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Biomedical subjects

A Braun

Publications and source records attributed to A Braun.

At least 109 records · Page 6Linked to original sources
PubMed1998

Fundamentals of quality assessment of molecular amplification methods in clinical diagnostics. International Federation of Clinical Chemistry Scientific Division Committee on Molecular Biology Techniques.

The increasing interest in molecular biology diagnostics is a result of the tremendous gain of scientific knowledge in genetics, made possible especially since the introduction of amplification techniques. High expectations have been placed on genetic testing, and the number of laboratories now using the relevant technology is rapidly increasing--resulting in an obvious need for standardization and definition of laboratory organization. This communication is an effort towards that end. We address aspects that should be considered when structuring a new molecular diagnostic laboratory, and we discuss individual preanalytical and analytical procedures, from sampling to evaluation of assay results. In addition, different means of controlling contamination are discussed. Because the methodology is in constant change, no general standards can be defined. Accordingly, this publication is intended to serve as a recommendation for good laboratory practice and internal quality control and as a guide to troubleshooting, primarily in amplification techniques.

Chemistry, Clinical↗
PubMed1998

Comprehensive analytical strategy for mutation screening in 21-hydroxylase deficiency.

Congenital adrenal hyperplasia (CAH) is an autosomal recessive disease with a wide range of clinical manifestations. It is most often caused by deficiency of steroid 21-hydroxylase, reflecting any of a wide range of mutations in the 21-hydroxylase (CYP21) gene. A major challenge in molecular diagnostics of CAH is the high homology between the CYP21 gene and the CYP21P pseudogene and the phenomenon of apparent gene conversion, which inactivates the functional gene. In this study we devised an improved stepwise diagnostic procedure involving nonradioactive Southern blotting and direct DNA sequencing. This strategy led to a successful elucidation of the molecular cause of the disease in 181 out of 182 unrelated alleles in a total of 91 clinically and biochemically characterized patients. We were able to identify all classical known disease-causing mutations of the 21-hydroxylase gene and a novel nonsense mutation (bp 670, A-->C, Y97X). Our method also allows the reliable, secure diagnosis of the heterozygous configuration and may therefore be used for pre-, peri-, and postnatal diagnosis of CAH, even when informative data of the index patient are lacking. Furthermore, it can be used to confirm the diagnosis of CAH in newborns detected in 17-hydroxyprogesterone screening programs.

Adrenal Hyperplasia, Congenital↗
PubMed1997

Improved analysis of microsatellites using mass spectrometry.

The primer oligo base extension reaction combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, recently introduced by our group for detection of single-point mutations and small insertions/deletions, has been applied to the reliable quantification of nucleotide repeat units in microsatellites. The AluVpA DNA marker within intron 5 of the interferon-alpha receptor gene was chosen as the model system. By varying the dNTP/ddNTP mixtures used, the assay could also be directed to detect the location of second-site mutations within the repeats, resulting in identification of alleles not detectable by electrophoretic sizing methods and thus an increase of the polymorphism information content for a sampling of 28 unrelated individuals. The method results in highly informative mass signals and has the potential to increase the polymorphism information content for systems containing second-site mutations; thus it is a very attractive alternative technique in statistics-based gene mapping, cancer diagnostics, and forensic applications.

Base Sequence↗
PubMed1997

Genomic organization and molecular characterization of a gene encoding HsPXF, a human peroxisomal farnesylated protein.

A protein modification essential for the cellular sorting of many biologically relevant proteins is the covalent attachment of prenyl lipids by specific transferases. Isoprenylation is known to render protein domains hydrophobic, thereby facilitating the interaction with lipid bilayers and/or membrane proteins. The target for the modification with farnesyl groups is the COOH-terminal sequence CaaX. Among the variety of farnesylated proteins the only one reported so far to be located to peroxisomes is the 37-kDa peroxisomal farnesylated hamster protein PxF. Recently we published data on the cDNA of the human gene HK33 (A. Braun et al., 1994, Gene 146: 291-295), which was revealed to be the human ortholog of PxF and was consequently renamed HsPXF. The genomic structure, molecular characterization, and evolutionary conservation of HsPXF are described herein. The exact location of the gene was defined as chromosome 1q22. The gene spans a region of approximately 9 kb, containing eight exons and seven introns. The 5' upstream region showed two potential Sp1-binding sites and an Alu repetitive sequence. Luciferase reporter activating capacity confirmed the presumed promoter activity of this region. On the transcriptional level, we detected four splice variants originating either from exon skipping or from alternative splicing events. For the HsPXF protein, a carboxyterminal farnesylation at cysteine residues was demonstrated. Through the use of HsPXF-specific antibodies, the protein was shown to be attached to the outer surface of peroxisomes. This localization together with the similarity to a peroxisomal assembly protein from Saccharomyces cerevisiae suggests HsPXF is involved in the process of peroxisomal biogenesis or assembly.

