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C Mamalaki

Publications and source records attributed to C Mamalaki.

30 records · Page 2Linked to original sources
PubMed1992

A self-reactive T cell population that is not subject to negative selection.

In male mice expressing a transgenic alpha beta TCR which recognizes a male antigen (HY), T cells which do not express normal levels of CD8 escape thymic deletion and appear in the periphery. These consist of two distinct populations, one which lacks expression of both CD4 and CD8, and one with low levels of CD8. Neither population has anti-HY reactivity, consistent with the known requirement of this TCR for CD8. We now describe the consequences of expression of both the anti-HY TCR transgene and a constitutive CD8.1 transgene on T cells of male mice. Peripheral T cells in these male 'double transgenic' mice express both the anti-HY TCR and normal levels of CD8, and can proliferate to male antigen in vitro. These cells do not express the endogenous allele of CD8 (CD8.2), suggesting that the increase in CD8 levels due to the CD8.1 transgene leads to the deletion of the CD8.2low population. In contrast, the CD8.1 transgene does not lead to the deletion of the CD8.2- population. This implies that, unlike the majority of alpha beta T cells, TCR+CD4-CD8- cells in TCR transgenic mice are not subject to deletion.

Animals↗
PubMed1991

Deletion analysis of the human CD2 gene locus control region in transgenic mice.

Genomic sequences located at the 3' flanking region of the human CD2 gene confer high level tissue-specific, position-independent expression of the gene when introduced in the germ line of mice. In order to further characterize these sequences a range of deletions, from the 3' end were produced and transgenic mice were generated with the human CD2 (hCD2) gene linked to these deleted fragments. This allowed us to establish the minimum sequences necessary for the copy-dependent transgene expression. 2.1 kb or 1.5 kb of 3' flanking sequences linked to a hCD2 mini-gene is sufficient to allow T-cell specific, copy-dependent, integration-independent expression in transgenic mice. 1.1 kb of 3' sequences results in the gene being expressed in a T-cell specific manner, but copy-dependent, integration-independent expression was not observed in a small number of transgenic animals. 0.2 or 0.5 kb of 3' flanking sequences were insufficient to allow expression above the level previously found with a human CD2 gene which lacked 3' flanking sequences. We conclude that the Locus Control Region (LCR) effect is caused by 1.5 kb of flanking sequences immediately 3' to the polyadenylation signal of the gene.

Animals↗
PubMed1989

Molecular size of the gamma-aminobutyric acidA receptor purified from mammalian cerebral cortex.

The hydrodynamic behaviour of both the soluble and purified gamma-aminobutyric acidA (GABAA) receptor of bovine or rat cerebral cortex has been investigated in solution in Triton X-100 or in 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulphonate (CHAPS). In all the hydrodynamic separations made, it was found that the binding activities for GABA, benzodiazepine, and (where detectable) t-butylbicyclophosphorothionate comigrated. Conditions were established for gel exclusion chromatography and for sucrose density gradient velocity sedimentation that maintain the GABAA receptor in a nonaggregated form. Using these conditions, the molecular weight of the bovine GABAA receptor in the above-mentioned detergents was calculated using the H2O/2H2O method. A value of Mr 230,000-240,000 was calculated for the bovine pure GABAA receptor purified in sodium deoxycholate/Triton X-100 media. A value of Mr 284,000-290,000 was calculated for the nonaggregated bovine or rat cortex receptor in CHAPS, but the Stokes radius is smaller in the latter than in the former medium and the detergent binding in CHAPS is underestimated. Thus the deduced Mr, 240,000, is the best estimate by this method.

Animals↗
PubMed1988

Retroviral-mediated gene transfer. Applications in neurobiology.

