Biomedical subjects

E Wingender

Publications and source records attributed to E Wingender.

At least 37 records · Page 2Linked to original sources

PubMed1995

MatInd and MatInspector: new fast and versatile tools for detection of consensus matches in nucleotide sequence data.

The identification of potential regulatory motifs in new sequence data is increasingly important for experimental design. Those motifs are commonly located by matches to IUPAC strings derived from consensus sequences. Although this method is simple and widely used, a major drawback of IUPAC strings is that they necessarily remove much of the information originally present in the set of sequences. Nucleotide distribution matrices retain most of the information and are thus better suited to evaluate new potential sites. However, sufficiently large libraries of pre-compiled matrices are a prerequisite for practical application of any matrix-based approach and are just beginning to emerge. Here we present a set of tools for molecular biologists that allows generation of new matrices and detection of potential sequence matches by automatic searches with a library of pre-compiled matrices. We also supply a large library (> 200) of transcription factor binding site matrices that has been compiled on the basis of published matrices as well as entries from the TRANSFAC database, with emphasis on sequences with experimentally verified binding capacity. Our search method includes position weighting of the matrices based on the information content of individual positions and calculates a relative matrix similarity. We show several examples suggesting that this matrix similarity is useful in estimating the functional potential of matrix matches and thus provides a valuable basis for designing appropriate experiments.

Base Sequence↗

PubMed1995

A compilation of composite regulatory elements affecting gene transcription in vertebrates.

Over the past years, evidence has been accumulating for a fundamental role of protein-protein interactions between transcription factors in gene-specific transcription regulation. Many of these interactions run within composite elements containing binding sites for several factors. We have selected 101 composite regulatory elements identified experimentally in the regulatory regions of 64 genes of vertebrates and of their viruses and briefly described them in a compilation. Of these, 82 composite elements are of the synergistic type and 19 of the antagonistic type. Within the synergistic type composite elements, transcription factors bind to the corresponding sites simultaneously, thus cooperatively activating transcription. The factors, binding to their target sites within antagonistic type composite elements, produce opposing effects on transcription. The nucleotide sequence and localization in the genes, the names and brief description of transcription factors, are provided for each composite element, including a representation of experimental data on its functioning. Most of the composite elements (3/4) fall between -250 bp and the transcription start site. The distance between the binding sites within the composite elements described varies from complete overlapping to 80 bp. The compilation of composite elements is presented in the database COMPEL which is electronically accessible by anonymous ftp via internet.

Animals↗

PubMed1995

Stimulation of creatine kinase activity in rat skeletal tissue in vivo and in vitro by protease-resistant variants of parathyroid hormone fragments.

We have reported that mid-region fragments of human parathyroid hormone (hPTH), exemplified by hPTH-(28-48), stimulated [3H]thymidine incorporation into DNA and increased the specific activity of the brain-type isoenzyme of creatine kinase (CK) in both skeletal-derived cell cultures (ROS 17/2.8 cells) and immature rat epiphyseal cartilage and diaphyseal bone, without stimulating cyclic AMP synthesis which is a prerequisite for bone resorption. In the present study, substitution of amino acids in hPTH-(28-48), which resulted in increased resistance to proteolysis, produced variants that stimulated skeletal systems at two orders of magnitude lower concentration than the wild-type fragment. We modified hPTH-(28-48) at Leu-37 by replacement with Met, Thr or Val. Under conditions in which 20% of the native hPTH-(28-48) resisted proteolysis by cathepsin D for 6 h, approx. 40% of the L37V mutant and 70% of the L37T mutant remained intact. Substitution of Met for Phe-34 in addition to Thr for Leu-37, or the substitution of Met for Phe-34 alone, produced 100%-resistant fragments. These variants at residue 34 caused maximal stimulation of CK in ROS 17/2.8 cells at 0.24 nM compared with 24 nM for hPTH-(28-48). The double mutant stimulated CK activity significantly in immature rats, at a minimum dose of 12.5 ng/rat, and caused maximal stimulation at 125 ng/rat, a 10-fold lower dose than for hPTH-(28-48). The effect of the double mutant lasted up to 24 h which differs from the stimulation by hPTH-(28-48) in which CK specific activity returns to the control level at 24 h. This same dose also significantly stimulated CK activity in gonadectomized rats. These results show the advantage of using protease-resistant mid-region variants of hPTH-(28-48) to stimulate bone cells, in terms of lower doses and longer duration of effectiveness, both in vitro and in vivo.

