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K C Breen

Publications and source records attributed to K C Breen.

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

The biochemical consequences of alpha2,6(N) sialyltransferase induction by dexamethasone on sialoglycoprotein expression in the rat H411e hepatoma cell line.

Previous studies have demonstrated sialyltransferase (ST) enzyme activity to be induced in hepatic cells by corticosteroids. In this study, we used the H411e rat hepatoma cell line to further characterise this induction with particular reference to the subsequent changes in the pattern of sialoglycoprotein (SGP) expression. The induction of total ST activity by dexamethasone was concentration dependent with maximum induction occurring 12 h subsequent to drug addition. Western blot analysis demonstrated that the induction was associated with an increase in the expression of the alpha2,6(N) ST enzyme with no change in the expression levels of the alpha2,3(N) enzyme. While the induction resulted in an increase in the reaction velocity (Vmax) of the enzyme for both the sugar donor (CMP-Neu5Ac) and the asialofetuin acceptor protein, there was no significant change in the enzyme affinity (Km) for the substrates, suggestive of either an increase in the expression or efficiency of the existing enzyme(s) rather than an induction of novel ST enzymes. Lectin blot analysis of cellular glycoprotein expression demonstrated no change in the expression patterns of either alpha2,3 or alpha2,6-linked SGP following enzyme induction. These results suggest that the available acceptor sites for the terminal sialic acid group(s) may be fully occupied in the control cells and therefore there are no further sites onto which the sialic acid can be transferred following induction of ST enzyme activity. This may be due to the high basal enzyme levels in the control cells already exhausting endogenous acceptor sites.

Animals↗
PubMed1997

Individual isoforms of the amyloid beta precursor protein demonstrate differential adhesive potentials to constituents of the extracellular matrix.

The amyloid beta precursor protein (AbetaPP) can exist as a membrane-bound glycoprotein which modulates neural cell adhesion. The adhesion of clones of the AtT20 mouse pituitary cell line, transfected with cDNA coding for the 695 (AbetaPP695) and 751 (AbetaPP751) amino acid forms of the protein, to individual components of the extracellular matrix was determined using a centrifugal shear assay. On laminin, poly-L-lysine, fibronectin, and uncoated glass substrata, the cells transfected with AbetaPP695 (6A1 cells) demonstrated a 50% increase in adhesivity over nontransfected cells, while those transfected with AbetaPP751 (7A1 cells) showed a significant decrease in adhesion. There was, however, a significant increase in the adhesive strength of the 7A1 cells to collagen type IV with no change in the adhesivity of the 6A1 cells when compared with control. These changes in adhesivity could be attributed to changes in the levels of the membrane-bound protein and were not due to the interaction of soluble AbetaPP with elements of the extracellular matrix. These studies provide evidence for differential adhesivities of the constituent AbetaPP isoforms and the possible role of the Kunitz protease inhibitor (KPI) domain in influencing the adhesive properties of the protein backbone.

Amyloid beta-Protein Precursor↗
PubMed1997

Plasma sialyltransferase levels in psychiatric disorders as a possible indicator of HPA axis function.

A dysfunction in the regulation of the hypothalamic-pituitary-adrenal (HPA) axis, possibly attributed to a change in glucocorticoid receptor (GR) functionality, has been implicated in depression. We have measured both lymphocyte GR receptor binding parameters and plasma sialyltransferase activity, as a biochemical marker of GR function, in two groups of patients suffering from depression or schizophrenia and in a group of age- and sex-matched controls. While there was a significant increase in plasma cortisol levels in the depressed group, there were no changes in the lymphocyte GR binding parameters (K(m) and Bmax). There was, however, a significant decrease in the plasma sialyltransferase: cortisol ratio in the depressed group suggesting an inability of the raised cortisol levels to induce enzyme expression and this ratio may provide a useful biochemical marker of cortisol receptor function. Although there was an increase in the plasma activity of the alpha 2,6 sialyltransferase isozyme in the schizophrenic group, no other changes were determined. Therefore, while the total plasma sialyltransferase:cortisol ratio reflects HPA axis function, alterations in specific isozyme activity may also be associated with other CNS disease states.

