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Biomedical subjects

E Wingender

Publications and source records attributed to E Wingender.

At least 55 records · Page 3Linked to original sources
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↗
PubMed1989

The central part of parathyroid hormone stimulates thymidine incorporation of chondrocytes.

The stimulation of DNA synthesis in primary cell cultures of chicken chondrocytes by parathyroid hormone was studied by assaying [3H]thymidine incorporation into DNA. Optimal assay conditions were determined by varying cell age, plating density, and incubation time. Under these conditions DNA synthesis was significantly stimulated by parathyroid hormone (PTH) and some of its fragments: cells treated with human (h)PTH(1-84), bovine (b)PTH(1-34) and [Nle8,18,Tyr34]bPTH(3-34)amide and hPTH(13-34) displayed 2.6-fold enhanced [3H]thymidine incorporation in a dose-dependent manner. The fragment hPTH(28-48) led to a similar stimulation, whereas [Tyr43]hPTH(43-68) and [Tyr52,Asp76]hPTH(52-84) had no effect. Using a series of synthetic hPTH peptides covering the central region of the hormone molecule (residues 25-47), we could delimitate further this putative mitogenic functional domain to a core region between amino acid residues 30 and 34. The effect of PTH on [3H]thymidine incorporation could not be mimicked by forskolin, indicating that the corresponding signal is not mediated by cAMP. It is, however, inhibited by EGTA and cannot be provoked in the absence of calcium ions in the medium. Therefore, the results presented indicate a hitherto unidentified functional domain of PTH in the central part of the molecule which exerts its mitogenic effect on chondrocytes in a cAMP-independent manner but seems to involve calcium ions for signal transduction.

Amino Acid Sequence↗
PubMed1989

Expression of human parathyroid hormone in Escherichia coli.

Human parathyroid hormone (PTH) has been expressed in Escherichia coli as a cro-beta-galactosidase-hPTH fusion protein under temperature-sensitive control of the lambda phage PR promoter. The lacZ gene has been truncated to a different extent revealing an optimal length of the prokaryotic peptide portion between 199 and 407 amino acid residues. Up to 250 mg of pure fusion protein have been obtained from 1-liter E. coli culture by stepwise solubilization with urea. The linkage between the prokaryotic and the eukaryotic protein moiety consists of an Asp-Pro peptide bond and therefore is easily cleavable by acid treatment. A simple procedure for the purification of the hormone is described. The resulting recombinant hormone reacts with anti-PTH antibodies and stimulates renal adenylate cyclase identically to bovine or human PTH.

Adenylyl Cyclases↗
PubMed1989

Purification of human transcription factor IIIA and its interaction with a chemically synthesized gene encoding human 5 S rRNA.

Transcription factor IIIA (TFIIIA) was purified from cytoplasmic extracts of HeLa cells by developing a simple and efficient procedure employing phosphocellulose under widely differing ionic conditions followed by affinity chromatography on immobilized human 5 S genes. This procedure yielded a fraction containing human TFIIIA activity and a protein of 35 kDa as its major component. Moreover, we succeeded in renaturing the activity of human transcription factor IIIA (hTFIIIA) isolated after preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis and in identifying a polypeptide of 35 kDa with the transcription activity. This value differs from that reported for Xenopus TFIIIA. It could be demonstrated by footprinting analyses that hTFIIIA specifically binds to the internal control region of the human 5 S rRNA gene. The limits of protection slightly differ at the 3' border of the internal control region from those imprinted by Xenopus TFIIIA on the same gene. Comparative footprint analyses of hTFIIIA on the human and frog somatic 5 S rRNA gene, measured in titration, competition, and salt-stability experiments, demonstrated a higher affinity of the human factor to the homologous gene. These results, together with the difference in molecular mass of these functionally analogous proteins, reemphasize the importance of homologous systems for the analysis of mechanisms involved in gene regulation.

Electrophoresis, Polyacrylamide Gel↗
PubMed1988

Complete synthesis and transcription in vitro of a gene coding for human ribosomal 5S RNA.

The gene coding for the major human ribosomal 5S RNA was chemically synthesized and cloned into a pUC13 vector. This approach was taken, because attempts to isolate the human 5S gene have thus far yielded either pseudogenes or variant 5S genes of unknown function. The synthetic human gene was transcribed by RNA polymerase III either in a crude HeLa cell extract or in a system reconstituted from partially purified transcription factors. Comparative studies with the Xenopus laevis somatic 5S gene show that the human gene is transcribed with similar fidelity and an efficiency of about 80% under optimal conditions. The time-course of transcription and optimal concentrations of template and transcription factors were found to be similar for both genes studied. The synthetic gene described may prove useful to study its interaction with human transcription factors in a homologous system.

Animals↗
PubMed1987

Transcription complexes for various class III genes differ in parameters of formation and stability towards salt.

RNA polymerase III faithfully transcribes the genes for ribosomal 5 S RNA, tRNA(1Met) or adenovirus VA RNA in vitro in the presence of required transcription factors. These genes display distinct differences in the kinetics of transcription complex formation and in their response to excess template. In contrast to tRNA and VA RNA synthesis, 5 S RNA synthesis displays a lag phase of 15 minutes before the onset of transcription and is clearly inhibited by high concentrations of template. Once formed, transcription complexes for the RNA polymerase III genes listed can be isolated by glycerol gradient centrifugation and display a remarkable stability against transient treatment with high salt concentrations. Complexes for 5 S RNA and tRNA remain functionally active up to 2.5 M-KCl. The activity of transcription complexes for VA RNA, however, is significantly diminished after treatment with high salt concentrations. This effect is shown to be due to an irreversible loss of transcription factors. RNA polymerase III is dissociated by high salt concentrations from all the transcription complexes studied but remains part of these complexes during the normal reinitiation cycle at 60 mM-KCl. An additional method for the purification of partial transcription complexes was developed that involves equilibrium centrifugation on cesium sulfate gradients. This method completely releases TFIIIB from 5 S complexes and a core complex, composed of the 5 S RNA gene, factors IIIA and IIIC, is retained. In the case of tRNA and VA RNA, core complexes are obtained that remain partly associated with TFIIIC and TFIIIB. These results indicate a qualitatively and/or quantitatively different interaction of individual factors in different polymerase III transcription complexes.