Amino Acid Sequence↗
PubMed1997

Intrathymic education of alpha beta and gamma delta T cells is accompanied by cell surface expression of RNA/DNA helicase [corrected].

Despite ubiquitous expression of the gene, RNA/DNA helicase protein was found to be expressed specifically in all cells of the T cell lineage. Interestingly, immature thymocytes that are rearranging T cell receptor (TCR) genes express the helicase strongly on the cell surface and the surface expression is terminated upon engagement of functional TCR by positively selecting ligands. This provides the first evidence that a protein that binds nucleic acids can directly contact the extracellular environment in a developmentally controlled manner. Our discovery of a novel molecular link between the cell surface and nuclear events specific for thymocytes suggests that thymic education is supervised by a previously unknown molecular mechanism, which can now be experimentally explored.

Amino Acid Sequence↗
PubMed1997

Detection of RET proto-oncogene codon 634 mutations using mass spectrometry.

Mutations located in the RET proto-oncogene at codon 634 associated with multiple endocrine neoplasia type 2A and medullary thyroid carcinoma are detected by low-resolution and high-resolution mass spectrometry schemes not requiring labeling or electrophoretic separation of diagnostic products. The former requires measurement by matrix-assisted laser desorption ionization time-of-flight mass spectrometry of 21- to 27-mer oligonucleotides generated by a primer oligo base extension reaction. The latter is based upon direct measurement of artificial products which include the mutation site using matrix-assisted laser desorption ionization Fourier transform mass spectrometry. In this feasibility study a synthetic 25-mer representing the wildtype allele (7660.3 Da) was easily distinguished from G to A (7644.3 Da) and G to T (7635.3 Da) mutant alleles; the mutant alleles, which differed in mass by only 9.0 Da, were easily resolved when analyzed as a mixture. The results of both detection schemes were highly accurate and reliable, indicating mass spectrometry to be a high-quality alternative for future DNA diagnostics performed in clinical laboratories and genetic profiling studies.

Base Sequence↗
PubMed1997

Development and use of enzyme-linked immunosorbent assays (ELISA) for the detection of protein aggregates in interferon-alpha (IFN-alpha) formulations.

PURPOSE: Protein aggregates are thought to be involved in the immunogenicity of recombinant proteins in humans. To probe human IFN-alpha formulations for the presence of soluble protein aggregates, enzyme-linked immunosorbent assays (ELISA) were developed. METHODS: For the detection of IFN-alpha-IFN-alpha and HSA-IFN-alpha aggregates, sandwich ELISAs were developed using a monoclonal anti-IFN-alpha antibody as a capture antibody and the same anti-IFN-alpha antibody and an anti-human serum albumin (HSA) antibody (HRP-labeled), respectively. RESULTS: Marketed freeze-dried, HSA-containing IFN-alpha-formulations tested in the ELISAs all contained IFN-alpha-IFN-alpha and/or HSA-IFN-alpha protein aggregates, although in varying amounts. These aggregates were predominantly IFN-alpha dimers and 1:1 conjugates of HSA with IFN-alpha. Test formulations revealed that aggregation of IFN-alpha was strongly affected by the presence of pharmaceutical excipients, pH of the formulation, lyophilisation procedure, and storage temperature and time. CONCLUSIONS: The ELISAs are rapid, highly specific for aggregates in the presence of both IFN-alpha and HSA monomers and allow the direct detection of both types of aggregates in formulations in the nanogram range. The new assays will assist the monitoring of the aggregate-inducing processes during IFN-alpha formulation and storage in an early phase and the development of aggregate-free IFN-alpha formulations.

Drug Storage↗
PubMed1997

Protein aggregates seem to play a key role among the parameters influencing the antigenicity of interferon alpha (IFN-alpha) in normal and transgenic mice.

