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D H Gutmann

Publications and source records attributed to D H Gutmann.

At least 109 records · Page 6Linked to original sources
PubMed1994

New insights into the neurofibromatoses.

The two neurofibromatoses, neurofibromatosis type 1 (NF1) and type 2 (NF2), have been greatly advanced by the cloning of their respective disease genes. Although NF1 and NF2 are clinically distinct disorders, they represent diseases caused by disruption of tumor suppressor genes. Tumor suppressor genes encode proteins involved in the proper regulation of cell differentiation and proliferation and their disruption presumably predisposes to tumor formation. This year it was demonstrated that the NF1 gene fulfills the genetic criteria for a tumor suppressor gene and that its expression is altered in a number of human malignancies unrelated to NF1. The protein product of the NF1 gene, neurofibromin, was shown to be associated with cytoplasmic microtubules and this interaction may modulate one of neurofibromin's functions. This year also witnessed the cloning of the NF2 gene and the identification of the predicted protein as a member of a family of cytoskeleton-associated proteins.

Cell Differentiation↗
PubMed1994

Specific expression of the neurofibromatosis type 1 gene (NF1) in the hamster Schwann cell.

The gene responsible for neurofibromatosis type 1 (NF1) has sequence homology to the GTPase-activating protein (GAP) and demonstrates GAP activity against ras p21. To study tissue-specific and/or tumor-specific expression of the NF1 gene product, now called neurofibromin, immunostaining and immunoblotting were applied to the N-nitroso-N-ethylurea (ENU)-induced Syrian hamster neurofibromatosis model using polyclonal antibodies against the NF1 fusion protein and a synthetic peptide. Strong expression was observed specific to the Schwann cells of the normal peripheral nerves by immunostaining. Neoplastic Schwann cells showed specific binding of anti-NF1; however, the frequency of positive cells was diminished. Immunoblotting also revealed positive expression of the 250-kd NF1 gene product in the brain, the normal peripheral nerves, and 7 of 14 ENU-induced neurofibromas. Although ENU-induced melanoma and Wilms' tumor were negative for neurofibromin, foci of Schwannian differentiation in both primary and transplanted melanomas were positive. These results suggest that neurofibromin plays some role in differentiation and growth regulation of the Schwann cell.

Animals↗
PubMed1994

Crosslinking of the surface immunoglobulin receptor in B lymphocytes induces a redistribution of neurofibromin but not p120-GAP.

The activation of Ras proteins is a key step in the signal transduction pathways triggered by ligand-bound cell surface receptors. The GTPase activating proteins (GAPs) p120-GAP and neurofibromin, the neurofibromatosis-type 1 (NF1) gene product, are thought to play an essential role in the regulation of Ras activity by increasing the GTPase activity of wild type, but not activated Ras in vitro. Both GAPs are widely expressed in mammalian tissues thus raising the question of whether or not they have different regulatory functions. In this study, we have analysed the distribution of p120-GAP and neurofibromin in splenic B lymphocytes by immunofluorescent staining. Crosslinking of surface immunoglobulin (slg), the B-lymphocyte antigen receptor, induced the redistribution of neurofibromin. In contrast, no apparent change in the cellular localization of p120-GAP occurred followed the cross-linking of slg. The redistribution of neurofibromin coincided both spatially and temporally with the relocalization of crosslinked slg and was inhibited by the cytoskeletal disrupting agents colchicine and cytochalasin D. These findings indicated that neurofibromin and p120-GAP can be differentially regulated in vivo and suggest that neurofibromin is a component of the signaling pathway initiated by crosslinking of B lymphocyte slg. Furthermore, our observations that cocapping neurofibromin with slg is independent of the p21ras redistribution suggests that the role of neurofibromin in B cells is not solely related to its ability to act as a Ras regulator.

Animals↗
PubMed1993

Modulation of the neurofibromatosis type 1 gene product, neurofibromin, during Schwann cell differentiation.

