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Biomedical subjects

A Petersen

Publications and source records attributed to A Petersen.

At least 91 records · Page 5Linked to original sources
PubMed1995

Isolation and characterization of the 18-kDa major apple allergen and comparison with the major birch pollen allergen (Bet v I).

The major allergen from birch pollen, Bet v I, and the cross-reacting 18-kDa major allergen from Golden Delicious and Granny Smith applies were isolated by micropreparative SDS-PAGE followed by electroelution. In the case of apples, highly active, low-temperature extracts were used. The purity of the allergens was checked by analytic SDS-PAGE and immunoblotting with allergic patients' sera, as well as by N-terminal amino acid microsequencing, and the allergens were found to be very pure. The strong immunologic activity of the isolates was determined by the enzyme allergosorbent test (EAST) and EAST inhibition assays; this activity was, in the case of Bet v I, similar to that of a preparation obtained by monoclonal antibody affinity chromatography. The allergenic potency of Bet v I and of the cross-reactive apple allergen was determined by EAST inhibition and dose-related histamine release. With both assay systems, the allergenic reactivity of Bet v I was considerably higher than that of the major apple allergen. Furthermore, skin prick tests with the purified allergens and with whole allergenic extracts were performed on a group of 33 patients suffering from birch-pollen and apple hypersensitivity, and on a control group of 10 patients. The frequency of positive prick test results in the allergic patient group ranged from 73% for the major allergen from Golden Delicious apples to 97% with Bet v I and whole birch pollen extract, respectively. In contrast to our low-temperature extracts, commercial prick test solutions of four different manufacturers were found to be unreliable for the diagnosis of apple allergy. The skin test results again indicated the strong immunologic activity of the allergen isolates and the predominance of the major allergens in context with birch-pollen and apple hypersensitivity. Taken together, the results support the view that the 18-kDa major allergen represents most of the allergenicity of the the apple fruit, and that all allergenic epitopes of the apple proteins are present on Bet v I.

Allergens↗
PubMed1995

Interaction of eukaryotic initiation factor eIF-4B with a picornavirus internal translation initiation site.

We studied the interaction of cellular proteins with the internal ribosome entry site (IRES) of foot-and-mouth disease virus by UV cross-linking and observed specific binding of a 80-kDa protein contained in cytosolic HeLa cell extract and in rabbit reticulocyte lysate. Binding of the protein was dependent on the presence of ATP. Immunoprecipitation with eIF-4B antiserum revealed that the protein is identical to the initiation factor eIF-4B. Deletions in the 3' part, but not in the 5' part, of the IRES interfered with UV cross-linking, indicating that the binding site of eIF-4B is located close to the end of the element. Attempts to separate ribosome-associated from non-ribosome-associated protein fractions of cytosolic cell extracts led to the loss of cross-linking activity. This finding suggests that additional protein factors contribute to this interaction of eIF-4B with the IRES of foot-and-mouth disease virus.

Adenosine Triphosphate↗
PubMed1995

Characterization of the allergen group VI in timothy grass pollen (Phl p 6). I. Immunological and biochemical studies.

For the identification and characterization of allergen grass group VI we performed two-dimensional immunoblotting of timothy grass pollen (Phleum pratense). Two intense 13-kD protein spots of pI 5.2 and 5.5 were found to be IgE reactive. By N-terminal microsequencing and amino acid analysis we identified them as Phl p 6 isoallergens. An antiserum was raised against Phl p 6 by immunizing BALB/c mice with allergen bearing nitrocellulose particles of one isoform. The antiserum revealed an intense reactivity to Phl p 6 isoforms, but also showed a weak cross-reactivity with Phl p 5 allergens. After immunoabsorption of patients' serum to Phl p 6 spots on the blotting membrane, we were able to demonstrate that the eluted human IgE antibodies cross-react with the grass group V allergens as well. Therefore, Phl p 5 and Phl p 6 possess one or more common IgE binding epitopes.

Allergens↗
PubMed1995

Characterization of the allergen group VI in timothy grass pollen (Phl p 6). II. cDNA cloning of Phl p 6 and structural comparison to grass group V.

The complete primary structure of the major allergen Phl p 6 was determined by cDNA cloning. A cDNA library of timothy grass pollen was screened by a Phl p 6 directed mouse antiserum. Six clones were obtained. From the cDNA data we deduced a protein sequence of 110 amino acids. While the size of the leader sequences varied considerably between the different clones, only one amino acid exchange affected the mature proteins. Sequence comparison of group VI and V allergens showed a high degree of homology in the N- and C-terminal regions. The structural data strongly indicate that group VI and V are independent groups of allergens, which have derived from a common precursor gene.

Allergens↗
PubMed1994

Major allergen Phl p Va (timothy grass) bears at least two different IgE-reactive epitopes.

