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J Apold

Publications and source records attributed to J Apold.

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

Expression of recombinant human phenylalanine hydroxylase as fusion protein in Escherichia coli circumvents proteolytic degradation by host cell proteases. Isolation and characterization of the wild-type enzyme.

Recombinant human phenylalanine hydroxylase (hPAH) was produced in high yields in Escherichia coli using the pET and pMAL expression vectors. In the pMAL system, hPAH was fused through the target sequences of the restriction protease factor Xa (IEGR) or enterokinase (D4K) to the C-terminal end of the highly expressed E. coli maltose-binding protein (MBP). The recombinant hPAH, recovered in soluble forms, revealed a high specific activity even in crude extracts and was detected as a homogeneous band by Western-blot analysis using affinity-purified polyclonal rabbit anti-(rat PAH) antibodies. The enzyme expressed in the pET system was subject to limited proteolysis by host cell proteases and was difficult to purify with a satisfactory yield. By contrast, when expressed as a fusion protein in the pMAL system, hPAH was resistant to cleavage by host cell proteases and was conveniently purified by affinity chromatography on an amylose resin. Catalytically active tetramer-dimer (in equilibrium) forms of the fusion protein were separated from inactive, aggregated forms by size-exclusion h.p.l.c. After cleavage by restriction protease, factor Xa or enterokinase, hPAH was separated from uncleaved fusion protein, MBP and restriction proteases by hydroxylapatite or ion-exchange (DEAE) chromatography. The yield of highly purified hPAH was approx. 10 mg/l of culture. The specific activity of the isolated recombinant enzyme was high (i.e. 1440 nmol of tyrosine.min-1.mg-1 with tetrahydrobiopterin as the cofactor) and its catalytic and physicochemical properties are essentially the same as those reported for the enzyme isolated from human liver. The recombinant enzyme, both as a fusion protein and as purified full-length hPAH, was phosphorylated in vitro by the catalytic subunit of cyclic AMP-dependent protein kinase. The phosphorylated from of hPAH electrophoretically displayed an apparently higher molecular mass (approximately 51 kDa) than the non-phosphorylated (approximately 50 kDa) form.

ATP-Binding Cassette Transporters↗
PubMed1995

The PKU mutation S349P causes complete loss of catalytic activity in the recombinant phenylalanine hydroxylase enzyme.

The mutation S349P in exon 10 of the phenylalanine hydroxylase (PAH) gene was identified in one Norwegian and one Polish phenylketonuria (PKU) allele on a haplotype 1.7 background. This missense mutation in PAH codon 349 is a T to C transition in cDNA position 1267. This mutation has been reported both on haplotype 1 and 4, suggesting recurrent mutation. In two different expression systems, the pET and the pMAL systems of Escherichia coli, it was shown that the S349P mutation, introduced by site directed mutagenesis, results in complete loss of enzymatic activity. Thus, protein instability alone does not seem to be the direct cause of the lack of activity of this PKU mutation as previously reported.

Base Sequence↗
PubMed1993

[Analysis of the Duchenne muscular dystrophy gene with PCR analysis in paraffin-embedded tissue. A new diagnostic possibility].

Analysis of DNA from archival, paraffin-embedded muscle tissue allowed tracing of the mutated dystrophin gene in two families with Duchenne muscular dystrophy. There were no living patients in these families. In one family this diagnosis contributed to the birth of a normal male, in the other family to the demonstration of carrier status. These analyses, based on the polymerase chain reaction, are relatively rapid and simple, and lend increased value to old tissue samples stored in pathology departments.

DNA↗
PubMed1993

[DNA diagnosis of fragile X syndrome in a family. A new type of heredity--dynamic mutations].

In typical cases, the diagnosis of fragile X syndrome can be made clinically, but has so far been based on cytogenetic detection of a fragile site on the X chromosome at Xq27.3. Cytogenetic analyses are time-consuming, and false negative results have been a problem. Following the successful molecular cloning of the gene causing the fragile X syndrome, a novel genetic principle was discovered: the "dynamic" DNA mutation. DNA analyses have now the power to distinguish between normal copies of the gene, carrier mutations and fragile X mutations. We have performed both cytogenetic and DNA analyses in an extended fragile X family. DNA fragment length variations in the gene were identified by Southern blot analysis, and the results were used diagnostically in the family. Instability of the mutation (dynamic mutation) occurred between generations and within individuals. DNA analysis, as performed here, improves diagnostic accuracy and genetic counselling in fragile X families.

Blotting, Southern↗
PubMed1993

The phenylketonuria G272X haplotype 7 mutation in European populations.

