Surface modifications of hydroxyapatite ceramics in aqueous media.

Hydroxyapatite (HA) surfaces exposed, at room temperature, to various aqueous media of different compositions (distilled water, saline solution, cell culture media, saturated HA solution) have been studied by X-ray photoelectron spectroscopy (XPS) angular analysis and IR reflectance spectroscopy. An important decrease of the Ca/P atomic ratio of the surface layer was seen on all exposed surfaces, reaching 1, the Ca/P ratio of dicalcium phosphate dihydrate (DCPD) for the uppermost surface layer. Furthermore, HPO4(2-) ions were observed by XPS and IR. These chemical changes seem to be related to an equilibration process of the surface independent of the dissolution mechanism. Despite the Ca/P ratio observed, no specific phosphate ion environments, such as that of DCPD, were seen by IR.

In vivo central actions of NPY(1-30), an N-terminal fragment of neuropeptide Y.

The purpose of the present study was to examine the possible central actions of a N-terminal fragment of NPY, NPY(1-30), on five measures typically influenced by the native peptide: decreased spontaneous activity, enhancement of muscle tone (increased grasping response), catalepsy, hypothermia, and stimulation of food intake. The peptides were administered ICV in doses ranging from 2.5 to 160 micrograms (0.75-48 nmol) and their effects on the three motor variables as well as thermal and feeding responses were evaluated and compared. Globally, results indicate that, similarly to NPY, the N-terminal fragment NPY(1-30), decreased spontaneous activity and induced hypothermia. However, the fragment displayed approximately half of the potency of NPY for producing these effects. On the other hand, contrary to NPY, NPY(1-30) did not affect muscle tone or food consumption and did not induced catalepsy in animals. These results demonstrate for the first time central actions of a N-terminal fragment of NPY and lend further support to the hypothesis that the receptors mediating the central actions of NPY are pharmacologically different.

Differential effects of pertussis toxin on body temperature changes induced by neuropeptide Y and NPY2-36.

Many effects of NPY have been attributed to a decrease in the activity of adenylate cyclase. Pre-treatment with pertussis toxin (PTx) has been shown to inhibit many pharmacological effects of NPY including increased feeding following administration in the paraventricular nucleus (PVN). In the present study, we examined the influence of PTx pretreatment on the effects of NPY on body temperature following administration in the preoptic area (POA), a region which seems to be the most sensitive to the effects of the peptide on body temperature. The effects of the same pre-treatment on the action of NPY2-36 was also studied since we have found previously that this fragment produced opposite effects on body temperature to that of NPY when injected in the POA. PTx was administered 3 days prior to the injection of NPY or NPY2-36. Results indicate that the hypothermic effect of NPY produced in the POA was blocked by PTx whereas the hyperthermic effect of NPY2-36 was not affected. These results are important as they provide evidence that, in the POA at least, the receptors mediating the hypothermic effect of NPY might be biochemically different from those mediating the hyperthermic effect of NPY2-36.

In vivo central actions of rat amylin.

The purpose of the present study was to examine and compare the profile of neurobehavioral effects of rat amylin (r-amylin) and rat calcitonin gene-related peptide (rCGRP), two peptides having a 50% structural homology. The effects of synthetic r-amylin and rCGRP administered in several doses (0.312-80.0 micrograms) into the lateral cerebro-ventricle of rats on spontaneous activity, muscular tone, body temperature, nociception, food intake as well as their potential for inducing catalepsy, were investigated. Intraventricular administration of r-amylin or rCGRP significantly reduced spontaneous motor activity and markedly increased body temperature of animals in a dose-dependent related fashion. rCGRP produced a significant increase in muscular tone and induced cataleptic effect in animals, but r-amylin had no effect on these variables. Furthermore, neither r-amylin nor rCGRP were able to induce any significant effect on nociceptive response time of animals in the tail immersion test even with doses as large as 80.0 micrograms. Finally, the two peptides did not affect ad libitum food intake, but significantly reduced food consumption in 22 h food-deprived animals. Together, the results of the present study suggest that amylin may be involved in a diversity of neurophysiological processes but displays a different profile of neurobehavioral effects to that of CGRP which may involve different receptors.

Neurochemical effects of neuropeptide Y (NPY) and NPY2-36.

