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P Longone

Publications and source records attributed to P Longone.

11 recordsLinked to original sources
PubMed1998

Aging-associated up-regulation of neuronal 5-lipoxygenase expression: putative role in neuronal vulnerability.

Aging is associated with neurodegenerative processes. 5-Lipoxygenase (5-LO), which is also expressed in neurons, is the key enzyme in the synthesis of leukotrienes, inflammatory eicosanoids that are capable of promoting neurodegeneration. We hypothesized that neuronal 5-LO expression can be up-regulated in aging and that this may increase the brain's vulnerability to neurodegeneration. We observed differences in the distribution of 5-LO-like immunoreactivity in various brain areas of adult young (2-month-old) vs. old (24-month-old) male rats. Greater 5-LO-like immunoreactivity was found in old vs. young rats, in particular in the dendrites of pyramidal neurons in limbic structures, including the hippocampus, and in layer V pyramidal cells of the frontoparietal cortex and their apical dendrites. The aging-increased expression of neuronal 5-LO protein appears to be due to increased 5-LO gene expression. Using a quantitative reverse transcription/polymerase chain reaction assay and 5-LO-specific oligonucleotide primers and their mutated internal standards, we observed about a 2.5-fold greater hippocampal 5-LO mRNA content in old rats. 5-LO-like immunoreactivity was also observed in small, nonpyramidal cells, which were positive for glutamic acid decarboxylase or glial fibrillary acid protein. This type of 5-LO immunostaining did not increase in the old rats. Hippocampal excitotoxic injury induced by systemic injection of kainate was greater in old rats. Neuroprotection was observed with the 5-LO inhibitor, caffeic acid. Together, these results suggest that aging increases both neuronal 5-LO expression and neuronal vulnerability to 5-LO inhibitor-sensitive excitotoxicity, and indicate that the 5-LO system might play a significant role in the pathobiology of aging-associated neurodegenerative diseases.

Aging↗
PubMed1997

7-Chloro-3-methyl-3,4-dihydro-2H-1,2,4-benzothiadiazine S,S-dioxide: a partial modulator of AMPA receptor desensitization devoid of neurotoxicity.

In cerebellar granule neurons of neonatal rats micromolar concentrations of 7-chloro-3-methyl-3,4-dihydro-2H-1,2, 4-benzothiadiazine S,S-dioxide (IDRA-21) and cyclothiazide, two negative modulators of the spontaneous agonist-dependent rapid desensitization of alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA)-gated ion channels, facilitate AMPA receptor function by increasing the content of free cytosolic Ca2+ as measured by single-cell fura-2 acetoxymethyl ester (Fura-2) Ca2+-dependent fluorescence and intracellular Na+ measured with the sodium-binding bezofuran isophthalate acetoxymethyl ester fluorescence indicator. IDRA-21 increases intracellular Na+ transient with a threshold (5 microM) that is approximately 10 times higher and has an intrinsic activity significantly lower than that of cyclothiazide. By virtue of its low intrinsic activity, IDRA-21 elicits a free cytosolic Ca2+ transient increase that is shorter lasting than that elicited by cyclothiazide even when the drug is left in contact with cultured granule cells for several minutes. Additionally, while dose dependently, 5-25 microM cyclothiazide in the presence of AMPA is highly neurotoxic, IDRA-21 (up to 100 microM) is devoid of neurotoxicity. The neurotoxicity elicited by cyclothiazide persists in the presence of dizocilpine (an antagonist of N-methyl-D-aspartate-selective glutamate receptors) but is blocked by 2,3-dihydroxy-6-nitrosulfamoylbenzo[f]quinoxaline (a competitive AMPA receptor antagonist) and the 1-(aminophenyl)-4-methyl-7, 8-methylendioxy-5H-2,3-benzodiazepine (GYKI 52466; a noncompetitive AMPA receptor antagonist). Since the doses of IDRA-21 that enhance cognitive processes in rats and monkeys are several orders of magnitude lower than those required to elicit marginal neurotoxicity in cultured neurons, it can be surmised that IDRA-21 is a potent cognition-enhancing drug virtually devoid of neurotoxic liability because it acts as a partial negative allosteric modulator of AMPA receptor desensitization.