It is established that most grass pollen allergens consist of several isoforms of which the function is mainly still unknown. A number of these allergens belonging to group V have been cloned, sequenced, and expressed. Antigenic sites and IgE-reactive epitopes of the major allergen Phl p Va, are unknown. We have identified the complete cDNA sequence of a Phl p Va isoallergen by immunoscreening of a timothy grass pollen cDNA library and mixed oligonucleotide primed amplification of N-terminal cDNA. Additionally, we found an incomplete isoallergenic cDNA clone of the same protein. Immunoreactivity of the fusion proteins with patients' sera and monoclonal antibodies showed that the clones represent group Va allergens. Comparison of deduced amino acid sequences with published sequences of Lol p V and Poa p IX revealed a homology of 81.1% and 86.9%, respectively. With affinity-purified IgE antibodies recognizing the recombinant fusion protein, we can demonstrate the existence of a common group V IgE-reactive epitope. By construction of both an N-terminal and a C-terminal peptide of the complete Phl p Va and cross-inhibition, we identified at least two different IgE epitopes. Eleven patients showed variable IgE immunoreactivities to both IgE-reactive epitopes.

Allergens↗
PubMed1994

Porcine reproductive and respiratory syndrome: temperature and pH stability of Lelystad virus and its survival in tissue specimens from viraemic pigs.

We investigated the growth of Lelystad virus (LV) in porcine alveolar macrophages, the thermal and pH stability of the virus in cell culture medium, and its survival in tissue specimens from viraemic pigs. Lelystad virus grew to titres of 10(6) TCID50/ml, which were found at 40 h after virus inoculation when the macrophage cultures showed a cytopathic effect of approximately 40%. In culture medium at pH 7.5, LV was stable for prolonged periods of storage at -70 degrees C and -20 degrees C. At higher temperatures the half-life of LV was 140 h at 4 degrees C, 20 h at 21 degrees C, 3 h at 37 degrees C and 6 min at 56 degrees C. The half-life of LV, both at 4 degrees C and 37 degrees C, changed considerably when the pH of the medium was varied. At 4 degrees C and pH 6.25 a maximum half-life of 50 h and at 37 degrees C and at pH 6.0 a maximum half-life of 6.5 h was observed. However, increasing or decreasing the pH of the medium rapidly decreased the half-life of LV at both temperatures. Although, LV proved to be more stable at pH 6.00 than at pH 7.5, it did not replicate at pH 6.0. We also tested various tissue specimens from viraemic pigs for the presence of LV. The virus was detected in tonsils, lymph nodes, lungs, serum, and sporadically, albeit at low titres, in muscle tissue. The titre of virus in muscle tissue and organs was only minimally affected by storage for up to 48 h at 4 degrees C.

Animals↗
PubMed1994

Epitope analysis of isoforms of the major allergen Phl p V by fingerprinting and microsequencing.

The major allergen of timothy grass pollen (Phleum pratense), designated as Phl p V, consists of isoallergenic components of 38 and 32 kDa with pl values of 5.2-7.5 and 4.8-5.9, respectively. The different-sized proteins reveal similarities in IgE reactivity, N-terminal sequence and protein staining. For epitope analysis of these allergens a combination of enzymatic cleavage of electrophoretically separated proteins and immunoblotting techniques with subsequent N-terminal sequencing was performed. After isolation of the components from two-dimensional PAGE gels, proteins were enzymatically cleaved and separated by SDS-PAGE. By endoproteinase Glu-C cleavage six IgE-reactive fragments of each 32 kDa protein and three of each 38 kDa allergen were obtained. Microsequencing of the fragments revealed internal sequences that did not show any similarities between the different-sized allergens. Therefore, we assume only slight structural variations among allergens of similar sizes, whereas the 32 and 38 kDa proteins reveal great differences.

Allergens↗
PubMed1994

Elucidation of the mechanism of CryIIIA overproduction in a mutagenized strain of Bacillus thuringiensis var. tenebrionis.

NB176 is a Bacillus thuringiensis mutant derived by gamma-irradiation of NB125 Bacillus thuringiensis var. tenebrionis (Krieg). It exhibits two interesting phenotypes: (i) oligosporogeny and (ii) twofold to threefold overproduction of the CryIIIA protein. Southern profiles of the NB176 strain showed an additional copy(s) of the cryIIIA gene located on a 4 kb HindIII fragment, in addition to the expected cryIIIA gene on a 3 kb HindIII fragment. Each cryIIIA gene-bearing HindIII fragment was cloned from NB176. The restriction map of the 3 kb HindIII fragment was identical to that published by Donovan and coworkers. Sequencing of the 4 kb HindIII fragment showed no alterations in the promoter region of the cryIIIA gene but did show replacement of the region immediately following the cryIIIA open reading frame with a sequence encoding a transposase with 50% amino acid homology to that of Tn1000. These findings suggest that the overproduction phenotype of NB176 results from extra copies of the cryIIIA gene produced from a transposition event(s) induced or stabilized by gamma-irradiation. Integration of additional copies of the cryIIIA gene into the native 90 MDa plasmid of the wild-type B. thuringiensis var. tenebrionis strain resulted in strains that made enormous crystals, many possessing greatly enhanced insecticidal activity.

