Biomedical subjects

C M Regan

Publications and source records attributed to C M Regan.

At least 73 records · Page 4Linked to original sources

PubMed1989

Antiproliferative action of benzodiazepines in cultured brain cells is not mediated through the peripheral-type benzodiazepine acceptor.

The benzodiazepines, Ro 5-4864, diazepam, clonazepam, and also PK-11195, inhibited, at micromolar concentrations, the proliferation of rat C6 glioma and mouse neuro-2A neuroblastoma cells in culture. The cells possessed high levels of "peripheral-type" high-affinity benzodiazepine binding sites as judged by binding assays and displacement potencies. However, the different potencies and specificities of compounds for the antiproliferative actions and binding affinities for the binding site suggest that the antiproliferative actions were not mediated through the peripheral-type binding site. In support of this, these compounds have also been shown to inhibit proliferation of some nonneuronal cultured cell lines, e.g., mouse SP2/O-Ag 14 hybridoma and rat NCTC epithelial cells, which have no detectable high-affinity peripheral-type benzodiazepine binding sites.

Animals↗

PubMed1988

The anticonvulsant sodium valproate specifically induces the expression of a rat glial heat shock protein which is identified as the collagen type IV receptor.

A potential mechanism for valproate (VPA)-induced increases in glial cell-substratum adhesivity has been demonstrated. Metabolically labelled glioma (C6) and primary astrocytes showed a statistically significant accumulation of protein when cultured in the presence of therapeutic concentrations of VPA (1 mM). This was mainly accounted for by a 10-fold increase in the production of a single polypeptide of 43 kDa molecular weight. Fractionation studies and metabolic labelling with N-acetyl-D-mannosamine showed this to be a sialoglycoprotein which was plasma membrane-bound. VPA-induction of the polypeptide was apparently specific to glioma and primary astrocytes and was not observed in neuroblastoma (neuro-2a), fibroblasts (3T3), pituicytes (GH3) and epithelial cells (NCTC). The 43 kDa component of glia was demonstrated to be the receptor for type IV collagen by binding metabolically labelled and solubilised cells to Sepharose beads which had been individually coated with laminin, fibronectin and type IV collagen. The protein has also been shown to be a heat shock product as metabolically labelled glioma showed a 10-fold increase in its expression when cultured at 42 degrees C. This heat shock induced expression was transient and was in marked contrast to that seen with VPA where it increased with time and was sustained. The expression of 43 kDa is suggested to arise by VPA and heat shock induced delays in cell cycle progression and this is discussed in relation to teratogenic action.

Anticonvulsants↗

PubMed1988

Neuronal and glial markers of the central nervous system.

This brief review evaluates the expression of cell-specific markers on differentiated neural cells and, where necessary, on their developing precursors. Within these limitations only the commonly used markers are discussed and those deemed unequivocal are only briefly appraised.

Animals↗

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↗

PubMed1988

The expression of an N-CAM serum fragment is positively correlated with severity of negative features in type II schizophrenia.

The expression of a 70-kD serum fragment of the neural cell adhesion molecule (N-CAM) in schizophrenia is described. Schizophrenic patients (n = 40) were found to have statistically significant increases (p less than 0.0001) in serum N-CAM levels when compared to normal individuals (n = 26), and this could not be associated with age or sex. This difference was more marked (p less than 0.0001) between type II schizophrenics (n = 13) and normal individuals (n = 26) than when patients in the overlap group between type I and type II schizophrenia (n = 18) were compared to normal individuals (p less than 0.001). This difference remained significant (p less than 0.01) when overlap patients were compared to those of type II schizophrenia. Furthermore, schizophrenic patients with lower serum N-CAM proved to be better responders to neuroleptic therapy. We suggest that these elevated serum N-CAM levels reflect an increased synaptic turnover rate in this psychotic state.

Adult↗

PubMed1988

Chronic low level lead exposure precociously induces rat glial development in vitro and in vivo.

Lead has been demonstrated to induce precocious glial differentiation both in vitro and in vivo. Lead-treated rat glioma (C6) and cerebellar astrocytes exhibited cytoplasmic extensions and the presence of glial endfeet after a 3-day exposure to 10(-6) to 10(-4) M PbCl2. In similar experiments no effect was noted in neuroblastoma (Neuro-2a) or on neurite outgrowth from chick spinal cord explants. This prodifferentiative effect on glia was also seen in the cerebella of postnatal rats in which the developmental expression of glial-specific glutamine synthetase activity was significantly increased up to postnatal day 12 after chronic exposure to lead from time of birth via their dam's drinking water (400 mg PbCl2/l).

Age Factors↗

PubMed1988

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↗

PubMed1988

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↗

PubMed1988

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↗

PubMed1987

Acquisition of a brief behavioural experience in the rat is inhibited by the brain-specific monoclonal antibody, F3-87-8.

Intraventricular infusion of the brain-specific monoclonal antibody F3-87-8 is demonstrated to inhibit the acquisition of a passive-avoidance paradigm in a rat model. Antibody was infused prior to, and following, training of a step-down task and recall was assessed after 24 and 48 h. The antibody only inhibited recall when infused prior to training and no effect was seen when administered 6 h after acquisition of the task. No effect was found with absorbed antibody or in sham-operated animals and open field studies showed no alterations in locomotor activity. Results are discussed considering available information on the antigen and current memory/learning theories.