Amino Acid Sequence↗

PubMed1995

Large-scale preparation and biological activity of recombinant human parathyroid hormone.

Human parathyroid hormone (hPTH) has been bacterially expressed in bioreactors as cro-beta-galactosidase-hPTH fusion protein. We have developed a large-scale purification scheme that exploits the pH-dependent differential solubility of hPTH and a two-step chromatographic procedure. We demonstrate that in a number of assay systems, the recombinant material obtained by this procedure is biologically active.

Animals↗

PubMed1995

Computer tool FUNSITE for analysis of eukaryotic regulatory genomic sequences.

We present the computer tool FUNSITE for description and analysis of regulatory sequences of eukaryotic genomes. The tool consists of the following main parts: 1) An integrated database for genomic regulatory sequences. The integrated database was designed on the basis of the databases TRANSFAC (Wingender 1994) and TRRD (Kel et al. 1995) that are currently under development. The following functions are performed: i) linkage to the EMBL database; ii) preparing samples of definite types of functional sites with their flanking sequences; iii) preparing samples of promoter sequences; iv) preparing samples of transcription factors classified with regard to structural and functional features of DNA binding and activating domains, functional families of the factors, their tissue specificity and other functional features; v) access to data on mutual disposition of cis-elements within the regulatory regions. 2) The second component of FUNSITE tool is the set of programs for analysis of the structural organization of regulatory sequences: i) Program for revealing of potential transcription factors binding sites based on their consensi; ii) program for revealing of the potential binding sites using homology search with nucleotide sequences of real binding sites; iii) program for analysis of oligonucleotide context features which are characteristic of flank sequences of the binding sites; iv) program for design of recognition method for the functional sites based on generalized weight matrix; v) program for revealing potential composite elements. The results of analysis of the promoter sequences of eukaryotic genes with the FUNSITE are presented, too.

Amino Acid Sequence↗

PubMed1994

Recognition of regulatory regions in genomic sequences.

For the functional interpretation of genomic sequences, effective algorithms have to be developed that will recognize regions of specific function and thus will suggest experiments for their verification. As a first step, relevant data have to be collected in an appropriate database from which suitable training sets can be extracted. In this paper, I discuss the requirements for a database that collects information about regulatory DNA sequences and describe the structure and contents of such a database (TRANSFAC). This compiled information will serve as a basis for comprehensive analysis of sites that regulate transcription, e.g., by statistical methods. It will thus facilitate the recognition of regulatory genomic sequence information and the assignment of the corresponding regulators. Moreover, it will provide all relevant data about the regulating proteins which will allow to trace back transcriptional control cascades to their origin.

Algorithms↗

PubMed1994

The structure of human parathyroid hormone from a study of fragments in solution using 1H NMR spectroscopy and its biological implications.