Amino Acid Sequence↗
PubMed1994

Characterization of the 18-kDa apple allergen by two-dimensional immunoblotting and microsequencing.

A low-temperature extract taken from Golden Delicious apples was separated by two-dimensional polyacrylamide gel electrophoresis. By means of two-dimensional immunoblotting with patients' serum containing IgE specific to Bet v I, a rabbit polyclonal antiserum raised against Bet v I, and two Bet v I specific monoclonal antibodies, epitopes cross-reactive to Bet v I were identified on an apple allergen with a molecular mass of 18 kDa and pI 5.5. Furthermore, certain antibody reactivities with 4 isoproteins of a molecular mass of 16 kDa and pIs ranging from 4.9 to 5.5 were observed, which may indicate the presence of Bet v I related epitopes on these proteins. Based on 26 amino acid residues, N-terminal sequencing of the 18-kDa apple allergen revealed 62% sequence identity between Bet v I from birch pollen and the apple allergen. Our results therefore support the view that both proteins express common as well as non-related IgE-reactive epitopes.

Allergens↗
PubMed1993

Characterization of grass group I allergens in timothy grass pollen.

Using Phl p V-depleted timothy grass pollen extract (Phleum pratense) as immunogen, we obtained a monoclonal antibody, QG 4, which recognized proteins of 33, 35, and 37 kd as determined by Western blotting. The antibody cross-reacted with pollen proteins of other grass species in the molecular weight range of 30 to 37 kd. By means of two-dimensional polyacrylamide gel electrophoresis blot of timothy grass pollen extract, we demonstrated at least seven protein spots: two of 37 kd with isoelectric points of 6.4 and 6.6; four of 35 kd with isoelectric points of 6.5, 6.8, 7.1, and 7.3; and one of 33 kd with an isoelectric point of 8.5. These protein spots were also detected by patients' pooled serum. Microsequencing of the 20 N-terminal amino acid residues revealed structures with sequence identities up to 90% to the well-established allergen, Lol p I of ryegrass (Lolium perenne). Therefore we assume that the monoclonal antibody QG 4 recognized the corresponding allergen Phl p I in timothy grass pollen.

Allergens↗
PubMed1993

Accumulated O2 deficit during intense exercise and muscle characteristics of elite athletes.

The accumulated O2 deficit (the difference between the estimated energy demand and the actual O2 uptake) was determined during intense exhaustive exercise (2-7 min) in elite athletes, and its relationship with muscle buffer capacity, muscle enzymes and muscle morphology was examined. Five oarsmen, fifteen soccer players, and fourteen distance runners ran, and three sprint cyclists cycled intensely to exhaustion (2-7 min). The oarsmen also performed exhaustive rowing. Blood lactate was measured immediately after several submaximal exercise bouts. A muscle biopsy was taken at rest from m. gastrocnemius of the soccer players and runners, and from m. vastus lateralis of the cyclists. The accumulated O2 deficit for the oarsmen, soccer players and runners during treadmill running was 47.3 (range: 29.6-62.4), 49.5 (34.3-73.7) and 51.9 (26.5-85.5) ml O2 equivalents ("O2-Eq").kg-1 b.w., respectively, and it was 56.5 (47.5-73.2) ml "O2-Eq".kg-1 for the cyclists during cycling. The O2 deficit was not related to blood lactate during submaximal exercise, muscle enzyme activity (citrate synthase, 3-hydroxyacyl-CoA-dehydrogenase, lactate dehydrogenase), number of muscle capillaries, %ST fibres or muscle buffer capacity. The accumulated O2 deficit was 36% higher (p < 0.05) during rowing compared to running. The present data suggest that the anaerobic energy production during intense exercise is related to the muscle mass involved. However, it appears that the anaerobic energy turnover is not determined by muscle fibre type distribution, muscle buffer capacity or muscle endurance capacity.

Adolescent↗
PubMed1993

Successful technique for the selective production of monoclonal antibodies against a major allergenic component in timothy pollen extract.