Animals↗

PubMed1987

The effect of a neuron-specific antiserum, BPM, on the in vitro development of cerebellar granule cells.

Anti-BPM is a neuron-specific antiserum which specifically recognizes the D2 cell adhesion molecule in crossed immunoelectrophoresis of Triton X-100-solubilized brain extracts. Here the effect of this antiserum on the in vitro development of cerebellar neuronal cultures is described. The initial adhesion of cells and neurite outgrowth were not influenced by immunoglobulin fractions of anti-BPM. However, after 5 days in vitro the cultures had become completely disorganized, with the majority of cells being dead at immunoglobulin concentrations greater than 0.5 mg/ml culture medium. This effect was seen only with immunoglobulins and their F(ab')2 fragments, the F(ab') fragments being without effect. The addition of anti-BPM to 8-day-old cultures resulted in a more rapid and pronounced rate of cell death. In many instances this was preceded by a rapid "destabilization" of culture organization. The cytotoxic effect of anti-BPM was neuron specific and the small numbers of astrocytes and fibroblasts found in the cultures were unaffected by prolonged exposure to this serum.

Animals↗

PubMed1987

Acquisition of a brief behavioral experience in the presence of neuron-specific and D2-CAM/N-CAM-specific antisera.

The effect of intraventricular infusion of D2-CAM/N-CAM directed antibodies prior to the acquisition of a passive-avoidance paradigm is described. The antisera used in this study were the neuron specific anti-BPM and a D2-CAM/N-CAM specific serum, anti-D2. Anti-BPM reliably inhibited paradigm acquisition when recall was ascertained at 24 and 48 hours and no effect was noted with absorbed anti-BPM or in sham-operated animals. This effect was time-dependent and no inhibition of memory formation was noted when the antiserum was administered at 6 and 10 hours after training. In contrast, infusion of anti-D2 had no effect on paradigm acquisition. These findings are discussed in relation to the potential synaptogenic events associated with memory formation.

Animals↗

PubMed1987

Chronic low-level lead exposure impairs embryonic to adult conversion of the neural cell adhesion molecule.

The neural cell adhesion molecule (N-CAM) is a complex of surface glycoproteins that are developmentally regulated and believed to be intimately involved in the orderly structuring of the CNS. Here the effect of chronic low-level lead exposure on their expression in the postnatally developing cerebellum is described. Rat pups were chronically exposed to lead via their dam's drinking water which contained either 200 or 400 mg PbCl2/L from time of birth. Pup postnatal blood lead levels ranged between 10 and 20 micrograms/dl until day 16 after which they became elevated to 40 micrograms/dl on day 20. During this period the developmental sialylation state of N-CAM, which is believed to regulate cell-cell interaction, fibre outgrowth, and synapse formation, was monitored by rocket immunoelectrophoresis. In control animals the expected desialylation of N-CAM was found to occur at times coincident with postnatal synaptogenesis. In contrast, desialylation in animals chronically exposed to lead was found to be significantly impaired when blood lead levels exceeded 20 micrograms/dl. This could not be attributed to lead-induced undernutrition or alterations in immunoprecipitate formation. These observations could account for the neurobehavioural deficits that are known to be induced at similar blood lead levels, and the potential contribution of impaired N-CAM desialylation to synaptic elaboration is discussed.

Aging↗

PubMed1987

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↗

PubMed1986

Purification and characterization of the D2 cell adhesion protein: analysis of the postnatally regulated polymorphic forms and their cellular distribution.

The developmentally regulated, D2 cell adhesion protein has been purified from 10-12 day old rat synaptosomes by sequential hydroxyapatite chromatography, wheat germ lectin affinity chromatography and gel filtration. The purified protein was found to be composed of two polypeptide components of 200 and 140 kd molecular weight which comprised 0.5-1.0% of total synaptosomal membrane protein. Lysine-Sepharose affinity chromatography could further separate the purified protein into sialic acid-rich and sialic acid-poor forms. Immunoblot analysis of whole brain homogenates and synaptosomes with an antiserum raised against the purified protein (anti-D2) revealed the presence of three immunologically related polypeptides of 200, 140, and 115 kd molecular weight. These polypeptides, which appeared as a diffuse zone (greater than 200 kd) in fetal material, were found to developmentally regulate by altering their relative expression. This was particularly marked in the 200 kd component. Furthermore, the 200 kd polypeptide appeared to be neuron-specific as both the 140 and 115 kd components were common to synaptosomes and primary cultures of astrocytes.

Animals↗

PubMed1986

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↗

PubMed1986

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↗

PubMed1986

Rapid drug haptenization procedure: application to gentamicin and quinidine.

A rapid and simple drug conjugation procedure which utilizes the heterobifunctional reagent N-hydroxysuccinimidyl 6-(4'-azido-2'-nitrophenylamino)hexanoate is described. With this protocol two drugs of diverse structure and solubility have been successfully haptenized. Rabbits immunized with these conjugates yielded drug-specific antibodies as judged by a solid phase radioimmunoassay. Furthermore, using this technique, no cross-reactivity could be detected. Such haptens also elicited an immune response in mice thus further extending their potential for the production of drug specific monoclonal antibodies.

Animals↗