In order to gain insight into the structure of human parathyroid hormone (hPTH), four fragments [hPTH(1-34), hPTH(18-48), hPTH(28-48), and hPTH(53-84)], which cover all regions of the intact hormone, have been investigated by CD and NMR spectroscopy in combination with distance geometry, and restrained molecular dynamics and energy minimization calculations, under a variety of solution conditions. Significantly, all fragments showed little propensity to form stable structures in aqueous solution alone, and it was only on the addition of trifluoroethanol (TFE) that defined structural features were observed. In an extension of earlier work [Klaus et al. (1991) Biochemistry 30, 6936-6942], hPTH(1-34) in 70% trifluoroethanol (TFE) showed two helices that were longer than in 10% TFE, but essentially showed the same characteristics. Although overlap in the 1H NMR spectra prevented the determination of quantitative NOE data for residues 26-30, the combination of the alpha-proton chemical shift data and quantitative NOE data indicated the helices extend from residues 3 to 13 and 15 to 29. No evidence was found for interaction of the two helical regions. The nature and extent of this second helix in the intact hormone were better defined from the data for hPTH(18-48). Under limiting solution conditions, where the fragment assumed its maximum helical content, a well-defined helix was observed between residues 21 and 38 with a possible discontinuity between Leu-28 and Gln-29. There was little evidence of any form of secondary structure between Gly-38 and the terminus of this fragment, Ser-48. In keeping with this result, the shorter fragment, hPTH(28-48), showed little evidence of stable secondary structure on addition of TFE. From the alpha-proton chemical shifts residues 23-27 appeared to sustain helical structure more readily than the rest of molecule under all solution regimes in both hPTH(1-34) and hPTH(18-48). In contrast to the other two longer fragments hPTH(53-84) showed little propensity for helical secondary structure even at the highest TFE concentrations. However, there was evidence that the molecule did adopt a defined three-dimensional structure. Various long-range NOE's were observed in 10% TFE that allowed the calculation of an open tertiary structure consisting of an initial series of turns surrounded by a loop structure of several loose turns.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗

PubMed1994

Production of human parathyroid hormone by recombinant Escherichia coli TG1 on synthetic medium.

Production of human parathyroid hormone (hPTH) by Escherichia coli TG1:I52cIts was studied. The hPTH is expressed as a fusion protein under control of the bacteriophage lambda pR promoter. The organism grows on glucose/mineral salt medium and the expression of the gene product was investigated under variation of temperature and growth rate prior to and after induction. hPTH formation largely depends on cultivation temperature and is optimal for a temperature shift from 30 to 38 degrees C. Product expression is growth coupled and specific hPTH concentration is independent of growth rate. The results are compared with a previous study on E. coli N4830:pEX-PPTH grown on complex media.

Culture Media↗

PubMed1994

The human immunodeficiency virus type 1 encoded Vpu protein is phosphorylated by casein kinase-2 (CK-2) at positions Ser52 and Ser56 within a predicted alpha-helix-turn-alpha-helix-motif.

The human immunodeficiency virus type 1 (HIV-1) encoded Vpu is a small integral membrane phosphoprotein that functions in the enhancement of viral particle release and has more recently been shown to cause degradation of CD4 at the endoplasmic reticulum. We have demonstrated earlier that Vpu is phosphorylated by the ubiquitous casein kinase-2 (CK-2) in HIV-1 infected cells. The phosphoacceptor sites targeted by CK-2 in Vpu, however, have not been demonstrated and it was unclear whether Vpu was phosphorylated at one or more of its four serine residues. In this study we characterized the CK-2 phosphoacceptor sites in Vpu using recombinant CK-2 for in vitro phosphorylation of recombinant Vpu protein as well as synthetic peptides of Vpu. Phosphorylation of both Ser52 and Ser56 was demonstrated by in vitro phosphorylation using three 54-residue peptides comprising the entire hydrophilic part of Vpu and containing single serine to asparagine transitions in either position 52 or 56. The Km values of CK-2 to these peptides were established, revealing a preferential phosphorylation of Ser56. The Km values are: Ser56 = 31 microM; Ser 52 = 156 microM; wild type = 27 microM. In addition, we studied phosphorylation of Vpu by endogenous CK-2 following in vitro translation in rabbit reticulocyte lysate of wild-type Vpu or a mutant, Vpum2/6, carrying serine to asparagine changes at amino acid positions 52 and 56. The in vivo phosphorylation of Vpu was studied in transiently transfected human embryonic kidney (293) cells. In this system, the mutant Vpum2/6 was not phosphorylated, indicating that the seryl residues of Vpu at amino acid positions 52 and 56, but not those at positions 23 and 61, are phosphorylated by CK-2. The two CK-2 phosphorylation sites are conserved in all known Vpu sequences and represent the consensus Ser52GlyAsn(Glu/Asp)Ser(Glu/Asp)Gly(Glu/Asp)59. Prediction of the secondary structure revealed a conserved alpha-helix-turn-alpha-helix motif for the hydrophilic C-terminal part of Vpu. A structural model for Vpu is proposed in which the membrane anchor precedes a region comprising two amphipathic alpha-helices of opposed polarity, joined by a strongly acidic turn that protrudes into the cytoplasm and contains the CK-2 phosphorylation sites. Possible functional and structural homologies of Vpu to the membrane channel-forming M2 protein of influenza A viruses are discussed.

