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J N Octave

Publications and source records attributed to J N Octave.

At least 37 records · Page 2Linked to original sources
PubMed1996

Missense mutations associated with familial Alzheimer's disease in Sweden lead to the production of the amyloid peptide without internalization of its precursor.

Production of soluble amyloid peptide precursor (APP) and amyloid peptide (A beta) was measured in CHO cells transfected by the wild-type APP 695 cDNA sequence or by the same sequence carrying missense mutations associated with familial Alzheimer's disease in Sweden. Deletion of the C-terminal domain of the protein corresponding to residues 654 to 695 of APP 695 not only inhibited very significantly the internalization of APP at 37 degrees C, but also led to the secretion of an uncleaved APP in the culture medium of CHO cells. This deletion did not affect A beta production from the Swedish APP but was able to inhibit the production of the wild-type APP. These results demonstrate that, in CHO cells, the internalization of the wild-type APP is needed for A beta production, while the production of the amyloid peptide from Swedish APP is independent of the internalization process.

Alzheimer Disease↗
PubMed1996

Interaction of the COOH-terminal domain of the neurotensin receptor with a G protein does not control the phospholipase C activation but is involved in the agonist-induced internalization.

The agonist-induced internalization of the neurotensin receptor was studied in transfected Chinese hamster ovary cells expressing either the wild-type or a truncated rat neurotensin receptor, lacking the complete intracellular COOH-terminal end. Incubation of cells expressing the wild-type neurotensin receptor in the presence of the peptide resulted in a dramatic decrease in the [3H]neurotensin binding at the cell surface. This disappearance of cell surface binding sites resulted from the internalization of the receptor after the binding of the peptide. The receptor/peptide complexes were internalized in an intracellular compartment resistant to acid washes. The truncated receptor displayed high affinity binding properties for neurotensin in cell homogenates and activated phospholipase C as did the wild-type receptor. However, in cells expressing the truncated receptor, incubation with neurotensin only induced a partial decrease in cell surface binding, and internalization of the bound peptide was also impaired. On cell homogenates, the GTP analogue Gpp(NH)p was found to decrease the affinity of [3H]neurotensin for the wild-type receptor, whereas no similar effect was observed with the truncated receptor. These results show that the intracellular COOH-terminal region of the rat neurotensin receptor is not required for its functional coupling with intracellular G protein but is involved in the shift of the affinity of the receptor for the agonist, which occurs as a consequence of receptor activation and coupling. Because the truncated receptor was shown to internalize poorly, it may be proposed that internalization is not directly related to the activation of G protein but rather is a consequence of modification of receptor affinity, after activation by the agonist.

Animals↗
PubMed1996

Gene therapy of rat C6 glioma using adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene: long-term follow-up by magnetic resonance imaging.

The herpes simplex virus thymidine kinase gene was transferred into C6 glioma cells by infection with a recombinant adenovirus. In vitro, a 10 microM ganciclovir concentration was able to kill 100% of the infected cells. For in vivo experiments, brain tumors were established by stereotactic injection of C6 glioma cells in the caudate nucleus of rats. Five days later, the recombinant adenovirus was inoculated into the tumors and the animals were treated by intraperitoneal injections of ganciclovir for 14 days. At the end of ganciclovir therapy, histological examination revealed a 28-fold decrease in tumor volumes in the treated animals, as compared with control animals. In long-term studies, the mean survival time of the treated animals were four-fold longer than that of control ones. Magnetic resonance imaging demonstrated an apparent complete tumor regression in 62% of the animals. However, late tumor recurrence was observed in the treated animals. Repeated inoculation of C6 glioma cells in the contralateral hemisphere of long-term surviving animals resulted in either tumor rejection or slowly growing tumors. These findings demonstrate the potential efficacy of adenovirus-mediated transfer of the herpes simplex virus thymidine kinase gene and ganciclovir administration in the treatment of rat gliomas.

