Cloning and characterization of Ph1 p 1 in different expression systems.
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Allergens↗
Biomedical subjects
A Petersen
Publications and source records attributed to A Petersen.
Synthesis of 9-deoxo-4''-deoxy-6,9-epoxyerythromycin derivatives: novel and acid-stable motilides.
In our quest toward the discovery of highly potent and acid-stable motilides, novel 4"-deoxy derivatives of 9-deoxo-6, 9-epoxyerythromycin were designed, synthesized, and evaluated for their gastrointestinal prokinetic activities. These compounds, in their 9R configuration, were more potent than their 6,9-enol ether homologues in inducing well-coordinated smooth muscle contractions in an in vitro rabbit duodenal assay: e.g., (9R), (8S)-9-deoxo-4"-deoxy-3'-N-desmethyl-3'-N-ethyl-6, 9-epoxyerythromycin A (10) and (9R), (8S)-9-deoxo-4"-deoxy-3'-N-desmethyl-3'-N-ethanol-6, 9-epoxyerythromycin A (15) had a pED50 of 8.54 and 8.11 compared to a pED50 of 7.22 for compound 2 (ABT-229). Reduction of the 6,9-enol ether, which was aimed at improving the acid stability, afforded the most stable motilides to date with t1/2 of 5.5 h for 10 and 15. Compounds 10 and 15 bind specifically to rabbit antral smooth muscle motilin receptors with pIC50 values of 8.52 and 8.70.
Animals↗
Cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius. A novel highly glycosylated, membrane-bound b-type hemoprotein.
In this study we re-examined the inducible cytochrome b558/566 from the archaeon Sulfolobus acidocaldarius (DSM 639), formerly thought to be a component of a terminal oxidase (Becker, M., and Schäfer, G. (1991) FEBS Lett. 291, 331-335). An improved purification method increased the yield of the protein and allowed more detailed investigations. Its molecular mass and heme content have been found to be 64,210 Da and 1 mol of heme/mol of protein, respectively. It is only detectable in cells grown at low oxygen tensions. The composition of the growth medium also exerts significant influence on the cytochrome b558/566 content of S. acidocaldarius membranes. The cytochrome exhibits an extremely high redox potential of +400 mV and shows no CO reactivity; a ligation other than a His/His-coordination of axial ligands appears likely. It turned out to be highly glycosylated (more than 20% of its molecular mass are sugar residues) and is probably exposed to the outer surface of the plasma membrane. The sugar moiety consists of several O-glycosidically linked mannoses and at least one N-glycosidically linked hexasaccharide comprising two glucoses, two mannoses, and two N-acetyl-glucosamines. The gene of the cytochrome (cbsA) has been sequenced, revealing an interesting predicted secondary structure with two putative alpha-helical membrane anchors flanking the majority of a mainly beta-pleated sheet structure containing unusually high amounts of serine and threonine. A second gene (cbsB) was found to be cotranscribed. The latter displays extreme hydrophobicity and is thought to form a functional unit with cytochrome b558/566 in vivo, although it did not copurify with the latter. Sequence comparisons show no similarity to any entry in data banks indicating that this cytochrome is indeed a novel kind of b-type hemoprotein. A cytochrome c analogous function in the pseudoperiplasmic space of S. acidocaldarius is discussed.
Amino Acid Sequence↗
Preparation of 9-deoxo-4"-deoxy-6,9-epoxyerythromycin lactams "motilactides": potent and orally active prokinetic agents.
A series of new, highly potent and orally active "motilactides", 9-deoxo-4"-deoxy-6,9-epoxyerythromycin lactams was designed, synthesized, and evaluated for their gastrointestinal motor stimulating activity. These compounds were acid stable and showed good oral efficacy.
Administration, Oral↗
Molecular and immunological characterization of group V allergen isoforms from velvet grass pollen (Holcus lanatus).
