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Biomedical subjects

G M Ross

Publications and source records attributed to G M Ross.

At least 37 records · Page 2Linked to original sources
PubMed1997

Tumorigenesis and a DNA repair defect in mice with a truncating Brca2 mutation.

Germline mutation of the BRCA2 gene carries a high risk of developing breast cancer. To study the function of this gene, we generated a mutation in Brca2 in mice. Unlike other mutations in the Brca2 gene, which are lethal early in embryogenesis when homozygous, some of our homozygous mutant mice survive to adulthood. These animals have a wide range of defects, including small size, improper differentiation of tissues, absence of germ cells and the development of lethal thymic lymphomas. Fibroblasts cultured from BrcaZ-/-embryos have a defect in proliferation that may be mediated by over-expression of p53 and p21Waf1/CIP1. We show that Brca2 is required for efficient DNA repair, and our results suggest that loss of the p53 checkpoint may be essential for tumour progression triggered by mutations in BRCA2.

Animals↗
PubMed1997

Zinc alters conformation and inhibits biological activities of nerve growth factor and related neurotrophins.

A role for Zn2+ in a variety of neurological conditions such as stroke, epilepsy and Alzheimer's disease has been postulated. In many instances, susceptible neurons are located in regions rich in Zn2+ where nerve growth factor (NGF) levels rise as a result of insult. Although the interaction of Zn2+ with this neurotrophin has previously been suggested, the direct actions of the ion on NGF function have not been explored. Molecular modeling studies predict that Zn2+ binding to NGF will induce structural changes within domains of this neurotrophin that participate in the recognition of TrkA and p75NTR. We demonstrate here that Zn2+ alters the conformation of NGF, rendering it unable to bind to p75NTR or TrkA receptors or to activate signal transduction pathways and biological outcomes normally induced by this protein. Similar actions of Zn2+ are also observed with other members of the NGF family, suggesting a modulatory role for this metal ion in neurotrophin function.

Animals↗
PubMed1997

Dopamine requires ascorbic acid to be the prolactin release-inhibiting factor.

A high concentration of dopamine (10(-6) mol/l) inhibited prolactin release for < 60 min during a 2-h perifusion period by use of primary cultured pituitary cells. However, when dopamine (10(-6) mol/l) and control medium were alternately perifused, dopamine inhibited prolactin release for a longer period, indicating that the inability of dopamine to sustain an inhibitory action is likely caused by decreased sensitivity of the lactotrophs to dopamine. When 3 x 10(-7) mol/l dopamine was perifused, prolactin release was inhibited for only 15 min, and the rate of prolactin release was decreased to a nadir by addition of ascorbic acid (10(-4) mol/l) 15 min after the start of dopamine perifusion. Dopamine decreased density of dopamine D2 receptors, and ascorbic acid inhibited the receptor downregulation in GH4ZR7 cells. These results support our hypothesis that dopamine requires a supplementary agent to be the prolactin release-inhibiting factor and that the supplementary agent is ascorbic acid.

Animals↗
PubMed1997

Immunochemical assay for recognition of 2-S-glutathionyl acetate, a glutathione conjugate derived from 1,1-dichloroethylene-epoxide.

Cytotoxicities induced by 1,1-dichloroethylene (DCE) are ascribed to cytochrome P450-dependent metabolism to an epoxide. Conjugation of the DCE-epoxide with glutathione (GSH) results in the formation of the conjugates 2-S-glutathionyl acetate (GTA) and 2-(S-glutathionyl) acetyl glutathione (GAG); GAG undergoes hydrolysis to form GTA, and thus GTA is a major metabolite of DCE metabolism. Our objective is to develop an antiserum against the chemically synthesized GTA, and for immunization, we have used a hapten that consists of GTA conjugated to bovine serum albumin (BSA) as the carrier protein and glutaraldehyde (GLUT) as a chemical cross-linker. The antisera were raised in rabbits and were characterized by using the following synthesized structural analogs: GTA, glycine-GLUT-BSA (GLY-GLUT-BSA), GTA-GLUT-ovalbumin (GTA-GLUT-OVB), GTA-1-ethyl-3-(3-dimethylaminopropyl) carbodiimide-BSA (GTA-EDC-BSA), TRIS-GLUT-BSA, glutathione-GLUT-BSA (GSH-GLUT-BSA). The enzyme-linked immunosorbent assay (ELISA) and slot immunoblotting were used to characterize the specificity of the antisera. Noncompetitive ELISA experiments showed that the reaction of the antiserum with the antigen was concentration-dependent. In the competitive ELISA, GTA-GLUT-BSA inhibited binding efficiently; in contrast, the unconjugated GTA did not inhibit binding to the antigen. Competitive studies with the other analogs indicated low or minimal reactivities with the antibodies, which were blocked by incubation with GLY-GLUT-BSA. However, there was residual reactivity with the antigen that was not competitively inhibited by either the GTA-EDC-BSA or the GSH-GLUT-BSA conjugates. Slot-blotting experiments confirmed the findings of the ELISA studies and revealed high specificity of the antiserum to detect the hapten. These results demonstrated the successful development of polyclonal antibodies to detect GTA and hence DCE-epoxide.

