Search PubMed⌕ Search

Biomedical subjects

J Apold

Publications and source records attributed to J Apold.

At least 55 records · Page 3Linked to original sources
PubMed1988

Primary structure, developmentally regulated expression and potential duplication of the zebrafish homeobox gene ZF-21.

We report the molecular cloning and characterization of a cDNA derived from a zebrafish gene (ZF-21) related to the mouse homeobox containing gene Hox2.1. Interesting information about the differential conservation of various domains was gained from comparisons between the putative protein sequences from ZF-21 (275 amino acids) and Hox2.1 (279 aa). A separate DNA binding domain including the ZF-21 homeodomain and 36 additional flanking residues is completely identical to the C-terminal part of Hox2.1. As a consequence, these two mouse and zebrafish proteins must have identical DNA binding properties. A lower level of sequence identity between the N-terminal coding regions of ZF-21 and Hox2.1 reduces the total protein homology to 81%. However, short stretches of perfect homology in these N-terminals suggests that the essential biochemical functions are the same. As expected for true homologues, the ZF-21 and Hox2.1 genes also share extensive similarities with respect to non-coding sequences and temporal expression during embryogenesis. The finding of a potential ZF-21 duplication is discussed in relation to functional and evolutionary aspects of vertebrate homeobox genes.

Amino Acid Sequence↗
PubMed1986

Antigenic and allergenic determinants of ovalbumin. I. Peptide mapping, cleavage at the methionyl peptide bonds and enzymic hydrolysis of native and carboxymethyl OA.

The effects of enzymic cleavage and perturbing the conformation of the allergenic and antigenic determinants of hens egg white albumin (OA) were examined. Hens egg white extract of a total protein concentration 8.43 g/l was prepared. Isoelectric focusing in sodium dodecyl sulfate and polyacrylamide gel peptide maps for the crude egg white extract showed 26 spots visualized by staining with Coomassie blue. The OA was purified using a TSK-2000 gel filtration chromatography column. The specific allergenic reactivity of the purified OA as measured by RAST inhibition and direct RAST was relatively high: 3 micrograms gave an inhibition of approximately 10%. The cleavage of OA with cyanogen bromide resulted in 4 fractions, all capable of binding specific IgE with the first peak showing the highest inhibition. Thermal denaturation of OA had no direct effect on the antigenic reactivity. RAST inhibition values for the denatured protein were similar to those of the native protein. Carboxymethylation of OA gave a product with only 20% of the inhibition reactivity. Further treatment with trypsin did not abolish the allergenic and antigenic reactivities as shown by RAST inhibition and by deflection of OA line in rocket line immunoelectrophoresis. On the other hand, limited pepsin hydrolysis destroyed the antigenic structure of the molecule. The reactivity of OA is thus relatively stable and could easily be retained making it possible to identify the allergenic determinants of enzymic hydrolysates used for elucidating the antigenic structure of the molecule.

Allergens↗
PubMed1983

Immunochemical analysis of cod fish allergen M: locations of the immunoglobulin binding sites as demonstrated by the native and synthetic peptides.

The major allergen of codfish (Allergen M) is a muscle protein belonging to the family of calcium binding parvalbumins. The primary structure of the molecule was established and the molecular weight was estimated from the sequence data to be 12,328. Allergen M consists of 113 amino acid residues and one residue of glucose. A molecular arrangement of three domains (AB, CD and EF--the latter two bind one Ca2+ ion each) was described for Allergen M, analogous to carp parvalbumin pI 4.25. The suggested structure was based on the extensive intramolecular amino acid homologies and the immunochemical cross-reactivities of the intact molecule and the two major isolated fragments. The immunological structure of Allergen M was studied by: 1. Modification of certain amino acids residues and study of the reactivity of the modified derivatives. 2. Examination of the immunochemical reactivity of a large number of overlapping peptides obtained by limited and selective tryptic hydrolyses. 3. Solid phase peptide synthesis (SPPS) of segments selected in regard to the reactivity of pre-examined native peptides. The immunological reactivity of the derivatives of Allergen M was assigned by: 1. Rocket line immunoelectrophoresis and quantitative precipitation using rabbit anti-Allergen M in precipitating antibody-mediated reactions and, 2. RAST/RAST-inhibition and PK test/PK-test inhibition using sera from individuals allergic to codfish in IgE-mediated reactions. The modification of Tyr-30 and Arg-75 in isolated and purified peptides indicated that the former was part of a reactive site whereas the latter did not contribute to the activity. Masking of Arg-residue or unchelating the two calcium ions from the native Allergen M, with the resulting perturbation of the tertiary structure, decreased the allergenicity by approximately 25%. Two major fragments of Allergen M were produced and purified: TM1 (residues 1-75) comprising domains AB and CD, and TM2 (residues 76-113) covering domain EF. Both were immunologically reactive; TM1 showing intermediate reactivity between Allergen M and TM2. A high degree of immunological cross reactivity was evident between TM1 and TM2. The finding was in concert with the high intramolecular amino acid homologies of Allergen M, and suggested that the reactive sites were repetitively distributed along the polypeptide chain. The immunological reactivity of several long-sequence overlapping peptides obtained by limited and selective trypsin hydrolysis of Allergen M was studied. The immunologically reactive sites were accordingly assigned to the following regions of the chain: 1. Residues 33-44 on the junction between the AB and CD domains.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1983