Group V grass pollen allergens belong to the major grass pollen allergens causing reactions of type I allergy. cDNAs coding for two isoforms of the group V allergen of velvet grass pollen (Holcus lanatus), a widespread grass species, were isolated from a cDNA library by hybridization with a 5'-terminal reverse-transcribed PCR-derived cDNA probe. Amino acid sequences of the two isoforms, designated rHol l 5.01 and rHol l 5.02, revealed high similarity between them (78% identity) and to group V allergens and their isoforms found in other grass species. Recombinant Hol l 5 isoforms were expressed in Escherichia coli and purified as fusion proteins. To compare their immunological reactivities with group-V-specific monoclonal antibodies and patients' IgE, immunoblotting, ELISA and histamine release assay were performed. Interestingly, monoclonal antibody Bo9, specific for group Vb isoforms of timothy grass, bound only to isoform rHol l 5.01, not to rHol l 5.02. On the other side, IgE reactivities of patients' sera revealed no differences between the two isoforms when investigated by immunoblotting and only slight differences when investigated by ELISA. In histamine release assay both isoforms released comparable amounts of histamine from basophils of four individual patients. Thus, the two group V isoforms of velvet grass pollen exhibit differential binding when tested with monoclonal antibodies, i.e. different structure of single epitopes, but negligible differences concerning overall IgE-binding capacity and histamine-releasing capacity.
Allergens↗
Post-translational modifications influence IgE reactivity to the major allergen Phl p 1 of timothy grass pollen.
BACKGROUND: Grass group I consists of very potent allergenic components which are found in the pollen of all temperate grasses. Several post-translational modifications are predicted from the cDNA data. OBJECTIVE: The aim of this study was to identify sequential IgE-binding sites on the allergen Phl p 1 and to determine their influence on IgE reactivity. METHODS: Based on cDNA data and microsequencing results we synthesized overlapping decapeptides covering the complete Phl p 1 molecule and tested them for immunological reactivity by means of the PEPSCAN technique. In a dot test we determined the frequency of IgE reactivities to post-translationally modified structures (hydroxylated proline residues, carbohydrate structure, and disulphide formations). RESULTS: Screening by overlapping peptides demonstrated an IgE binding site on the 10 N-terminal amino acids. Comprehensive studies showed that the two hydroxyproline residues of the native Phl p 1 allergen (at positions 5 and 8) and the N-glycan (at position 9) can result in an increased IgE reactivity; 3.3% of the sera exclusively bound to the hydroxyproline bearing peptide, while only 0.4% bound to the proline containing peptide. With regard to glycosylation, we estimated that 20% of sera recognized protein and carbohydrate epitopes, while one serum exclusively bound to the glycan. The formation of disulphide bonds has no detectable effect on the IgE reactivity to Phl p 1. CONCLUSION: Our results indicate that the post-translational modifications, the carbohydrate structure and the hydroxylation of proline residues, can enhance the IgE reactivity of Phl p 1.
Allergens↗
Hepatocellular carcinoma presenting as a tumour of the hilar and extrahepatic bile ducts.
A case of hepatocellular carcinoma extending within the large extra- and intrahepatic bile ducts is reported. No primary tumour was found in the liver parenchyma by abdominal ultrasound, spiral computed tomography or magnetic resonance, but transduodenal cholangioscopy showed tumour in the common hepatic ducts and the two main branches. Endoscopic biopsy showed highly differentiated hepatocellular carcinoma. The patient was treated with endoscopic biliary drainage and died at home 7 months after admittance.
Aged↗
Antibiotic resistance in Acinetobacter spp. isolated from sewers receiving waste effluent from a hospital and a pharmaceutical plant.
The possible increase of antibiotic-resistant bacteria in sewage associated with the discharge of wastewater from a hospital and a pharmaceutical plant was investigated by using Acinetobacter species as environmental bacterial indicators. The level of susceptibility to six antimicrobial agents was determined in 385 Acinetobacter strains isolated from samples collected upstream and downstream from the discharge points of the hospital and the pharmaceutical plant. Results indicated that while the hospital waste effluent affected only the prevalence of oxytetracycline resistance, the discharge of wastewater from the pharmaceutical plant was associated with an increase in the prevalence of both single- and multiple-antibiotic resistance among Acinetobacter species in the sewers.
Acinetobacter↗
Calcium-dependent proteolysis in rabbit lens epithelium after oxidative stress.
