The amidated PACAP1-23 fragment is a potent reduced-size neuroprotective agent.

BACKGROUND: Neurodegenerative disorders, such as Parkinson's disease (PD), are characterized by neuronal death involving, among other events, mitochondrial dysfunction and excitotoxicity. Along these lines, several attempts have been made to slow this pathology but none have been yet discovered. Based on its capacity to cross the blood-brain barrier and provide neuronal protection in vitro and in vivo, the pituitary adenylate cyclase-activating polypeptide (PACAP) represents a promising lead molecule. Pharmacological studies showed that PACAP interacts with three different G protein-coupled receptors, i.e. PAC1, VPAC1 and VPAC2. However, only PAC1 is associated with neuronal anti-apoptotic actions, whilst VPAC activation might cause adverse effects. In the context of the development of PAC1-selective agonists, PACAP(1-23) (PACAP23) appears as the shortest known PACAP bioactive fragment. METHODS: Hence, the capacity of this peptide to bind PACAP receptors and protect neuroblastoma cells was evaluated under conditions of mitochondrial dysfunction and glutamate excitotoxicity. In addition, its ability to activate downstream signaling events involving G proteins (Gαs and Gαq), EPAC, and calcium was also assessed. RESULTS: Compared to the endogenous peptide, PACAP23 showed a reduced affinity towards PAC1, although this fragment exerted potent neuroprotection. However, surprisingly, some disparities were observed for PACAP23 signaling compared to full length PACAP, suggesting that downstream signaling related to neuroprotection is distinctly regulated following subtle differences in their PAC1 interactions. CONCLUSIONS: Altogether, this study demonstrates the potent neuroprotective action of amidated PACAP23. GENERAL SIGNIFICANCE: PACAP23 represents an attractive template for development of shorter PACAP-derived neuroprotective molecules.

Discovery of new antagonists aimed at discriminating UII and URP-mediated biological activities: insight into UII and URP receptor activation.

BACKGROUND AND PURPOSE: Recent evidence suggested that urotensin II (UII) and its paralog peptide UII-related peptide (URP) might exert common but also divergent physiological actions. Unfortunately, none of the existing antagonists were designed to discriminate specific UII- or URP-associated actions, and our understanding, on how these two endogenous peptides can trigger different, but also common responses, is limited. EXPERIMENTAL APPROACH: Ex vivo rat and monkey aortic ring contraction as well as dissociation kinetics studies using transfected CHO cells expressing the human urotensin (UT) receptors were used in this study. KEY RESULTS: Ex vivo rat and monkey aortic ring contraction studies revealed the propensity of [Pep(4)]URP to decrease the maximal response of human UII (hUII) without any significant change in potency, whereas no effect was noticeable on the URP-induced vasoconstriction. Dissociation experiments demonstrated the ability of [Pep(4)]URP to increase the dissociation rate of hUII, but not URP. Surprisingly, URP, an equipotent UII paralog, was also able to accelerate the dissociation rate of membrane-bound (125)I-hUII, whereas hUII had no noticeable effect on URP dissociation kinetics. Further experiments suggested that an interaction between the glutamic residue at position 1 of hUII and the UT receptor seems to be critical to induce conformational changes associated with agonistic activation. Finally, we demonstrated that the N-terminal domain of the rat UII isoform was able to act as a specific antagonist of the URP-associated actions. CONCLUSION: Such compounds, that is [Pep(4)]URP and rUII(1-7), should prove to be useful as new pharmacological tools to decipher the specific role of UII and URP in vitro but also in vivo.

Biochemical and pharmacological characterization of nuclear urotensin-II binding sites in rat heart.

BACKGROUND AND PURPOSE During the past decade, a few GPCRs have been characterized at the nuclear membrane where they exert complementary physiological functions. In this study, we investigated (1) the presence of a functional urotensin-II (U-II) receptor (UT) in rat heart nuclear extracts and (2) the propensity of U-II and U-II-related peptide (URP) to cross the plasma membrane in a receptor-independent manner. EXPERIMENTAL APPROACH Biochemical and pharmacological methods including competitive binding assays, photoaffinity labelling, immunoblotting as well as de novo RNA synthesis were used to characterize the presence of functional UT receptors in rat heart nuclei. In addition, confocal microscopy and flow cytometry analysis were used to investigate the cellular uptake of fluorescent U-II and URP derivatives. KEY RESULTS The presence of specific U-II binding sites was demonstrated in rat heart nuclear extracts. Moreover, such subcellular localization was also observed in monkey heart extracts. In vitro transcription initiation assays on rat, freshly isolated, heart nuclei suggested that nuclear UT receptors are functional, and that U-II, but not URP, participates in nuclear UT-associated gene expression. Surprisingly, hU-II and URP efficiently crossed the plasma membrane in a receptor-independent mechanism involving endocytosis through caveolin-coated pits; this uptake of hU-II, but not that of URP, was dependent on extracellular pH. CONCLUSION Our results suggest that (1) U-II and URP can differentially modulate nuclear UT functions such as gene expression, and (2) both ligands can reach the internal cellular space through a receptor-independent mechanism.

[Third molar surgery under general anesthesia: a review of 180 patients]. / Extraction des dents de sagesse sous anesthésie générale : à propos de 180 patients.

