Biomedical subjects

P Nicolas

Publications and source records attributed to P Nicolas.

At least 55 records · Page 3Linked to original sources

PubMed2000

Peptide secretion in the cutaneous glands of South American tree frog Phyllomedusa bicolor: an ultrastructural study.

The development of the dermal glands of the arboreal frog Phyllomedusa bicolor was investigated by immunocytochemistry and electron microscopy. The 3 types of glands (mucous, lipid and serous) differed in size and secretory activity. The mucous and serous glands were apparent in the tadpole skin, whereas the lipid glands developed later in ontogenesis. The peptide antibiotics dermaseptins and the D-amino acid-containing peptide opioids dermorphins and deltorphins are abundant in the skin secretions of P. bicolor. Although these peptides differ in their structure and activity they are derived from precursors that have very similar preproregions. We used an antibody to the common preproregion of preprodermaseptins and preprodeltorphins and immunofluorescence analysis to show that only the serous glands are specifically involved in the biosynthesis and secretion of dermaseptins and deltorphins. Scanning and transmission electron microscopy revealed that the serous glands of P bicolor have morphological features, especially the secretory granules, which differ from those of the glands in Xenopus laevis skin.

Amphibian Proteins↗

PubMed2000

The 1998 Senegal epidemic of meningitis was due to the clonal expansion of A:4:P1.9, clone III-1, sequence type 5 Neisseria meningitidis strains.

Between January and April 1998, a meningitis outbreak due to serogroup A meningococcus took place in Senegal. The outbreak began in Gandiaye, 165 km to the east of Dakar, and progressed towards the towns of Gossas, Niakkhar, Guinguineo, Fatik, Foundiougne, Dioffior, Sokone, Kaolack, and Nioro. At the same time, the outbreak reached regions of Kaffrine, Koungheul, and Tambacounda in the east of Senegal. A total of 1,350 cases and 200 deaths were reported. The WHO Collaborating Center in Marseilles received 24 strains for analysis. All were serogroup A Neisseria meningitidis, type 4 and subtype P1.9. Multilocus enzyme electrophoresis, performed by Institut Pasteur Paris, showed that the strains belonged to clone III-1. DNA restriction fragments generated by endonuclease BglII and analyzed by pulsed-field gel electrophoresis showed 24 indistinguishable fingerprint patterns similar to those of meningococcus strains isolated from African outbreaks since 1988. Three strains were studied by multilocus sequence typing (MLST) with seven loci. The comparison between sequences and existing alleles on the MLST website () allowed us to assign these strains to sequence type 5 (ST5), as their sequences were identical to the consensus at seven loci. All 24 strains were susceptible to penicillin, amoxicillin, chloramphenicol, and rifampin. Subgroup III is finishing its spread towards west of the meningitis belt of Africa. To our knowledge, this is the first time subgroup III, and more precisely ST5, strains are reported as being responsible for a meningitis outbreak in Senegal.

Bacterial Typing Techniques↗

PubMed1999

The pituitary-skin connection in amphibians. Reciprocal regulation of melanotrope cells and dermal melanocytes.

In amphibians, alpha-MSH secreted by the pars intermedia of the pituitary plays a pivotal role in the process of skin color adaptation. Reciprocally, the skin of amphibians contains a number of regulatory peptides, some of which have been found to regulate the activity of pituitary melanotrope cells. In particular, the skin of certain species of amphibians harbours considerable amounts of thyrotropin-releasing hormone, a highly potent stimulator of alpha-MSH release. Recently, we have isolated and sequenced from the skin of the frog Phyllomedusa bicolor--a novel peptide named skin peptide tyrosine tyrosine (SPYY), which exhibits 94% similarity with PYY from the frog Rana ridibunda. For concentrations ranging from 5 x 10(-10) to 10(-7) M, SPYY induces a dose-related inhibition of alpha-MSH secretion. At a dose of 10(-7) M, SPYY totally abolished alpha-MSH release. These data strongly suggest the existence of a regulatory loop between the pars intermedia of the pituitary and the skin in amphibians.

Amino Acid Sequence↗

PubMed1999

The dermaseptin precursors: a protein family with a common preproregion and a variable C-terminal antimicrobial domain.

