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Biomedical subjects

K C Breen

Publications and source records attributed to K C Breen.

18 recordsLinked to original sources

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

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

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

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

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

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

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

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

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

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

Lead stimulates Golgi sialyltransferase at times coincident with the embryonic to adult conversion of the neural cell adhesion molecule (N-CAM).

The neural cell adhesion molecule N-CAM is believed to be intimately involved in the structuring of the central nervous system. During post-natal development the molecule exists in 2 forms--a sialic acid-rich form which is preferentially expressed during cell acquisition and fibre outgrowth and a sialic acid-poor form which appears at times coincident with synaptogenesis. The developmental changes between these 2 forms have been demonstrated to be impaired by chronic low-level lead exposure and this is consistent with the reduced synaptic elaboration associated with this action. Here is described the effect of lead on the Golgi-associated sialyltransferase which regulates N-CAM sialylation state. Lead chloride was found to markedly stimulate sialyltransferase with an ED50 of 5 X 10(-7) M in adult Golgi fractions. This was not observed in fractions derived from 12-day old animals. At the concentration of 5 X 10(-5) M lead was found to have a differential effect on the developmental expression of this enzyme. During the early phases of development (days 4-16) sialyltransferase activity was inhibited. However, in coincidence with periods of N-CAM desialylation (days 16-30), it was significantly stimulated. These findings are related to the perturbations of N-CAM function during chronic low-level lead exposure.

Aging

Differentiation-dependent sialylation of individual neural cell adhesion molecule polypeptides during postnatal development.

The postnatal sialylation of individual neural cell adhesion molecule (N-CAM) polypeptides by a developmentally regulated sialyltransferase in Golgi-enriched fractions isolated from rat brain is described. The 120-kilodalton polypeptide of N-CAM was found to be sialylated at each developmental age examined. This was in contrast to the 140- and 180-kilodalton N-CAM polypeptides which were only sialylated until postnatal day 10 and from postnatal day 12, respectively. Immunoblotting procedures demonstrated that all N-CAM polypeptides were expressed in the Golgi fractions at each developmental stage examined. The heavily sialylated "embryonic" form of N-CAM was found to be reexpressed at postnatal days 10 and 12, a time coincident with extensive fibre outgrowth. The "embryonic" form of N-CAM incorporated similar amounts of [14C]sialic acid into its constituent polypeptides reflecting the difference in sialic acid to protein ratio, as this form of N-CAM was virtually undetectable in the immunoblots of postnatal material.

Animals

Developmental control of N-CAM sialylation state by Golgi sialyltransferase isoforms.

A rat brain Golgi sialyltransferase activity capable of the differentiation-dependent control of N-CAM sialylation state is described. The specific activity of Golgi sialyltransferase was found to be developmentally regulated with respect to both endogenous and exogenous protein acceptors, with a particular elevation on postnatal days 10-12 when the heavily sialylated or 'embryonic' form of N-CAM is re-expressed. The subsequent developmental decrease in activity was associated with a significant decrease in apparent Km for the CMP-NeuNAc substrate, but not for the asialofetuin exogenous acceptor, which could not be attributed to the temporal expression of an endogenous competitive inhibitor. The apparent Vmax remained constant for CMP-NeuNAc but was significantly reduced for asialofetuin. Sialyltransferase activity, which was optimal at pH 7.0-7.5, was also modulated by various cations. Zinc abolished enzyme function, in contrast to ferric ions which stimulated activity fourfold-sevenfold. The marked activation of the adult form of the enzyme by potassium and magnesium ions, together with the alterations in kinetic constants, suggested this activity to be distinct from that derived from postnatal day-12 tissue. The kinetics of [14C]sialic acid incorporation into immuno-precipitated N-CAM demonstrated the individual polypeptides to be sialylated, possibly by addition of polysialosyl units, in a developmental sequence. The presence of four distinct sialyltransferase activities was demonstrated by non-denaturing gel electrophoresis followed by solid-phase enzyme assay. These isoforms were temporally expressed during development, two being correlated with the postnatal reexpression of the 'embryonic' form of N-CAM.

Animals

Postnatal D2-CAM/N-CAM sialylation state is controlled by a developmentally regulated Golgi sialyltransferase.