We have compiled data on the frequencies of the phenylketonuria G272X mutation in European populations. This mutation occurs north of the Alps. It has a particularly high frequency in the Oslo Fjord region of Norway with the adjacent Bohuslän region of Sweden. An intermediate frequency was noted in a separate area, the eastern part of Germany with the adjacent western part of Czechoslovakia. The G272X mutation was associated with phenylalanine hydroxylase haplotype 7, except for one case with haplotype 3. Genealogical studies going back eight to nine generations revealed no common source for this mutation, but there was some geographical convergence to the Bohuslän region. These findings suggest a single origin for this mutation, with at least one founding population in south-eastern Norway/adjacent Sweden.

Europe↗
PubMed1992

A de novo phenylketonuria mutation: ATG (Met) to ATA (Ile) in the start codon of the phenylalanine hydroxylase gene.

We here describe the detection of a de novo mutation in the phenylalanine hydroxylase gene in a Norwegian phenylketonuria (PKU) patient. This novel mutation, M1I, disrupts the start codon of the gene by a G to A transition. The compound heterozygote genotype (IVS-12/M1I) of this patient predicts that no phenylalanine hydroxylase enzyme is formed, thus leading to a severe classical PKU. Determination of haplotypes and DNA fingerprint patterns indicates a paternal origin of the de novo mutation.

Adult↗
PubMed1992

PKU mutations R408Q and F299C in Norway: haplotype associations, geographic distributions and phenotype characteristics.

Details are given concerning the phenylketonuria (PKU) mutations R408Q and F299C. Both mutations were identified among 47 PKU patients, derived from the Norwegian PKU registry. A novel PKU mutation (R408Q) was identified, by single-strand conformation polymorphism analysis, on six out of eight mutant haplotype 12 chromosomes and on none of the other PKU chromosomes. The F299C mutation occurred exclusively on mutant haplotype 8, and was the only mutation associated with this haplotype (on six chromosomes). One patient homozygous for each mutation was found. The patient homozygous for F299C manifested severe PKU, whereas the R408Q homozygote exhibited a mild PKU variant. Pedigree analysis of these families has not, so far, revealed consanguinity. Information on the place of birth of the relevant grandparents of the PKU patients with these mutations suggests that each of these mutations in Norway has originated from a common gene source.

Adolescent↗
PubMed1991

Application of natural and amplification created restriction sites for the diagnosis of PKU mutations.

PCR amplification, either conventional, or as site directed mutagenesis using primers with mismatched 3'-ends, followed by restriction endonuclease digestion, provides rapid, non-isotope assays of known mutations in the human phenylalanine hydroxylase gene. Such assays were shown to have the potential to detect all of the 18 presently reported phenylketonuria mutations. The practical applicability of this approach was demonstrated for eight mutations in Norwegian phenylketonuria patients, among them the most common ones.

Base Sequence↗
PubMed1991

The structural requirements of epitopes with IgE binding capacity demonstrated by three major allergens from fish, egg and tree pollen.

Three major allergens from cod fish, egg white and tree pollen, were characterized by studies on their allergenic and antigenic structures. The major allergen of cod fish, Allergen M "parvalbumins pI 4.75", is composed of 113 amino acid residues with a molecular weight of 12,328 daltons. It comprised three domains, AB, CD and EF, consisting of 3 helices interspaced by one loop. Each of the loops of the CD and EF domains each coordinates one Ca2+. The antigenicity and allergenicity of Allergen M was deduced from studying the modified protein and some particular synthetic peptides. Three sites were encompassing IgE binding epitopes namely peptides 33-44, 65-74 and 88-96. A novel peptide (49-64), of the CD-domain, was demonstrated to be allergenically/antigenically active and cross reactive with birch pollen allergen, which incidentally was used as a negative control. This site encompassed two repetitive sequences (D-E-D-K) and (D-E-L-K), suggested to be mutually critical for the specificity of antibody binding. This hypothesis was reconfirmed by SPPS of several analogous peptides of region 39-64. Furthermore, peptide 88-103 of the EF-domain was similarly synthesized; it functioned as a monovalent hapten, blocking and not eliciting allergic reaction. Moreover, peptide 13-32 of domain AB, the non-calcium binding domain, was thoroughly tested. The results of PK inhibition showed clear activity and the peptide was found to function at the level of a divalent determinant. Ovalbumin (OA) is the most dominant of five major allergens of egg white and universally used as model protein. OA allergenic epitopes were shown to be mainly determined by the primary structure and depend on certain peptide chain length. The N-terminal decapeptide (OA 1-10) was shown to react with reaginic IgE. Direct skin test on egg allergic patients, showed no activity and the site was therefore concluded to encompasses one single Ig binding haptenic epitope. Peptide OA 323-339, was demonstrated to be valuable in studies of T-cell recognition of protein antigens. Three analogous peptides of this region were prepared and clearly shown to be immunogenic in rabbits and to bind specific IgE from patients allergic to egg. OA 323-339 was concluded to encompass an allergenic and antigenic epitope which was recognized by human and rabbit B-lymphocytes. Eight peptides in the region 11-122 were similarly synthesized. A test battery was performed to study this region using rabbit polyclonal antibodies and human specific IgE. Some of these sites were involved in binding of particular Ig paratopes. Five immunogenic peptides from the major allergens of tree pollen extracts (segment 23-38), were synthesized. The selection of those peptides was setteled using two algorithms for providing the optimal hydrophobicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Allergens↗
PubMed1990

