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Biomedical subjects

G D Offner

Publications and source records attributed to G D Offner.

At least 37 records · Page 2Linked to original sources
PubMed1995

Interaction of bovine gallbladder mucin and calcium-binding protein: effects on calcium phosphate precipitation.

BACKGROUND & AIMS: Gallstones consist of calcium salts and cholesterol crystals, arrayed on a matrix of gallbladder mucin (GBM), and regulatory proteins like calcium-binding protein (CBP). To determine if interactions between CBP and GBM follow a biomineralization scheme, their mutual binding and effects on CaHPO4 precipitation were studied. METHODS: Binding of CBP to GBM was assessed by inhibition of the fluorescence of the complex of GBM with bis-1,8-anilinonaphthalene sulfonic acid (bis-ANS). The effects of the proteins on precipitation of CaHPO4 were assessed by nephelometry and gravimetry. Precipitates were analyzed for calcium, phosphate, and protein. RESULTS: CBP and bis-ANS competitively displaced each other from 30 binding sites on mucin, with a 1:1 stoichiometry and similar affinity. The rate of precipitation of CaHPO4 was retarded by mucin and CBP. Precipitate mass was unaffected by GBM alone but decreased with the addition of CBP. Complexing CBP with GBM abolished or moderated this latter effect, altered precipitate morphology, and changed the stoichiometric ratios of Ca to PO4 in the precipitates from 1:1 to 3:2. Mucin and CBP were incorporated into the precipitates. CONCLUSIONS: These studies suggest that the formation of calcium-containing gallstones is a biomineralization process regulated by both GBM and CBP.

Anilino Naphthalenesulfonates↗
PubMed1995

Mucin-vesicle interactions in model bile: evidence for vesicle aggregation and fusion before cholesterol crystal formation.

Nucleation of cholesterol monohydrate crystals from bile is a critical step in the formation of cholesterol gallstones. Measurement of nucleation in model bile system and the characteristics of the initial nucleus have proven elusive. In this study we have used three separate physical chemical techniques to examine vesicle aggregation and fusion, including dynamic light scattering (DLS), transmission electron microscopy (TEM), and fluorescent biochemical assays. These assays enabled us to quantify the effect of biliary proteins, such as gallbladder mucin, on vesicle fusion and aggregation. In the absence of mucin, fusion is a relatively slow process occurring over 24 hours, whereas physiological concentrations of mucin are able to accelerate almost complete fusion of vesicles within 6 hours. Vesicle fusion and aggregation as characterized by TEM result in the formation of aggregates of multilamellar vesicles and giant fusion bodies associated with a background of mucin. These mucin-vesicle aggregate bodies may represent true nuclei and precede cholesterol monohydrate crystal nucleation. In future studies, these vesicle fusion assays can be used to quantitatively examine the effect of putative pro- and anti-nucleating proteins on the earliest steps of cholesterol crystal nucleation.

Animals↗
PubMed1994

Identification of a 130-kilodalton human biliary concanavalin A binding protein as aminopeptidase N.

BACKGROUND/AIMS: Human gallbladder bile contains a group of nonmucin glycoproteins that binds to the lectin concanavalin A (con A) and has been reported to promote cholesterol monohydrate crystal nucleation, an event preceding the formation of gallstones. Several of these proteins, including a 130-kilodalton protein, have been isolated and shown to promote nucleation in vitro. The aim of this study was to identify this and other major biliary con A binding glycoproteins. METHODS: Gallbladder bile was chromatographed on con A agarose, and the eluted proteins were electrophoresed, blotted, and subjected to amino-terminal sequence analysis. RESULTS: The major con A binding proteins were identified as aminopeptidase N (a 130-kilodalton protein), alpha 2 macroglobulin, hemopexin, immunoglobulin heavy chains, and the beta chain of haptoglobin. After further purification, aminopeptidase N was found to be enzymatically active and to promote cholesterol crystallization at its approximate physiological concentration in bile. CONCLUSIONS: It is likely that aminopeptidase N is the previously characterized 130-kilodalton biliary crystallization promoting protein. Aminopeptidase N is probably released from the biliary canalicular membrane by the detergent activity of bile salts and may be one factor that promotes cholesterol crystallization in the gallbladder.

