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.

'Concord' grapevine nutritional status and chlorosis rank associated with fungal and bacterial root zone microbiomes.

Leaf chlorosis in vineyards is associated with reduced crop yields and quality. While iron (Fe) is understood to play a crucial role in chlorosis, total plant and soil Fe are not always indicative of chlorosis in grapevines. Physiology of chlorosis in vineyards has been well-studied, but the soil microbial consequences of and contributions to chlorosis have received little attention. We used next-generation sequencing (NGS) to examine the bacterial and fungal communities associated with grapevines demonstrating varying degrees of visual chlorosis symptoms. Additionally, chemical analyses of soils and grape leaves were used to explore the influence of plant nutritional status and soil chemistry on microbial community composition. Finally, factors influencing bacterial community composition were correlated with predicted bacterial community function. Leaf tissue magnesium (leaf Mg) concentrations and chlorosis rank were correlated with bacterial community composition as determined via dbRDA (distance-based Redundancy Analysis) using Bray-Curtis dissimilarities. Non-metric multidimensional scaling (NMDS) revealed a significant correlation between fungal community composition and soil Fe and pH, along with leaf N, Mg, and Ca (mg.kg-1). Chlorosis rank was moderately correlated with KEGG Orthology (KO) terms associated with nitrogen (N) and carbon (C) metabolism in soils, while leaf Mg was associated with a spectrum of KO terms including glycosphingolipid biosynthesis, glycan degradation, transporters, and porphyrin and chlorophyll metabolism. Additionally, abundance of many bacterial operational taxonomic units was significantly correlated with leaf Mg, including those from the following orders: Rhodobacterales, Acidobacteriales, Opitutales, Sphingomonadales, Burkholderiales, Saprospirales, and Flavobacteriales. Our findings suggest grapevine chlorosis is interrelated with soil microbial community structure and function, plant nutrition, and soil chemistry.

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.

Development of photolabile caged analogs of endothelin-1.

Photoactivable caged analogs of endothelin-1 (ET-1) were obtained after derivatization with the photolabile 4,5-dimethoxynitrobenzyl (DMNB) group. This was achieved by the incorporation of N-alpha-Fmoc caged building blocks of Lys, Asp, Glu and Tyr during the solid phase peptide synthesis step. The C-terminal carboxylic function was also derivatized. However, difficulties were encountered with the introduction of the Asp and Glu photoactivable building blocks. As a matter of fact, formation of an aminosuccinyl derivative, through cyclization of the Asp(ODMNB) residue, and the formation of a pyrrolidone ring from the Glu(ODMNB) residue were highly favored by the electronic properties of the photocleavable function. ET-1 analogs were also tested in the ET(A) and ET(B) paradigms and specific pharmacological profiles were obtained for each peptide.

N-[3-(2-Dimethylaminoethyl)-2-methyl-1H- indol-5-yl]-4-fluorobenzamide: a potent, selective, and orally active 5-HT(1F) receptor agonist potentially useful for migraine therapy.

Recent studies have demonstrated that selective 5-HT(1F) receptor agonists inhibit neurogenic dural inflammation, a model of migraine headache, indicating that these compounds may be effective therapies for the treatment of migraine pain. This communication describes the synthesis and discovery of a novel compound, N-[3-(2-(dimethylamino)ethyl)-2-methyl-1H-indol-5-yl]-4-fluorobenzamide (4), which possesses high binding affinity and selectivity at the 5-HT(1F) receptor relative to more than 40 other serotonergic and nonserotonergic receptors examined.

New short peptide substrates of endothelin-converting enzyme and characterization of the enzyme.

Endothelin (ET) is a 21 amino acid peptide produced following the cleavage of its precursor, big ET, by a metalloprotease, the endothelin-converting enzyme (ECE). In the study reported here we determined the minimal peptide sequence of big ET necessary for enzyme recognition and cleavage at the P1-P1' site. Furthermore, we have explored the role of the amino acids found at the boundaries of the cleavage site. To reach these goals. we synthesized a series of fragments, all containing the P1-P1' cleavage site, Trp21-Val22. Following the incubation of peptide fragments with a partly purified bovine ECE preparation and after analyzing the cleavage pattern by high-performance liquid chromatography (HPLC), we were able to identify big ET(18-23) amide as the minimal peptide core recognized and cleaved by the enzyme. This hydrolysis was inhibited by phosphoramidon but not by thiorphan, a characteristic of the ECE metalloprotease. However, none of the shorter peptides was able to inhibit the cleavage of big ET-1 by ECE, suggesting that they are not recognized by the enzyme. Particularly, it appears that aspartic acid 18 is a key residue for the recognition phenomenon. The delineation of the minimal structure will be a useful tool to further characterize ECE.