Animals↗
PubMed1997

Full-length and N-terminally truncated chicken intestinal diazepam-binding inhibitor. Purification, structural characterization and influence on insulin release.

Two forms of diazepam-binding inhibitor (DBI) have been purified from chicken intestine and identified as the intact avian polypeptide (residues 1-86) and a truncated variant (residues 35-86). At 10 nM concentration, both the intact and the truncated peptide suppress in vitro-monitored glucose-induced insulin release by 50 (p < 0.02) and 64% (p < 0.01) respectively. The truncation starts at a segment. -Thr-Val-Gly-Asp-, that is strictly conserved between characterized DBI species, indicating special restrictions on the structure. However, overall DBI conservation appears to be complex. A number of differently bioactive fragments with separate processings and tissue distributions have been observed, suggesting multiple functions of DBI and its sub-segments.

Amino Acid Sequence↗
PubMed1997

Increased hippocampal 5-lipoxygenase mRNA content in melatonin-deficient, pinealectomized rats.

Melatonin is neuroprotective because of its antioxidative action, but it also can modify neuronal vulnerability by altering gene expression. 5-Lipoxygenase (5-LO) gene expression is suppressed by the binding of melatonin to its high-affinity nuclear receptors. Recently, we reported that in rats the melatonin deficiency elicited by pinealectomy increases hippocampal susceptibility to excitotoxic injury. Here we have hypothesized that pinealectomy may increase hippocampal vulnerability by eliminating the tonic inhibitory action of melatonin on 5-LO gene expression. Sham-pinealectomized controls and pinealectomized rats were killed 15 days after surgery. Their hippocampi were dissected, and total RNA was extracted and processed for quantitative reverse transcription-polymerase chain reaction assay of 5-LO and cyclophilin mRNAs. Mutated primers were used as internal standards to assay attomole quantities of these two specific mRNAs per microgram of total RNA; the ratio 5-LO/cycophilin was used to compare samples from control and pinealectomized rats. Pinealectomy increased hippocampal 5-LO mRNA content by about threefold. These results support our hypothesis that melatonin deficiency may abate the tonic inhibition of 5-LO mRNA expression and thereby up-regulate 5-LO gene expression, which in turn would increase the brain's synthesis rate of potentially harmful eicosanoids, leukotrienes.

Animals↗
PubMed1996

Reversible modification of GABAA receptor subunit mRNA expression during tolerance to diazepam-induced cognition dysfunction.

Benzodiazepines (BZs) that are endowed with full positive allosteric modulatory (FAM) activity on GABAA receptors cause anterograde amnesia in both animals and humans. In rats subjected to a delayed object recognition test, diazepam, endowed with FAM activity, exerted an amnesic action, whereas BZs endowed with partial allosteric modulatory (PAM) activity on GABAA receptors, such as imidazenil, failed to induce amnesia, even if administered at doses five times higher than those equipotent to a standard anticonvulsant dose of diazepam (17.6 mumol/kg/os). After discontinuation of 14 days' treatment with vehicle, diazepam, or imidazenil (three times daily with increasing doses starting from 17.6 mumol/kg/os for diazepam and 2.5 mumol/kg/os for imidazenil), we compared the persistence of tolerance to the amnesic effect of diazepam with the persistence of the changes in the context of four (alpha 1, alpha 5, gamma 2L, gamma 2S) GABAA receptor subunit mRNAs in the fronto-parietal motor (FrPaM) cortex and the hippocampus. Rats receiving the long-term treatment with diazepam developed a tolerance to the amnesic effect of this drug and showed a decrease (30-50%) in the expression of mRNAs encoding for alpha 1 gamma 2L, gamma 2S GABAA receptor subunits, an increase, by approximately 30%, of the expression of mRNA of the alpha 5 subunit in the FrPaM cortex and a decrease, by approximately 25%, in the expression of mRNA, of the alpha 1 subunit in the hippocampus. These changes of subunit mRNA expression and the tolerance to the amnesic effect of diazepam returned to control values 72 hr after termination of the long-term treatment with diazepam. No tolerance to the amnesic effect of diazepam and no changes in GABAA receptor subunit mRNA expression were found in rats undergoing long-term treatment with imidazenil.