The purpose of this study was to examine changes in calcium-dependent proteolytic activity in the lens epithelium from whole rabbit lenses exposed to long-term oxidative stress at near physiological levels. Rabbit lenses, incubated in 50 microM H2O2 for 1 or 24 h, were checked for clarity and morphological changes in the epithelium. Proteolytic activity was measured in the epithelium using a fluorogenic synthetic substrate; N-succinyl-Leu-Tyr-7-amino-4-methylocoumarin, both in the presence and the absence of calcium (1 mM Ca2+ and 5 mM EDTA respectively). The effect on transparency and morphology of the epithelium following a 1-hour incubation in 100 microM H2O2 was also studied. Lenses incubated in 50 microM H2O2 were clear even after 24h. After a 1-hour incubation in 50 microM H2O2 the epithelium of the exposed lens appeared normal. However, after 24 h the epithelium cells appeared swollen and microscopical examination showed extensive intracellular and subepithelial vacuolization. Incubation in 100 microM H2O2 for 1 h caused loss of transparency; vacuole formation, globulization of the superficial lens fibers and death of the epithelial cells. There was a 55% increase in calcium-dependent proteolytic activity after 1 h in 50 microM H2O2, implying a role for the calcium-activated protease calpain in oxidatively induced cataract.
Animals↗
Investigation of different recombinant isoforms of grass group-V allergens (timothy grass pollen) isolated by low-stringency cDNA hybridization--antibody binding capacity and allergenic activity.
A cDNA library of timothy grass pollen was screened for homologous isoforms of major group-V allergens by low stringency hybridization with a Phl p 5 (Phleum pratense) probe. After restriction analysis of the 40 clones obtained, 17 were selected for cDNA sequencing. Of these clones, two were unrelated to group-V allergens, six showed high similarity but an incomplete open reading frame and nine had high similarity with a complete open reading frame. Comparison of deduced amino acids of ten complete cDNA clones confirmed the presence of two major isoforms, a and b. Within these two subgroups, only minor sequence variations were observed. Eight isoforms were expressed in Escherichia coli K12 and purified to homogeneity. Although the subgroups a and b could be distinguished by their molecular masses and by binding constants towards monoclonal antibodies, all isoforms turned out to be biochemically similar. Ribonuclease activity as a marker for the biological function of group-V allergens was shown to be in the same range for both subgroups. Analysis of allergenic B-cell responses towards the isoforms in 26 grass pollen allergic patients revealed that the IgE reactivities to the different isoforms were identical for each individual. IgE reactivities and allergenic activities of three isovariants and an allergen of a different group were compared in a selected group of four grass pollen allergic patients by immunoblot, histamine-release and skin-prick tests. The IgE reactivity does not necessarily mirror the allergenic activity of the single molecule, and the variability of allergenic activity between the isovariants does not, in every case, depend on the structural differences of these allergens. We conclude that group-V isoallergens in grass pollen, although they can be structurally different, induce a similar B-cell response but can show variable allergenic activity. Thus, the most allergenic isoform of each important group of allergens should be sufficient for the diagnosis of type-I allergy. Whether the isoallergenic variation has any significant influence on the outcome of immunotherapy in allergic disease still has to be elucidated.
Allergens↗
Expression of the Solfolobus acidocaldarius Rieske iron sulfur protein II (SOXF) with the correctly inserted [2FE-2S] cluster in Escherichia coli.
The Rieske protein II (Schmidt et al., 1996, FEBS Lett. 388, 43-46) from the thermoacidophilic crenarcheon Sulfolobus acidocaldarius (DSM 639) was expressed in E. coli cells. The full length protein was strictly bound to the E. coli membranes and could only be removed by detergent treatment indicating the presence of a membrane anchor. The iron sulfur cluster was correctly inserted into a fraction of the full length protein and much more effectively into a soluble form created by the deletion of the 45 N-terminal amino acids. The soluble form of the protein displayed the typical spectroscopic properties of a respiratory Rieske protein. The midpoint potential was +375 mV determined by CD redox potentiometry. The presented data demonstrate that the structure of the recombinant protein is very similar or identical to the authentic protein making this a powerful model system for the studies of Rieske proteins by site directed mutagenesis.
Amino Acid Sequence↗
The signal recognition particle receptor alpha subunit of the hyperthermophilic archaeon Acidianus ambivalens exhibits an intrinsic GTP-hydrolyzing activity.
