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Biomedical subjects

B Rapp

Publications and source records attributed to B Rapp.

At least 19 recordsLinked to original sources
PubMed2003

Gene structure of human carbamylphosphate synthetase 1 and novel mutations in patients with neonatal onset.

Carbamylphosphate synthetase 1 (E.C. 6.3.4.16) deficiency is a rare autosomal recessive disorder of the urea cycle that can result in severe neonatal hyperammonemia. Since the genomic structure of the CPS1 gene was not yet elucidated, mutation detection was performed by analysis of transcripts in the past. Here, we present the entire DNA sequence of the human CPS1 gene including all exon-intron boundaries. Moreover, mutation analysis was performed in six patients leading to the detection of 9 novel mutations including the missense mutations c.2528T>C and c.2623A>G, the nonsense mutations c.712C>T and c.2115ins35bp, the splice site mutations c.1263+5G>C, c.3558+1G>C and c.4101+2T>C, and a small deletion c.3036_3038delGGT. The mutations c.2528T>C and c.2623A>G were identified on a double mutated allele. New data on the genomic structure of the CPS1 gene provided in this study are useful to characterize the heterogenous molecular basis of the disease in patients deficient for carbamylphosphate 1 deficiency.

Age of Onset↗
PubMed2002

[Value of fetal cerebral magnetic resonance imaging for the prenatal diagnosis and prognosis of corpus callosum agenesis].

OBJECTIVE: To evaluate MR contribution to prenatal diagnosis and prognosis of corpus callosum agenesis suspected by ultrasound and to ascertain how and when this examination should be included part in prenatal management of such malformation. PATIENTS AND METHODS: During a six-year period from January 1st 1994 and December 31st 2000, fifteen patients (and fifteen fetuses) were referred to our fetal medicine unit with suspicion of corpus callosum agenesis on prenatal ultrasound. Cerebral MRI was performed in all cases to confirm prenatal diagnosis. In our study, prenatal MRI examinations were retrospectively studied and compared with neuropathological examinations (n=8) or postnatal imaging (n=6). RESULTS: Corpus callosum agenesis were either complete (n=13) or partial (n=1). All were visible on prenatal MRI but only six on prenatal ultrasound. In one case, ultrasound suspicion of corpus callosum agenesis was ruled-out (false positive on prenatal ultrasound) by fetal cerebral MRI. In five cases corpus callosum agenesis was an isolated finding whereas in 12 cases associated malformations were encountered (cerebral n=7 or extra-cerebral n=5). MR depicted 7 of the 12 associated neurologic abnormalities. CONCLUSION: Prenatal MRI is a valuable complementary technique for either diagnosis of corpus callosum agenesis and depiction of associated neurologic abnormalities. Superiority of MR on prenatal sonography and its help in post-mortem examination of the brain (help in the choice of the pathologic technique and localisation of the samples) makes it essential even when pregnancy termination is considered.

Adult↗
PubMed2001

The Stat1 binding motif of the interferon-gamma receptor is sufficient to mediate Stat5 activation and its repression by SOCS3.

Signal transduction via the interferon-gamma (IFN-gamma) receptor requires the tyrosine phosphorylation of signal transducers and activators of transcription (Stats). Whereas tyrosine phosphorylation of Stat1 occurs in all cells, activation of Stat5 by IFN-gamma is cell type-restricted. Here we investigated the mechanism of Stat5 activation by the IFN-gamma receptor. In transfection assays both Stat5 isoforms, Stat5a and Stat5b, were phosphorylated on tyrosine in response to IFN-gamma. Stat5 activation required the presence of tyrosine 420 (Tyr-420) in the murine IFNGR1 receptor chain, which also serves as the Stat1 binding site. Moreover, a peptide including Tyr-440, the Stat1 binding site of the human IFNGR1 chain, conferred the ability upon a synthetic receptor to activate Stat5. Suppressor of cytokine signaling 3 (SOCS3) inhibited the activation of Stat5 by the IFN-gamma receptor, and the Tyr-440-containing peptide stretch was sufficient for repression. SOCS3 expression had little effect on the activity of Jak kinases not associated with cytokine receptors. In IFN-gamma-treated, Stat1-deficient fibroblasts Stat5 was inefficient in inducing transcription of a Stat-dependent reporter gene, suggesting it does not per se make a major contribution to the expression of IFN-gamma-responsive genes.

Animals↗
PubMed2001

Genetic analysis of carbamoylphosphate synthetase I and ornithine transcarbamylase deficiency using fibroblasts.

