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R F Troxler

Publications and source records attributed to R F Troxler.

At least 55 records · Page 3Linked to original sources
PubMed1995

Nucleotide sequence and expression of the genes for the alpha and beta subunits of phycocyanin in Cyanidium caldarium.

The nucleotide sequence of the plastid-encoded operon containing genes for the alpha (cpcA) and beta (cpcB) subunits of phycocyanin in the unicellular red alga Cyanidium caldarium is described. cpcB is located 5' to cpcA and the two genes are separated by a 102-bp spacer region. The transcription start site of cpcBA was mapped to 80 bp upstream of the ATG initiation codon of cpcB. Promoter-like elements similar to the -10 (TATAAT) and -35 (TTGACA) consensus promoters in bacteria were found 6 and 31 bp upstream of the transcription initiation site. Northern blotting revealed an abundant 1.3-kb cpcBA transcript in illuminated cells, but this transcript was undetectable in dark-grown cells. Expression levels of cpcBA in cells incubated with 10(-6) M heme in the dark were similar to those in cells illuminated for 24 h. Cells illuminated with 150 microM gabaculine (an inhibitor of delta-aminolevulinate synthesis) or 10 mM levulinic acid (an inhibitor of delta-aminolevulinate dehydrase) lacked detectable cpcBA transcripts. In cells illuminated with 200 microM N-methyl-mesoporphyrin IX (an inhibitor of ferrocheletase), inhibition of cpcBA expression and phycocyanin synthesis was similar. These results provide strong evidence that light induction of the cpcBA operon is dependent on synthesis of heme.

Amino Acid Sequence↗
PubMed1995

Functional comparison of native and recombinant human salivary histatin 1.

Histatin 1 is a histidine-rich phosphoprotein present in human parotid saliva that possesses candidacidal activity and functions in mineralization by adsorbing to hydroxyapatite. The objective of the present study was to develop a system for recombinant production of histatin 1 and to examine the role of phosphorylation in the functional activities of this molecule. Native histatin 1 (containing a phosphoserine at residue 2) was purified from parotid saliva, whereas a bacterial expression system was used to produce a recombinant form of histatin 1 (re-Hst1) that lacked phosphorylated serine. Histatin 1 cDNA was inserted into the vector pGEX-3X, which expresses foreign genes as soluble fusion proteins attached to the carboxyl-terminus of glutathione S-transferase (GST). The GST/re-Hst1 fusion protein was isolated from cell lysates by affinity chromatography on glutathione (GSH)-Sepharose and digested with cyanogen bromide to separate re-Hst1 from the GST fusion partner. The digest was subjected to reversed-phase high-performance liquid chromatography on a C18 column, and re-Hst1 was eluted as a well-defined peak. The yield of re-Hst1 was 4 mg/L of bacterial culture. Amino-terminal sequencing and amino acid analysis confirmed the final product as re-Hst1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that native histatin 1 and re-Hst1 had the same apparent molecular weights, while cationic PAGE showed that re-Hst1 was more basic. Phosphate analysis indicated 1 mol phosphate/mol of native histatin 1, while re-Hst1 lacked any detectable phosphate. Re-Hst1 demonstrated candidacidal activity comparable to that of native histatin 1, but displayed substantially lower binding to hydroxyapatite. These results show that phosphorylation of histatin 1 at residue 2 contributes significantly to its ability to bind to hydroxyapatite.

Antifungal Agents↗
PubMed1994

Regulation of endothelial cell glyceraldehyde-3-phosphate dehydrogenase expression by hypoxia.

Exposure of endothelial cells (EC) to hypoxia results in the increased expression of a distinct set of proteins with molecular masses of 56, 47, 39, 36, and 34 kDa. Their induction appears to be unique to EC and the stress of decreased oxygen tension. To understand the mechanism(s) and significance of the up-regulation of these proteins we have identified the 36-kDa protein by limited amino-terminal amino acid sequencing. The 21-amino acid sequence from the bovine protein exhibited 90.5% identity with the human sequence of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Northern blot analysis showed that the time course and extent of EC GAPDH mRNA up-regulation correlated with the increase in 36-kDa protein synthesis. Nuclear runoff analysis demonstrated that this increase in GAPDH expression is regulated, in part, at the transcriptional level; however, the increase in the rate of transcription did not account for the entire mRNA accumulation, suggesting that GAPDH, like other hypoxia-regulated proteins, is posttranscriptionally regulated. Subcellular fractionation of hypoxic EC showed up-regulation of the 36-kDa protein in the cytoplasmic fraction and, to a lesser extent, in the nuclear fraction. The up-regulation of GAPDH in EC may be related to their relative hypoxia tolerance. Alternatively, the up-regulation of GAPDH in EC during hypoxia may be related to the potential nonglycolytic functions of this enzyme.

Amino Acid Sequence↗
PubMed1994

Evidence that sigma factors are components of chloroplast RNA polymerase.