PURPOSE: During long-term treatment of various malignant or viral diseases with IFN-alpha up to 20% of patients develop anti-IFN-alpha antibodies for as yet unknown reasons. METHODS: To address this issue, a mouse model using Balb/C mice was established and the relevance of several potentially anti-IFN-alpha antibodies inducing factors was studied. RESULTS: The model revealed that both a higher frequency of injections and a higher dosage of IFN-alpha were more immunogenic and that the route of administration affected the antibody response to IFN-alpha. The intrinsic immunostimulatory activity of IFN-alpha itself also enhanced the immune response. IFN-alpha protein aggregates (IFN-alpha-IFN-alpha and human serum albumin (HSA)-IFN-alpha aggregates), which were recently identified in all marketed IFN-alpha products, were significantly more immunogenic than IFN-alpha monomers. These aggregates broke the tolerance against human IFN-alpha monomers in human IFN-alpha transgenic mice. CONCLUSIONS: Based on these animal studies it is proposed that the immune response to IFN-alpha in humans is most probably elicited by a combination of several factors among which IFN-alpha protein aggregates seem to play a key role.

Adjuvants, Immunologic↗
PubMed1997

Synthesis of kininogen and degradation of bradykinin by PC12 cells.

1. In this study, the abilities of PC12 cells to synthesize and degrade kinins were investigated. Kinin formation was assessed as kinin and kininogen content of cells and supernatants in serum-free incubations by use of a bradykinin-specific radioimmunoassay. Expression of kininogen mRNA was demonstrated by reverse-transcriptase PCR. Kinin degradation pathways of intact PC12 cells were characterized by identification of the kinin fragments generated from tritiated bradykinin either in the absence or presence of the angiotensin I-converting enzyme inhibitor ramiprilat. 2. Kinin immunoreactivity in the supernatant of PC12 cell cultures accumulated in a time-dependent fashion during incubations in serum-free media. This effect was solely due to de novo synthesis and release of kininogen (35 pg bradykinin h-1 mg-1 protein) since it could be suppressed by cycloheximide. Continuous synthesis of kininogen was a specific property of PC12 cells, as it was not observed in cultured macro- or microvascular endothelial cells. PC12 cells contained only minor amounts of stored kininogen. The rate of kininogen synthesis was not affected by ramiprilat, bacterial lipopolysaccharide, nerve growth factor or dexamethasone, but was stimulated 1.4 fold when cells were pretreated for 1 day with 1 microM desoxycorticosterone. 3. By use of cDNA probes specific for kininogen subtype mRNAs, expression of low-molecular-weight kininogen and T-kininogen in PC12 cells was confirmed. Expression of high molecular weight kininogen mRNA was also shown, though only at the lowest limit of detection of the assay. 4. Degradation of tritiated bradykinin by PC12 cells occurred with a half-life of 48 min resulting in the main fragments [1-7]- and [1-5]-bradykinin. The degradation rate of bradykinin decreased to 15% in the presence of ramiprilat (250 nM). Apart from angiotensin I-converting enzyme direct cleavage of bradykinin to [1-7]- and [1-5]-bradykinin still occurred under this condition as a result of additional kininase activities. 5. Along with previous findings of B2-receptor-mediated catecholamine release, these results now confirm the hypothesis that a cellular kinin system is expressed in PC12 cells. The presence of such a system may reflect a role of kinins as local neuromodulatory mediators in the peripheral sympathetic system.

Animals↗
PubMed1997

Drosophila immunity: analysis of larval hemocytes by P-element-mediated enhancer trap.

Our aim was to identify new genes involved in the cellular aspects of defense mechanism of Drosophila, as well as in melanotic tumor formation processes that are linked to blood cell disregulation. We have screened 1341 enhancer detector fly lines for expression of the lacZ reporter gene in larval hemocytes at the end of the third instar. We have selected 21 lines in which we observed a reproducible lacZ expression in blood cells. These lines were classified according to the subsets of hemocytes in which lacZ was expressed, and we identified five lines that can be used as lamellocyte markers. Three lines were selected for further analysis. The first exhibited strong lacZ expression in all lamellocytes. The second expressed lacZ in plasmatocytes and lamellocytes, and exhibited a melanotic tumor phenotype in larvae homozygous for the insertion. A third line showed a striking insertion-linked phenotype of melanized lymph glands (the hematopoietic organ), which resulted in the total absence of circulating hemocytes in the mutant larvae. We anticipate that this mutation, which we named domino, will prove a useful tool in the analysis of the role of hemocytes during the various aspects of immune response and melanotic tumor formation.