BACKGROUND: Third molar surgery is an important part of the activity in a maxillofacial surgery department. This common activity is often under-evaluated by patients who forget its surgical aspect. The aim of this study was to evaluate our practice, and especially complications, with special consideration given to medicolegal aspects. MATERIALS AND METHODS: All the patients operated between September 2004 and July 2006 were enrolled in a retrospective study. This population is described, with the indications, follow-up, and complications. RESULTS: One hundred and eighty patients were reviewed (sex-ratio 1, mean age 27 years). The most frequent indications were impaction and pain. The mean duration of hospitalization was 1.7 days and temporary disability, one week. Local infection occurred in 8%; there was neurological complication in 2% for the inferior alveolar nerve, and 1% for the lingual nerve. These were all transient cases. DISCUSSION: Third molar surgery is an important and profitable part of the activity in a maxillofacial surgery department. Standardized information is necessary even if the rate of complications remains low.

Comparative sensitivity of sea urchin sperm bioassays to metals and pesticides.

A simple sperm/fertilization bioassay, primarily using sea urchin gametes, has been developed and used by a variety of laboratories. This assay was recently refined into a standard test and is now being used by the U.S. Environmental Protection Agency and others for toxicity testing in marine waters. One factor that has lagged behind the development of this assay is the comparison of its sensitivity to various common toxicants as compared to other bioassay systems and life stages of other marine organisms. The objective of this study was to compare the sensitivity of a standardized sea urchin sperm/fertilization assay to the responses of embryo, larval, and adult marine organisms to metals (Ag, Cd, Cu, Pb, Zn) and pesticides (DDT, Dieldrin, Endrin, Endosulfan) added to natural seawater. The results, although highly variable, generally showed that sperm/fertilization and embryo assays were quite sensitive to the metals tested, but that the larval and adult assays were more sensitive to the pesticides. These comparative data, together with other studies of complex effluents, show that the standardized sperm/fertilization bioassay is an especially quick and useful tool for biomonitoring of marine waters.

Sensitivity of cultured skin fibroblasts from patients with neurofibromatosis to DNA-damaging agents.

Neurofibromatosis (NF) is an autosomal dominant disorder associated with various constitutional abnormalities as well as a striking predisposition for malignant and nonmalignant neoplasms, both in cells originating in and not originating in the neural crest. We have examined the sensitivity of cultured skin fibroblasts from patients with neurofibromatosis to several types of DNA damage. Fibroblasts in Dulbecco's modified Eagle's medium were plated at 10(2) to 2 X 10(4) cells per 75 cm2 tissue culture plates, and exposed to various doses of gamma radiation (leads to DNA scission), actinomycin D (a DNA intercalating agent), or mitomycin C (a bifunctional alkylating agent leading to DNA cross-links). Cells were reincubated for 15 to 40 days until surviving colonies exhibited greater than 30-50 cells. Plates were then stained with 1% methylene blue and the colonies counted, with surviving fraction determined relative to plating efficiency. Nine skin fibroblast cell strains from normal individuals were studied as controls. One neurofibromatosis (NF) cell strain, SB23, exhibited normal sensitivity to all three DNA-damaging agents studied in early (7-8) and middle (12-13) in vitro passage. Strain GM0622, on the other hand, exhibited normal sensitivity to the three DNA-damaging agents studied at early passage, but showed a significant decrease in survival after exposure to both gamma radiation (D0 = 106 rad) and actinomycin D (D0 = 0.024 mcg/ml) with increasing passage. Strain GM1639 exhibited decreased survival after actinomycin D exposure at early passage (D0 = 0.017 mcg/ml), with normal survival after exposure to gamma radiation and mitomycin C at the same passage. Cell strains exhibited decreasing low density plating efficiencies and growth rates with increasing passage such that study of cytotoxicity was not feasible after middle passage in strains SB23 and GM0622, and after early passage in strain GM1639. The results suggest that cultured fibroblast cell strains from patients with NF exhibit early in vitro senescence which sometimes is associated with an inability to handle certain DNA-damaging agents.

Pyogenic osteomyelitis versus pseudo-osteomyelitis in Gaucher's disease. Report of a case and review of the literature.

Presented is a young girl with Gaucher's disease who developed acute bone pain accompanied by signs of inflammation and who was felt to have possible pyogenic osteomyelitis. The lack of significant pathogenic bacterial growth on culture and the findings at orthopedic surgery led the authors to conclude that this child probably represented a case of pseudo-osteomyelitis, but the isolation of an anaerobe from the operative culture of the involved bone leaves the exact diagnosis unclear. Since this child underwent an open surgical procedure, she was treated with antibiotics to prevent the possible development of chronic osteomyelitis. This anaerobic growth on culture, although strongly felt to be a contaminant, also played a role in this decision. She had an uneventful hospital course and subsequently has done well. It is suggested that great caution be taken before subjecting a patient with Gaucher's disease to orthopedic surgical procedures. If pyogenic osteomyelitis is strongly suspected, obtaining multiple blood cultures and culture by needle aspirate may be preferred over an open surgical procedure. The use of empiric antibiotic therapy without an attempt at further diagnosis is not recommended. If an orthopedic surgical procedure is necessary in a patient with Gaucher's disease, antibiotic coverage is indicated and long-term observation of the operative sight for drainage and/or other signs of chronic inflammatory changes in mandatory.