Golgi-enriched fractions have been isolated from rat brain of increasing postnatal age and defined by electron microscopy and distribution of marker enzymes. The expression of sialyltransferase activity associated with these fractions has been demonstrated to developmentally decrease and this appeared to be, in part, dependent on endogenous competitive inhibition. The developmental regulation of this activity paralleled the sialylation state of the neural cell adhesion molecule (D2-CAM/N-CAM) and could be demonstrated to be capable of endogenously sialylating this protein in the isolated Golgi fractions. In 12-day-old animals the majority of the transferred [14C]sialic acid was found to be associated with the high-molecular-weight [greater than 200 kilodaltons (kd)] form of D2-CAM/N-CAM, indicative of the protein having been heavily sialylated. Sialylation of the individual D2-CAM/N-CAM polypeptides was also demonstrated in both 12-day and adult animals and transfer was evident only in the 180-kd and 115-kd components and not in the 140-kd component. In contrast, Golgi-enriched fractions prepared from adult animals showed little capability of heavily sialylating D2-CAM/N-CAM to any significant extent.

Animals

Synaptosomal sialyltransferase glycosylates surface proteins that are inaccessible to the action of membrane-bound sialidase.

Sialyltransferase has been characterized in P2 pellets derived from animals of increasing age. The enzyme was found to be associated with the plasma membrane and to be developmentally regulated at times coincident with cell migration and fibre outgrowth. This regulation appeared to be due, in part, to an endogenous competitive inhibitor in the P2 pellet but not in the synaptosome. Optimal transfer of [14C]N-acetylneuraminic acid to endogenous synaptosomal acceptors was achieved only in the absence of detergent. Furthermore, the transferred sialic acid was found to be inaccessible to the action of membrane-bound sialidase. The significance of these findings is discussed.

Aging

Characterization and cellular localization of a developmentally regulated rat neural sialidase.

A developmentally regulated neural sialidase has been identified in particulate, subcellular fractions of rat brain. Enzyme activity, measured using a [3H]sialoganglioside substrate, was linear with time and had a pH optimum of 4.0-4.5. Protein linearity was only observed at low protein concentrations. This appeared to be caused by enzyme access to a lipophilic substrate, as activity was significantly stimulated by membrane-fluidizing agents. Enzyme activity was developmentally expressed in P2 pellets coincident with in vivo synaptogenesis. It was located on the synaptosome and was particularly high in myelin-containing fractions. Its cellular distribution was confined to neuronal cells and centrally derived oligodendrocytes.

Animals

APP-collagen interaction is mediated by a heparin bridge mechanism.

The amyloid precursor protein (APP) is a glycoprotein consisting of at least four isoforms derived from a single gene by a process of alternative splicing. The membrane-bound forms of APP have been suggested to have adhesive properties and to mediate neural cell adhesion. Previous studies have demonstrated the ability of Fab' fragments of antibodies to extracellular domains of APP to inhibit neural cell binding to a collagen substrate, suggesting a physiological role for the collagen-binding properties of APP. The binding of APP has been demonstrated to be specific for type IV collagen, and no binding to other extracellular matrix components, including fibronectin and laminin, was detected. The APP-collagen binding appeared to be mediated by a heparin-bridge mechanism, since the binding was abolished by the addition of excess heparan or heparinase. These results were observed by both a homogenate-collagen binding assay and a cell-surface adhesion assay, thus providing further evidence for the adhesion role of APP. They also pose the question of the possible role of the heparin-binding properties of APP in the genesis of the neuritic plaques characteristic of Alzheimer's disease.

Alzheimer Disease

A decrease in neural sialyltransferase activity in Alzheimer's disease.

The activities of both a particulate and soluble form of the sialyltransferase enzyme have been examined in post-mortem brain samples from Alzheimer's disease patients and age-matched controls. There was a considerable decrease in the activity of both the soluble and membrane-bound forms of the enzyme in the frontal and temporal cortical lobes, although no change was observed in the hippocampus. There was, however, no change in activity of the Golgi marker enzyme thiamine pyrophosphatase. Therefore, it is suggested that the decrease in sialyltransferase enzyme activity may be a specific biochemical event associated with the AD-like neurodegeneration.

Aged