Animals↗
PubMed1997

Pimozide-induced depression in men who stutter.

BACKGROUND: Neuroleptic-related dysphoric reactions are well recognized in the context of psychiatric disorders, especially in association with extrapyramidal side effects. Very few controlled data exist regarding the effects of neuroleptics on the mood of psychiatrically "normal" subjects. In this study, the depressogenic effect of the neuroleptic drug pimozide was assessed in men without psychiatric disorders. METHOD: Eight men with developmental stuttering but no past or present psychiatric illness participated in a double-blind, placebo-controlled study assessing the effect of 6 weeks of pimozide treatment on speech fluency and mood. RESULTS: Four of the seven subjects who were compliant with the treatment developed marked depressive symptoms. No clear association was found between these reactions and pimozide dose, blood level, or degree of neurologic side effects. Symptoms abated soon after drug discontinuation. CONCLUSION: Pimozide induced significant depressive symptoms in this group of psychiatrically normal men who stutter. Neuroleptic drugs may have a causal effect in the induction of depression in psychiatrically normal subjects, ostensibly independent of dose or severity of neurologic side effects.

Adolescent↗
PubMed1997

Detecting CFTR gene mutations by using primer oligo base extension and mass spectrometry.

A new method for the reliable identification of localized variations in DNA by detection of associated diagnostic products with matrix-assisted laser desorption ionization time-of-flight mass spectrometry is described. The diagnostic products are generated by the primer oligo base extension (PROBE) reaction, which requires a single detection primer complementary to a region down-stream of a target strand's variable site. On addition of a polymerase, three dNTPs, and the fourth nucleotide in dideoxy form, the primer is extended through the mutation region until the first ddNTP is incorporated; the mass of the extension products determines the composition of the variable site. Tests for five cystic fibrosis mutations, including two exon 11 sites measured in a biplex reaction, and for differentiating between three common alleles of the poly(T) tract at the intron 8 splice acceptor site of the CFTR gene are presented. All experimental steps required for PROBE are amenable to the high degree of automation desirable for a high-through-put diagnostic setting. Furthermore, it requires no fluorescent, chemiluminescent, or radioactive labeling; the mass signals measured offer a far more analytically definitive signal, leading in all cases to high-quality unambiguous and easily interpreted results.

Alleles↗
PubMed1997

Interferon immunogenicity: preclinical evaluation of interferon-alpha 2a.

A preclinical evaluation of the immunogenicity of various preparations of interferon-alpha (IFN-alpha) was performed with in vitro and in vivo animal models. The distribution of genes for IFN-alpha 2a, IFN-alpha 2b, and IFN-alpha 2c in various cell populations and the response of human T cell clones to IFN-alpha peptides were investigated. The immunogenicity of IFN-alpha in IFN-alpha 2b transgenic mice and factors that influence the immunogenicity of IFN-alpha in normal mice were also studied. The genes for IFN-alpha 2a and IFN-alpha 2b were found in KG-1 cells, whereas IFN-alpha 2b and IFN-alpha 2c genes were present in Namalwa cells. No difference in proliferation of human T cells, T cell lines, or T cell clones could be obtained with IFN-alpha peptides. In transgenic mice bearing the human IFN-alpha 2b gene, no antibody response was obtained following immunization with either IFN-alpha 2a or IFN-alpha 2b. Normal mice immunized with either IFN-alpha 2a or IFN-alpha 2b produced equivalent titers of antibodies, which cross-reacted with both IFNs. Studies evaluating the relative immunogenicity of IFN-alpha in normal mice demonstrated that a number of treatment and host variables can modulate immunogenicity of IFN-alpha preparations.

Amino Acid Sequence↗
PubMed1997

Identification of apolipoprotein E polymorphisms using temperature cycled primer oligo base extension and mass spectrometry.