UNLABELLED: Deficiencies of carbamoylphosphate synthetase or of ornithine transcarbamylase, two urea cycle enzymes located within mitochondria, often present as severe neonatal hyperammonaemic crises and have a poor prognosis. While genetic analysis of the X-chromosomal transmitted ornithine transcarbamylase deficiency (OTC) is performed by exon-wise mutation screening of genomic DNA in most cases, identification of mutations in the autosomal inherited carbamoylphosphate synthetase (CPS 1) deficiency requires analysis of transcripts due to the unknown genomic structure. We tested the hypothesis that CPS 1 and OTC are expressed at low levels in fibroblasts and indeed were able to amplify full-length cDNA from that source. Using a reverse transcriptase polymerase chain reaction based procedure we completely characterised the genetic background in five patients and identified three novel mutations and a novel polymorphism of the CPS 1 gene (deletion/insertion 2170delGCTCinsCCA, nonsense mutation 2359C > T, missense mutation 3161T > G and Thr1406Asn, respectively), as well as the missense mutations 482A > G and 994T > A of the OTC gene. CONCLUSION: Cultured fibroblasts are an easily accessible source for genetic analysis of inborn errors of urea cycle enzymes which are functionally expressed only in liver and gut.

Carbamoyl-Phosphate Synthase (Ammonia)↗
PubMed2000

Attention-referenced visual representations: evidence from impaired visual localization.

Spatial representations in the visual system were probed in 4 experiments involving A. H., a woman with a developmental deficit in localizing visual stimuli. Previous research (M. McCloskey et al., 1995) has shown that A. H.'s localization errors take the form of reflections across a central vertical or horizontal axis (e.g., a stimulus 30 degrees to her left localized to a position 30 degrees to her right). The present experiments demonstrate that A. H.'s errors vary systematically as a function of where her attention is focused, independent of how her eyes, head, or body are oriented, or what potential reference points are present in the visual field. These results suggest that the normal visual system constructs attention-referenced spatial representations, in which the focus of attention defines the origin of a spatial coordinate system. A more general implication is that some of the brain's spatial representations take the form of coordinate systems.

Adult↗
PubMed2000

Discreteness and interactivity in spoken word production.

Five theories of spoken word production that differ along the discreteness-interactivity dimension are evaluated. Specifically examined is the role that cascading activation, feedback, seriality, and interaction domains play in accounting for a set of fundamental observations derived from patterns of speech errors produced by normal and brain-damaged individuals. After reviewing the evidence from normal speech errors, case studies of 3 brain-damaged individuals with acquired naming deficits are presented. The patterns these individuals exhibit provide important constraints on theories of spoken naming. With the help of computer simulations of the 5 theories, the authors evaluate the extent to which the error patterns predicted by each theory conform with the empirical facts. The results support a theory of spoken word production that, although interactive, places important restrictions on the extent and locus of interactivity.

Brain↗
PubMed1998

Dissociable coordinate frames of unilateral spatial neglect: "viewer-centered" neglect.

Detailed analyses of reading and nonlexical tasks by three patients with unilateral spatial neglect (USN) secondary to stroke indicate that the USN in each of these patients affects the left side (contralateral to brain damage) of the viewer, with respect to the viewer's head, mid-sagittal plane of the body, or line of sight. In one case, the neglect was further specified as concerning the left side of the viewer's line of sight (the left half of her residual visual field). Thus, the frame of reference of USN in these three cases appears to have viewer-centered (in at least one case, specifically retinotopic) coordinates. The performance of these patients is contrasted to that of other patients in the literature whose USN appears to have a frame of reference with stimulus-centered or object-centered coordinates. These results are interpreted within a model of visual processing (adapted from Marr, 1980 and others) with at least three coordinate frames. It is argued that USN can affect one or more of these coordinate frames independently.

Aged↗
PubMed1998

Unilateral spatial neglect in dissociable frames of reference: a comment on Farah, Brunn, Wong, Wallace, and Carpenter (1990)

We comment on a paper by Farah et al. in which it is claimed that averaged performance on a letter naming task by a group of patients with left unilateral spatial neglect (USN) provides evidence that spatial allocation of attention is defined by environment-centered and viewer-centred coordinates, but not by object-centred coordinates. We re-analyze the raw data from that paper, and present statistical analyses of the data from individual subjects which show dissociations in the coordinate frames used by the individual subjects with USN. The data from individual cases demonstrate that object-centered USN occurs alone (without evidence of USN in other reference frames) in some cases, and in association with USN in other reference frames other cases. In contrast to the conclusions drawn from the group performance, results from individual subjects provide evidence for the role of dissociable frames of reference, including an object-centred coordinate frame, in the spatial allocation of attention.

Attention↗
PubMed1997

The modality-specific organization of grammatical categories: evidence from impaired spoken and written sentence production.

We describe the case of a brain-damaged individual whose speech is characterized by difficulty with practically all words except for elements of the closed class vocabulary. In contrast, his written sentence production exhibits a complementary impairment involving the omission of closed class vocabulary items and the relative sparing of nouns. On the basis of these differences we argue: (1) that grammatical categories constitute an organizing parameter of representation and/or processing for each of the independent, modality-specific lexicons, and (2) that these observations contribute to the growing evidence that access to the orthographic and phonological forms of words can occur independently.