Structural characterization and effects on corticosteroid secretion of endothelin-1 and endothelin-3 from the frog Rana ridibunda.

ABSTRACT Despite the intensive study of endothelin (ET) in mammals, the primary structure and biological activity of the peptide is not known for any species of non-mammalian tetrapod. Extracts of the stomach and the liver of the European green frog Rana ridibunda contained ET-like immunoreactivity measured by RIA using an antiserum raised against human ET-1. The amino acid sequence of the peptide that was isolated in pure form from the stomach extract was identical to that of human ET-1 and the peptide purified from the liver extract was identical to human ET-3 except for a single amino acid substitution (Phe(4)-->Tyr). These observations demonstrate that the amino acid sequences of ET family peptides have been very strongly conserved during evolution of tetrapods and suggest that the pathway of post-translational processing of preproendothelin in the frog is similar to that in mammals. Both frog/human ET-1, frog ET-3 and human ET-3 produced a concentration-dependent increase in the production of corticosteroids from perifused slices of the frog interrenal gland. The maximum responses produced by the peptides (approximately 2-fold increase over basal levels for both corticosterone and aldosterone production) were not significantly different. The potency of ET-1 (-log EC(50)=9.81+/-0.01 (s.e.m.) for corticosterone and 9.52+/-0.29 for aldosterone production) was significantly (P<0.01) greater than that of frog ET-3 (-log EC(50)=8.13+/-1.6 for corticosterone and 8.15+/-0.33 for aldosterone production) but the potencies of frog ET-3 and human ET-3 (-log EC(50)=8.29+/-0.34 and 7.87+/-0.18) were not significantly different.

Sexual Assault Response Team: overcoming obstacles to program development.

After several years of planning, The SART at Immanuel St. Joseph's--Mayo Health Systems became a reality in August 1997. The nurses who were trained for this program were already providing 24-hour coverage in the emergency department for psychiatric emergencies and patients with chemical dependency. The SANE responsibilities were added to their on-call duties. Five nurses participated in a 40-hour training program by SANE specialists and experts in the local community. As expected, nurses were apprehensive as they conducted their first examinations; however, all has gone well. Over time, the providers' and clients' satisfaction with the program has improved. The examination is completed in less time, and the person assaulted does not have to wait as long for the SANE to arrive. Members of law enforcement and the prosecutor's office are especially pleased with the quality of evidence collected and the procedures followed to maintain chain of evidence so the evidence obtained can be used in prosecution. The program has resulted in kind and compassionate care for persons who have been sexually assaulted. The providers are continuing to meet monthly as an interdisciplinary, interagency team and are addressing concerns as they arise. Members of the SART are developing a good working relationship. Everyone involved agrees that developing this program has been a worthwhile effort and that the hospital is providing a valued service for the community.

Retinal findings after head trauma in infants and young children.

BACKGROUND: Many authorities believe that the finding of retinal hemorrhages in a child younger than 3 years of age with a history of head trauma, in the absence of an obvious cause for the injury, is pathognomonic of child abuse. To date, no studies have examined the prospective retinal examination of children who have had head trauma. The authors undertook such a study because the presence of retinal hemorrhage from any head trauma in children may have medicolegal diagnostic significance in differentiating accidental from nonaccidental trauma. METHODS: Seventy-nine children younger than 3 years of age, each of whom experienced head injury, underwent an ophthalmologic assessment, which included a dilated funduscopic examination. RESULTS: Seventy-five children sustained accidental head injuries and had normal funduscopic examinations. Three children had nonaccidental head injuries and all were found to have varying degrees of retinal hemorrhages. One child, with a normal fundus examination, had injuries that were of indeterminate cause. CONCLUSION: The finding of retinal hemorrhages in a child with a head injury suggests a nonaccidental cause.

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.