Genes↗
PubMed1986

Association of RNA polymerase III with transcription factors in the absence of DNA.

The gene for tRNAMet1 from Xenopus oocytes was transcribed in a cell free system with components isolated from a HeLa cell-free extract. It was found that, apart from the established assembly of transcription factors IIIB and IIIC on tRNA genes into stable transcription complexes, these factors can also associate with the enzyme in the absence of DNA to form a functional polymerase III complex. These complexes can be isolated in a highly active form from the bulk of other cellular proteins by mild methods such as gel filtration or density gradient centrifugation. When associated with RNA polymerase III into a functional complex, the transcription factors IIIB and IIIC can clearly be differentiated from free transcription factors, which individually display a much lower relative molecular mass. The polymerase complexes are stable against 1 M KCl, rendering unlikely that they represent fortuitous aggregates including RNA polymerase III and transcription factors IIIB and IIIC. These complexes are sensitive to dilution and, whereas transcription factor IIIC binds to the enzyme more tightly, factor IIIB tends to leak from the complex upon dilution of the protein concentration. From these results it is clear that in addition to their function as DNA-binding protein(s), transcription factors IIIB and IIIC can directly interact with RNA polymerase III without prior binding to the promoter region of the gene to be transcribed.

Animals↗
PubMed1984

Zinc ions are differentially required for the transcription of ribosomal 5S RNA and tRNA in a HeLa-cell extract.

Chelation of divalent cations by 5 mM EDTA and subsequent removal by dialysis from a cytoplasmic HeLa cell extract leads to a complete loss of 5S rRNA transcription without affecting tRNA synthesis. Transcription complexes for 5S RNA can no longer be assembled in such a zinc-depleted extract and this ability can be fully restored only by the re-addition of 5 microM zinc. Reconstitution experiments with isolated protein fractions show that transcription factor A from HeLa-cells requires zinc to exert its specific function. Pre-formation of transcription complexes partially protects the metal ion against removal by chelation even in the presence of 1.8 M KCl. These results indicate that the zinc ions are bound to mammalian transcription factor IIIA which, in a transcription complex, binds very strongly to the 5S RNA gene. Cation depletion with 75 mM EDTA also suppresses tRNA transcription; an effect which is reversible by zinc addition. We conclude that beside for the binding of TF IIIA, zinc is also bound with a different affinity to a transcription component common to 5S and tRNA synthesis, most likely polymerase III itself.

Edetic Acid↗
PubMed1984

Isolation of a transcription complex for ribosomal 5S RNA.

Cloned 5S rRNA genes from Xenopus borealis oocytes can be used to assemble functional transcription complexes from cytoplasmic HeLa cell extracts as a source for polymerase III and all factors additionally required for faithful 5S RNA transcription. Such complexes can be isolated by glycerol gradient ultracentrifugation and non-denaturing gel electrophoresis. They contain less than 1% of the cellular protein and retain their fidelity to synthesize 5S rRNA. The assembly of the complex is unaffected by KCl concentrations up to 140 mM whereas the transcription of 5S rRNA by the isolated complex is significantly reduced at this ionic strength. This indicates that the latter process, involving re-initiation by RNA polymerase III, is more sensitive to elevated salt concentrations than is the assembly of the transcription complexes. Furthermore, we show that complex formation also takes place in the absence of exogenously added nucleoside triphosphates, although this results in a slight shift in the sedimentation position which can be reversed by addition of the initial nucleotides GTP and CTP. We have analyzed the isolated transcription complexes by the protein blotting technique in an attempt to characterize their DNA-binding components. The results show a single component, corresponding to a protein with a mol. wt. of approximately 45 kd, which binds selectively, but not exclusively to a DNA fragment containing the 5S gene. The possible relationship of this protein to transcription factor IIIA from Xenopus oocytes is discussed.

Animals↗
PubMed1983

Faithful transcription of ribosomal 5-S RNA in vitro depends on the presence of several factors.

Cytoplasmic extracts from HeLa cells, capable of transcribing the cloned genes for ribosomal 5-S RNA, were employed to study the factors involved in this process. Two factors can be isolated, by gel filtration through Sephadex G-100, which are devoid of RNA polymerase activity. They significantly enhance the extent and specificity of the transcription of 5-S rRNA. Both proteins can jointly be purified by affinity chromatography on immobilized DNA containing the genes for ribosomal 5-S RNA from Xenopus borealis. Besides a protein of approximately 45 kDa, possibly corresponding to TF IIIA isolated from Xenopus oocytes, a second protein with a molecular mass of 22 +/- 1 kDa stimulates the formation of 5-S RNA. This protein is contained in the breakthrough of DEAE-cellulose; it binds to and is eluted from phosphocellulose with 0.6 M KCl. In addition, it was found that the exclusion volume obtained after gel filtration on Sephadex G-100 contains functional complexes, which are capable of transcribing the cloned 5-S genes and hence contain all the required factors. Direct evidence is presented that the protein of 22 kDa described above is contained in and can be isolated from such complexes. It is postulated from indirect evidence that an additional factor with a molecular mass in excess of 100 kDa is required which can be removed from functional polymerase complexes by gel filtration through Bio-Gel A5m.

Chromatography, DEAE-Cellulose↗