Binding Sites↗
PubMed1996

Evidence for a correlation between ambient cholesterol levels and soluble plasma sialyltransferase enzyme activity.

While soluble forms of the sialyltransferase (sialyl-T) enzyme have been detected in significant quantities in serum, the exact source(s) of the enzyme, or the factors controlling its secretion are poorly understood. In this study, we have examined the relationship between ambient plasma cholesterol concentrations and sialyl-T-activities and also levels of constituent plasma sialoglycoproteins (SGP). There was an inverse relationship between levels of the alpha 2, 6 sialyl-T enzyme and both total plasma cholesterol and HDL, although no such relationship was observed for the alpha 2,3 enzyme. While there was no correlation between total cholesterol and the levels of plasma SGPs, there was an inverse relationship between the HDL component and alpha 2,3 SGPs.

Cholesterol↗
PubMed1996

Tissue-specific regulation of sialyltransferase activities in the rat by corticosteroids in vivo.

In this study we have examined the effects of corticosteroids on both the total sialyltransferase (sialyl-T) activity and on two individual isozymes in neural, hepatic, and renal tissues using an in vivo model system. Rats were adrenalectomised to deplete their endogenous stores of steroid hormones, and some subsequently received steroid replacement with dexamethasone or aldosterone. Adrenalectomy resulted in a significant decrease in total neural sialyl-T activity when compared with sham-operated animals in the four brain regions examined, indicating that total sialyl-T activity is normally under positive corticosteroid control. The subsequent effects of exogenous corticosteroids exhibited regional specificity with the enzyme activities in the cortex, cerebellum, and brainstem being stimulated by both dexamethasone and aldosterone and enzyme activity in the hippocampus being stimulated by aldosterone alone. In general, the changes in total enzyme activity could be attributed to the alpha 2,6 sialyl-T isozyme, although the changes in the cerebellum appeared to coincide with alpha 2,3 sialyl-T activity. In the liver, adrenalectomy resulted in an increase in enzyme activity which was not altered by administration of corticosteroids. There were no changes in total renal sialyl-T activity in any of the four experimental groups although certain changes were observed at the level of individual sialyl-T isozymes. These results demonstrate that sialyl-T activity in certain tissues is under the control of corticosteroids and that this is both a tissue-specific and region-specific effect.

Adrenal Cortex Hormones↗
PubMed1995

The control of sialyltransferase activity in tumor-cell lines derived from different tissues in multifactorial.

The activities of the sialyltransferase enzymes and the resulting expression of sialoglycoproteins were examined in tumor cells derived from different tissues in order to gain a greater understanding of the factors controlling the cell glycosylation state. Cell-cell contact, which is dependent on cell confluency state, was shown to influence glycosylation in the neurally-derived mouse neuro-2A neuroblastoma and the C6 glioma cell lines. Both showed a relatively high level of cell sialyltransferase activity under sub-confluent conditions with activity decreasing upon the formation of cell-cell contacts associated with confluency. A parallel decrease in the expression of sialoglycoproteins, as determined by lectin blot analysis, was observed under these conditions. In contrast, the H411e hepatoma cell line showed an increase in enzyme activity with confluency with the susceptibility of the enzyme in this cell line to glucocorticoid induction only being detected in sub-confluent cell cultures. The number of trypsinisation cycles of the cells was also shown to affect the enzyme activity of the neuro-2A and C6 cells with an increase in enzyme activity coincident with passage number being observed in the neuro-2A cells, and a decrease in the C6 glioma cell line. Trypsinisation had no effect on enzyme activity in the H411e cells. These results demonstrate that the control of sialyltransferase activity in tumor cells is multifactorial with the tissue of origin playing a key role.

Animals↗
PubMed1994

The effect of cell confluency state on the expression of neural cell surface glycoconjugates.

The expression of neural cell surface glycoconjugates during development reflects the precise control of cellular adhesivity that is required for the exact positioning of the developing cells. We have investigated the effect of cell confluency state on the expression of key cell surface glycoconjugates using the mouse neuroblastoma cell line, neuro-2A. There was an up-regulation of expression of the neural cell adhesion molecule (NCAM) coincident with an increase in cell confluency state and a parallel decrease in the expression of the amyloid precursor protein, beta APP. The expression pattern of cell surface glycoconjugates in neuro-2A cells is therefore similar to that observed during the early embryonic period of development during which cells aggregate to form collectives.