Adenoviridae↗
PubMed1995

Interaction of fluorescein derivatives with glibenclamide binding sites in rat brain.

In rat brain, [3H]glibenclamide binds with high affinity to sulfonylurea receptors associated with ATP-sensitive potassium (KATP) channels. KATP channels may play a modulatory role in neurotransmitter release and are involved in acute pathological events occurring in the brain. Fluorescein derivatives, which are suitable tools for the labelling of nucleotide binding sites, influence KATP channels and sulfonylurea receptors properties in insulinoma and cardiac cells. In this study, a negative allosteric action of fluorescein derivatives on glibenclamide binding sites has been shown in rat cortical neurons. This supports the hypothesis of interactions between nucleotide- and sulfonylurea-binding sites within the sulfonylurea receptor.

Animals↗
PubMed1995

Ganciclovir mediated regression of rat brain tumors expressing the herpes simplex virus thymidine kinase imaged by magnetic resonance.

Using a rat C6 brain tumor model, we studied the antitumor effects of Herpes simplex virus type 1 thymidine kinase (HSV-tk) gene transfer followed by ganciclovir treatment. C6 glioma cells were transfected in vitro with the HSV-tk gene, and tested for their sensitivity to ganciclovir. Although there was no surviving cell at a 30 microM ganciclovir concentration, unmodified C6 cells were not affected by the drug. For in vivo experiments, intracerebral tumors were induced in rats by stereotactic injection of 10(4) HSV-tk-modified C6 cells. Ten days later, the animals were treated with intraperitoneal injections of ganciclovir for 21 days. The tumors evolution was evaluated by high resolution magnetic resonance imaging. In 33% of the rats, the signal intensity of the tumors became heterogeneous, with development of highly hyperintense areas, and a complete tumor regression was subsequently noted. Histological examination of successfully treated tumors revealed progressive necrosis with formation of cysts. The survival time of the HSV-tk/ganciclovir treated animals was consistently increased, all rats surviving more than 30 days and 33% of them being still alive after 80 days.

Animals↗
PubMed1995

Lack of rapid desensitization of Ca2+ responses in transfected CHO cells expressing the rat neurotensin receptor despite agonist-induced internalization.

Transfected Chinese hamster ovary cells were used as a model for the study of the desensitization of the neurotensin receptor at the second messenger level. Stimulation with nanomolar concentrations of neurotensin elicited rapid rises in the cytosolic calcium concentration ([Ca2+]i), which remained elevated throughout the peptide application. A significant response was already detected with neurotensin concentrations as low as 0.01 nM. This high efficiency of neurotensin in mediating this calcium response contrasts with the nanomolar affinity of the peptide for its receptor measured in binding experiments. Evidence indicated that the initial elevation of the [Ca2+]i resulted from release of Ca2+ from intracellular stores, whereas the sustained response involved an influx of extracellular origin. Return to the basal level was only reached after extensive washing of the peptide or its displacement with the neurotensin receptor antagonist SR48692. After washing, further stimulations were still able to mediate an increase in the [Ca2+]i, indicating an apparent absence of rapid desensitization of the intracellular signaling pathway that mediates calcium mobilization. In contrast with this absence of response desensitization, the neurotensin receptors were found to internalize after stimulation with the peptide. This internalization was maximal after 30 min and accounted for approximately 70% of the number of neurotensin binding sites located at the cell surface. These results indicate that despite the functional properties of the rat neurotensin receptor present in Chinese hamster ovary cells after transfection, the intracellular signaling pathway triggered by stimulation with neurotensin seems to be resistant to desensitization. This might be related to the high efficiency of the intracellular signaling pathway coupled to the neurotensin receptor observed in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

The amyloid peptide precursor in Alzheimer's disease.