Amino Acid Sequence↗
PubMed1993

Cholesterol cholelithiasis in the prairie dog: role of mucin and nonmucin glycoproteins.

The aims of this study were to examine the effect of exogenous prostaglandin on mucin secretion and to determine the role of nonmucin glycoproteins on gallstone formation in the prairie dog model of cholesterol cholelithiasis. The concentration of total glycoprotein and nonmucin glycoproteins was measured in gallbladder bile from four groups of prairie dogs fed a control diet or a diet supplemented with 1.2% cholesterol with or without simultaneous subcutaneous administration of prostaglandin E1. Cholesterol feeding resulted in an increased concentration of concanavalin-A binding-proteins in gallbladder bile associated with an increase in pronucleating activity in vitro. Treatment with prostaglandin E1 and cholesterol feeding was associated with a significant increase in the incidence of cholesterol gallstone formation. Prostaglandin E1 treatment in the cholesterol-fed animals increased biliary concentrations of total glycoprotein and concanavalin-A-binding glycoproteins. Therefore the increased biliary glycoprotein level in cholesterol-fed, prostaglandin E1-treated prairie dogs, which reflects higher levels of mucin and nonmucin glycoproteins, appears to be an important factor in gallstone formation.

Alprostadil↗
PubMed1993

Localization of the gene for human heart fatty acid binding protein to chromosome 1p32-1p33.

Heart fatty acid binding protein (hFABP) is an abundant 14-kDa cytosolic protein thought to be involved in trafficking of fatty acids from the plasma membrane to sites of beta-oxidation in mitochondria and peroxisomes and to the endoplasmic reticulum for lipid synthesis. A human hFABP cDNA isolated by polymerase chain reaction was used as a probe for in situ hybridization to metaphase chromosomes. A fragment of the gene for human hFABP was used as a probe for fluorescence in situ hybridization to metaphase chromosomes. The cDNA and genomic probes both localized the gene for human hFABP to chromosome 1p32-1p33.

Amino Acid Sequence↗
PubMed1992

Amino acid sequence of human plasma galactoglycoprotein: identity with the extracellular region of CD43 (sialophorin).

The amino acid sequence of galactoglycoprotein purified from human plasma was elucidated to 75% completeness by using chemical degradation of peptides and glycopeptides derived from digests of the protein with seven specific proteases. This sequence represents a polypeptide chain of approximately 220 amino acid residues including a high content of serine, threonine, alanine, and proline with one N-linked and multiple O-linked glycans. Comparison of peptide sequences from the native galactoglycoprotein and the deglycosylated derivative demonstrated the locations of 25 sites of O-glycosylation and three serine sites that are not glycosylated. The homogeneous N terminus was established as serine. C-terminal analysis revealed multiple C-terminal residues, suggesting that galactoglycoprotein molecules are of varying lengths. A search of a protein data base revealed that the galactoglycoprotein polypeptide is identical to the N-terminal (extracellular) polypeptide region of the blood-cell surface molecule CD43 (sialophorin, leukosialin). Further support of the relatedness of these molecules was obtained by immunoprecipitation of 125I-labeled galactoglycoprotein by monoclonal anti-CD43 antibodies. The composition and properties of the molecules together with the known structure of the gene encoding CD43 suggest that galactoprotein is derived by proteolytic cleavage from transmembrane "hexasaccharide CD43," known to be expressed on neutrophils, activated T lymphocytes, and platelets.

Amino Acid Sequence↗
PubMed1990

Structural studies on bovine spleen heme oxygenase. Immunological and structural diversity among mammalian heme oxygenase enzymes.