Plastid genes are transcribed by DNA-dependent RNA polymerase(s), which have been incompletely characterized and have been examined in a limited number of species. Plastid genomes contain rpoA, rpoB, rpoC1, and rpoC2 coding for alpha, beta, beta', and beta" RNA polymerase subunits that are homologous to the alpha, beta, and beta' subunits that constitute the core moiety of RNA polymerase in bacteria. However, genes with homology to sigma subunits in bacteria have not been found in plastid genomes. An antibody directed against the principal sigma subunit of RNA polymerase from the cyanobacterium Anabaena sp. PCC 7120 was used to probe western blots of purified chloroplast RNA polymerase from maize, rice, Chlamydomonas reinhardtii, and Cyanidium caldarium. Chloroplast RNA polymerase from maize and rice contained an immunoreactive 64-kD protein. Chloroplast RNA polymerase from C. reinhardtii contained immunoreactive 100- and 82-kD proteins, and chloroplast RNA polymerase from C. caldarium contained an immunoreactive 32-kD protein. The elution profile of enzyme activity of both algal chloroplast RNA polymerases coeluted from DEAE with the respective immunoreactive proteins, indicating that they are components of the enzyme. These results provide immunological evidence for sigma-like factors in chloroplast RNA polymerase in higher plants and algae.

Animals↗
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↗
PubMed1993

Localized amyloidosis of the larynx: evidence for light chain composition.

We report the biochemical characterization of amyloid fibrils from a patient with localized amyloidosis of the epiglottis and larynx. Biopsy specimens showed amorphous material consistent with amyloid deposits with a plasmacytic infiltrate. Both plasma cells and amyloid deposits stained positively by immunohistochemistry for kappa light chains. Amyloid fibrils were isolated. The major constituent resolved as a 13 kd band was sequenced and found to be consistent with a kappa 1 light chain. A tryptic digest was carried out and 3 tryptic peptides were sequenced defining the first 45 residues of the protein and residues 110 through 119. Four amino acid substitutions were found, 3 of which have not been described previously. This study defines the immunoglobulin origin of amyloid deposits in localized amyloidosis. The benign nature of localized amyloidosis suggests that a localized clone of plasma cells producing an amyloidogenic light chain may represent the pathogenetic mechanism of this disease, which appears to be a form of plasma cell dyscrasia.

Amino Acid Sequence↗
PubMed1991

Salivary cystatin SA-III, a potential precursor of the acquired enamel pellicle, is phosphorylated at both its amino- and carboxyl-terminal regions.

Cystatin SA-III was purified from human submandibular/sublingual glandular secretions by adsorption to hydroxyapatite, gel filtration chromatography, and reversed-phase HPLC. The amino acid sequence of its amino-terminus was deduced by sequential Edman degradation and found to be identical to the first 10 residues of cystatin HSP-12. The purified protein was digested with endoproteinase Asp-N and the digestion products were subjected to fast atom bombardment mass spectroscopy. m/z values corresponding to 12 peptides were aligned to the sequence of cystatin S preceded by the eight-residue amino-terminal peptide detected in HSP-12. This process resulted in the assignment of peptides corresponding with 118 out of the 121 amino acid residues predicted from the nucleotide sequence for cystatin SA-III. In order to align several peptides, it was necessary to substitute four residues of phosphoserine for four residues of serine. Fast atom bombardment mass spectrometry and additional Edman degradation procedures localized the phosphate moieties to Ser-3, Ser-99, Ser-112, and Ser-116. This is the first report of the structure of cystatin SA-III deduced by amino acid sequencing techniques and indicates the sites of phosphoserine within the molecule. Based on these assignments, cystatin SA-III is unique among salivary proteins in that it possesses phosphate groups at its amino-terminus as well as its carboxyl-terminus.

Amino Acid Sequence↗
PubMed1991

Maize chloroplast RNA polymerase: the 78-kilodalton polypeptide is encoded by the plastid rpoC1 gene.

The 180-, 120- and 38-kDa polypeptides found in highly purified maize plastid RNA polymerase preparations are encoded by the maize plastid genes rpoC2, rpoB, and rpoA, respectively [Hu, J. and Bogorad, L. (1990) Proc. Natl. Acad. Sci. USA. 87, pp. 1531-1535]. These genes have segments that specify amino acid sequences homologous to those of E. coli RNA polymerase subunits. The plastid gene products are designated b", b and a, respectively. We report here that the amino-terminal amino acid sequence of a 78-kDa polypeptide also found in highly purified maize plastid RNA polymerase preparations matches precisely the sequence deduced from the maize plastid rpoC1 gene which has segments homologous to the 5' end of the E. coli rpoC gene. Thus, the 78-kDa polypeptide is likely to be a functional component of maize plastid DNA-dependent RNA polymerase. This polypeptide is designated subunit b'. Three polypeptides unrelated to RNA polymerase have also been identified in this preparation.

Amino Acid Sequence↗
PubMed1991

Multiple forms of statherin in human salivary secretions.

Sequential chromatography of hydroxyapatite-adsorbed salivary proteins from submandibular/sublingual secretions on Sephadex G-50 and reversed-phase HPLC resulted in the purification of statherin and several statherin variants. Amino acid analysis, Edman degradation and carboxypeptidase digestion of the obtained protein fractions led to the determination of the complete primary structures of statherin SV1, statherin SV2, and statherin SV3. SV1 is identical to statherin but lacks the carboxyl-terminal phenylalanine residue. SV2, lacking residues 6-15, is otherwise identical to statherin. SV3 is identical to SV2 but lacks the carboxyl-terminal phenylalanine. These results provide the first evidence for multiple forms of statherin which are probably derived both by post-translational modification and alternative splicing of the statherin gene.

Adsorption↗
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

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↗