Comparative studies on tree-pollen allergens. V. Immunochemical mapping of the antigens and allergens of birch pollen extract (Betula verrucosa).

A comparative electrophoretic and autoradiographic analysis of the different fractions of birch pollen crude extract was performed. This extract included a minimum of 17 reproducible and distinct antigens located in the gel filtration fractions BV2-BV4. Only one of these precipitates had the ability to bind IgE, as demonstrated by immunoelectrophoretic and autoradiographic techniques using several poly- and monospecific rabbit antibodies. The allergenicity of the different fractions was examined in vitro by RAST and RAST inhibition, and in vivo by passive cutaneous transfer and skin prick tests. The data suggested that Betula verrucosa pollen extract contains a group of isoallergens with related antigenicity but with variable molecular sizes. The findings have also confirmed the presence of identical antigenic properties of the previously isolated pI 5.18 and BV45 as deduced from their immunoelectrophoretic and autoradiographic studies.

Allergens↗
PubMed1982

Comparative studies on tree pollen allergens. II. Isolation of alder (Alnus incana) pollen allergens: purification and some characteristics of the major allergen pI 4.78.

The aqueous extract of alder (Alnus incana) pollen was found to consist of a protein mixture amounting to 3% of the dry pollen weight. Separation by gel permeation chromatography on an AcA-54 column resulted in 6 fractions designated chronologically to their relative elution volumes (AI 1 to AI 6). The immunological reactivity of these fractions was demonstrated in both IgE and IgG test systems. Being both immunochemically and quantitatively dominant fraction AI 4 was selected for further purification by a semipreparative isoelectric focusing column. A well-resolved and apparently homogeneous component with a pI value of 4.78 was eluted from the IEF column. The homogeneity of this fraction was demonstrated by one sharp band in thin layer IEF, one single band in HVE, one distinct sharp peak in HPLC and a symmetrical precipitate in CIE. The molecular weight was estimated to 22,500 daltons. Fraction pI 4.78 was shown to be a major allergen of alder pollen, it elicited a 1.17 histamine wheal reaction in skin prick test at a concentration of 1 mg . ml-1 and showed a significantly high RAST inhibition in all of the reaginic sera used. Fraction pI 4.78 was found to be an adequate model allergen of alder pollen well suited for further immunochemical characterization.

Allergens↗
PubMed1982

Comparative studies on tree pollen allergens. IV. Evaluation of two commercially available allergen extracts of alder (Alnus incana) and birch (Betula verrucosa) pollen.