Preprodermaseptins are a group of antimicrobial peptide precursors found in the skin of a variety of frog species. Precursors of this family have very similar N-terminal preprosequences followed by markedly different C-terminal domains that correspond to mature antimicrobial peptides. Some of these peptides are 24-34 amino acids long and form well-behaved amphipathic alpha-helices, others are disulfide-linked peptides of 20-46 residues, still others, highly hydrophobic, are the smallest antimicrobial peptides known so far being only 10-13 residues in length. All these peptides are broad-spectrum microbicides that kill many bacteria, protozoa, yeasts and fungi by destroying or permeating the microbial membrane. In frogs belonging to the genus Phyllomedusinae, preprodermaseptins encoded peptides also include dermorphins and deltorphins, D-amino acid-containing heptapeptides which are very potent and specific agonists of the mu- or delta-opioid receptors. The remarkable similarity between preproregions of precursors that give rise to peptides with very different primary structures, conformations and activities suggests that the corresponding genes originate from a common ancestor. The high conservation of the precursor prepropart indicates that this region must have an important function.

Amino Acid Sequence↗

PubMed1999

The delta-selective opioid peptide dermenkephalin and the mu-selective hybrid peptide dermenkephalin-[1-4]-dermophin-[5-7] display strikingly different conformations despite identical tetrapeptide N-termini. A quantitative 2-D NMR and molecular modeling analysis.

The selective recognition of the aminoterminal binding pharmacophore Tyr-D-Xaa-Phe of the opioid heptapeptide dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 (DRM)1, and of dermenkephalin, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (DREK), by the mu-opioid receptor and delta-opioid receptor, respectively, depends upon the constitution / conformation of the C-terminal tripeptide. The hybrid peptide DREK-[1-4]-DRM-[5-7] is very potent at, and exquisitely selective for the mu-opioid receptor, and differs only from dermenkephalin by its C-terminal tripeptide. Comparison of the structural features of DREK-[1-4]-DRM-[5-7] and dermenkephalin by nmr analysis and molecular modeling revealed striking differences, as well in the trans (Tyr5 - Pro6) isomer (population 75%) than in the cis isomer.. Whereas the folded C-terminal tail of dermenkephalin influenced the tertiary structure of the N-terminal tetrapeptide and placed the Tyr1 and Phe3 aromatic rings in definite orientations that are best suited for the delta-receptor, there were only weak contacts, as shown by NOE data, between the aminoterminal and carboxyterminal parts of the hybrid peptide. This promoted increased flexibility of the whole backbone and relaxed orientations for the side-chains of Tyr1 and Phe3 that are compatible with the mu-receptor but unsuitable for the delta-receptor. The steric hindrance introduced by Pro6 in DREK-[1-4]-DRM-[5-7], plus the absence of large hydrophobic side-chains in positions 5 and 6 may prevent close contacts between the N-terminal and C-terminal domains and reorientation of the main pharmacophoric elements Tyr1 and Phe3.

Amino Acid Sequence↗

PubMed1999

Development of a polymerase chain reaction diagnostic test for the detection of the biotrophic pathogen Plasmopara halstedii in sunflower.

The obligate parasitic fungus-like organism Plasmopara halstedii (Farl.) Berl. et De Toni, is the causal agent of downy mildew disease in sunflower (Helianthus annuus). New races of this economically important parasite are regularly detected throughout the world. In addition, fungicide-resistant isolates have been reported in Europe and North America. These observations of parasite evolution, as well as the risk of propagation of the disease by infected seeds, means that it is necessary to guarantee the absence of Plasmopara halstedii in seed shipments. We report here the development of a rapid assay that can be used to detect infection by Plasmopara halstedii in plant tissues. Based on the nucleotide sequence information obtained from one cloned random amplified polymorphic DNA fragment, specific oligonucleotides were designed and used as primers for in vitro DNA amplification by polymerase chain reaction. An amplification product was detected on agarose gel stained with ethidium bromide when DNA from various Plasmopara halstedii races was tested, whereas no amplified DNA was detected when DNA from other origins was tested, including DNA from the host plant. The sensitivity of the technique was evaluated. The assay successfully reveals the presence of Plasmopara halstedii in infected sunflower plants prior to sporulation.

DNA, Fungal↗

PubMed1999

Cloning of thyrotropin-releasing hormone precursor and receptor in rat thymus, adrenal gland, and testis.