Animals↗
PubMed1996

Modifications of gamma-aminobutyric acidA receptor subunit expression in rat neocortex during tolerance to diazepam.

We evaluated whether tolerance to the antagonism of bicuculine-induced seizures by diazepam is associated with changes (i) in the content of mRNAs encoding for gamma-aminobutyric acidA (GABAA) receptor subunits, (ii) in the expression density of these subunits, and (iii) in the 1,4-benzodiazepine binding site characteristics in discrete neocortical structures. We found that in diazepam-tolerant rats, the content of the mRNA encoding for the alpha 1 subunit of the GABAA receptor decreased in the frontoparietal motor (FrPaM) cortex and in the hippocampus (42% and 20%, respectively) but not in the frontoparietal somatosensory (FrPaSS) cortex, striatum, olfactory bulb, and cerebellum. In the FrPaM cortex, gamma 2S and gamma 2L subunit mRNA contents were also decreased (48% and 30%, respectively), whereas that of alpha 5 was increased (30%). In the FrPaM and FrPaSS cortices as well as in cerebellum of diazepam-tolerant rats, the content of alpha 2, alpha 3, alpha 6, beta 2, and delta subunit mRNA was unchanged, as was the content of alpha 2, alpha 5, gamma 1, and gamma 2S subunit mRNA in the hippocampus. Furthermore, the reduction in alpha 1 subunit mRNA content in the FrPaM cortex and the anticonvulsant tolerance to diazepam returned to control values 72 hr after termination of the protracted diazepam treatment. Rats receiving a treatment with imidazenil in doses equipotent and with a schedule identical to that of diazepam failed to exhibit tolerance to the anticonvulsant action of this drug or cross-tolerance to diazepam. In these rats, the content of mRNA encoding for alpha 1, alpha 2, alpha 3, alpha 5, alpha 6, gamma 1, gamma 2S, gamma 2L, and delta GABAA receptor subunits failed to change in the FrPaM and FrPaSS cortices, in the hippocampus, and in the other brain areas that were studied in diazepam-tolerant rats. Although the density and affinity of [3H]flumazenil and [3H]imidazenil binding failed to change in the FrPaM and FrPaSS cortices of diazepam-tolerant rats, the expression density of alpha 1 subunit immunogold labeling decreased by 37%, whereas that of alpha 5, gamma 2L/S, and beta 2/3 increased by 158%, 50%, and 47%, respectively, in the FrPaM cortex, and the density of the alpha 5 subunit selectively increased (209%) in the FrPaSS cortex. In contrast, the immunogold labeling density of the alpha 1, alpha 5, gamma 2L/S, and beta 2/3 subunits failed to change in either the FrPaM or FrPaSS cortex of rats receiving protracted imidazenil treatment.

Animals↗
PubMed1995

Identification of three transcriptional regulatory elements in the rat mitochondrial benzodiazepine receptor-encoding gene.

The sequence upstream from the first exon in the rat mitochondrial benzodiazepine receptor-encoding gene (MBR) was analyzed for transcriptional promoter activity by three techniques: promoter deletion analysis in vectors containing the gene cat encoding chloramphenicol acetyltransferase, electrophoretic mobility shift analysis (EMSA) and DNase I protection assay. All three methods are in uniformity with the identification of at least three regulatory elements corresponding to locations -51/-33, -267/-249 and -555/-526. The most distal and proximal domains are positive-acting, whereas the element at -267/-249 acts in a negative manner. The positive-acting -51/-33 element contains the middle of three consensus Sp1-recognition sequences found in this region of the gene. Binding of Y1 cell nuclear protein to a DNA fragment corresponding to this region of the gene is competed by a synthetic oligodeoxyribonucleotide bearing the consensus Sp1-binding site sequence. These studies provide the first reported functional evidence localizing transcriptional elements of MBR.