beta A4, an hydrophobic peptide containing from 39 to 43 amino acids, is the major constituent of the amyloid core of characteristic lesions of Alzheimer's disease (AD) known as senile plaques. By cDNA cloning, it was demonstrated that beta A4 is derived from a much larger precursor named the amyloid peptide precursor or APP. The isolation of cDNA clones and the characterization of their nucleotide sequence has shown that several APP proteins containing from 365 to 770 amino acids are produced by alternative splicing of a single primary transcript. The major APP isoforms contain a large extracellular N-terminal domain and a short intracellular C-terminal end. The beta A4 sequence itself is contained for 15 amino acids in the transmembrane domain while 28 amino acids are protruding into the extracellular space. The gene encoding APP is located on human chromosome 21, which is involved in the autosomal dominant inheritance of some early onset of familial AD (FAD). In a few families, mutations of the APP gene have been described. Although they can explain less than 3% of all FAD cases, these mutations clearly demonstrate that APP metabolism is involved in the development of AD. The overexpression of APP in several cultured cells allowed to characterize two catabolic pathways of the protein. A non amyloidogenic pathway precludes formation of beta A4, because APP is cleaved by an alpha-secretase within the beta A4 sequence, leading to the extracellular release of a C-terminal truncated protein. The amyloidogenic pathway produces soluble extracellular beta A4, by cleavage of APP by beta- and gamma-secretases after endocytosis of the transmembrane protein. Although soluble beta A4 is non toxic, it becomes very neurotoxic as soon as it makes fibrils. It is therefore essential to characterize the different factors which favor the organization of beta A4 into fibrils. They have to be considered as risk factors for AD, as well as targets for new therapies. Another therapeutic approach could consist in developing molecules able to inhibit beta A4 production by stimulating the non amyloidogenic pathway of APP.

Aging↗
PubMed1994

The amyloid peptide of Alzheimer's disease is not produced by internal initiation of translation generating C-terminal amyloidogenic fragments of its precursor.

The molecular mechanisms of the amyloid peptide (A beta) production from the amyloid precursor protein (APP) remain unclear and it has been suggested that initiation of translation at methionine 596, which immediately precedes the A beta sequence, could generate soluble amyloidogenic fragments. We show that the amyloid peptide is actually produced by expression of the C-terminal 100 residues of the APP, using methionine 596 as an initiation codon. However, the amyloid peptide is no longer detectable when a stop codon is introduced in the APP mRNA, before the A beta coding region. These results strongly suggest that A beta is produced by degradation of APP and not by local translation of its mRNA.

Alzheimer Disease↗
PubMed1994

Quantification of two splicing events in the L-type calcium channel alpha-1 subunit of intestinal smooth muscle and other tissues.

cDNA fragments encoding a representative region of the L-type calcium channel alpha-1 subunit of rabbit intestine smooth muscle were amplified by polymerase chain reaction (PCR). The nucleotide sequences of these intestine clones shared a high similarity with aorta, lung and heart calcium channels. However, in the extracellular loop between the third and fourth segments of domain IV and in the transmembrane IVS3 segment itself, we observed primary sequence variations corresponding to alternative splicing phenomenons. Since structural differences of L-type calcium channel alpha-1 subunits could result in functional variations, the respective expression frequency of these isoforms was determined in various tissues and species, and in the embryonic A7r5 cell line. The ontogeny of these splicing events was also examined from tissues of different ages. From this quantitative study, carried out by PCR of reverse-transcribed mRNA, it clearly appears that the observed splicing processes in the IVS3-IVS4 region are not only tissue-dependent but also regulated during development.

Amino Acid Sequence↗
PubMed1994

Heparin treatment increases 9-kb MAP2 mRNA levels in neuronal cell cultures.

When neuronal adhesion is reduced by heparin, cultured neurons detach from the substratum and form clusters connected by neuritic fascicles. 24 h after treatment, Northern blots analysis revealed an increase in the expression of the 9-kb MAP2 mRNA in the heparin-treated neuronal cultures. MAP2 being associated with the cross-bridges between microtubules, this increased expression could be an attempt of the cells to rigidify their cytoskeleton to compensate for the reduced attachment. However, the MAP2 protein content was not increased after a 24-h heparin treatment. We discuss possible explanations for this observation and their implications on MAP2 metabolism.