Heme oxygenase is an Mr 32,000 microsomal enzyme which catalyzes the rate-limiting step in the oxidative catabolism of heme to yield equimolar quantities of biliverdin IX alpha, carbon monoxide, and iron. In the present investigation, evidence is presented suggesting that immunochemical and structural differences exist between bovine spleen heme oxygenase and heme oxygenase enzymes from other mammalian species. Using an antibody directed against bovine spleen heme oxygenase, enzyme-linked immunosorbent assays, Western blotting experiments, and cell-free translation immunoprecipitation studies showed that bovine spleen heme oxygenase is only weakly immunochemically related to heme oxygenase from rat spleen. This observation was supported by the fact that a rat spleen heme oxygenase cDNA probe did not hybridize significantly to bovine spleen heme oxygenase mRNA in Northern analyses nor to restriction fragments containing the bovine heme oxygenase gene in Southern analyses. Tryptic peptides were prepared from bovine spleen heme oxygenase and the amino acid sequences of nine peptides comprising 94 amino acid residues were determined, providing the first information on the primary structure of bovine spleen heme oxygenase. Comparison of the sequences of these tryptic peptides with regions of the deduced amino acid sequences of rat spleen and human macrophage heme oxygenase revealed sequence similarities ranging from 55 to 100%. Several peptides displaying the highest degree of sequence similarity were found to occur in regions of the heme oxygenase molecule postulated to contain the heme binding site, indicating that despite the immunochemical and apparent structural differences between bovine spleen heme oxygenase and the rat and human enzymes, functionally important amino acid residues have been conserved in the evolution of mammalian heme oxygenase genes.

Amino Acid Sequence↗
PubMed1990

Cloning of rat aorta lysyl oxidase cDNA: complete codons and predicted amino acid sequence.

Lysyl oxidase cDNA clones were identified by their reactivity with anti-bovine lysyl oxidase in a neonatal rat aorta cDNA lambda gt11 expression library. A 500-bp cDNA sequence encoding four of six peptides derived from proteolytic digests of bovine aorta lysyl oxidase was found from the overlapping cDNA sequences of two positive clones. The library was rescreened with a radiolabeled cDNA probe made from one of these clones, thus identifying an additional 13 positive clones. Sequencing of the largest two of these overlapping clones resulted in 2672 bp of cDNA sequence containing partial 5'- and 3'-untranslated sequences of 286 and 1159 nucleotides, respectively, and a complete open reading frame of 1227 bp encoding a polypeptide of 409 amino acids (46 kDa), consistent with the 48 +/- 3 kDa cell-free translation product of rat smooth muscle cell RNA that was immunoprecipitated by anti-bovine lysyl oxidase. The rat aorta cDNA-derived amino acid sequence contains the sequence of each of the six peptides isolated and sequenced from the 32-kDa bovine aorta enzyme, including the C-terminal peptide with sequence identity of 96%. Northern blots screened with lysyl oxidase cDNA probes identified hybridizing species of 5.8 and 4.5 kb in mRNA of rat aorta and lung, while dot blot analyses were negative for lysyl oxidase mRNA in preparations of rat brain, liver, kidney, and heart. A 258-bp segment of the 3'-untranslated region of lysyl oxidase cDNA is 93% identical with a highly conserved region of the 3'-untranslated sequence of rat elastin cDNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1990

Molecular cloning of human submandibular histatins.

Histatins are a group of histidine-rich polypeptides found in human parotid and submandibular gland secretions. These polypeptides are microbiocidal, possibly involved in maintaining the acquired enamel pellicle, and enhance the glycolytic activity of certain oral micro-organisms. Histatins 1, 3 and 5 are homologous proteins with 38, 32 and 24 amino acid residues, respectively; the cDNAs coding for histatins 1 and 3 have now been isolated and sequenced. The cDNA sequences were highly homologous but contained differences throughout their length, indicating that they arise from different genes that may be derived from a common ancestral gene. Northern blots were hybridized to a series of oligonucleotide probes, designed on the basis of histatin cDNA sequences, and these positively identified mRNAs for histatins 1 and 3. In addition, there was a third mRNA, which hybridized to several histatin oligonucleotide probes, suggesting that histatin 5 might be derived from a distinct mRNA and not by proteolytic processing of histatin 3. A Northern blot of macaque parotid gland total RNA also showed three histatin mRNAs, indicating that similar histatins exist in a non-human primate.

Amino Acid Sequence↗
PubMed1990

Isolation and characterization of peptides from the protein core of bovine gallbladder mucin.