Amino Acid Sequence↗

PubMed1994

TRANSFAC retrieval program: a network model database of eukaryotic transcription regulating sequences and proteins.

DNA sequences that are involved in the control of gene expression in eukaryotes have been collected in conjunction with the proteins binding to and acting through them (TRANSFAC data set). To make these data accessible, the TRANSFAC retrieval program (TRP) has been developed as a database management system which is based upon the network model. This database model possesses particular advantages for data management of a complex structure. The aim of TRP is to provide an easily handled statistical basis for a computational approach to transcription control.

Amino Acid Sequence↗

PubMed1993

Organization and sequence of the gene encoding the human acrosin-trypsin inhibitor (HUSI-II).

A complete cDNA encoding the acrosin-trypsin inhibitor, HUSI-II, was used as a probe to isolate genomic clones from a human placenta library. Three clones which cover the entire HUSI-II gene were isolated and characterized. The exon-intron organization of the gene was determined and found to be identical to other known Kazal-type inhibitor-encoding genes. The striking similarity in the amino acid sequences which was found previously in HUSI-II and glycoprotein hormone beta-subunits, is neither reflected in codon usage nor in the exon-intron arrangement of the genes. A 1.8-kb segment 5' of the gene was sequenced. The analysis of this sequence showed that HUSI-II contains a G + C-rich region upstream from the transcription start point (tsp) which fulfills the criteria for a CpG island. Furthermore, in the first intron, a potential glucocorticoid-responsive element was found as a half-palindrome flanked by two CACCC elements. Determination of the tsp by S1 mapping revealed that HUSI-II has multiple tsp. Genomic Southern hybridization was used to show that HUSI-II is a single-copy gene. The localization of the gene to chromosome 4 was determined by hybridization of a 5' genomic fragment to the DNA of a panel of somatic hybrids between human and rodent cells.

Acrosin↗

PubMed1993

Studies on the production of human parathyroid hormone by recombinant Escherichia coli.

Expression of human parathyroid hormone, hPTH(-1-84), by Escherichia coli N4830:pEX-PPTH was studied in controlled bioreactors. The hPTH is expressed as a fusion protein under control of the bacteriophage lambda PR promoter. In batch runs, low biomass concentrations but high specific hPTH productivities were obtained with complex TY (bactotryptone and yeast extract) medium whereas high biomass concentration and low specific productivities were found when fructose was used instead of bactotryptone (YF medium). The preinduction temperature was always 30 degrees C; the temperature shift to induce production of fusion protein was varied from 36 to 42 degrees C. Formation of hPTH passed a pronounced maximum as a function of induction temperature when using YF medium. However, the optimum temperature shift was 38 degrees C for both media used. For this temperature increase both media yielded about the same volumetric hPTH productivity (approx. 30 mg hPTH/1 per hour). By applying a fed-batch strategy for the YF medium, the productivity of the recombinant protein could be further increased more than fourfold. Compared to shake-flask experiments, the hPTH yield could be increased by a factor larger than 20.