Two adjacent genes of the acidophilic and hyperthermophilic crenarchaeon Acidianus ambivalens were cloned and sequenced. The 1.6 kb genomic nucleotide sequence under investigation consists of the 1.12 kb SRa gene encoding the putative signal recognition particle receptor alpha subunit (SR alpha, 42.2 kDa) and the 186 basepair secE gene coding for the putative secretory component secE subunit (6800 Da). The SR alpha protein is structured by three distinct regions: the N-terminal hydrophilic H-region, the following X-region and the C-terminal GTP-binding domain. A polyclonal anti-E. coli lacZ/A. ambivalens SR alpha antiserum detects a 51 kDa cell protein (p51) on immunoblots. Proteolysis of the recombinant SR alpha protein by Proteinase K produces a 31.6 kDa protease-resistant protein fragment comprising X-region and G-domain. The protein binds tightly to the GTP-agarose affinity matrix in a temperature-dependent manner. It hydrolyzes GTP readily at higher temperatures only in the presence of Mg2+. Point mutations (T326N) and (D329A) in the G-4 element of A. ambivalens SR alpha G-domain diminish the GTPase activity significantly. In contrast, the deletion mutant protein SR alpha (delta1-92) lacking the hydrophilic H-region displays a higher GTP-hydrolyzing activity when compared to the unmodified recombinant protein. Addition of GDP greatly inhibits GTP hydrolysis in mutant and unmodified A. ambivalens SR alpha.
Amino Acid Sequence↗
Human vascular smooth muscle cells express interleukin-1beta-converting enzyme (ICE), but inhibit processing of the interleukin-1beta precursor by ICE.
Local immunoregulatory processes during normal vascular biology or pathogenesis are mediated in part by the production of and response to cytokines by vessel wall cells. Among these cytokines interleukin (IL)-1 is considered to be of major importance. Although vascular smooth muscle (SMC) and endothelial cells (EC) expressed both IL-1alpha and IL-1beta as cell-associated, 33-kilodalton (kD) precursors, SMC neither contained detectable mature IL-1beta, nor processed recombinant IL-1beta precursor into its mature 17-kD form. Thus, we investigated the expression and function of IL-1beta-converting enzyme (ICE) in vascular cells. We demonstrate in processing experiments with recombinant IL-1 precursor molecules that EC processed IL-1beta, in contrast to SMC. Despite the failure of SMC to process IL-1beta, these cells expressed ICE mRNA, immunoreactive ICE protein, and the expected IL-1beta nucleotide sequence. The lack of processing was explained by our finding that extracts of SMC specifically and concentration dependently blocked processing of IL-1beta precursor by recombinant or native ICE. The initial biochemical characterization of the inhibitory activity showed that it is heat-labile, has a molecular size of 50-100 kD, and is associated to the cell membrane compartment. Inhibition of processing, i.e., activation of IL-1beta precursor by SMC may constitute a novel regulatory mechanism during normal vascular biology or pathogenesis of vascular diseases.
Caspase 1↗
Comparison of natural and recombinant isoforms of grass pollen allergens.
More than 95% of grass pollen allergic patients possess IgE antibodies against grass group I, a heterogeneous group of glycoproteins found in all temperate grasses. We studied the structural variability of the group I allergens in single species and among different grasses. By 2-DE blotting using patients' IgE and monoclonal antibodies, we detected IgE-reactive isoforms with molecular masses between 32 and 37 kDa and focusing in a wide pI ranging from 4.7 to 7.6. While the group I allergens of timothy grass (Phl p 1) were composed of 37 and 35 kDa components, only single isoforms were found for ryegrass (Lol p 1) and velvet grass (Hol l 1): 32 and 34 kDa, respectively. By N-terminal microsequencing we determined single amino acid substitutions in different-sized group I allergens. The post-translational modifications (one N-glycosylation site, two hydroxylated proline residues and seven cysteine residues for potential disulfide formations), which contribute to IgE reactivity, were identical in all. From the cDNA sequences we deduced protein sequence homologies > 90%, a result which might explain the high IgE cross-reactivity among the grasses. In order to test whether recombinant group I grass allergens can act as substitutes for the natural forms, we expressed rPhl p 1 in E. coli and in P. pasteuris. 2-DE immunoblotting again demonstrated a microheterogeneity in molecular mass and pI. While the E. coli products were free from post-translational modifications, rPhl p 1 from Pichia is a heterogeneous glycoprotein fraction with a carbohydrate content of about 15%. This rPhl p 1 is hyperglycosylated compared to the nPhl p 1, which only has a 5% carbohydrate content.