Our previous in vivo structure-activity studies suggested that the putative receptors mediating the effects of NPY and NPY2-36 on food intake and body temperature following ICV administration are pharmacologically different. In the present study, we examined and compared dose related effects of NPY and NPY2-36 on levels of norepinephrine (NE), dopamine (DA) and its main metabolites, dihydroxyphenylacetic acid (DOPAC) and homovanilic acid (HVA), as well as serotonin (5-HT) and its metabolite, 5-hydroxyindolacetic acid (5-HIAA), in several brain regions of the rat, including: frontal cortex, hypothalamus, amygdala, septum, nucleus accumbens, corpus striatum, globus pallidus, substantia nigra and hippocampus. NPY and NPY2-36 (10 or 20 micrograms) were administered intraventricularly and the regional levels of the amines and metabolites were assessed 30 min following administration. Results indicate that both doses of NPY decreased NE levels within the hypothalamus. Furthermore, DOPAC concentrations were increased in this region while DA and HVA remained unchanged. The most pronounced neurochemical effects of NPY were found in the hippocampus, where the peptide produced dose related increases in DA, DOPAC and HVA. On the other hand, NPY2-36 significantly increased NE, DA and its metabolite DOPAC in both the amygdala and septum. The metabolism of DA was most obviously affected in the hippocampus and frontal cortex where levels of DA and DOPAC were significantly increased. 5-HT was affected in both the hypothalamus and globus pallidus where DA and its metabolite HVA were also increased.(ABSTRACT TRUNCATED AT 250 WORDS)

Enhanced cell growth inhibition following PTEN nonviral gene transfer using polyethylenimine and photochemical internalization in endometrial cancer cells.

PTEN is a tumor suppressor gene mapped on chromosome 10q23.3 and encodes a dual specificity phosphatase. PTEN has major implication in PI3 kinase (PI3K) signal transduction pathway and negatively controls PI3 phosphorylation. It has been reported to be implicated in cell cycle progression and cell death control through inhibition of PI3K-Akt signal transduction pathway and in the control of cell migration and spreading through its interaction with focal adhesion kinase. Somatic mutations of PTEN are frequently detected in several cancer types including brain, prostate and endometrium with more than 30% of tumor tissue specimens bearing PTEN mutations and/or deletions. Because of its high frequency of mutations and its important function as tumor suppressor gene, PTEN is a good candidate for gene therapy. Inducible expression of PTEN has been also reported. In cancer cells bearing PTEN abnormalities, the reversion of PTEN function by external gene transfer becomes more and more investigated in cancer treatment research. Several technologies including the photochemical internalization (PCI) and aiming at improving the transfection efficiency have been reported. PCI is an innovative procedure based on light-induced delivery of macromolecules such as DNA, proteins and other therapeutic molecules from endocytic vesicles to the cytosol of target cells. PCI has been reported to enhance the gene delivery potential of viral and nonviral vectors. The present study was designed to evaluate the influence of photochemical internalization on polyethylenimine (PEI)-mediated PTEN gene transfer and its effects on the cellular viability in Ishikawa endometrial cancer cells bearing PTEN abnormalities. PCI was found to significantly (P < 0.01) enhance PTEN mRNA expression (4.2 fold increase). Subsequently, following PEI-mediated PTEN gene transfer, the restoration of the PTEN protein expression was observed. As a consequence, significant cell growth inhibition (44%) was observed in Ishikawa endometrial cells. Using PCI for PEI-mediated PTEN gene transfer was found to further enhance PTEN mRNA and protein expression as well as PTEN-related cell growth inhibition reaching 89%.

Mapping of hypothalamic sites involved in the effects of NPY on body temperature and food intake.

The objective of the present study was to identify hypothalamic sites that might be implicated in the effects of neuropeptide Y (NPY) on both body temperature and food intake. For this purpose, the effects of direct microinjections of NPY in several doses (0.156-20 micrograms) into discrete hypothalamic nuclei on body temperature were examined in rats. To examine specificity of effects, food consumption of animals following injections was also measured. Results indicate that the influence of NPY on body temperature varies with the hypothalamic region where the peptide is administered. NPY had no effect on temperature after administration into the ventromedial (VMH) and the perifornical hypothalamus (PeF). However, a significant hypothermia was seen following administration into the preoptic (POA) and arcuate nucleus (Arc), and hyperthermia was seen after injection into the paraventricular nucleus (PVN). Finally, a biphasic effect was observed after injection into the lateral hypothalamus (LH): hyperthermia with relatively small doses and hypothermia with higher doses. Similar effects were obtained when administered into the third ventricle (3V) but in an inverted dose-related fashion: hypothermia at low and hyperthermia at higher doses. For feeding, NPY consistently increased food intake in all regions examined, with the strongest effect obtained after administration into the PeF. The present results clearly dissociate the effects of NPY on food intake and body temperature, and demonstrate that these effects are related to specific hypothalamic nuclei.

