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Biomedical subjects

F Collins

Publications and source records attributed to F Collins.

At least 19 recordsLinked to original sources
PubMed1996

N-terminal sequences contained in the Src homology 2 and 3 domains of p120 GTPase-activating protein are required for full catalytic activity toward Ras.

The p120 GTPase-activating protein (GAP) is a negative regulator of Ras, which has a central role in signal transduction pathways that control cell proliferation. p120 GAP accelerates the conversion of activated Ras-GTP to its inactive form, Ras-GDP, thereby inhibiting mitogenic signaling. To examine potential contributions of p120 N-terminal sequences to regulation of its C-terminal catalytic domain, we constructed deletion mutants lacking defined regions, including the variable hydrophobic region as well as the Src homology 2 (SH2) and 3 (SH3) domains. These mutant proteins were expressed in infected Sf9 insect cells from recombinant baculoviruses and assayed in vitro for their ability to stimulate the intrinsic GTPase activity of purified Ras. While deletion of the variable hydrophobic region had no effect on p120 GAP activity, deletion of the entire SH2/SH3/SH2 region severely impaired catalytic activity toward Ras. Deletion of individual SH2 and SH3 domains within this region partially inhibited p120 GAP activity. Moreover, p120 N-terminal sequences enhanced the Ras GTPase-stimulating activity of the neurofibromin GAP-related domain. These results demonstrate that sequences in the SH2/SH3/SH2 region of p120 GAP are required for full catalytic activity toward Ras. Together with earlier findings that the p120 GAP SH2 domains mediate interactions with several GAP-associated proteins, our results suggest multiple roles for the N-terminal sequences in regulating p120 GAP catalytic activity and mitogenic signaling pathways. In addition, our results raise the possibility that SH2 domain point mutations in p120 GAP detected in some basal cell carcinomas reduce catalytic activity toward Ras and thereby contribute to oncogenesis.

Animals↗
PubMed1995

Effects of the obese gene product on body weight regulation in ob/ob mice.

C57BL/6J mice with a mutation in the obese (ob) gene are obese, diabetic, and exhibit reduced activity, metabolism, and body temperature. Daily intraperitoneal injection of these mice with recombinant OB protein lowered their body weight, percent body fat, food intake, and serum concentrations of glucose and insulin. In addition, metabolic rate, body temperature, and activity levels were increased by this treatment. None of these parameters was altered beyond the level observed in lean controls, suggesting that the OB protein normalized the metabolic status of the ob/ob mice. Lean animals injected with OB protein maintained a smaller weight loss throughout the 28-day study and showed no changes in any of the metabolic parameters. These data suggest that the OB protein regulates body weight and fat deposition through effects on metabolism and appetite.

Adipose Tissue↗
PubMed1995

Microdissection and microcloning of chromosomal alterations in human breast cancer.

The recognition of recurring sites of chromosome changes in malignancies has greatly facilitated the identification of genes implicated in the pathogenesis of human cancers. Based especially upon recent studies [1-4], it appears increasingly likely that a subset of recurring chromosome alterations will be recognized in human breast cancer. Currently recognized chromosome changes characterizing breast carcinoma include the recognition of cytologic features of gene amplification (e.g. double minutes [dmins] and homogeneously staining regions [HSRs]) [5-8]. As these and other chromosome regions are implicated in recurring abnormalities in breast cancer, it will become increasingly important to have band- or region-specific genomic libraries and probes in order to facilitate high resolution physical mapping and ultimately to clone breast cancer related genes [9]. Toward this end an important recent development in physical mapping has been the establishment of chromosome microdissection as a rapid and reproducible approach to rapidly isolate and characterize chromosome region-specific DNA, greatly facilitating the initial steps in positional cloning of disease-related genes [10-13]. In this brief report, we will highlight the application of chromosome microdissection to the generation of region-specific probes for both fluorescent in situ hybridization (FISH) and the generation of genomic microclone libraries. Additionally, efforts using this methodology to generate a microclone library encompassing the early onset breast/ovarian cancer (BRCA1) gene will be presented.