Allergens↗
Latex allergen database.
Two-dimensional (2-D) electrophoresis followed by immunoblotting and N-terminal protein microsequencing were used to characterize and identify the IgE-reactive proteins of Hevea latex that are the main cause of the latex type I allergy affecting especially health care workers and spina bifida children. This approach generated a comprehensive latex allergen database, which facilitated the integration of most of the latex allergen data presented in the literature. The major latex allergens Hev b 1, Hev b 3, Hev b 6 and Hev b 7 have been localized on our 2-D maps. Moreover, we were able to identify six previously undescribed IgE-binding latex proteins, namely enolase, superoxide dismutase, proteasome subunit C5, malate dehydrogenase, triosephosphate isomerase and endochitinase. The generated latex 2-D maps will provide valuable information to develop strategies for the isolation of the novel IgE binding proteins in order to study the frequency of sensitization among both risk groups. Detailed knowledge of all proteins involved in latex allergy will allow better diagnosis of latex allergy and to monitor the success of prevention strategies that are needed to reduce the high prevalence of latex allergy among both risk groups.
Allergens↗
Are speech perception deficits associated with developmental dyslexia?
Phonological awareness and phoneme identification tasks were administered to dyslexic children and both chronological age (CA) and reading-level (RL) comparison groups. Dyslexic children showed less sharply defined categorical perception of a bath-path continuum varying voice onset time when compared to the CA but not the RL group. The dyslexic children were divided into two subgroups based on phoneme awareness. Dyslexics with low phonemic awareness made poorer /b/-/p/ distinctions than both CA and RL groups, but dyslexics with normal phonemic awareness did not. Examination of individual profiles revealed that the majority of subjects in each group exhibited normal categorical perception. However, 7 of 25 dyslexics had abnormal identification functions, compared to 1 subject in the CA group and 3 in the RL group. The results suggest that some dyslexic children have a perceptual deficit that may interfere with processing of phonological information. Speech perception difficulties may also be partially related to reading experience.
Child↗
Mapping of IgE-binding epitopes on the recombinant major group I allergen of velvet grass pollen, rHol l 1.
BACKGROUND: New and more successful approaches to diagnosis and therapy of allergic diseases require a more subtle understanding of the structure and the epitopes on the allergen molecule. OBJECTIVE: This study was done to obtain more information on the structure and the IgE-binding epitopes of a major allergen of velvet grass pollen, Hol l 1. METHODS: We cloned Hol l 1 from a complementary DNA library and performed B-cell epitope mapping with 21 recombinant fragments expressed as fusion proteins in Escherichia coli. The fragments were analyzed by Western blotting with sera from 50 different patients. RESULTS: The patients' sera individually recognized at least four different IgE-binding regions (amino acids 1 to 27, 61 to 76, 84 to 105, and 158 to 240). According to their binding patterns with these epitopes, they were divided into five groups. Most sera (92%) bound to the C-terminal peptide (158 to 240), which consists of more than 80 amino acids, whereas there was virtually no binding to smaller fragments covering this region. In contrast to the C-terminal peptide, the IgE-binding peptides on the N terminus and on the middle region of the molecule were of a smaller size (15 to 30 amino acids). CONCLUSIONS: The major group I allergen of velvet grass bears at least four different IgE-binding epitopes, which were individually recognized by sera from different patients. The C terminus represents the major IgE-binding region and contains at least one discontinuous IgE-binding epitope, whereas the N terminus and middle region of Hol l 1 seem to contain continuous IgE-binding epitopes.
Allergens↗
A family with chronic haemolysis and selective accumulation of erythrocyte CDP-choline.
In this paper, we report a family with compensated chronic haemolysis where the only erythrocyte abnormality detected was an increased level of erythrocyte CDP-choline. Using 31P-NMR spectroscopy and enzymatic analysis the possibility of a pyrimidine 5'-nucleotidase deficiency was excluded. Thus, this family represents the first evidence for a hereditary haemolytic anaemia where the inferred enzymatic defect is located to choline phosphotransferase, the enzyme catalysing the final step in lecithin synthesis. The family history indicates an autosomal dominant mode of transmission with incomplete penetrance.
5'-Nucleotidase↗