Biotechnology↗

PubMed1992

Structural changes in condylar cartilage following prolonged exposure to the human parathyroid hormone fragment (hPTH) 1-34 in vitro.

This investigation presents the structural changes in condylar cartilage incubated in the presence of human parathyroid hormone (1-34) in an organ culture system for 6 to 12 days. Control cultures maintained their cartilaginous characteristics whereas human parathyroid hormone (1-34)-treated cultures revealed the following modifications: (1) The chondroprogenitor cell zone at the apical region of the explant underwent a substantial enlargement. The cells changed from a mesenchyme-like morphology into polygonal, glycogen-rich cells that were tightly attached to each other by a fibrillar intercellular matrix, but even by 12 days the apical region was comprised of healthy cells. (2) The mineralizing zone in the hypertrophic cartilage revealed a change in its cellular population. Hypertrophic chondrocytes were replaced by cells with amoeboid extensions and large numbers of secretory granules or vesicles. Based upon the above findings it appears that the chondroprogenitor cells that are initially stimulated to proliferate, are being suppressed from subsequent differentiation into chondroblasts; and that hypertrophic chondrocytes apparently undergo a dedifferentiation process followed by development into an as yet unknown cell population.

Animals↗

PubMed1992

New perspectives in the differentiation of bone-forming cells.

Bone formation comprises a complex but ordered sequence of events which involves the proliferation and differentiation of chondrogenic and osteoblastic precursor cells ultimately leading to the formation of a calcified extracellular matrix. This process can be observed in vivo but under these conditions is difficult to study at the molecular level. A number of in vitro models have been developed which recapitulate discrete elements of this process. Using these models, detailed information has been obtained regarding the differentiation of bone forming cells and the molecular biology of the mineralization process. It has been shown that, in vitro, osteoblastic precursor cells can form a mineralized matrix similar to that seen in vivo. This calcification process was shown to consist of three interdependent phases: proliferation, matrix maturation and mineralization. Each of these phases was characterized by the expression of particular genes. Osteoblast precursors have been cloned and consequently shown to be able to differentiate in vitro into a number of other mesenchymal cells, supporting the theory that osteoblasts are derived from multipotent mesenchymal cells. It is possible that markers derived from these models could be used in the future to extend our knowledge of bone formation in vivo.

Animals↗

PubMed1991

Stimulation of cell proliferation in skeletal tissues of the rat by defined parathyroid hormone fragments.

We have found, in previous studies in vitro using skeletal derived cell cultures, that mid-region fragments of human parathyroid hormone (hPTH) stimulate [3H]thymidine incorporation into DNA and increase the specific activity of the brain-type isoenzyme of creatine kinase (CK). These changes occurred without an increase in cyclic AMP formation which is linked to bone resorption. In this study, we found that the mid-region fragment hPTH-(28-48) stimulated CK activity in diaphysis, epiphysis and kidney in a time- and dose-dependent manner, parallel to the effects of the whole molecule bovine (b)PTH-(1-84) and the fully active fragment hPTH-(1-34). The increase caused by hPTH-(28-48) at a dose of 1.25 micrograms/rat was not less than the 2-fold increase caused by a roughly equimolar concentration bPTH-(1-84). A significant increase was reached at 1 h after intraperitoneal injection in all cases. All three sequences of PTH caused an increase in [3H]thymidine incorporation into DNA in diaphysis and epiphysis, but not in kidney, 24 h after injection. A fragment further towards the C-terminal, hPTH-(34-47), was inactive compared with an equimolar concentration of the fragment hPTH-(25-39), which stimulated both CK activity and DNA synthesis. These results in vivo are in line with previous findings in vitro; they provide further support for the suggestion that mid-region fragments of the PTH molecule could be used to induce bone formation without incurring the deleterious effect of bone resorption.