Breast Neoplasms↗
PubMed1995

Glial cell line-derived neurotrophic factor augments midbrain dopaminergic circuits in vivo.

Recently, a novel glial cell line-derived neurotrophic factor (GDNF) has been identified, cloned, and shown to have potent survival- and growth-promoting activity on fetal rat midbrain dopaminergic neurons in cell culture. In this study, we document marked and long-lasting effects on adult rat midbrain dopaminergic neurons in vivo after intracranial administration. A single injection of this factor into the substantia nigra elicited a dose-dependent increase in both spontaneous and amphetamine-induced motor activity, and a decrease in food consumption, lasting 7-10 days. Using immunocytochemistry, we found sprouting of tyrosine hydroxylase-positive neurites towards the injection site, and increased tyrosine hydroxylase immunoreactivity of the ipsilateral striatum was produced by GDNF. There was also a marked and dose-dependent increase in dopamine turnover in the substantia nigra and striatum, and in ipsilateral dopamine levels in the substantia nigra. Little or no effects of GDNF were seen on norepinephrine or serotonin levels. The neurochemical changes on dopaminergic afferents persist for at least 3 weeks after a single intracranial injection of 10 micrograms. Taken together, these data suggest that this glial cell line-derived factor has a potent influence on adult rat dopamine neurons and may have a potentially important role as a trophic factor for these neurons.

Animals↗
PubMed1995

An amino acid substitution in the pyruvate dehydrogenase E1 alpha gene, affecting mitochondrial import of the precursor protein.

A mutation in the mitochondrial targeting sequence was characterized in a male patient with X chromosome-linked pyruvate dehydrogenase E1 alpha deficiency. The mutation was a base substitution of G by C at nucleotide 134 in the mitochondrial targeting sequence of the PDHA1 gene, resulting in an arginine-to-proline substitution at codon 10 (R10P). Pyruvate dehydrogenase activity in cultured skin fibroblasts was 28% of the control value, and immunoblot analysis revealed a decreased level of pyruvate dehydrogenase E1 alpha immunoreactivity. Chimeric constructs in which the normal and mutant pyruvate dehydrogenase E1 alpha targeting sequences were attached to the mitochondrial matrix protein ornithine transcarbamylase were synthesized in a cell free translation system, and mitochondrial import of normal and mutant proteins was compared in vitro. The results show that ornithine transcarbamylase targeted by the mutant pyruvate dehydrogenase E1 alpha sequence was translocated into the mitochondrial matrix at a reduced rate, suggesting that defective import is responsible for the reduced pyruvate dehydrogenase level in mitochondria. The mutation was also present in an affected brother and the mildly affected mother. The clinical presentations of this X chromosome-linked disorder in affected family members are discussed. To our knowledge, this is the first report of an amino acid substitution in a mitochondrial targeting sequence resulting in a human genetic disease.

Amino Acids↗
PubMed1994

Differential phylogenetic footprinting as a means to identify base changes responsible for recruitment of the anthropoid gamma gene to a fetal expression pattern.

Expression of the anthropoid (simian) gamma gene in fetal life contrasts with the exclusively embryonic expression pattern of the gamma-like genes of other eutherian mammals. To elucidate the factors responsible for this change in expression pattern, we utilized a strategy called differential phylogenetic footprinting (DPF). This strategy entails the following: (a) identification, within regulatory regions, of the gamma promoter, of individual nucleotides that differ between human (fetal expression), and galago (embryonic expression) gamma genes, (b) analysis of the effect of these nucleotide differences on the binding of nuclear proteins to human and galago sequences, and (c) assessment of the functional consequences of these binding changes in expression assays. The DPF analysis revealed several proteins that bind upstream from the CCAAT motif in the galago gamma promoter but do not bind to the corresponding region of the human gamma promoter. In transfection assays, binding of these proteins is associated with erythroid-specific repression of promoter strength. Binding sites for these proteins also occur near the CCAAT box of other embryonically expressed genes, including rabbit, mouse, and dwarf lemur gamma genes and the human epsilon globin gene. These data are consistent with the hypothesis that sequence changes near the proximal CCAAT box in the ancestral simian gamma gene may have facilitated a novel expression pattern by reducing the binding of repressors that act in the fetal stage.