Neurofibromin, the product of the neurofibromatosis type 1 (NF1) gene, is a approximately 250 kDa protein expressed predominantly in cortical neurons and oligodendrocytes in the central nervous system (CNS) and sensory neurons and Schwann cells in the peripheral nervous system (PNS). To gain insight into the biological role of neurofibromin in Schwann cells, the modulation of NF1 gene expression in a Schwann cell line (MT4H1) stimulated to either proliferate or differentiate in response to agents that elevate intracellular cAMP was examined. Untreated cells and cells exposed to mitogenic doses of forskolin (1-10 microM) or 8-bromo-cAMP (0.1 mM) expressed low levels of NF1 mRNA and the protein was barely detectable. High doses of forskolin (100 microM) or 8-bromo-cAMP (1 mM) induced the expression of both myelin P0 protein and neurofibromin with an identical time course. Although NF1 mRNA levels peaked within 1-6 hr, the rise in neurofibromin was not apparent until 24-48 hr and peaked 72 hr after treatment. P0 and neurofibromin were also coinduced by cell-cell contact in high density, untreated cultures. Moreover, differentiation initiated by either cAMP stimulation or high density culture conditions was associated with predominant expression of the type 2 NF1 mRNA isoform. In contrast, type 1 NF1 mRNA isoform expression was observed in untreated Schwann cells or those stimulated with mitogenic doses of forskolin or 8-bromo-cAMP. A switch from the type 1 neurofibromin that can efficiently downregulate p21-ras to the type 2 isoform with reduced activity may facilitate a p21-ras signaling pathway associated with Schwann cell differentiation.

Animals↗
PubMed1993

Neurofibromatosis type 1. Beyond positional cloning.

Recent advances in molecular genetics have enabled researchers to more rapidly identify human disease genes. The identification of these genes by positional cloning has opened the door to a better understanding of such diseases through a more complete appreciation of the molecular biologic processes that underlie them. In this review, the approaches taken to dissect the function of the gene for von Recklinghausen neurofibromatosis are presented. These general approaches involve identification of the protein product, determination of its relation to other known proteins, analysis of its distribution in tissues, within cells, and over development, and dissection of its role in producing the disease phenotype. Last, the insights gained from studying the molecular biology of the neurofibromatosis type 1 gene have direct impact on other biologic processes, such as neoplasia, cellular differentiation, and growth factor-mediated signal transduction as well as potential application to improved treatments for neurofibromatosis type 1 and cancer.

Cloning, Molecular↗
PubMed1993

Neurofibromatosis type 1 gene product (neurofibromin) associates with microtubules.

The neurofibromatosis type 1 (NF1) gene was recently identified by positional cloning and found to encode a protein with structural and functional homology to mammalian and yeast GTPase-activating proteins (GAPs). Using antibodies directed against the NF1 gene product, a protein of approximately 250 kDa was identified and termed neurofibromin. Double-indirect immunofluorescent labeling with anti-neurofibromin and anti-tubulin antibodies demonstrates that neurofibromin associates with cytoplasmic microtubules. Immunoblotting of microtubule-enriched cytoplasmic fractions, using antibodies generated against neurofibromin, shows that neurofibromin copurifies with microtubules. When portions of neurofibromin are expressed in Sf9 insect cells they associate with polymerized microtubules; furthermore, the critical residues for this interaction reside within the GAP-related domain of neurofibromin. The unexpected association of neurofibromin with microtubules suggests that neurofibromin is involved in microtubule-mediated intracellular signal transduction pathways.

3T3 Cells↗
PubMed1993

Mutations in the neurofibromatosis 1 gene in sporadic malignant melanoma cell lines.

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder characterized by progressive and variable involvement of tissues predominantly derived from the neural crest and a predisposition toward malignancies. The NF1 gene encodes neurofibromin, a GTPase-activating protein containing a GAP-related domain (NF1-GRD) that is capable of down-regulating ras by stimulating its intrinsic GTPase activity. We report a homozygous deletion of most of NF1 in one of eight malignant melanoma cell lines leading to loss of detectable mRNA and protein, as well as the apparent absence of protein and mRNA in another melanoma. This data suggests that NF1 can function as a tumour suppressor gene in the development or progression of malignant melanoma.