There are now many examples of the successful expression of genes transduced by retroviruses in studies from outside the field of neuroscience. Retroviruses will undoubtedly also prove to be effective tools for neuro-scientists interested in expressing cloned neurotransmitter and receptor genes. There are also other less obvious applications of retroviruses, such as their insertional mutagenic effects, which may be useful in studies of the genetic factors and biochemical mechanisms involved in, for example, neurotoxicity. Strong cellular promoters have been identified by retroviral infection and subsequent rescue of the flanking genomic DNA. Retroviruses can be employed again to reintroduce these regulatory sequences back into cells. In this way the complexities of gene expression in the many subpopulations of neurons may be unraveled. Retroviruses can also serve as very useful genetic markers in studies of development and lineage relationships. Retroviruses may be used to efficiently transfer oncogenes into neuronal cells to create new cell lines. This application exploits one of the natural traits of retroviruses--oncogenesis--which led to their original discovery. Finally, there are neurotropic retroviruses that could serve as important vectors for delivering genes into neurons. Studying these retroviruses may lead to an understanding of how they cause neuropathologic changes in the CNS.

Animals↗
PubMed1987

The GABAA/benzodiazepine receptor is a heterotetramer of homologous alpha and beta subunits.

The GABAA receptor has been purified to homogeneity from bovine cerebral cortex. Under stringent conditions of isolation, the GABAA receptor was shown to consist only of alpha (Mr 53 000) and beta (Mr 57 000) subunits. A densitometric scan of SDS-PAGE gels under reducing conditions showed that these subunits were present in a 1:1 ratio. A model of the receptor as a heterologous tetramer alpha 2 beta 2 is proposed. Monoclonal antibodies have been raised to the purified bovine GABAA receptor. One of these antibodies, 1A6, was shown to react with both the alpha and beta subunits of the purified receptor. The subunits were still positive in immunoblots following the removal of the carbohydrate moieties of the respective polypeptides by endoglycosidase F treatment. This antibody has been employed to demonstrate antigenic cross-reactivity between the GABAA receptors of three vertebrate species. It is further proposed that there is partial amino acid sequence homology between the alpha and beta polypeptides and hence that they are derived from a single ancestral gene.

Animals↗
PubMed1986

Antibodies recognising the GABAA/benzodiazepine receptor including its regulatory sites.

Polyclonal antibodies have been raised against the GABA/benzodiazepine receptor purified to homogeneity from bovine cerebral cortex in deoxycholate and Triton X-100 media. Radioimmunoassay was applied to measure specific antibody production using the 125I-labelled gamma-aminobutyric acid (GABA)/benzodiazepine receptor as antigen. The antibodies specifically immunoprecipitated the binding sites for [3H]muscimol and for [3H]flunitrazepam from purified preparations. In addition, when a 3-[(3-cholamidopropyl)dimethylammonio] 1-propanesulphonate (CHAPS) extract of bovine brain membranes was treated with the antibodies, those sites as well as the [3H]propyl-beta-carboline-3-carboxylate binding, the [35S]t-butylbicyclophosphorothionate binding (TBPS), the barbiturate-enhanced [3H]flunitrazepam binding, and the GABA-enhanced [3H]flunitrazepam binding were all removed together into the immunoprecipitate. Western blot experiments showed that these antibodies recognise the alpha-subunit of the purified GABA/benzodiazepine receptor. These results further support the existence in the brain of a single protein, the GABAA receptor, containing a set of regulatory binding sites for benzodiazepines and chloride channel modulators.

Allosteric Regulation↗
PubMed1986

The GABAA receptor and its antibodies.

The GABAA/benzodiazepine receptor has been purified to homogeneity from bovine and rat cerebral cortex. Under optimum conditions, the purified receptor has been shown to possess four distinct drug-binding sites, for GABA, benzodiazepine, barbiturate and Cl- channel gating classes of ligands. The receptor is a multi-subunit membrane glycoprotein with an oligomeric size of 230,000 Da. It contains at least two subunits, alpha and beta, the first of which can be photoaffinity-labelled with the benzodiazepine, flunitrazepam. Polyclonal and monoclonal antibodies have been raised to the native receptor and both have been used in an immunological characterization of the receptor protein in detergent extracts and likewise in purified preparations from bovine cerebral cortex.