TRH is a hypophysiotropic peptide that acts mainly via the hypothalamic-pituitary-thyroid axis, but TRH immunoreactivity is also detected in several peripheral tissues. PCR with two pairs of primers enabling amplification of three fragments of TRH complementary DNA (cDNA) was used to demonstrate local production of TRH. Products of the expected size were detected in the testis, adrenal gland, lymphoid organs, thymus, and spleen. The amplified cDNA fragments were cloned and sequenced to show that the TRH gene is expressed in the thymus, spleen, and adrenal gland. Competitive RT-PCR showed that the TRH messenger RNA content of the testis was about one third that of the hypothalamus, whereas the adrenal gland contained 2% and the thymus 6%. HPLC analysis of thymus and spleen extracts showed small amounts of TRH, with a particular processing pattern of pro-TRH in lymphoid organs. The expression of the TRH receptor gene in peripheral organs was investigated to determine whether TRH had an autocrine or a paracrine action. cDNA fragments that encompassed the coding region of the receptor were identified in the testis, adrenal gland and thymus. No signal was detected in the spleen. These findings indicate that TRH may have a biological activity in extrapituitary organs and may act locally in the testis, adrenal gland, and thymus.

Adrenal Glands↗

PubMed1999

[Neisseria meningitidis and meningitis].

Meningococcal meningitis epidemics can occur anywhere in the world. However this risk is particularly high during the dry season in the sub-Saharan zone of Africa known as the Lapeyssonnie meningitis belt. This area characterized by hyperendemicity that regularly gives rise to epidemics. Multilocus enzyme electrophoresis has made possible identification and monitoring of the progression of virulent clones of Neisseria meningitidis strains in the world. Monitoring is now possible by multilocus sequence typing and data bank on the Internet. Vaccination is a major prophylactic modality. The usefulness of plain group A plus C polysaccharide vaccines is limited because of poor effectiveness in young children who constitute the highest risk group. During epidemics, mass vaccination should be carried out as early as possible according to the state of alert defined for the area. More recent conjugate vaccines against group A and C, which are effective in young children and provide long-term protection by induction of immunologic memory, may allow routine vaccination in the future. Although clinical signs are often apparent, not all cases are diagnosed by clinical examination unless gravity is taken into account. Untreated the disease is always fatal. The only hope of survival is early institution of appropriate antimicrobial therapy (even prior to hospitalization). Several strains resistant to chloramphenicol have been reported and the number of strains with reduced sensitivity to penicillin is rising constantly. Although treatment remains feasible, the existence of resistant forms raises the need to monitor the sensitivity of meningococci using standardized of antibiograms.

Adult↗

PubMed1999

[Inter-laboratory reproducibility of pulsed-field electrophoresis for the study of 12 types of Pseudomonas aeruginosa].

The increasing hospital-to-hospital transmission of multiple drug-resistant bacteria is a major concern for bacteriology laboratories involved in nosocomial infection control. The interlaboratory reproducibility of pulsed-field gel electrophoresis (PFGE) for Pseudomonas aeruginosa typing was evaluated by asking four hospital laboratories (two in Lyon, one in Brest, and one in Marseille) to study 11 P. aeruginosa isolates, some of which were epidemiologically related, and the reference strain ATCC 27853. Two laboratories used the Genepath system, one the Chef DR II, system, and one the Chef Mapper system, Bio-Rad, restriction/Spe I. Profiles were read visually and by computerized comparison of restriction band molecular weights (Taxotron, software, PAD Grimont, Pasteur Institute, Paris, France). These two methods led to similar epidemiological conclusions. However, centralization of the data showed poor center-to-center reproducibility due to inadequate standardization of the procedure.

Electrophoresis, Gel, Pulsed-Field↗

PubMed1998

Dermaseptin, a peptide antibiotic, stimulates microbicidal activities of polymorphonuclear leukocytes.

Dermaseptin (DRs S1), a 34-amino acid residue cationic antimicrobial peptide was studied for its effects on the production of reactive oxygen species (respiratory burst) and exocytosis of polymorphonuclear leukocytes (PMN). Treatment of PMN with DRs S1 (10-100 nM) stimulated significant production of reactive oxygen species (approximately a 2-fold increase relative to control) and release of myeloperoxidase. In addition, low DRs S1 concentrations (1-10 nM) primed the stimulation of respiratory burst induced by zymosan particles. In contrast to the native peptide, a dermaseptin fragment without either the COOH-terminal (DRs 1-10) or NH2 terminal (DRs 16-34) portion was inactive. The DRs S1-induced respiratory burst was inhibited by a selective protein kinase C inhibitor, GF 109203X, and was associated with early signalling events such as a rapid and transient elevation of cytosolic-free calcium concentration and phospholipase D activity. These data provide the first evidence of stimulating and priming properties of a peptide antibiotic on microbicidal activities of neutrophils, suggesting a potential role of dermaseptin in modulating host-defense mechanisms.