The zebrafish homeobox gene hox-2.2: transcription unit, potential regulatory regions and in situ localization of transcripts.

The data presented in this report strongly suggest that the genome of the zebrafish, Brachydanio rerio, has a homeobox cluster which is equivalent to the murine Hox-2 locus. In support of this conclusion, we have found two closely linked zebrafish genes which are true homologues of the mouse Hox-2.1 and Hox-2.2 genes. Here we describe structural and functional properties of the zebrafish Hox-2.2 homologue hox-2.2. Furthermore, we have identified another zebrafish gene related to hox-2.2 which appears to correspond to the Hox-6.1 gene of the murine Hox-3 locus. In order to characterize the zebrafish hox-2.2 gene we have determined the genomic DNA sequence of a 3.4 kb SalI fragment. This revealed that the hox-2.2 transcription unit encodes a putative protein of 228 amino acids. The homeodomains of the murine Hox-2.2 and the zebrafish hox-2.2 proteins are almost identical and extensive sequence identity exists in other regions of the two proteins, which share 160 (70%) of the amino acid residues. Also in terms of expression, strong similarities were observed relative to the murine Hox-2.2 gene. Transcripts derived from zebrafish hox-2.2 start to accumulate when somite formation is initiated. Later in development these transcripts are detected mainly in the central nervous system. Reminiscent of Hox-2.2, the rostral boundary of zebrafish hox-2.2 expression is located in the posterior region of the hindbrain. Notably, untranslated regions of the hox-2.2 gene contain several short sequences closely related to a known homeodomain recognition sequence.

Amino Acid Sequence↗
PubMed1990

A termination mutant prevalent in Norwegian haplotype 7 phenylketonuria genes.

RFLPs in the phenylalanine hydroxylase (PAH) gene locus were determined in 47 Norwegian nuclear families that had at least one child with phenylketonuria (PKU). The PKU haplotype distribution differed somewhat from that of other European populations. Mutant haplotype 7 is relatively rare in other populations but constituted 20% of all mutant haplotypes in Norway. In 14 of the 17 mutant haplotypes 7, a previously unreported deletion of the BamHI restriction site in exon 7 of the PAH gene was observed. The abrogation of the BamHI site was shown to be due to a G-to-T transversion, changing Gly 272 to Ter 272 in exon 7 of the gene, thus directly identifying the PKU mutation. Unlike the families of the other PKU patients, the families with this mutation clustered along the southeastern coast of Norway, suggesting a founder effect for this mutation.

Adolescent↗
PubMed1989

Dust from carpeted and smooth floors. I. Comparative measurements of antigenic and allergenic proteins in dust vacuumed from carpeted and non-carpeted classrooms in Norwegian schools.

Dust samples from fitted-carpets and linoleum floors in 12 schools in Norway were collected by vacuum cleaning. The presence of antigens and allergens of alder (Alnus incana), birch (Betula verrucosa), timothy (Phleum pratense), mould (Cladosporium herbarum), house dust mite (Dermatophagoides farinae), cat and dog dander, codfish, hen egg white and human dander were investigated by crossed immunoelectrophoresis (CIE), crossed radio-immunoelectrophoresis (CRIE) and radio-allergosorbent test (RAST) inhibition. No qualitative differences in allergen contents of dust from both types of floor tested were noted. Similarly, no relationship could be demonstrated between floor-type and allergen concentration under identical experimental conditions. Antigens and allergens of both cat and dog were frequently demonstrated in dust extracts. All extracts included human dander and mould allergens. In addition, most dust samples from both carpeted and smooth floors contained hen egg white and codfish allergens. Furthermore, the study demonstrated that dust from smooth floors and fitted-carpets was relatively free of mite and pollen from alder, birch and timothy.

Allergens↗