Acetates↗
PubMed1996

Survival of cholinergic forebrain neurons in developing p75NGFR-deficient mice.

The functions of the low-affinity p75 nerve growth factor receptor (p75(NGFR)) in the central nervous system were explored in vivo. In normal mice, approximately 25 percent of the cholinergic basal forebrain neurons did not express TrkA and died between postnatal day 6 and 15. This loss did not occur in p75(NGFR)-deficient mice or in normal mice systemically injected with a p75(NGFR)-inhibiting peptide. Control, but not p75(NGFR)-deficient, mice also had fewer cholinergic striatal interneurons. Apparently, p75(NGFR) mediates apoptosis of these developing neurons in the absence of TrkA, and modulation of p75(NGFR) can promote neuronal survival. Cholinergic basal forebrain neurons are involved in learning and memory.

Animals↗
PubMed1996

Modulation of brain catecholamine absorbing proteins by dopaminergic agents.

Catecholamine absorbing proteins (CATNAPs) are localized in the brain and thus far have no known biochemical and pharmacological characteristics consistent with other receptor proteins or metabolic enzymes in the central nervous system. The oxidative metabolism of catecholamines in the brain, especially the catabolism of dopamine and its conjugation with metabolic brain proteins, results in the production of highly toxic free radicals. Since such processes are implicated in the pathophysiology of various neurodegenerative diseases, including parkinsonism, and since CATNAPs bind catecholamines with high affinity, there is a need to further investigate if these novel proteins could play a protective role against these harmful catecholamine metabolites. In this study, we demonstrate the purification, pharmacological characterization and modulation of CATNAPs, as the first steps necessary to elucidate the function of these proteins in the brain. First, CATNAPs were identified from tissues using [3H]N-n-propylnorapomorphine (a specific dopamine receptor agonist) and [125I]6-hydroxy-5-iodo[N(N-2,4-dinitro-phenyl)- aminopropyl]1,2,3,4-tetrahydronaphthalene ([125I]DATN; a highly specific ligand synthesized in our laboratory). Three proteins, with molecular masses of 47, 40 and 26 kDa, were identified and purified, which allowed for the subsequent production of antibodies against each of these CATNAPs. The effects of in vivo chronic administration of several dopaminergic agents on CATNAPs were also examined by Western immunoblotting. L-3,4-Dihydroxyphenylalanine (L-DOPA) treatment in rats resulted in the increase of all of the three proteins, as compared to controls. Treatment in rats with the dopamine depleting agent, reserpine, produced a significant decrease in all of the three CATNAPs. In addition, the effects of direct administration of apomorphine, dopamine, epinephrine, isopropylnorepinephrine, norepinephrine, N-n-propylnorapomorphine and 6-hydroxydopamine on CATNAP levels in rats were examined. Interestingly, we observed an increase (as compared to control) of the 47, 40 and 26 kDa proteins in animals treated with dopamine, norepinephrine, N-n-propylnorapomorphine and apomorphine. In contrast, animals treated with 6-hydroxydopamine showed significant decreases in the levels of all three proteins. It is evident that as the concentration of catecholamines increases, there is a corresponding increase in the levels of CATNAPs in the brain. These results clearly demonstrate the pharmacological modulation of CATNAPs by dopaminergic agents and suggest their possible role in the cytoprotection against damage caused by free radicals generated by oxidative stress.

Adrenergic Uptake Inhibitors↗
PubMed1996

Characterization of a distinctive motif of the low molecular weight neurotrophin receptor that modulates NGF-mediated neurite growth.