Monoclonal antibodies (MAbs) were selectively raised against a major allergenic component of 38 kD in timothy grass pollen (Phleum pratense). We used a special prefractionating technique to isolate the 38 kD allergen, because immunizations with crude pollen extract had resulted in a wide variety of antibodies against other determinants, and cross-reactions between different-sized proteins occurred. Pollen extract was separated by Western blotting. Strips of the nitrocellulose membrane containing the allergen were excised, dispersed into allergen bearing particles, and used for immunization of BALB/c mice. Five MAbs were obtained that reacted with the 38 kD allergen. Four of these antibodies (BF 1, DC 9, EB 4, GE 2) exclusively detected the 38 kD protein, while one MAb (EB 6) additionally bound to a 32 kD allergen. Epitope mapping with the 3 IgG 1 antibodies (BF 1, EB 6, DC 9) was performed by ELISA inhibition tests using the purified 38 kD component. The antibodies did not interfere with each other. This confirms that they bind to different sites of the 38 kD molecule. Only a weak inhibitory effect of the MAbs on the binding of patient's IgE was determined, we suppose that the MAbs do not bind to IgE reactive epitopes. For standardization and further characterization of the 38 kD protein by peptide mapping the MAbs will be useful tools.

Allergens↗
PubMed1993

Comparison of four grass pollen species concerning their allergens of grass group V by 2D immunoblotting and microsequencing.

The identification and characterization of allergenic components is a vital step towards improving diagnosis and therapy. Members of the grass family (Poaceae) reveal a high cross-reactivity among each other caused by the close phylogenetical relationship. In order to investigate the variability between allergenic components, we studied the allergen grass group V, one of the major allergens. Pollen extracts of 4 different tribes (timothy grass (Phleum pratense)--Agrostidae, perennial rye grass (Lolium perenne)--Festuceae, meadow velvet (Holcus lanatus)--Aveneae, and rye (Secale cereale)--Triticeae) of the Festucoideae subfamily were separated by 2D PAGE and investigated by immunoblotting using patients' poolserum and monoclonal antibodies (raised against group V allergens of timothy grass pollen). The antibodies identify different allergens in the four grass species. The components vary from 30-50 kDa and pI 4.8-7.0. The eight NH2-terminal amino acids were determined and indicated high similarities between the different components. These results cast doubt on the suitability of classifying allergens into groups based only on their molecular mass, isoelectric point and N-terminal sequence analysis. It suggests to classify allergens according to their IgE-reactive epitopes.

Allergens↗
PubMed1993

Influence of labor and neonatal hypoxia on sympathoadrenal activation and methionine enkephalin release in calves.

Labor and delivery stimulate increased release of catecholamines and endogenous opioid peptides in neonates. Catecholamines promote adaptation to the extrauterine environment after birth. Enkephalins are stored together with catecholamines in the adrenal medulla and have an inhibitory effect on catecholamine release. We investigated the influence of labor and neonatal hypoxia on epinephrine, norepinephrine, and met-enkephalin release in calves. Blood samples were taken from the umbilical artery before rupture of the umbilical cord and from the jugular vein repeatedly after birth. Highest plasma norepinephrine concentration was found in calves delivered at the end of gestation (term calves) before umbilical cord rupture. In calves delivered before the physiologic end of gestation (preterm calves), norepinephrine values increased after cord rupture, but remained lower than values in term calves. Epinephrine release followed a similar pattern, but norepinephrine was clearly predominant. In term calves, met-enkephalin values were significantly higher than values in preterm calves. In calves of both groups, met-enkephalin release increased after cord rupture. During birth, the increase in catecholamine release seems to take place earlier than that of enkephalins. Norepinephrine-dominated stimulation during expulsion of the calf might be followed by increasing enkephalinergic inhibition after cord rupture and onset of respiration. Reduced release of catecholamines and enkephalins in preterm calves may be connected with delayed adaptation to the extrauterine environment.

Acidosis↗
PubMed1993

[Characterization of allergens of timothy grass (Phleum pratense)].

By means of 2-D electrophoresis and subsequent immunoblotting allergens of timothy grass pollen are separated as single components. Via N-terminal sequencing, it is possible to classify these components as isoallergens of defined groups according to the allergen nomenclature. We are able to identify the allergens as Phl p I, Phl p IV, Phl p V and Phl p VI.

Allergens↗
PubMed1992

Fructose metabolism in the human erythrocyte. Phosphorylation to fructose 3-phosphate.

In human erythrocytes, the first step in the metabolism of fructose is generally thought to be phosphorylation to fructose 6-phosphate catalysed by hexokinase. In variance with this assumption, we show here that fructose in these cells is metabolized primarily to fructose 3-phosphate by a specific 3-phosphokinase. This process has an overall estimated Km of 30 mM with respect to extracellular fructose and an apparent Vmax. of 0.6 mumol/h per ml. At a fixed concentration of fructose in the medium, the accumulation of fructose 3-phosphate was linearly dependent on the duration of incubation up to 5 h and was not affected by glucose. Once accumulated, fructose 3-phosphate appears to be degraded and/or relatively slowly metabolized, decreasing by only approximately 30% after a 12 h incubation in a fructose-free medium.

Erythrocytes↗