The isothermal Primer Oligo Base Extension (PROBE) reaction combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for diagnostic product detection as recently introduced by our group is modified to incorporate temperature cycling during the primer extension step, resulting in enhanced levels of diagnostic product generation. Utilizing temperature cycled PROBE, the identities of two apolipoprotein E polymorphisms (codons 112 and 158) for differentiation of epsilon 2/epsilon 3, epsilon 3/epsilon 3, epsilon 3/epsilon 4, and epsilon 4/epsilon 4 genotypes were simultaneously determined. Primers specific for each site are extended by a series of bases unique to the identity of that variable site, producing low mass diagnostic products (M(r) < 9000) highly amenable to detection by mass spectrometry. The temperature cycled PROBE method has yielded unambiguous and correct diagnoses for all samples tested thus far. The increased amount of diagnostic product generated per primer by the cycling method makes possible faster spectrum acquisition due to the increased signal intensity, critical for future automated measurement of such samples.

Apolipoproteins E↗
PubMed1996

Cloning of the gene encoding the antibacterial peptide drosocin involved in Drosophila immunity. Expression studies during the immune response.

A potent inducible antibacterial peptide carrying an O-glycosylated substitution has recently been isolated from Drosophila [Bulet, P., Dimarcq, J. L., Hetru, C., Lagueux, M., Charlet, M., Hegy, G., Van Dorsselaer, A. and Hoffmann, J. A. (1993) J. Biol. Chem. 268, 14893-14897]. Here we report cloning studies that show that Drosophila contains a single, intronless gene, located at position 51C1-6, which encodes the precursor protein from which drosocin is processed. The upstream and the downstream sequences of the drosocin gene contain putative cis-regulatory elements similar to mammalian regulatory motifs, namely three kappa B-related decameric sequences. The drosocin gene is silent in naive animals, and is strongly induced with acute phase kinetics after immune challenge in larvae and in adults. We have established several transgenic fly lines in which reporter genes were placed under the control of various drosocin promoter sequences. Our results indicate that 2.5 kb of upstream sequences confer inducibility and tissue specificity to the transgene, but that the level of its expression in the fat body after immune challenge is low. Addition of genomic regions downstream of the drosocin transcribed sequences results in increased transcription levels, which are similar for the fusion and the resident drosocin genes upon infection. Analysis of transgenic fly lines showed that the drosocin reporter gene is constitutively expressed in the oviducts of egg-laying females.

Amino Acid Sequence↗
PubMed1996

Structure of the bradykinin B2 receptors' amino terminus.

The peptide hormone bradykinin exerts important biological functions by binding to and activating bradykinin B2 receptors. B2 receptors belong to the seven transmembrane domain (7TM) receptor family. Cloning of the cDNA sequences for the rat, human, and mouse bradykinin B2 receptor revealed several in-frame AUG triplets as potential initiation sites for translation. Due to "Kozak-like" consensus nucleotides, the AUG codon closest to transmembrane domain 1 was assumed the preferred initiation site for translation, but the real amino terminus of the B2 receptor protein was unknown. The amino terminus of several 7TM receptors has been shown to be essentially involved in receptor activation and/or ligand binding. Therefore we determined the amino terminus of the human and of the rat B2 receptor using domain-specific antipeptide antibodies, amino acid sequence analysis, and in vitro transcription/translation. We report that the human and rat B2 receptor protein start with the methionine which is translated from the first in-frame AUG. This start site extends the known amino terminus of the human and rat B2 receptors by 27 or 30 amino acid residues, respectively. Antibodies raised against a peptide of the initial 27 amino acids of the human B2 receptor stained B2 receptors on intact cells. This finding excludes the existence of a signal sequence for this receptor.

Amino Acid Sequence↗
PubMed1996

Characterization of a partial pseudogene homologous to the adrenoleukodystrophy gene and application to mutation detection.

The gene for the most common peroxisomal disorder, X-linked adrenoleukodystrophy (X-ALD, McKusick #300100), encodes a peroxisomal membrane transporter protein (ALDP), and comprises 10 exons spanning approximately 21 kb. So far, however, the mutation analysis at the genomic level was handicapped by the coamplification, in PCR reactions, of sequences related to the distal exons, also detected by Southern blot hybridization on genomic DNA. We isolated one clone from a human genomic phage library, which represents a partial ALD pseudogene, spanning exon 7 to exon 10 and exhibits approximately 93% sequence homology with the ALD gene in this region. Primers designed in the region of maximum mismatch between the pseudogene and the functional gene allowed the amplification of the functional exons without any contaminating sequences of the pseudogene or other related sequences. This information will greatly facilitate the detection of mutations in distal exons of the ALD gene and increase the reliability of the mutation analysis.

ATP Binding Cassette Transporter, Subfamily D, Mem↗