Effects of NPY and NPY2-36 on body temperature and food intake following administration into hypothalamic nuclei.

Our previous in vivo structure-activity studies suggested that the putative receptors mediating the effects of NPY and NPY2-36 on food intake and body temperature are pharmacologically different [17]. In the present study, we examined and compared dose-related effects of NPY and NPY2-36 on ad lib food intake and rectal temperature after administration into discrete hypothalamic nuclei of the rat. Results indicate that NPY and NPY2-36 have opposite effects on body temperature to those of NPY when injected in the preoptic area (POA): hypothermia and hyperthermia, respectively. When administered in the paraventricular nucleus (PVN), both increased body temperature. When injected into the third ventricle (3V), NPY produced a biphasic effect: hypothermia at low doses and hyperthermia at high doses. Similar effects were obtained with NPY2-36, but in an inverted dose-related fashion: hyperthermia at low and hypothermia at higher doses. In the arcuate nucleus (Arc), NPY induced a significant hypothermia whereas NPY2-36 had no effect. Finally, neither peptide affected body temperature when injected into the ventromedial (VMH) and perifornical (PeF) nuclei. Both NPY and NPY2-36 increased food intake after injection in all regions examined. In general, NPY was more potent and efficacious than NPY2-36. The present results clearly dissociate the effects of NPY on food intake and body temperature. Furthermore, the data support the hypothesis that the putative receptors underlying the effects of NPY and NPY2-36 on food intake are similar, whereas those mediating the effects on body temperature are pharmacologically different.

Structure-activity analysis of the motor effects of neuropeptide Y.

The purpose of the present study was to examine structure-activity relationships for three known motor effects of neuropeptide Y (NPY): decreased spontaneous activity, enhancement of muscle tone, and catalepsy. Various NPY fragments and structural analogues were synthesized and administered intracerebroventricularly in several doses (2.5-160 micrograms); their effects on these three motor variables were evaluated and compared. Globally, results indicate that the C-terminal portion of the peptide is responsible for the three motor effects of NPY. However, the distributions of potencies of the various fragments and analogues for each effect were clearly different, suggesting that the putative receptors mediating each motor effect are pharmacologically different. The findings of the present work are discussed in relation to those obtained in previous structure-activity studies.

Influence of ambient temperature on the effects of NPY on body temperature and food intake.

Because thermoregulation and food consumption are interrelated, and because thermoregulation processes are influenced by ambient temperature, we examined the effects of neuropeptide Y (NPY) on both body temperature and food intake in various thermal environments after intracerebroventricular administration of 20 micrograms. Results reveal that the prominent effects of NPY on body temperature and food intake in relatively thermoneutral environments are drastically altered at more extreme ambient temperatures. NPY produced hypothermia in animals placed at 4, 12, and 21 degrees C, and actually increased body temperature in animals subjected to 30 and 38 degrees C temperature. On the other hand, in comparison with ambient temperatures of 12 and 21 degrees C, ambient temperatures of 4 and 30 degrees C significantly reduced the stimulatory effect of NPY on food consumption. Moreover, at 38 degrees C the effect of NPY on food intake was totally abolished. These data demonstrate that ambient temperature has a critical influence on central actions of NPY.

P53 and PTEN expression contribute to the inhibition of EGFR downstream signaling pathway by cetuximab.

Cetuximab (Erbitux) is an anti-epidermal growth factor receptor (EGFR) monoclonal antibody whose activity is related to the inhibition of EGFR downstream signaling pathways. P53 and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) have been reported to control the functionality of PI3K/AKT signaling. In this study we evaluated whether reintroducing P53 using non-viral gene transfer enhances PTEN-mediated inhibition of PI3K/AKT signaling by cetuximab in PC3 prostate adenocarcinoma cell line bearing p53 and pten mutations. Signaling phosphoproteins expression was analyzed using Bio-Plex phosphoprotein array and western blot. Apoptosis induction was evaluated from BAX expression, caspase-3 activation and DNA fragmentation analyses. The results presented show that p53 and pten gene transfer additionally mediated cell growth inhibition and apoptosis induction by restoral of signaling functionality, which enabled the control of PI3K/AKT and MAPKinase signaling pathways by cetuximab in PC3 cells. These results highlight the interest of the analysis of signaling phosphoproteins expression as molecular predictive markers for response to cetuximab and show that p53 and pten mutations could be key determinants of cell response to cetuximab through the functional impact of these mutations on cell signaling.