCs (tetrodotoxin). A 260-kd protein representing VASCs was found only in the PC-3 cell line, and these were shown to be membrane expressed on flow cytometry. Patch clamping studies indicated that functional VASCs were present in 10% of PC-3 cells and blocking these by tetrodotoxin (600 nmol/L) reduced their invasiveness by 31% (P = 0.02) without affecting the invasiveness of the LNCaP cells. These results indicate that the reduction of invasion is a direct result of VASC blockade and not a nonspecific action of the drug. This is the first report of VASCs in a human prostatic cell line. VASCs are present in PC-3 but not LNCaP cells as determined by both protein and functional studies. Tetrodotoxin reduced the invasiveness of PC-3 but not LNCaP cells, and these data suggest that ion channels may play an important functional role in tumor invasion.

Animals↗
PubMed2004

Biochemical purification and functional analysis of complexes between the G-protein subunit Gbeta5 and RGS proteins.

Regulator of G-protein signaling (RGS) proteins of the R7 subfamily (RGS6, 7, 9, and 11) contain a unique Ggamma-like (GGL) domain that enables their association with the G-protein beta subunit Gbeta5. The existence of these complexes was demonstrated by their purification from native tissues as well as by reconstitution in vitro. According to pulse-chase analysis, Gbeta5 and RGS7 monomers undergo rapid proteolytic degradation in cells, whereas the dimer is stable. Studies of the functional role of Gbeta5-RGS dimers using GTPase activity, ion channel, and calcium mobilization assays showed that, similarly to other RGS proteins, they can negatively regulate G-protein-mediated signal transduction. Protein-protein interactions involving the Gbeta5-RGS7 complex can be studied in cells using fluorescence resonance energy transfer utilizing Gbeta5, RGS, and Galpha subunits fused to the cyan and yellow versions of green fluorescent protein.

Animals↗
PubMed1996

Structure-function analysis of P-selectin-sialyl LewisX binding interactions. Mutagenic alteration of ligand binding specificity.

P-selectin is a vascular cell adhesion molecule that is expressed on the surface of platelets and endothelial cells in response to inflammatory stimuli. It is believed to aid in the binding and recruitment of leukocytes to inflamed tissue. P-selectin adhesion to leukocytes is mediated by the amino-terminal lectin domain that binds the sialyl LewisX (sLeX) carbohydrate (Neu5Acalpha2-3Galbeta1-4(Fucalpha1-3)GlcNAc). Neither the three-dimensional structure of P-selectin nor the protein-carbohydrate interactions that mediate the binding of P-selectin to the sLeX carbohydrate have been determined. The most closely related protein for which a ligand-bound three-dimensional structure has been resolved is the rat mannose-binding protein (Weis, W. I., Drickamer, K., and Hendrickson, W. A. (1992) Nature 360, 127-134). Using the known binding interactions that occur between the rat mannose-binding protein and its ligand (oligomannose) as a template, we have used site-directed mutagenesis to substitute Ala-77 with lysine. This substitution changed P-selectin-carbohydrate binding specificity from sLeX to oligomannose. Further substitution altered the binding preference from mannose to galactose in a predictable manner. These results indicate that P-selectin binds sLeX in a shallow cleft that is similar to the mannose-binding protein saccharide-binding cleft. Additionally, we present an extensive mutagenic analysis of P-selectin Lys-113, a residue that has previously been implicated in P-selectin binding to both sLeX and 3-sulfated galactosylceramide (sulfatide). Our analysis demonstrates that Lys-113 is probably not involved in P-selectin binding to either sulfatide or sLeX. Functionally, it appears that P-selectin has retained a conserved carbohydrate and calcium coordination site that enables it to bind carbohydrate in a manner that is quite similar to that which has been determined for the rat mannose-binding protein.

Alanine↗
PubMed1996

The application of K-function analysis to the geographical distribution of road traffic accident outcomes in Norfolk, England.

One method applicable to the examination of spatial point patterns of disease, the calculation of K-functions, is presented. The technique is used to determine the degree of clustering exhibited by the residuals from a spatially referenced logit model constructed to ascertain the factors influencing the likelihood of death in a road traffic accident. This was done to test if there was some systematic geographical factor influencing outcome not adequately controlled for in the model. K-functions are extremely versatile, overcoming many of the problems of incorporating the notion of scale associated with traditional methods of spatial autocorrelation. Recently software has become available which allows their calculation in an easy to use Geographical Information System style environment. This study illustrates the relevance of the method, not only to the analysis of data on mortality and morbidity, but also to the examination of the residuals from any spatial regression.

Accidents, Traffic↗