Animals↗
PubMed1994

Detection of fusion transcripts generated by the inversion 16 chromosome in acute myelogenous leukemia.

Pericentric inversion of chromosome 16 [inv(16)(p13q22)] and the related t(16;16)(p13;q22) are seen in a subset of acute myelogenous leukemia (AML) phenotypically and prognostically differing from other cases. We have recently shown that inv(16) results in fusion of CBFB/PEBP2B, a gene encoded at 16q22 to MYH11, a smooth muscle myosin heavy chain gene encoded at 16p13. Chimeric transcripts consisting of upstream CBFB fused to downstream MYH11 coding sequences result from this fusion. In this study we have examined a series of 37 of these cases using reverse transcriptase-polymerase chain reaction (RT-PCR) to detect expression of a hybrid CBFB/MYH11 transcript. Chimeric cDNAs were detected in all but 1 of 37 leukemias with typical inv(16) or t(16;16). Such chimeric products were not seen in a case with inv(16)(p13q24) (ie, a variant q arm breakpoint) or any of 10 cases of AML without these chromosomal changes. Four different chimeric transcripts were found, representing differing fusion points within MYH11 spliced to position 495 of CBFB. Primer sets are described for efficient amplification of these different cDNA forms. Amplification of cDNA showed that all but 17 codons of the CBFB coding sequence are included in the abnormal transcripts. RT-PCR was shown to be highly sensitive and potentially useful for detection of leukemic cells during morphologic remission.

Base Sequence↗
PubMed1994

Neurons of the hippocampal formation express glial cell line-derived neurotrophic factor messenger RNA in response to kainate-induced excitation.

Glial cell line-derived neurotrophic factor (GDNF) is a novel member of the transforming growth factor-beta superfamily with potent trophic effects on dopamine neurons. Kainate-induced epileptic seizures have been shown to induce gene expression of trophic factors, particularly members of neurotrophin or fibroblast growth factor families, in the hippocampus. In this study, we examined the effects of kainate (12 mg/kg, i.p.)-induced epileptic seizures on the expression of the novel neurotrophic factor GDNF in the hippocampus. While GDNF messenger RNA was not detected during development or in normal adult rats in the hippocampus, kainate-induced epileptic seizures markedly increased GDNF messenger RNA in scattered neurons in the dentate granule layer 3 h after injection. Six hours after kainate almost all dentate granule cells and expressed GDNF messenger RNA. The increase in GDNF messenger RNA in the dentate granule layer returned almost to control levels 24 h after kainate; however, there was still expression of GDNF messenger RNA in the hilus/CA4 and also in pyramidal neurons in areas CA1-CA3. We conclude that GDNF messenger RNA is regulated, in part, via glutamate-mediated excitation and may play a role in long-lasting structural and/or functional reorganization in the hippocampal formation.

Animals↗
PubMed1994

Purification and initial characterization of rat B49 glial cell line-derived neurotrophic factor.

The rat glial cell line B49 releases into its culture medium a potent neurotrophic factor that exhibits relative specificity for the dopaminergic neurons in dissociated cultures of rat embryonic midbrain. This factor is a heparin-binding, basic protein that is heterogeneously glycosylated and migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and on molecular sieve chromatography with an apparent mass of approximately 33-45 kDa. The factor behaves like a disulfide-bonded homodimer, whose biological activity is destroyed by reduction of disulfide bonds but not by SDS-PAGE or reversed-phase (RP)-HPLC. The apparent mass of the monomer is approximately 16 kDa after deglycosylation with N-Glycanase. This factor has been purified 34,000-fold to apparent homogeneity by a combination of heparin-affinity chromatography, molecular sieving chromatography, SDS-PAGE, and RP-HPLC. The purified rat protein promotes the survival, morphological differentiation, and high-affinity dopamine reuptake of dopaminergic neurons in midbrain cultures, without obvious effects on total neurons or glia and without increasing high-affinity GABA or serotonin reuptake. The purified protein exhibits an EC50 in midbrain cultures at approximately 40 pg/ml, or 1 pM, and has unique amino-terminal and internal amino acid sequences. The sequences provide a basis for cloning and expression of the gene for rat and human glial cell line-derived neurotrophic factor (GDNF), confirming that the protein purified as reported here is GDNF.