Gallbladder mucin may promote cholesterol gallstone formation by accelerating cholesterol monohydrate crystal nucleation in supersaturated bile. In this study, peptides were isolated from the mucin protein core by protease digestion and molecular-sieve high-performance liquid chromatography. Tryptic peptides were purified by anion exchange or reverse-phase high-performance liquid chromatography, and amino acid compositions were determined. Tryptic peptides were (a) nonglycosylated, (b) selectively enriched in serine, glutamic acid plus glutamine, and glycine, and (c) depleted in threonine and proline compared with native gallbladder mucin. Bilirubin derivatized with Woodward's reagent K covalently bound to purified mucin. Tryptic digestion of the mucin-bilirubin complex yielded low-molecular-weight nonglycosylated peptides with covalently bound bilirubin. These data indicate that the mucin protein core contains at least two distinct domains. One domain is rich in threonine and proline and contains the majority of covalently bound carbohydrate. A second domain, possibly internally located, is nonglycosylated, enriched in serine, glutamic acid plus glutamine, and glycine, and binds hydrophobic ligands such as bilirubin and 1-anilino-8-naphthalene sulfonate. Hydrophobic domains on the mucin protein core may contribute to the pathogenesis of cholesterol cholelithiasis.

Amino Acids↗
PubMed1990

Characterization of bovine gallbladder mucin. Amino acid sequences of tryptic peptides from the glycosylated domain of the protein core.

Gallbladder mucin is a densely glycosylated macro-molecule that promotes cholesterol gallstone formation in experimental animals and in humans. Bovine gallbladder mucin structure was studied after chemical deglycosylation by treatment with anhydrous hydrogen fluoride at 23 degrees C for 3 hours. Deglycosylated mucin contained less than 5% of the amino sugar and neutral hexose content of native mucin. Electrophoretic and molecular sieve chromatographic analyses indicated that significant cleavage of the mucin polypeptide core had occurred during deglycosylation. Deglycosylated mucin was separated into three major fractions by reverse-phase chromatography, one of which was enriched with respect to threonine and proline. Tryptic peptides prepared from this fraction were purified by molecular sieve and reverse-phase chromatography, and the amino acid sequences (8-20 residues) of the four principal tryptic peptides were determined. These peptides contained 65%-75% threonine and proline residues and demonstrated 80%-100% sequence similarity. These data provide the first information on the primary structure of gallbladder mucin and suggest that repeating amino acid sequences occur in this protein. Comparison of gallbladder mucin peptide structure with the consensus repeat sequence of human intestinal mucin showed approximately 60% sequence similarity. It was concluded that mammalian gastrointestinal mucins may be derived from a common ancestral gene.

Amino Acid Sequence↗
PubMed1990

Studies on Cyanidium caldarium Phycobiliprotein Pigment Mutants.

Phycobiliprotein biosynthesis was investigated in four strains of the unicellular rhodophyte, Cyandium caldarium, with different pigment phenotypes. All strains were incapable of synthesizing phycobiliproteins when grown in the dark. Western blotting experiments showed that dark-grown cells of the wild-type and mutant GGB synthesized the alpha and beta subunit polypeptides of allophyocyanin and phycocyanin after exposure to light for 24 hours, whereas cells of mutant IIIC and GGBY did not. Similarly, light promoted the appearance of allophycocyanin and phycocyanin mRNAs in the wild-type and GGB but not in IIIC and GGBY. However, Southern blots of restricted genomic DNA from the wild type, IIIC, GGBY, and GGB, all hybridized with heterologous phycobiliprotein gene probes and revealed that all four strains contained identical Pst, EcoRI, and Dral restriction fragments containing allophycocyanin and phycocyanin genes. Cells of the wild type and GGB incubated in the dark with the heme precursor. delta-aminolevulinate, synthesized allophycocyanin and phycocyanin apoproteins providing strong evidence for the role of a tetrapyrrole in regulation of phycobiliprotein gene expression. However, cells of IIIC and GGBY incubated in the dark with delta-aminolevulinate did not contain detectable quantities of allophycocyanin or phycocyanin apoproteins. The possible role of a tetrapyrrole in phycobiliprotein gene expression and basis for the genetic lesion in mutants IIIC and GGBY is discussed.

Journal Article↗
PubMed1990

Structural relationship between human salivary histatins.