Aphasia↗
PubMed1997

From graphemes to abstract letter shapes: levels of representation in written spelling.

The letter substitution errors of 2 dysgraphic subjects who, despite relatively intact oral spelling, made well-formed letter substitution errors in written spelling, were studied. Many of these errors bear a general physical similarity to the intended target. Analyses revealed that this similarity apparently was based on the features of the component strokes of letters rather than on visuospatial characteristics. A comparison of these subjects' letter substitution errors with those of 2 other individuals with brain damage, whose damage was at a different level of processing, revealed that the latter subjects' errors are not explicable in terms of stroke-feature similarity. Strong support was found for the computation of multiple representational types in the course of written spelling. This system includes a relatively abstract, effector-independent representational level that specifies the features of the component strokes of letters.

Aged↗
PubMed1997

Cell-type-specific separate regulation of the E6 and E7 promoters of human papillomavirus type 6a by the viral transcription factor E2.

Gene expression of human papillomaviruses (HPV) is tightly controlled by cellular factors and by the virally encoded E2 protein through binding to distinct sites within the regulatory noncoding region. While for the high-risk genital papillomaviruses a single promoter drives the expression of all early genes, a second promoter present in the E6 open reading frame of the low-risk HPV type 6 (HPV6) would allow an independent regulation of E6 and E7 oncogene expression. In this report, we provide the first evidence that E2 regulates both early promoters of HPV6 separately and we show that promoter usage as well as E2 regulation is cell type dependent. Among the different epithelial cell lines tested, only RTS3b cells allowed an expression pattern similar to that observed in naturally infected benign condylomas. While the E6 promoter was repressed by E2 to 50% of its basal activity, the E7 promoter was simultaneously stimulated up to fivefold. Activation of the E7 promoter was mediated predominantly by the binding of E2 to the most promoter-distal E2 binding site. Repression of the E6 promoter depended on the presence of two intact promoter-proximal binding sites. Mutation of both of these repressor binding sites reversed the effect of E2 on the E6 promoter from repression to activation. In contrast, in HT3 cells we observed an E2-mediated activation of the E6 promoter in the context of the wild-type noncoding region. This indicated that repression of the E6 promoter by binding of E2 to both promoter-proximal binding sites did not function in the cellular environment provided by HT3 cells. These data suggest that the separate regulation of the E6 and E7 promoters of HPV6 is mediated through successive occupation of binding sites with different affinities for E2 depending on the intracellular concentration of E2 and on the cellular environment provided by the infected cell.

Animals↗
PubMed1996

HPV6 variants from malignant tumors with sequence alterations in the regulatory region do not reveal differences in the activities of the oncogene promoters but do contain amino acid exchanges in the E6 and E7 proteins.

Human papillomavirus type 6 (HPV6) causes benign epithelial proliferations of the anogenital and aerodigestive tract, which usually tend to regress spontaneously. The low incidence of HPV6 in carcinomas and the rare progression of the benign tumors has led to the classification of HPV6 as "low-risk" virus. A series of reports, however, described the isolation of HPV6 variants from malignant tumors characterized by sequence rearrangements in the noncoding regulatory region (NCR). It was speculated that these sequence alterations play a role in tumor progression by enhancing the promoter activity and thereby increasing the expression of the viral oncogenes E6 and E7. To elucidate if HPV6 isolates from malignancies do regularly exhibit sequence alterations in the regulatory region we first determined and complied the sequences of the NCRs of a number of isolates from benign and malignant lesions. This analysis revealed in general a high degree of sequence conservation between the individual isolates. Most of the isolates, however, differed, independently of origin, by a major and one or two minor insertions from the prototype HPV6b sequence. When tested in a functional assay these altered NCR sequences did not result in significantly different activities of the promoters responsible for the expression of the E6 and E7 genes. Further analysis of the E6 and E7 coding region revealed a surprisingly high sequence variability within the E6 ORF and allowed the detection of amino acid exchanges unique for isolates from carcinomas.

Base Sequence↗
PubMed1996

Identification of a differentiation-inducible promoter in the E7 open reading frame of human papillomavirus type 16 (HPV-16) in raft cultures of a new cell line containing high copy numbers of episomal HPV-16 DNA.