The cytoplasmic region of the common neurotrophin receptor (p75(NGFR)) (rat, human, chick) contains a putative membrane-associating domain implicated in intracellular signalling. A peptide (R3) identical to this domain (p75(NGFR) 367-379) and various analogues of this peptide displayed circular dichroism spectra in aqueous and non-polar environments identical to the amphiphilic tetradecapeptide mastoparan (MP) and were internalized by PC12 rat pheochromocytoma cells. The R3 peptide enhanced neurite growth in PC12 cells, embryo chick primary sensory neurons and fetal rat primary sensory neurons in vitro in the presence of sub-saturating concentrations of NGF. Peptide analogues of R3 not faithful to the distance and angular relationships of ionic groups and the putative amphiphilic structure of p75(NGFR)367-379 displayed reduced potency to enhance p75(NGFR) (PC12(nnr5)), had no influence on neurite growth. The R3 peptide had no effects on cell survival, cell binding or uptake of [125]NGF, affinity cross-linking of [125]NGF to p75(NGFR) or trkA monomers and homodimers, of NGF-mediated trkA monomer tyrosine phosphorylation. The studies implicate a role for a highly conserved motif of p75(NGFR) in the downstream modulation of NGF-mediated neurite growth.

Amino Acid Sequence↗
PubMed1995

A nerve growth factor peptide retards seizure development and inhibits neuronal sprouting in a rat model of epilepsy.

Kindling, an animal model of epilepsy wherein seizures are induced by subcortical electrical stimulation, results in the upregulation of neurotrophin mRNA and protein in the adult rat forebrain and causes mossy fiber sprouting in the hippocampus. Intraventricular infusion of a synthetic peptide mimic of a nerve growth factor domain that interferes with the binding of neurotrophins to their receptors resulted in significant retardation of kindling and inhibition of mossy fiber sprouting. These findings suggest a critical role for neurotrophins in both kindling and kindling-induced synaptic reorganization.

Animals↗
PubMed1995

DNA strand break rejoining defect in xrs-6 is complemented by transfection with the human Ku80 gene.

The radiosensitive mutant xrs-6, derived from Chinese hamster ovary cell line CHO-K1, has been demonstrated to be defective in DNA double-strand break repair and also in its proficiency to undergo V(D)J recombination. Recent work has provided both genetic and biochemical evidence that the M(r) 80,000 subunit of the Ku protein is able to complement the radiosensitivity and the V(D)J recombination defect in the xrs-6 mutant. We demonstrate here that complementation of the radiosensitive phenotype in xrs-6 cells by the introduction of Ku80 cDNA is accompanied by the concomitant restoration of DNA double-strand break rejoining proficiency to almost that of the parental CHO-K1 cells, as measured both by neutral single-cell microgel electrophoresis (Comet) technique and by pulsed-field gel electrophoresis. These results provide further biochemical evidence for the involvement of the Ku protein in the repair of DNA double-strand breaks.

Animals↗
PubMed1995

Covalent affinity labeling of brain catecholamine-absorbing proteins using a high-specific-activity substituted tetrahydronaphthalene.

The recently described catecholamine-absorbing proteins (CATNAPs) are expressed within the CNS and have been shown to participate in neurochemical processes involving dopamine and several structurally related catecholamines. Specifically, CATNAPs have been implicated in participating directly in oxidative mechanisms involving reactive species (such as free radicals) derived from these compounds. Toxic free radicals generated from endogenous catecholamines have been identified as a major cause of neuronal tissue injury and are implicated in several disease processes. CATNAPs were first identified by their ability to react covalently with tritiated dopaminergic compounds, incorporating low levels of radioactivity under appropriate reaction conditions. The biochemical characterization of CATNAPs has until now been hampered by the lack of a suitable high-specific-activity probe to allow the rapid detection of these proteins. We describe here the synthesis and labeling characteristics of a high-specific-activity substituted tetrahydronaphthalene derivative (6-hydroxy-[125I]iodo-[N-(N-2',4'- dinitrophenyl)aminopropyl]-2-amino-1,2,3,4-tetrahydronaphthalene), which covalently incorporates into CATNAPs with the same tissue distribution, molecular weight patterns, and pharmacology as observed for the previously studied tritiated catecholamines. This compound greatly enhances the detection of CATNAPs and will facilitate further biochemical characterization of these proteins.

Absorption↗
PubMed1995

The comet moment as a measure of DNA damage in the comet assay.