Animals↗
PubMed1994

Rapid desensitization of agonist-induced calcium mobilization in transfected PC12 cells expressing the rat neurotensin receptor.

The effect of neurotensin on intracellular calcium mobilization was measured in PC12 cells transfected with the cDNA sequence encoding the rat neurotensin receptor. Stimulation with nanomolar concentrations of neurotensin induced a rapid increase of the [Ca++]i. This response was transient and the [Ca++]i returned to basal level within 2 minutes, despite the continuous presence of the agonist. The response was also observed in the absence of extracellular calcium, indicating the intracellular origin of the released calcium. Successive stimulations with the same concentration of the peptide failed to produce similar responses. ATP was also found to mediate the release from the same intracellular store of calcium in PC12 cells. The amplitude of the response to ATP was not affected by previous stimulation with neurotensin. These results demonstrate the existence of a rapid and homologous desensitization of the neurotensin-induced calcium release in PC12 transfected cells.

Animals↗
PubMed1994

Receptor mediated internalization of neurotensin in transfected Chinese hamster ovary cells.

After association with intact Chinese hamster ovary (CHO) cells expressing the rat neurotensin receptor, tritiated neurotensin was rapidly internalized. Internalization was maximal after 30 min and accounted for about 90% of the total associated ligand. Neurotensin internalization was not observed at 0-4 degrees and was inhibited by an excess of unlabelled neurotensin or by the neurotensin non peptide antagonist, SR 48692. Moreover, the incubation of intact cells for 30 min with 10 nM neurotensin resulted in a significant decrease in the number of the cell surface neurotensin receptors. These results indicate that the endocytosis of membrane bound neurotensin in transfected CHO cells resulted from the internalization of the ligand-receptor complex inside the cell, through an agonist-induced process.

Animals↗
PubMed1994

Distribution of the phosphorylated microtubule-associated protein tau in developing cortical neurons.

During brain development, the microtubule-associated protein tau presents a transient state of high phosphorylation. We have investigated the developmental distribution of the phosphorylated fetal-type tau in the developing rat cortex and in cultures of embryonic cortical neurons, using antibodies which react with tau in a phosphorylation-dependent manner. The phosphorylated fetal-type tau was present in the developing cortex at 20 days but not at 18 days of embryonic life and was not detected before four to five days in neuronal culture. The cyclin-dependent kinase p34cdc2 was expressed only in germinal layers in the embryonic brain and was not co-localized with phosphorylated tau. After 10 days of postnatal life, the phosphorylated tau progressively disappeared from cortical neurons, disappearing first from the deepest cortical layers where neurons are ontogenetically the oldest. Phosphorylated tau was found in axons and dendrites of cortical neurons at all developmental stages whereas unphosphorylated tau tended to disappear from dendrites during development. The timing of appearance of phosphorylated tau in the cortex, by comparison with the expression of other developmental markers, indicates that phosphorylated tau is present at a high level only during the period of intense neuritic outgrowth and that it disappears during the period of neurite stabilization and synaptogenesis, concomitantly to the expression of adult tau isoforms. In control cultures and in cultures treated with colchicine, the phosphorylated tau was not associated to cold-stable and to colchicine-resistant microtubules. These in vivo results suggest that the high expression of phosphorylated tau species is correlated with the presence of a dynamic microtubule network during a period of high plasticity in the developing brain.

Animals↗
PubMed1993

A new monoclonal antibody against the anionic domain of the amyloid precursor protein of Alzheimer's disease.

A new mouse monoclonal antibody was raised to a bacterial fusion protein between beta-galactosidase and the extracellular domain of the human amyloid protein precursor (APP). In immunoblotting experiments, this monoclonal antibody labelled the bacterial fusion protein used as an immunogen, the human brain APP, and different full-length APP isoforms expressed by transfected cells. In immunocytochemistry, the monoclonal antibody stained the dystrophic neurites of abundant senile plaques found in the cerebral cortex of patients with Alzheimer's disease. Using bacterial expression of several cDNA fragments, the epitope was mapped to an amino acid sequence of APP not investigated before.