Animals↗
PubMed1985

Reconstitution of the purified gamma-aminobutyric acid-benzodiazepine receptor complex from bovine cerebral cortex into phospholipid vesicles.

The purified gamma-aminobutyric acid-benzodiazepine receptor complex from bovine cerebral cortex has been reconstituted into phospholipid vesicles by a cholate dialysis procedure. The reconstituted receptor bound [3H]flunitrazepam at a single class of sites with dissociation constant Kd = 2.9 +/- 0.3 nM, an increase in affinity to the membrane level from the 4-fold weakening found in detergent solution. It also bound [3H]muscimol with a Kd for the high-affinity sites of approximately 50 nM. [35S]tert.-Butyl-bicyclophosphorothionate, for which there is evidence in membranes for binding to a channel gating site on this receptor, showed similar binding to the reconstituted receptor.

Animals↗
PubMed1984

The purified GABA/benzodiazepine/barbiturate receptor complex: four types of ligand-binding sites, and the interactions between them, are preserved in a single isolated protein complex.

A GABA/benzodiazepine/barbiturate receptor complex has been purified from bovine cerebral cortex by affinity chromatography on a benzodiazepine column. Depending on the detergent present during the isolation of the receptor (deoxycholate/Triton X-100 or CHAPS/Asolectin), and during the binding assays (Triton X-100 or CHAPS), the receptor displays different binding properties for the GABAA agonist [3H]muscimol and for the chloride ion channel blocking agent [35S]t-butylbicyclophosphorothionate (TBPS), whereas the binding properties for the benzodiazepine [3H]flunitrazepam are independent of isolation and assay conditions. Both methods of isolation yield a protein complex consisting of the same two subunits of Mr 53 000 and Mr 57 000. Therefore the different binding properties reflect different conformations of the isolated receptor protein. [3H]flunitrazepam binding to the CHAPS-purified receptor is stimulated by GABA and the barbiturate pentobarbital in a dose-dependent manner. Photo-affinity labeling of the purified receptor with [3H]flunitrazepam leads to incorporation of radioactivity into both subunits, but predominantly into the Mr 53 000 band, as shown by fluorography. Proteolytic degradation by trypsin of the isolated photo-affinity labeled receptor in detergent solution proceeds via a labeled Mr 48 000 polypeptide. Proteolytic destruction of the reversible [3H]flunitrazepam and [3H]muscimol binding activities requires greater than 100 fold higher concentrations of trypsin than the decomposition of the receptor polypeptides into fragments less than Mr 10 000.

Animals↗
PubMed1983

A gamma-aminobutyric acid/benzodiazepine receptor complex of bovine cerebral cortex.

The gamma-aminobutyric acid/benzodiazepine receptor from bovine cerebral cortex was solubilized with sodium deoxycholate and purified by affinity chromatography on benzodiazepine-agarose and ion exchange chromatography. The benzodiazepine binding protein was enriched 1800-fold. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol showed the presence of two major bands of Mr = 57,000 and 53,000. [3H]Flunitrazepam, after UV irradiation, was incorporated irreversibly into both bands of the isolated protein. A high affinity binding site for gamma-aminobutyric acid was co-purified with the benzodiazepine binding site and the two sites were shown to reside on the same physical structure. The dissociation constants were 10 +/- 4 nM for [3H] flunitrazepam and 12 +/- 3 nM for the gamma-aminobutyric acid agonist [3H]muscimol. The maximum specific activity for [3H] muscimol binding was 4.3 nmol/mg of protein. The ratio of [3H]muscimol to [3H]flunitrazepam binding sites was between 3 and 4. Gel filtration and sucrose density gradient sedimentation studies gave a Stokes radius of 7.3 +/- 0.5 nm and a sedimentation coefficient of 11.1 +/- 0.3 S, respectively. The purified complex had a pharmacological profile that corresponds to the receptor specificity found in membranes and crude soluble extracts.

Animals↗