Amyloid beta-Protein Precursor↗
PubMed1994

Expression of the neural cell adhesion molecule (NCAM) in Alzheimer's disease.

Expression of the nerve cell adhesion molecule, NCAM, was examined in post-mortem brain samples from patients with Alzheimer's disease (AD) and from age-matched controls. Four major isoforms of NCAM were identified by Western blot analysis 195, 185, 145 and 120 kDa with no differences being observed in either the intensity of individual band staining or in the expression of the constituent NCAM isoforms in AD when compared with controls. Immunohistochemical staining revealed star-shaped NCAM-positive structures with numerous radiating 'processes' or a more protoplasmic shape with short and thick 'processes'. These structures were not obviously more abundant in AD and there was no NCAM immunoreactivity in neuritic plaques or neurofibrillary tangles. This evidence suggests there is no change in NCAM expression in AD.

Aged↗
PubMed1993

Neurofilament expression in human T lymphocytes.

The expression of intermediate filaments in normal cells is mainly determined by their embryonal developmental origin. Flow cytometry using monoclonal antibody RT97 demonstrated that neurofilament was detectable in the human HuT 78 T-cell line and on resting T lymphocytes. Expression was greatly increased on lymphocytes activated for 3 days with phorbol ester. Western blotting confirmed the presence of the 200,000 MW form of neurofilament in T lymphocytes. Stimulation of peripheral blood T cells with phorbol myristate acetate (PMA) or with anti-CD3 monoclonal antibodies resulted in a marked increase in detection of phosphorylated neurofilament on Western blotting. Stimulation of HuT 78 cells with anti-LFA-1 resulted in redistribution of neurofilament from a perinuclear spheroid core into dendritic processes. These data indicate that T cells activated through the T-cell receptor associated complex express an intermediate filament usually associated with neurally derived cells. The finding that neurofilament expression and organization are regulated by T-cell surface molecules suggests a role for this intermediate filament in T-cell function.

Blotting, Western↗
PubMed1991

Beta amyloid precursor protein mediates neuronal cell-cell and cell-surface adhesion.

The beta-amyloid precursor protein (APP) is a membrane-bound glycoprotein which has been proposed to play a role both as a growth factor and a mediator of cell adhesion. Using the Neuro-2A neuroblastoma cell line, we have investigated the capacity of APP to mediate neural cell adhesion. The cells express the protein at a high level, the immunohistochemical staining pattern at the level of the membrane having a punctate pattern. Fab' fragments of antibodies to the extracellular portion of the molecule were found to inhibit cell binding to a collagen substrate, but not to laminin, fibronectin, or poly-l-lysine. Fab' fragments of antibodies to the nerve cell adhesion molecule N-CAM also inhibited binding of Neuro-2A cells specifically to collagen. This inhibition of cell-surface binding was accompanied by a repression of neurite outgrowth in differentiating cells in the presence of antibodies. APP antibodies also inhibited neuron-neuron and neuron-glial binding, but not glial-glial cell adhesion. These data suggest that the APP, which is expressed primarily on differentiated neuronal cells, may play a role in the mediation of both cell-cell and cell-substrate adhesion.

Amyloid beta-Peptides↗
PubMed1991

Temporal expression of neurofilament polypeptides in differentiating neuroblastoma cells.

NEURO-2A neuroblastoma cells were differentiated by the addition of dibutyryl-cyclic AMP, resulting in an increase in transcription of mRNA coding for the two neurofilament polypeptides NF-L and NF-M. The mRNAs for these two polypeptides appeared to be co-expressed with both being switched on at 24 h after the addition of drug. This was in contrast to NF-H whose induction was only seen at 60 h following addition of drug. These results are in good agreement with the expression time course previously reported in-vivo and suggest that the neurofilament triplet polypeptides can be divided into two subsets which appear to be under different genetic control mechanisms according to their time course of expression.

Animals↗
PubMed1990

Intraventricular infusions of antibodies to amyloid-beta-protein precursor impair the acquisition of a passive avoidance response in the rat.