The development of rapid assays of radiation-induced DNA damage requires the definition of reliable parameters for the evaluation of dose-response relationships to compare with cellular endpoints. We have used the single-cell gel electrophoresis (SCGE) or 'comet' assay to measure DNA damage in individual cells after irradiation. Both the alkaline and neutral protocols were used. In both cases, DNA was stained with ethidium bromide and viewed using a fluorescence microscope at 516-560 nm. Images of comets were stored as 512 x 512 pixel images using OPTIMAS, an image analysis software package. Using this software we tested various parameters for measuring DNA damage. We have developed a method of analysis that rigorously conforms to the mathematical definition of the moment of inertia of a plane figure. This parameter does not require the identification of separate head and tail regions, but rather calculates a moment of the whole comet image. We have termed this parameter 'comet moment'. This method is simple to calculate and can be performed using most image analysis software packages that support macro facilities. In experiments on CHO-K1 cells, tail length was found to increase linearly with dose, but plateaued at higher doses. Comet moment also increased linearly with dose, but over a larger dose range than tail length and had no tendency to plateau.

Animals↗
PubMed1994

Putative cytoplasmic amphiphilic domains in the nerve growth factor/tumour necrosis factor receptor superfamily.

Potential alpha-helical regions in cytoplasmic domains of the NGF/TNF receptor superfamily were searched to identify amphiphilic sequences favouring association with membrane surfaces, analogous to the predicted secondary structure of mastoparan (MP). Similar to MP, NGFR (rat, chick, human), human TNFR-1, and human 4-1BB have domains with putative surface membrane associating sequences. The circular dichroism spectra of mastoparan and a peptide homologous to the putative amphiphilic domain of NGFR were identical in an aqueous milieu, and both adopted an alpha-helical conformation in trifluoroethanol.

Amino Acid Sequence↗
PubMed1994

Cell cycle arrests and radiosensitivity of human tumor cell lines: dependence on wild-type p53 for radiosensitivity.

Loss of p53 function has been shown to cause increased resistance to ionizing radiation in normal murine cells; however, the role of p53 in radioresistance of human tumor cells is less clear. Since wild-type p53 function is required for radiation-induced G1 arrest, we measured G1 arrest in 12 human tumor cell lines that have a wide range of radiosensitivities (surviving fraction at 2 Gy, 0.11-0.8). We observed a significant correlation between the level of ionizing radiation-induced G1 arrest and radiosensitivity. Cell lines having G1 arrest are more radiosensitive. There is no correlation between maximal G2 arrest and radiosensitivity. Expression of a dominant-negative mutant of p53 (codon 143, Val to Ala) in transfectants of the radiosensitive human ovarian cell line A2780 abrogates the radiation-induced G1 arrest. Such mutant p53 transfectants are more resistant to ionizing radiation than the parental line and vector-alone transfectants, as measured by clonogenic assays. These results support the concept that wild-type p53 function is required for sensitivity of tumor cells to DNA-damaging agents, such as ionizing radiation, and that the loss of p53 function in certain human tumor cells can lead to resistance to ionizing radiation.

G1 Phase↗
PubMed1993

Identification of novel catecholamine absorbing proteins in the central nervous system.

Several pharmacologically active catecholamines have been shown to react covalently with CNS proteins, namely species of 47, 40, 22, and 20 kDa. Of these, the 47-kDa protein showed the greatest incorporation of tritium following treatment with [3H]dopamine, [3H]ADTN, or [3H]N-propyl-norapomorphine. Labeling was accomplished by incubating the tritiated ligands with crude membrane preparations in the absence of reducing agents. These proteins displayed several unique characteristics: 1. The proteins are distributed throughout the CNS, but no evidence was found for their presence in other tissues; 2. The proteins have a unique pharmacological profile, interacting with dopamine, ADTN, N-propyl-norapomorphine, and apomorphine, but not with ligands specific for other proteins known to interact with these compounds; 3. The labeling of these proteins is not inhibited by several similar catecholamines and other catechols, suggesting specific structural requirements; and 4. These proteins exhibited stereoselectivity with respect to this labeling. These results demonstrate the existence of novel CNS proteins capable of covalently absorbing several physiologically important catecholamines in vitro.

Animals↗
PubMed1990

Solubilization and reconstitution of dopamine D1 receptor from bovine striatal membranes: effects of agonist and antagonist pretreatment.