Animals↗
PubMed1993

GDNF: a glial cell line-derived neurotrophic factor for midbrain dopaminergic neurons.

A potent neurotrophic factor that enhances survival of midbrain dopaminergic neurons was purified and cloned. Glial cell line-derived neurotrophic factor (GDNF) is a glycosylated, disulfide-bonded homodimer that is a distantly related member of the transforming growth factor-beta superfamily. In embryonic midbrain cultures, recombinant human GDNF promoted the survival and morphological differentiation of dopaminergic neurons and increased their high-affinity dopamine uptake. These effects were relatively specific; GDNF did not increase total neuron or astrocyte numbers nor did it increase transmitter uptake by gamma-aminobutyric-containing and serotonergic neurons. GDNF may have utility in the treatment of Parkinson's disease, which is marked by progressive degeneration of midbrain dopaminergic neurons.

Amino Acid Sequence↗
PubMed1993

Glial cell line-derived neurotrophic factor is expressed in the developing but not adult striatum and stimulates developing dopamine neurons in vivo.

The potential role of glial cell line-derived neurotrophic factor (GDNF) as a trophic molecule for midbrain dopamine neurons was examined using two different approaches: in situ hybridization and intraocular transplantation. The presence of mRNA for GDNF was noted in striatal and ventral limbic dopaminergic target areas in the developing (E20-P7) rat, but not the adult rat. Signals were also found in nondopaminergic areas during maturation, such as the cerebellar anlage, spinal cord, and thalamus. Lesions of the nigrostriatal pathway in neonatal or adult rats, using 6-hydroxydopamine injected into the medial forebrain bundle, did not elicit upregulation of mRNA for GDNF. Grafts of fetal ventral mesencephalon in the anterior eye chamber were exposed to repeated injections of GDNF, which elicited a marked and dose-dependent increase in transplant volume. A low (0.1 microgram/eye) and high (1 microgram/eye) dose of GDNF both led to a somewhat larger mean area of dopamine fiber outgrowth into host irides. In the transplants, cell counts of tyrosine hydroxylase (TH)-immunoreactive neurons revealed a doubling of cell numbers in the low-dose group and about four times as many cells in the high-GDNF-dose group compared to controls. Moreover, the density of TH-immunoreactive nerve fibers was markedly and significantly higher in transplants treated with the high GDNF dose. Since the volumes of these transplants were also larger, the total amount of both TH-positive cells and TH-positive nerve fibers was many-fold greater in the high-GDNF group than that in the controls. Taken together, these data support the concept that GDNF functions as a dopaminotrophic factor in vivo.

Aging↗
PubMed1992

National working conference on smoking and body weight. Task Force 1: Mechanisms relevant to the relations between cigarette smoking and body weight.

Careful, comprehensive, and empirical observations provide the building blocks of the sciences, whereas theory and mechanisms provide the "cement" to hold the blocks together and serve as blueprints to direct future building. This article resulted from several days of discussion regarding theories that may underlie the relation between cigarette smoking and body weight and the relation between smoking cessation and body weight. The working group composed of social and biological scientists who addressed this assignment considered what is already known within the smoking and body weight literature and also considered relevant findings from studies of smoking or body weight regulation that have not directly addressed the interaction of these variables. As expected, we were successful at listing some of what is not known and what is worth knowing. We also tried to identify fruitful possibilities for research activity that might clarify mechanisms of action and eventually lead to theoretical development. Because we do not believe that the present state of our deliberations merits the label of theories, we decided, instead, to report the summary of these deliberations as potential mechanisms relevant to the relation between smoking and body weight.

Adult↗