Animals↗
PubMed1993

Characterization of a decrease in muscarinic m2 mRNA in cerebellar granule cells by carbachol.

Studies involving carbachol (100 microM) treatment of cerebellar granule cells for 1, 3, 6, 9, 12 and 24 hr show a decrease in the mRNA encoding for the muscarinic m2 receptor. The response was transient, decreasing m2 mRNA by 25 to 50% in 6 and 9 hr, respectively. The data presented in this work were quantified by ribonuclease protection assay, using a [32P]-cRNA probe corresponding to nucleotide +1138 to 1650 of the rat m2 muscarinic receptor. Because cerebellar granule cells express muscarinic m2 and m3 receptors, we tested whether the carbachol-mediated decrease in m2 mRNA resulted from a homologous or heterologous activation of muscarinic receptors. At a 1 microM concentration, methoctramine specifically blocked the muscarinic m2 receptor and reversed carbachol's action. These data suggested that carbachol acts via a possible homologous activation of muscarinic m2 receptors. The half-life of the receptor mRNA measured in the presence of actinomycin D with and without carbachol were similar. Because carbachol treatments decrease the steady-state levels of m2 mRNA without changing the half-life of the message, we suggest that a carbachol treatment induces a decrease in the transcription of the gene for the muscarinic m2 receptor.

Animals↗
PubMed1989

Retinoic acid inhibits phosphatidylinositol turnover only in RA-sensitive while not in RA-resistant human neuroblastoma cells.

Phosphatidylinositol (PI) turnover has recently been implicated in the regulation of cell proliferation and transformation. We have investigated its role in differentiation using LAN-1 cells, a human neuroblastoma cell line which can be induced to differentiate along the neuronal pathway by retinoic acid (RA), and a derivated RA-resistant subline of it (LAN-1-res). We have found that treatment of LAN-1 cells with RA is followed by a rapid decrease of inositol phospholipid metabolism, using myo-[1,2-3H] inositol or [1,(3)-3H] glycerol. Analysis of labelled phosphatidylinositol metabolites from prelabelled LAN-1 cells indicated a rapid decrease of inositol (1,4,5)-trisphosphate and (1,2) diacylglycerol within 1 min. of induction of differentiation by RA, while no changes were observed in RA-treated LAN-1-res cells. These findings indicate that phosphoinositides-derived metabolites may be directly implicated in the induction processes of RA-triggered NB cell differentiation.

Cell Differentiation↗
PubMed1989

"In situ" characterization of guanine nucleotide-binding properties of erythrocyte membranes.

Unsealed membranes from human erythrocytes bind GTP and GTP analogs according to first order kinetics, a single rate constant being observed. With [35S]GTP gamma S this is 0.15 +/- 0.2 min-1. Treatment of the membranes with detergents decreases binding considerably. Scatchard plots reveal uncomplicated patterns of ligand association, with Kd values of 10.2 +/- 2.3 nM [35S]GTP gamma S, of 18.2 +/- 4.3 nM [alpha-32P]GTP and of 28.6 +/- 3.5 nM [alpha-32P]GDP, respectively. The stoichiometry with the three ligands is strictly comparable, i.e. 65 +/- 7 picomoles/mg of membrane protein. Binding of each labeled nucleotide is competitively inhibited by the other two unlabeled ligands, the inhibition constants being very close to the corresponding Kd values. Metabolic depletion and subsequent repletion of intact erythrocytes result in membrane preparations still active in guanine nucleotide binding, with unmodified Kd values. However, the stoichiometry falls to 35 picomoles/mg protein with the "depleted" erythrocyte membranes and regains higher values (50 picomoles/mg protein) with the "repleted" cell membranes. Accordingly, the "in situ" characterization of guanine nucleotide-binding properties of erythrocyte membranes seems to represent a new tool for monitoring the metabolic state of intact erythrocytes.

Binding, Competitive↗