Animals↗

PubMed1991

Investigation of the solution structure of the human parathyroid hormone fragment (1-34) by 1H NMR spectroscopy, distance geometry, and molecular dynamics calculations.

The structure of human parathyroid hormone fragment (1-34) in a solvent mixture of water and trifluoroethanol has been determined by 1H nuclear magnetic resonance spectroscopy and a combination of distance geometry and molecular dynamic simulations. After complete assignment of the 1H signals, the nuclear Overhauser enhancement data imply the existence of two alpha-helices, comprising residues 3-9 and 17-28, joined by a nonstructured region. The absence of any long-range NOEs and the relative magnitudes of the sequential NOEs and the 3J(HNH alpha) values reflect an inherent flexibility within the entire fragment. The final structures refined by molecular dynamics further support the above results and allow discussion of structural-activity relationships.

Amino Acid Sequence↗

PubMed1990

Stimulation by defined parathyroid hormone fragments of cell proliferation in skeletal-derived cell cultures.

We have reported previously that parathyroid hormone (PTH) acts on cultured bone cells to stimulate creatine kinase (CK) activity and [3H]thymidine incorporation into DNA via phosphoinositide turnover, in addition to its other actions via increased cyclic AMP production. We also found that mid-region fragments of PTH stimulate [3H]thymidine incorporation into avian chondrocytes. In the present study of mammalian systems, we demonstrate differential effects of defined synthetic PTH fragments on CK activity and DNA synthesis, as compared with cyclic AMP production, in osteoblast-enriched embryonic rat calvaria cell cultures, in an osteoblast-like clone of rat osteosarcoma cells (ROS 17/2.8) and in chondroblasts from rat epiphysial cartilage cell cultures. Unlike full-length bovine (b)PTH-(1-84) or the fully effective shorter fragment human (h)PTH-(1-34), fragments lacking the N-terminal region of the hormone did not increase cyclic AMP formation, whereas they did stimulate increases in both DNA synthesis and CK activity. Moreover, the PTH fragment hPTH-(28-48) at 10 microM inhibited the increase in cyclic AMP caused by 10 nM-bPTH-(1-84). The increase of CK activity in ROS 17/2.8 cells caused by bPTH-(1-84) or hPTH-(28-48) was completely inhibited by either cycloheximide or actinomycin D, as was shown previously for rat calvaria cell cultures. These results indicated the presence of a functional domain of PTH in the central part of the molecule which exerts its mitogenic-related effects on osteoblast- and chondroblast-like cells in a cyclic AMP-independent manner. Since cyclic AMP formation by PTH leads to bone resorption, specific mid-region fragments of PTH might prove suitable for use in vivo to induce bone formation without concomitant resorption.

Animals↗

PubMed1990

The promoter of the human parathyroid hormone gene contains a functional cyclic AMP-response element.

We have screened the sequence of the 394 base pairs upstream of the main transcriptional start site of the promoter of the human parathyroid hormone (PTH) gene for well-known protein recognition motifs with the aim to identify potential positive or negative regulatory elements. Within this region we found a potential cAMP-response element (CRE) besides several other putative binding sites for transcription factors. We fused promoter regions that contain this element and extend beyond the transcription start site to an appropriate reporter gene (CAT) and transfected different cell lines with these constructs. Transient expression of the CAT gene from these hybrid genes could be shown to be significantly stimulated by forskolin or isoproterenol thus proving the responsiveness of the whole promoter region towards elevated cAMP levels. DNase I protection studies revealed protein binding around the putative CRE (PTH-CRE) and an adjacent CCAAT element. Gel retardation assays with the PTH-CRE as well as the well-characterized CRE from the rat somatostatin promoter indicated specific binding of the same protein to both elements, although with a slightly reduced affinity of the PTH-CRE. Both of these elements were also able to confer cAMP-responsiveness to a heterologous promoter.

Animals↗