Chromosome Mapping↗
PubMed1993

A conserved alternative splice in the von Recklinghausen neurofibromatosis (NF1) gene produces two neurofibromin isoforms, both of which have GTPase-activating protein activity.

Sequence analysis has shown significant homology between the catalytic regions of the mammalian ras GTPase-activating protein (GAP), yeast Ira1p and Ira2p (inhibitory regulators of the RAS-cyclic AMP pathway), and neurofibromin, the protein encoded by the NF1 gene. Yeast expression experiments have confirmed that a 381-amino-acid segment of neurofibromin, dubbed the GAP-related domain (GRD), can function as a GAP. Using the RNA polymerase chain reaction with primers flanking the NF1-GRD, we have identified evidence for alternative splicing in this region of the NF1 gene. In addition to the already published sequence (type I), an alternative RNA carrying a 63-nucleotide insertion (type II) is present in all tissues examined, although the relative amounts of types I and II vary. The insertion is conserved across species but is not present in GAP, IRA1, or IRA2. GenBank searches have failed to identify significant similarity between the inserted sequence and known DNA or protein sequences, although the basic amino acid composition of the insertion shares features with nuclear targeting sequences. Expression studies in yeasts show that despite the partial disruption of the neurofibromin-IRA-GAP homology by this insertion, both forms of the NF1-GRD can complement loss of IRA function. In vivo assays designed to compare the GAP activity of the two alternatively spliced forms of the NF1-GRD show that both can increase the conversion of GTP-bound ras to its GDP-bound form, although the insertion of the 21 amino acids weakens this effect. The strong conservation of this alternative splicing suggests that both type I and II isoforms mediate important biological functions of neurofibromin.

Amino Acid Sequence↗
PubMed1993

Analysis of the neurofibromatosis type 1 (NF1) GAP-related domain by site-directed mutagenesis.

The gene for von Recklinghausen neurofibromatosis type 1 (NF1) was recently identified by positional cloning and found to encode a protein with sequence similarity to a family of eucaryotic GTPase-activating proteins (GAPs). Expression of the NF1-GAP-related domain (NF1GRD) has been shown to complement yeast strains deficient in the yeast GAP homologs, IRA1 and IRA2, to interact with human RAS proteins and to accelerate the conversion of ras-GTP to ras-GDP. Further analysis of this region has revealed a number of residues that are highly conserved between members of the GAP family. Mutational analysis of a representative number of these residues produced one of three effects: (1) no change in NF1GRD function, (2) complete disruption of NF1GRD function and (3) intermediate retention of NF1GRD function. One of these mutations at residue 1423 was shown to have reduced ability to negatively regulate ras in yeast, which is interesting in light of a recent report demonstrating a similar naturally occurring mutation in human malignancies.

Amino Acid Sequence↗
PubMed1992

Aberrant regulation of ras proteins in malignant tumour cells from type 1 neurofibromatosis patients.

Defects in the NF1 gene have been implicated in the inherited disorder neurofibromatosis type 1, which is characterized by several developmental abnormalities including an increased frequency of benign and malignant tumours of neural crest origin (neurofibromas and neurofibrosarcomas respectively). The NF1 gene encodes a ubiquitous protein homologous to p120GAP, the GTPase-activating protein (GAP) for the products of the ras protooncogenes. When expressed in non-mammalian systems, the region of the NF1 gene homologous to p120GAP produces a protein with GAP-like activity. Here we present evidence that the ras proteins in malignant tumour cell lines from patients with type 1 neurofibromatosis are in a constitutively activated state, as judged by the guanine nucleotide bound to them, and are necessary for cellular proliferation. These cells contain p21ras and p120GAP that are both functionally wild type, but barely any functional NF1 protein. Our results show that the NF1 protein is normally essential for correct negative regulation of ras proteins in the cell, even in the presence of normal p120GAP, and they support the hypothesis that NF1 is a tumour-suppressor gene whose product acts upstream of ras.