Histatins are a group of electrophoretically distinct histidine-rich polypeptides with microbicidal activity found in human parotid and submandibular gland secretions. Recently, we have shown that histatins 1, 3, and 5 are homologous proteins that consist of 38, 32, and 24 amino acid residues, respectively, and that these polypeptides kill the pathogenic yeast, Candida albicans. We now describe the isolation and structural characterization of histatins 2, 4, 6, and 7-12, the remaining members of this group of polypeptides. Histatin 2 was found to be identical to the carboxyl terminal 26 residues of histatin 1; histatin 4 was found to be identical to the carboxyl terminal 20 residues of histatin 3; and histatin 6 was found to be identical to histatin 5, but contained an additional carboxyl terminal arginine residue. The amino acid sequences of histatins 7-12 formally correspond to residues 12-24, 13-24, 12-25, 13-25, 5-11, and 5-12, respectively, of histatin 3, but could also arise proteolytically from histatin 5 or 6. These results establish, for the first time, the complete structural relationships between all members of this group of microbicidal proteins in human parotid saliva. The relationship of histatins to one another is discussed in the context of their genetic origin, biosynthesis and secretion into the oral cavity, and potential as reagents in anti-candidal studies.

Amino Acid Sequence↗
PubMed1989

Heme regulates expression of phycobiliprotein photogenes in the unicellular rhodophyte, Cyanidium caldarium.

Allophycocyanin and phycocyanin in the red alga (Cyanidium caldarium) are chloroplast-encoded, light-harvesting accessory pigments composed of alpha and beta subunit polypeptides (17-19 kDa) to which 1 or more residues of the heme-derived bile pigment chromophore phycocyanobilin are attached by cysteinyl thioether linkages (Offner, G.D., and Troxler, R.F. (1983) J. Biol. Chem. 258, 9931-9940). Western blot experiments utilizing phycobiliprotein antisera revealed that immunoreactive allophycocyanin and phycocyanin apoproteins were absent in cells grown in the dark and present in cells exposed to light. Northern blot experiments using genomic DNA hybridization probes indicated that phycobiliprotein mRNAs were absent in the dark, whereas cells exposed to light contained two allophycocyanin mRNA transcripts, 1.4 and 1.6 kilobases in length, and one phycocyanin mRNA transcript, 3.0 kilobases in length, providing evidence that phycobiliproteins are encoded in photogenes which are only transcriptionally active in the light. Northern and Western analyses demonstrated that cells incubated in the dark with the heme precursor delta-aminolevulinate contained allophycocyanin and phycocyanin mRNAs and apoproteins, indistinguishable in size, number, and quantity from those made in the light. Cells incubated in the dark with delta-aminolevulinate, protoporphyrin IX, or heme, but not biliverdin or phycocyanobilin, synthesized allophycocyanin and phycocyanin alpha and beta apoproteins, suggesting a role for heme in the control phycobiliprotein gene expression. Cells incubated with heme in the dark produced allophycocyanin and phycocyanin mRNA transcripts, but did not produce mRNAs for four other photogenes coding for a P-700 reaction center protein, a 32-kDa herbicide-binding protein, and the large and small subunits of ribulose-bisphosphate carboxylase. These results show, for the first time, that heme is a regulatory factor specifically involved in transcriptional regulation of chloroplast genes for phycobiliproteins.

DNA Probes↗
PubMed1989

Role of tropoelastin fragmentation in elastogenesis in rat smooth muscle cells.

