Biomedical subjects

G Kresbach

Publications and source records attributed to G Kresbach.

5 recordsLinked to original sources

PubMed1998

A quantitative study of optical mapping surfaces by atomic force microscopy and restriction endonuclease digestion assays.

Many new techniques in biomolecular chemistry and genomic analysis require the immobilization of molecular reagents on specially prepared surfaces. However, the process of molecular fixation often interferes with or precludes the use of standard in vitro biochemical assays. Optical mapping is an emergent technology for genomic analysis which relies on the biochemical activity of DNA fixed to silanized glass surfaces. Optical mapping surfaces have been shown to be compatible with restriction endonucleases and a variety of DNA polymerases. The essential properties of biochemically active surfaces are poorly understood in most of the current technologies which utilize molecular fixation, including optical mapping. The purpose of this study is to use the powerful technique of atomic force microscopy, in combination with informative enzymatic assays, to correlate biochemical activity with microscopic surface structure. The results presented provide meaningful insight into the effect of surface preparation on the biochemical accessibility of surface-bound molecules. Novel analysis which may facilitate the automation of optical mapping is presented.

DNA↗

PubMed1995

Evaluation of the cell specificity and sulfate dependence of glomerular extracellular matrix proteoglycan synthesis.

Homogeneous cultures of epithelial, endothelial, and mesangial cells from calf glomeruli were radiolabeled with [35S]sulfate in order to evaluate their capacity for the biosynthesis of the proteoglycan (PG) components present in the glomerular extracellular matrix. Although each cell type was observed to incorporate into its matrix predominantly immunologically related heparan sulfate (HS) PGs (M(r) approximately 500 kDa), endothelial and mesangial cells also deposited substantial amounts of PGs with chondroitin sulfate (CS) and dermatan sulfate (DS) chains. The limited capacity of epithelial cells to synthesize PGs other than those containing HS was also evident from the immunologically distinct components (M(r) approximately 300 kDa) shed into the medium which in contrast to those from the endothelial and mesangial cells contained no CS and only small amounts of DS glycosaminoglycans. While the matrix proteoglycan HS chains differed in length depending on cell type, they were similar in containing the six mono- and disulfated disaccharide species previously found in bovine glomerular basement membrane, including the distinctive iduronic-GlcNSO3 (3-SO4) sequences. While the addition of sulfate to medium free of this ion brought about no change in HS PG production by any of the three cell types and the formation of CS and DS chains by epithelial and mesangial cells was unaffected, the formation of CS/DS PGs by endothelial cells was altered to a pronounced extent through the conversion of an undersulfated PG to a more polyanionic molecule. Our findings are consistent with the concept that the glomerular extracellular matrix is made up of two biosynthetically distinct regions (mesangium and basement membrane) and are relevant to an understanding of various diseases affecting the renal filter.

Animals↗

PubMed1995

Stable expression in Chinese hamster ovary cells of a homogeneous recombinant active fragment of human platelet glycoprotein Ib alpha.

A fragment (residues His1-Val289) of the alpha chain of human platelet glycoprotein Ib containing the von Willebrand factor and thrombin binding sites has been expressed in Chinese hamster ovary cells. The secreted soluble recombinant protein had an apparent molecular mass of 42 kD and reacted with a conformation-dependent monoclonal antibody that only binds to native GP Ib, thus demonstrating its proper folding. The rather broad band obtained after immobilization of the recombinant fragment on nitrocellulose could be resolved into a very sharp band of molecular weight of about 35 kD by growing the cells in the presence of tunicamycin, an inhibitor of N-linked glycosylation. The recombinant GP Ib alpha fragments (with or without glycosylation) were purified by immunoaffinity chromatography. Both truncated forms bound vWF in the presence of botrocetin with comparable affinity as a proteolytic 42 kD fragment of purified human platelet GP Ib-IX. They were also retained on thrombin-Sepharose. We then selected a cell clone (B1) that produced over at least three months about 1.5 micrograms of recombinant protein per million cells per day. Using this clone a large-scale production finally yielded milligram amounts of the functionally active recombinant human GP Ib alpha fragment.

Animals↗

PubMed1993

Expression and characterization of functionally active fragments of the platelet glycoprotein (GP) Ib-IX complex in mammalian cells. Incorporation of GP Ib alpha into the cell surface membrane.

The platelet glycoprotein Ib-IX complex (GP Ib-IX) is essential for the initial attachment of platelets to the wall of damaged arteries. In this study, an N-terminal fragment of human GP Ib alpha (residues 1-318), containing the ligand binding sites for von Willebrand factor (vWF) and thrombin, as well as the entire human GP Ib alpha were expressed in Chinese hamster ovary cells. The transfected cells secreted a 48- and a 110-kDa protein, respectively, into the supernatant. Both recombinant proteins were purified by immunoaffinity chromatography. The purified proteins bound soluble vWF in the presence of botrocetin as demonstrated in solid-phase binding assays. The dissociation constant (Kd) for 125I-vWF binding to the recombinant 110-kDa protein was 1.2 +/- 0.2 nM as compared with 1.0 +/- 0.3 nM for vWF binding to purified platelet GP Ib-IX. Both recombinant proteins were also retained on thrombin-Sepharose 4B. The 48-kDa protein contained two N-linked oligosaccharide chains. A 125-kDa protein was identified in the lysate of cells transfected with the coding sequence for the entire GP Ib alpha. Trypsin treatment of this protein generated a 110-kDa fragment, whereas the secreted 110-kDa protein remained unchanged. Post-translational removal of the C-terminal transmembrane domain of recombinant GP Ib alpha might have facilitated the secretion of the soluble glycocalicin-like 110-kDa fragment. In addition, flow cytometry and immunofluorescence microscopy demonstrated that the expression of GP Ib alpha alone is sufficient for its incorporation into the cell surface membrane. These data indicate that two soluble fragments of human GP Ib alpha with binding activity for vWF and thrombin can be expressed in mammalian cells and that the incorporation of GP Ib alpha into the surface membrane does not depend on co-expression with GP Ib beta and/or GP IX.

Animals↗

PubMed1988

Pentafluorobenzylation of alkyl and related DNA base adducts facilitates their determination by electrophore detection.

The DNA adduct O4-ethylthymine can be alkylated under mild conditions with pentafluorobenzyl bromide. The product has good gas chromatographic characteristics and also forms a structurally characteristic anion in high yield when subjected to electron capture mass spectrometry. Related adducts for other DNA bases behave similarly. These properties stimulated us to develop a general analytical method based on the derivatization reaction. Important for this method is an oxidation-elimination reaction that mildly releases a base from a nucleoside. Thus, a general analytical method based on pentafluorobenzylation is now available for determining many alkyl and related DNA adducts.

Alkylation↗