Antibodies, Monoclonal↗
PubMed1992

Recent progress toward understanding the molecular biology of von Recklinghausen neurofibromatosis.

The gene for von Recklinghausen neurofibromatosis (NF1) was recently identified by positional cloning and found to code for a large, ubiquitously expressed protein. This protein has both structural and functional similarity to a family of proteins with guanosine triphosphatase-activating properties, involved in the regulation of the protooncogene ras. One of the postulated functions of the NF1 gene product may relate to its ability to regulate ras-mediated cell proliferation. Selective pharmacotherapy directed at downregulating ras may be of benefit to patients with NF1.

Forecasting↗
PubMed1991

Identification of the neurofibromatosis type 1 gene product.

The gene for neurofibromatosis type 1 (NF1) was recently identified by positional cloning. The complete cDNA encodes a polypeptide of 2818 amino acids. To study the NF1 gene product, antibodies were raised against both fusion proteins and synthetic peptides. Initial characterization of two anti-peptide antibodies and one fusion-protein antibody demonstrated a specific protein of approximately 250 kDa by both immunoprecipitation and immunoblotting. This protein was found in all tissues and cell lines examined and is detected in human, rat, and mouse tissues. To demonstrate that these antibodies specifically recognize the NF1 protein, additional fusion proteins containing the sequence specific to the synthetic peptide were generated. Both peptide antisera recognize the proper specific fusion proteins so generated. Immunoprecipitates using the peptide antisera were shown to recognize the same protein detected by immunoblotting with either the other peptide antiserum or the fusion-protein antiserum. Immunoblotting using antiserum specific to spatially distinct epitopes conducted on tissue homogenates demonstrated the NF1 protein in all adult tissues. Based on the homology between the NF1 gene product and members of the GTPase-activating protein (GAP) superfamily, the name NF1-GAP-related protein (NF1GRP) is suggested.

Amino Acid Sequence↗
PubMed1991

Oculodentodigital dysplasia syndrome associated with abnormal cerebral white matter.

Oculodentodigital dysplasia (ODDD) syndrome is an uncommon inherited disorder with eye and facial abnormalities, syndactyly, and defects in tooth enamel. Some of the previously reported patients with ODDD syndrome also manifested spastic quadriparesis. We describe a patient with sporadic ODDD syndrome referred for evaluation of progressive spastic paraparesis. Magnetic resonance imaging of the brain demonstrated abnormal white matter, which suggests an explanation for the observed spastic paraparesis.

Adult↗
PubMed1991

Congenital nystagmus in a (46,XX/45,X) mosaic woman from a family with X-linked congenital nystagmus.

X-linked congenital nystagmus is a rare disorder in which affected males manifest binocular uniplanar nystagmus with associated head oscillation. In the families previously reported, affected females have been described. We report on a multigeneration family with X-linked congenital nystagmus with an affected woman. She was a (46,XX/45,X) mosaic. Magnetic resonance images of the brain of affected individuals were normal.

Aged↗
PubMed1991

cDNA cloning of the type 1 neurofibromatosis gene: complete sequence of the NF1 gene product.

Von Recklinghausen neurofibromatosis, or type 1 neurofibromatosis (NF1), is a common autosomal dominant disorder characterized by abnormalities in multiple tissues derived from the embryonic neural crest. Portions of the gene have been recently identified by positional cloning, and sequence analysis has shown homology to the GTPase activating protein (GAP) family. In this report we present the results of an extensive cDNA walk resulting in the cloning of the complete coding region of the NF1 transcript. Analysis of the sequences reveals an open reading frame of 2818 amino acids, although alternatively spliced products may code for different protein isoforms. The gene extends for approximately 300 kb on chromosome 17, with its promoter in a CpG-rich island.

Adult↗