Two commercially available extracts of alder and birch pollen usually used in clinical allergy were evaluated and correlated to two corresponding reference extracts 'S' produced at the authors' laboratory. The set of extracts produced by ALK, Denmark are referred to as 'A', while those produced by Nyco, Norway are referred to as 'N'. The results of the CIE analysis demonstrated that, upon using the same antibodies, both the morphology and the intensity of the precipitates of the different extracts differed considerably. In the alder pollen extract, the 'N' preparation contained more immunoprecipitate lines than the two others. Extract 'S' contained the least number of antigenic lines. Only two of the antigenic lines bound specific IgE in CRIE and were represented in variable concentrations in the three extracts. These two allergenic precipitation lines were demonstrated to have an immunological reaction of identity as shown by tandem-CIE and tandem-CRIE systems, respectively. In the birch pollen system variable numbers of reproducible antigenic lines of precipitation were demonstrated. Preparation 'A' contained more antigens than the two other extracts. This extract lacked a cathodic antigen with slow electrophoretic mobility represented in 'N' and 'S'. One dominant antigenic line was commonly present in the three extracts and could bind IgE antibodies in the CRIE system. A minor cathodic allergen was shown by CRIE in extracts 'S' and 'N', but not in extract 'A'. The RAST and RAST-inhibition titration experiments in all extracts showed qualitative and quantitative similarities. The amino acid compositions and the carbohydrate analyses showed that extracts 'S' and 'N' were similar while extract 'A' contained higher concentrations of certain amino acids and carbohydrates. In both alder and birch pollen, the reference extract 'S' contained lower concentrations of the nonallergenic antigens and of the low molecular weight contaminants.

Allergens↗
PubMed1981

Allergenic synthetic peptide corresponding to the second calcium-binding loop of cod allergen M.

A peptide with the sequence of the second calcium-binding loop (EF loop) of cod Allergen M was synthesized by automatic solid-phase technique. The synthetic peptide corresponded to residues 88-103 of the known primary structure of Allergen M. The immunochemical reactivity of this loop, previously demonstrated for the overlapping enzymic fragments, was confirmed by using the synthetic preparation. The purified hexadecapeptide was shown to bind specifically to reaginic IgE from sera of cod-allergic individuals, in both in vivo and in vitro tests systems. It could also bind rabbit anti-Allergen M, as shown by rocket line immunoelectrophoresis and quantitative precipitation inhibition techniques. The findings emphasized that the immunological reactivity of the synthetic peptide (88-103) was compatible with a monovalent haptenic function: blocking and not eliciting allergic reactions.

Allergens↗
PubMed1981

Comparative studies on tree-pollen allergens. I. Isolation and partial characterization of a major allergen from birch pollen (Betula verrucosa).

A major allergen from birch pollen (Betula verrucosa) was isolated by a combination of gel permeation chromatography and preparative isoelectric focusing, and was found adequate for further immunological and chemical characterization. The crude aqueous pollen extract was eluted in 6 UV-absorbance peaks from an Ultrogel AcA-54 column. The main allergenic components were detected in two peaks of molecular weights 29,000 (BV4) and 40,000 daltons, respectively. BV4 contained one quantitatively dominating antigen as assessed by crossed immunoelectrophoresis, and was selected for further purification by isoelectric focusing. Column isoelectric focusing of BV4 gave four protein fractions with pI values corresponding to 5.18, 5.42, 5.76, and 5.94. The pI 5.18 protein was quantitatively dominating. This protein inhibited the radio-allergosorbent test in 8 out of 10 sera from birch pollen-sensitive individuals. Using a serum pool, the inhibition curve of BV4 pI 5.18 was parallel to that of the crude extract and gave a similar maximum inhibition. This allergen was shown to be homogeneous as judged by a single, symmetrical precipitate in crossed immunoelectrophoresis, one band in high-volt electrophoresis and a sharp strip in thin-layer analytical electrofocusing. Amino acid analysis showed a high content of serine. The N-terminal analysis as well as the carbohydrate concentration were preliminarily reported and will be confirmed in future studies.

Allergens↗
PubMed1980

A synthetic hexadecapeptide derived from allergen M imposing allergenic and antigenic reactivity.

A solid phase synthesis (SPPS) hexadecapeptide corresponding to residues 49-64 of allergen M from cod was found to bind specifically IgE antibodies from sera of cod-allergic individuals. The SPPS peptide was similarly reactive in IgG-mediated reactions; it could interfere with Allergen M line precipitate in rocket-line immunoelectrophoresis, giving a distinct deflection. It is the only reported synthetic polypeptide imposing allergenic and antigenic reactivity. The hexadecapeptide is composed of two homologous tetrapeptides located at its terminai, tentatively concluded to be mutually critical for the immunological specificity. The data obtained confirmed the predicted reactivity of the Ca2+-binding CD loop of the intact Allergen M.

Allergens↗