Alzheimer Disease↗
PubMed1993

Postnatal ontogeny of the rat brain neurotensin receptor mRNA.

Total RNA was purified from rat forebrain at different postnatal ages and analyzed by Northern blot using a specific neurotensin receptor RNA probe. The rat neurotensin receptor mRNA was present in high amount during the first 10 days of life. Thereafter, it rapidly decreased and was undetected after 20 days. [3H]neurotensin binding experiments performed on the same tissues indicated that the total amount of neurotensin receptors increased during the first week and was maximal between day 7 and day 10. This plateau was followed by an important loss (70%) of neurotensin receptors. These results indicate that an important reduction in the genetic expression of the neurotensin receptor after day 10 may probably account for the [3H]neurotensin binding profile observed in rat forebrain during the postnatal ontogeny.

Aging↗
PubMed1993

Activation of protein kinase C increases the extracellular release of the transmembrane amyloid protein precursor of Alzheimer's disease.

The beta A4 peptide is the major constituent of the amyloid core of abundant senile plaques found in the cerebral cortex of patients with Alzheimer's disease. This amyloid peptide is synthesized as part of a large transmembrane amyloid protein precursor or APP. In addition to the highly expressed transmembrane APP isoforms, an mRNA encoding a secreted APP lacking the transmembrane domain has been identified. A cleavage of the transmembrane protein also yields an extracellular soluble APP fragment. The effect of phorbol esters on the release of the extracellular APP was studied in transfected Chinese hamster ovary cells which stably express either a transmembrane or a secreted APP isoform. The activation of protein kinase C by phorbol-12,13-dibutyrate increased the extracellular release of the transmembrane APP resulting from its proteolytic cleavage, while 4-beta-phorbol, which does not activate protein kinase C, did not significantly affect the recovery of the soluble APP. On the contrary, the recovery of APP secreted in the culture medium without proteolytic cleavage was not increased by protein kinase C-mediated phosphorylation.

Alkaloids↗
PubMed1993

Interaction of the substance P receptor antagonist RP 67580 with the rat brain NK1 receptor expressed in transfected CHO cells.

In the present study, we describe the effects of RP 67580, a substance P non-peptide antagonist, in binding and second messenger experiments performed using transfected Chinese hamster ovary cells expressing the rat NK1 receptor. The cDNA sequence encoding the rat brain substance P receptor was transfected in Chinese hamster ovary cells, and cellular clones which stably express the corresponding protein were isolated. [3H]Substance P binding was performed in homogenates of these transfected cells and revealed the presence of NK1 receptors in displacement experiments, using peptide analogs of three mammalian tachykinins (substance P, neurokinin A, neurokinin B). Scatchard analysis indicated a KD value of 0.33 +/- 0.13 nM and a Bmax value of 5.83 +/- 1.16 pmol/mg of protein. RP 67580, a selective NK1-receptor antagonist was found to displace the specific binding of [3H]substance P. When [3H]RP 67580 was used as a ligand, it displayed a high affinity (KD value: 1.22 +/- 0.27 nM) in transfected cell homogenates and only competed with NK1 receptor ligands. Substance P stimulated the hydrolysis of phosphoinositide in a time- and concentration-dependent manner and this effect was mimicked by selective agonists of the NK1 receptor ([Pro9]SP and septide). RP 67580 did not induce any accumulation of inositol phosphates, but was found to inhibit the inositol phosphate increase mediated by substance P, without affecting the maximal response. From these results, one may conclude that the receptor expressed by the transfected Chinese hamster ovary cells revealed similar binding characteristics as the NK1 receptor present in the rat brain and also confirmed the high affinity and the antagonist properties of RP 67580.

Animals↗