The bovine striatal dopamine D1 receptor was solubilized with a combination of sodium cholate and NaCl in the presence of phospholipids, following treatment of membranes with a dopaminergic agonist (SKF-82526-J) or antagonist (SCH-23390). The solubilized receptors were subsequently reconstituted into lipid vesicles by gel-filtration. A comparison of ligand-binding properties shows that the solubilized and reconstituted receptors bound [3H]SCH-23390 to a homogeneous site in a saturable, stereospecific and reversible manner with a Kd of 0.95 and 1.1 nM and a Bmax of 918 and 885 fmol/mg protein respectively for agonist- and antagonist-pretreated preparations. These values are very similar to those obtained for membrane-bound receptors. The competition of antagonists for [3H]SCH-23390 binding exhibited a clear D1 dopaminergic order in the reconstituted preparation obtained from either agonist or antagonist-pretreated membranes, except that (+)butaclamol was about four-fold more potent than cis-flupentixol in displacing [3H]SCH-23390 binding in preparation obtained from agonist-pretreated membranes compared to antagonist-pretreated membranes. The agonist/[3H]SCH-23390 competition studies revealed the presence of a high-affinity component of agonist binding in both the reconstituted receptor preparations. The number of high-affinity agonist binding sites, however, is 40-80% higher in reconstituted preparation obtained from antagonist-treated membrane compared to that obtained from the agonist-treated membrane. In both the preparations, 100 microM guanylylimidodiphosphate (Gpp(NH)p) completely abolished the high-affinity component of agonist binding compared to partial abolition in the native membranes, indicating a close association of a G-protein with the solubilized receptors. Whether the receptor was solubilized following agonist or antagonist preincubation of the membranes, the receptor-detergent complex eluted from a steric-exclusion HPLC column with an apparent molecular size of 360,000. Preincubation of the solubilized preparations with Gpp(NH)p had virtually no effect on the elution profile suggesting a lack of guanine nucleotide-dependent dissociation of G-protein receptor complex.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1988

Sulfated cholecystokinin octapeptide (CCK8) failed to modulate basal or dopamine-stimulated adenylate cyclase activity in the rat striatum.

1. In view of previously demonstrated modulatory effects of CCK8 on DA-sensitive adenylate cyclase activity in the nucleus accumbens, we examined the effects of this neuropeptide in the striatum. 2. Adenylate cyclase activity was measured by conversion of alpha-[32P]ATP into [32P]cAMP. 3. CCK8, when added to the adenylate cyclase assay in concentrations up to 100 microM, failed to significantly alter, either positively or negatively, basal or DA-stimulated (30 or 100 microM) adenylate cyclase activity. Similar results were obtained in the presence of various peptidase inhibitors. 4. Under the assay conditions employed in these experiments, it would appear that there is no effect of CCK8 on DA-sensitive adenylate cyclase in the striatum.

Adenylyl Cyclases↗
PubMed1986

Characterization of high-affinity dopamine D2 receptors and modulation of affinity states by guanine nucleotides in cholate-solubilized bovine striatal preparations.

3,4-Dihydroxyphenylethylamine (dopamine) D2 receptors, solubilized from bovine striatal membranes using a cholic acid-NaCl combination, exhibited the typical pharmacological characteristics of both agonist and antagonist binding. The rank order potency of the agonists and antagonists to displace [3H]spiroperidol binding was the same as that observed with membrane-bound receptors. Computer-assisted analysis of the [3H]spiroperidol/agonist competition curves revealed the retention of high- and low-affinity states of the D2 receptor in the solubilized preparations and the proportions of receptor subpopulations in the two affinity states were similar to those reported in membrane. Guanine nucleotide almost completely converted the high-affinity sites to low-affinity sites for the agonists. The binding of the high-affinity agonist [3H]N-n-propylnorapomorphine ([3H]NPA) was clearly demonstrated in the solubilized preparations for the first time. Addition of guanylyl-imidodiphosphate completely abolished the [3H]NPA binding. When the solubilized receptors were subjected to diethylaminoethyl-Sephacel chromatography, the dopaminergic binding sites eluted in two distinct peaks, showing six- to sevenfold purification of the receptors in the major peak. Binding studies performed on both peaks indicated that the receptor subpopulation present in the first peak may have a larger proportion of high-affinity binding sites than the second peak. The solubilized preparation also showed high-affinity binding of [35S]guanosine-5'-(gamma-thio)triphosphate, a result suggesting the presence of guanine nucleotide binding sites, which may interact with the solubilized D2 receptors. These data are consistent with the retention of the D2 receptor-guanine nucleotide regulatory protein complex in the solubilized preparations and should provide a suitable model system to study the receptor-effector interactions.

Animals↗