Neonatal rat aortic smooth muscle cell cultures produce two major soluble elastin molecules termed protropoelastin (77 kDa) and tropoelastin (71 kDa). Cell layer extracts are protroproelastin-enriched, while protropoelastin, tropoelastin, and significant amounts of discrete elastin fragments (Mr of 66,000, 61,000, 56,000, and 45,000) are present in preparations from the medium of these cultures. To determine the role of the various elastin molecules in the metabolism of elastin in neonatal rat aortic smooth muscle cell cultures, the amino termini of these proteins were sequenced. All soluble elastin components present in the medium were purified as a single peak by high performance liquid chromatography; further separation of the components was achieved by polyacrylamide gel electrophoresis and electroblotting. The bands were excised and sequenced. The amino-terminal sequences of protropoelastin, tropoelastin, and the 66-kDa, 61-kDa, and 56-kDa fragments were identical: Gly-Gly-Val-Pro-Gly-Ala-Val-Pro-Gly-Gly. This sequence is identical with published amino-terminal sequences of tropoelastins from several other species. As expected, when the cell cultures were pulsed with [3H]valine, all the soluble elastin molecules were radioactive, while only protropoelastin appeared radioactive after [35S] cysteine pulsing. Since cysteine is present only in the carboxyl-terminal end of the molecule, all the data indicate that the cleavage of the elastin fragments identified in the culture are occurring at the carboxyl end of protropoelastin. These results are consistent with the original hypothesis that a precursor-product relationship exists between the 77-kDa and 71-kDa soluble elastin molecules. Based on known tropoelastin sequences and the molecular weights of the discrete fragments, additional fragmentation of protropoelastin and/or tropoelastin most likely occurs at the lysine/alanine-enriched domains presumably involved in cross-link formation.

Amino Acid Sequence↗
PubMed1988

Histatins, a novel family of histidine-rich proteins in human parotid secretion. Isolation, characterization, primary structure, and fungistatic effects on Candida albicans.

Histatins 1, 3, and 5 from human parotid secretion were isolated by gel filtration on Bio-Gel P-2 and reverse phase high performance liquid chromatography. The complete amino acid sequences of histatins determined by automated Edman degradation of the proteins, Staphylococcus aureus V8 protease, and tryptic peptides, are as follows: (Sequence: see text). Histatins 1, 3, and 5 contain 38, 32, and 24 amino acid residues, have molecular weights of 4929, 4063, and 3037, respectively, and contain 7 residues of histidine. Histatin 1 contains 1 mol of phosphate/mol of protein; histatins 3 and 5 lack phosphate. With the exception of Glu (residue 4) and Arg (residue 11) in histatin 1, the first 22 amino acid residues of all three histatins are identical, and the carboxyl-terminal 7 residues of histatins 1 and 3 are also identical. The sequence, -Glu-Phe-Pro-Phe-Tyr-Gly-Asp-Tyr-Gly- (residues 23-29), in histatin 1 is absent in histatin 3; and the sequence, -Gly-Tyr-Arg- (residues 23-25), in histatin 3 is absent in histatin 1. The complete sequence of histatin 5 is contained within the amino terminal 24 residues of histatin 3. The structural data suggest that histatins 1 and 3 are derived from different structural genes, whereas histatin 5 is a proteolytic product of histatin 3. All three histatins exhibit the ability to kill the pathogenic yeast, Candida albicans.

Amino Acid Sequence↗
PubMed1988

Characterization and amino acid sequence of a fatty acid-binding protein from human heart.

The complete amino acid sequence of a fatty acid-binding protein from human heart was determined by automated Edman degradation of CNBr, BNPS-skatole [3'-bromo-3-methyl-2-(2-nitrobenzenesulphenyl)indolenine], hydroxylamine, Staphylococcus aureus V8 proteinase, tryptic and chymotryptic peptides, and by digestion of the protein with carboxypeptidase A. The sequence of the blocked N-terminal tryptic peptide from citraconylated protein was determined by collisionally induced decomposition mass spectrometry. The protein contains 132 amino acid residues, is enriched with respect to threonine and lysine, lacks cysteine, has an acetylated valine residue at the N-terminus, and has an Mr of 14768 and an isoelectric point of 5.25. This protein contains two short internal repeated sequences from residues 48-54 and from residues 114-119 located within regions of predicted beta-structure and decreasing hydrophobicity. These short repeats are contained within two longer repeated regions from residues 48-60 and residues 114-125, which display 62% sequence similarity. These regions could accommodate the charged and uncharged moieties of long-chain fatty acids and may represent fatty acid-binding domains consistent with the finding that human heart fatty acid-binding protein binds 2 mol of oleate or palmitate/mol of protein. Detailed evidence for the amino acid sequences of the peptides has been deposited as Supplementary Publication SUP 50143 (23 pages) at the British Library Lending Division, Boston Spa, Yorkshire LS23 7BQ, U.K., from whom copies may be obtained as indicated in Biochem. J. (1988) 249, 5.

Amino Acid Sequence↗