Gene expression of human papillomaviruses (HPV) is tightly linked to differentiation processes within the pluristratified epithelium. To analyze changes in the transcription pattern of HPV-16 during epithelial cell differentiation, we established a permanently growing HPV-16 positive cell line, designated KG, from a vulvar intraepithelial neoplasm. KG cells of early passages harbored multiple copies of the HPV-16 DNA as episomes and were able to form a stratified epithelium in an organotypic raft culture system. Analysis of viral gene expression revealed the known transcription pattern of the early region of HPV-16 with the exception of a so far undefined mRNA class with start sites in the E7 open reading frame. Quantitative analysis of primer extension experiments with RNA from KG cells grown in monolayer and raft culture showed a strong induction of this transcript in differentiated KG cells, whereas the level of the mRNAs initiated at the early promoter P97 remained almost constant. Primer extension analyses with four different primers and direct sequencing of the extension product revealed that the differentiation-inducible transcript initiated at a novel promoter with a major start site around nucleotide position 670 (P670) in the E7 open reading frame of HPV-16. Sequence analysis of cDNAs derived from RNA of KG cells grown in raft culture suggested that the transcripts initiated at P670 have a coding potential for an E1E4 fusion protein and for the E5 protein.

3T3 Cells↗
PubMed1994

Comparison of the properties of the E6 and E7 genes of low- and high-risk cutaneous papillomaviruses reveals strongly transforming and high Rb-binding activity for the E7 protein of the low-risk human papillomavirus type 1.

A comparative analysis of different properties of the E6 and E7 proteins of high-risk and low-risk cutaneous papillomaviruses was performed. The corresponding genomic regions of human papillomavirus types 1 and 8 (HPV1 and HPV8) and of the cottontail rabbit papillomavirus (CRPV) were cloned into the eucaryotic expression vector pZipNeo-SV(X)-1 and into vectors for in vitro transcription and translation. With the help of these vectors, the individual proteins were investigated for their ability to transform C127 and NIH 3T3 rodent fibroblasts, bind the Rb protein in vitro, transactivate the adenovirus E2 promoter, and cooperate in the immortalization of primary human keratinocytes. Expression vectors for HPV16 E6 and E7 were used as a positive control. A highly transformed phenotype could be observed with rodent cell lines expressing HPV8 E6, HPV16 E6 and E7, and, surprisingly, HPV1 E7. In contrast, no transformation was detected with CRPV long E6 and HPV8 E7, whereas cells expressing HPV1 E6 and CRPV short E6 exhibited a weakly transformed phenotype. Although neither CRPV E6 nor CRPV E7 caused morphological transformation of C127 cells, CRPV E6 was able to induce anchorage-independent growth in both rodent cell lines, whereas CRPV E7 led to high cloning efficiencies only in NIH 3T3 cells. The in vitro Rb-binding affinities relative to that of HPV 16 E7 were 66% for HPV1 E7, 34% for HPV8 E7, and 11% for CRPV E7. In spite of its high Rb-binding affinity, HPV1 E7 did not trans activate the adenovirus E2 promoter, whereas HPV8 E7 and CRPV E7 showed low activities. Complementation studies in primary human keratinocytes revealed a weak immortalizing potential for HPV8 E7 and indicated a low degree of cooperativity between CRPV E7 and CRPV or HPV16 E6.

3T3 Cells↗
PubMed1989

Locomotion of white blood cells: a biophysical analysis.

We determined some biophysical properties of human granulocytes, monocytes, and lymphocytes in respect to their locomotion. Granulocytes were exposed to plasma and were allowed to crawl on uncoated or glycol methacrylate coated glass plates. Monocytes did not migrate on uncoated glass, but did so on glycol methacrylated glass. Lymphocytes did not move on glass or glycol methacrylated glass, but moved on plexiglas coverslips. Granulocytes and monocytes showed a pronounced, directed movement towards a lysed erythrocyte (necrotaxis), lymphocytes showed no necrotactic response. The information collected by the granulocytes and monocytes in the necrotactic gradient was between 1 and 2 bits. This small amount of information indicated that the cellular decision in favor of a new direction of migration is based on a mechanism involving instability. We showed that the necrotactic response of granulocytes and monocytes is the product of the chemokinetic activity and the polar order parameter (= McCutcheon index) indicating that the cellular decision for a new direction of migration is independent of the speed of the cell movement. The movement of monocytes can be characterized in a similar way to that of granulocytes: the angle of deviation from a straight line path is nearly a fixed value (+/- 35 degrees). Lymphocytes stay in a restricted area after straight line movement. Particular attention was focused on cellular properties involved in locomotion. The characteristic time of the internal clock controlling the locomotion was 0.9 minutes for granulocytes and 2 minutes for monocytes. We were not able to determine the characteristic time of lymphocytes. We were able to determine the internal program responsible for the change in direction of movement. The directional memory time for granulocytes was 0.9 minutes. Monocytes had two directional memory times, short (2 minutes) and long (greater than 18 minutes). Lymphocytes had a very short directional memory time of 40 seconds. The distribution of the track velocities of migrating granulocytes and monocytes was described by bell shaped curves indicating homogeneous populations of cells. The distribution for lymphocytes had two maxima.

Biophysical Phenomena↗