Intraventricular infusions of an antiserum raised against a 14 amino acid residue in the extracellular domain of amyloid-beta-protein precursor significantly decreased stepdown latency, at both 24 h and 48 h recall times, in rats trained to avoid an electroshock by remaining on a platform. The antiserum was effective when infused up to 2.5 h following training and no retention deficit was noted when it was administered at 4h or 6h after training. An antiserum generated against a 17 amino acid residue of the A4 amyloid peptide had no effect on learning. Thus the amyloid precursor protein, which is aberrantly processed in Alzheimer's disease, appears to be directly involved in memory formation.

Adult↗
PubMed1990

Cyclic AMP-dependent expression of the heavy neurofilament (NF-H) polypeptide in differentiating neuroblastoma cells.

Dibutyryl cyclic AMP (dbcAMP) treatment of Neuro 2A neuroblastoma cells induces cell differentiation and neurite outgrowth. Undifferentiated cells express the heavy neurofilament polypeptide (NF-H) at a low level. Following differentiation, there is a large increase in its expression due to an increase in the corresponding cellular mRNA. A similar increase is seen following forskolin treatment of the cells indicating that the increase in NF-H expression is cyclic AMP-dependent. The increase in mRNA is due to an increase in gene transcription as demonstrated by a nuclear run-off assay.

Animals↗
PubMed1989

The developmental regulation of the L2/HNK-1 and L3 carbohydrate epitopes in mouse brain. Evidence for separate control of lipid- and protein-bound epitopes.

The carbohydrate epitopes L2/HNK-1 and L3 have previously been identified on various neural cell adhesion molecules and have been suggested to play a role in the mediation of cell-cell adhesion. In this study, the developmental expression of the two epitopes in soluble, membrane-bound and chloroform/methanol-extracted fractions of the constituent mouse brain regions was examined by enzyme-linked immunosorbent assay (ELISA). The protein-bound epitopes were shown to be uniformly developmentally regulated, with levels peaking at postnatal day 20 (P20). The epitopes in a crude chloroform/methanol fraction, however, demonstrated a different pattern, with L2 peaking earlier at postnatal day zero (P0). These results suggest a possible interaction between the control of the two pools of the epitope.

Aging↗
PubMed1988

Partial sequence of the rat heavy neurofilament polypeptide (NF-H). Identification of putative phosphorylation sites.

A 3 kb cDNA clone has previously been isolated in this laboratory corresponding to the rat heavy neurofilament polypeptide (NF-H). This clone, equivalent to approximately 70% of the total mRNA of the protein has been sequenced and shown to contain the carboxy-terminal region of the message. This contains 51 of the Lys-Ser-Pro repeat triplets which are reported to be the site of neurofilament phosphorylation. The sequence obtained was subsequently compared to those of mouse and human NF-H, showing a homology of approximately 85%. There is, however, one region which is variable between the species, this being the highly phosphorylated region of the protein containing the Lys-Ser-Pro triplet repeat.

Amino Acid Sequence↗
PubMed1988

Soluble rat brain sialidase does not influence intracellular glycosylation of Golgi sialyltransferase or its constitutive glycoproteins.

Cytosol- and Golgi-enriched fractions were obtained from whole rat brain homogenates by density gradient centrifugation. Using a 4-methylumbelliferyl neuraminic acid substrate a soluble neural sialidase has been identified and characterised. The enzyme had optimal activity at pH 6.0 and a Km of 0.44 +/- 0.18 mM. The specific activity increased during postnatal development and this was in parallel with the described temporal changes in total brain neuraminic acid turnover. The potential of this enzyme to influence the intracellular processing of sialoglycoconjugates was also investigated. Cytosol fractions were incapable of releasing [14C]NeuNAC [( 14C]N-acetylneuramic acid) transferred to the glycoproteins of isolated Golgi membranes by their associated sialyltransferase. Further preincubation of Golgi membranes with soluble sialidase had no effect on their intrinsic sialyltransferase activity. These results demonstrate that no epigenetic regulation of processed sialoglycoconjugates occurs intracellularly and these finding are related to post-translational control of neural cell adhesion molecule (N-CAM) sialylation state.

Aging↗