Amino Acid Sequence↗

PubMed1998

Structure, synthesis, and molecular cloning of dermaseptins B, a family of skin peptide antibiotics.

Analysis of antimicrobial activities that are present in the skin secretions of the South American frog Phyllomedusa bicolor revealed six polycationic (lysine-rich) and amphipathic alpha-helical peptides, 24-33 residues long, termed dermaseptins B1 to B6, respectively. Prepro-dermaseptins B all contain an almost identical signal peptide, which is followed by a conserved acidic propiece, a processing signal Lys-Arg, and a dermaseptin progenitor sequence. The 22-residue signal peptide plus the first 3 residues of the acidic propiece are encoded by conserved nucleotides encompassed by the first coding exon of the dermaseptin genes. The 25-residue amino-terminal region of prepro-dermaseptins B shares 50% identity with the corresponding region of precursors for D-amino acid containing opioid peptides or for antimicrobial peptides originating from the skin of distantly related frog species. The remarkable similarity found between prepro-proteins that encode end products with strikingly different sequences, conformations, biological activities and modes of action suggests that the corresponding genes have evolved through dissemination of a conserved "secretory cassette" exon.

Amino Acid Sequence↗

PubMed1998

Synthesis, antimicrobial activity and gene structure of a novel member of the dermaseptin B family.

Dermaseptins are a family of cationic (Lys-rich) antimicrobial peptides that are abundant in the skin secretions of the arboreal frogs Phyllomedusa bicolor and P. sauvagii. In vitro, these peptides are microbicidal against a wide variety of microorganisms including Gram-positive and Gram-negative bacteria, yeasts, protozoa and fungi. To date, 6 dermaseptin B mature peptides, 24-34 residues long, 2 dermaseptin B cDNAs and 2 gene sequences have been identified in P. bicolor. To assess dermaseptin related genes further, we screened a P. bicolor genomic library with 32P-labeled cDNAs coding either for prepro-dermaseptins B1 or B2 (adenoregulin). A gene sequence was identified that coded a novel dermaseptin B, termed Drg3, which exhibits 23-42% amino acids identities with other members of the family. Analysis of the cDNAs coding precursors for several opioid and antimicrobial peptides originating from the skin of various amphibian species revealed that the 25-residue preproregion of these preproforms are all encoded by conserved nucleotides encompassed by the first coding exon of the Drg3 gene. Synthetic dermaseptin Drg3 exhibited a bactericidal activity towards several species of mollicutes (wall-less eubacteria), firmicutes (Gram-positive eubacteria), and gracilicutes (Gram-negative eubacteria), with minimal inhibitory concentrations (MICs) ranging from 6.25 to 100 microM. Experiments performed on Acholeplasma laidlawii cells revealed that this peptide is membranotropic and that if efficiently depolarizes the plasma membrane.

Amino Acid Sequence↗

PubMed1998

Opioid peptides from frog skin.

The skin of the South American frogs Phyllomedusa secretes, in addition to numerous mammalian-like hormones and neuropeptides, several gene-encoded opioid peptides that contain a D-amino acid in position 2 of their sequence. Dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2, dermenkephalin/deltorphin A, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 and the deltorphins, Tyr-D-Ala-Phe-Xaa-Val-Val-Gly-NH2 (where Xaa is either Asp or Glu) are highly potent at, and exquisitely selective, for the mu- and delta-opioid receptors. D-Ala and D-Met present in dermorphin and related peptides are coded for by the usual codons in the corresponding messenger RNAs. Prepro-dermorphin/dermenkephalin and prepro-deltorphins have considerable sequence identities to precursors encoding 10-46-residue-long antimicrobial peptides--dermaseptins, brevinins, temporins, esculentins and gaegurins--originating from various amphibian species. The similarity between the prepro-regions of precursors encoding end products with strikingly different structures and biological activities supports the suggestion that the genes encoding these peptides are all members of the same family.

Amino Acid Sequence↗

PubMed1998

Cloning, sequence and characterization of a sunflower (Helianthus annuus L.) pathogen-induced gene showing sequence homology with auxin-induced genes from plants.

The establishment of a plant-pathogen interaction involves changes in gene expressions in both organisms. To isolate Helianthus annuus genes whose expression is induced during processes of resistance to Plasmopara halstedii, a comparison of the expression pattern of healthy sunflowers was made with sunflowers infected with 2 races of P. halstedii, either virulent or avirulent, using differential display of mRNA. A full-length cDNA, HaAC1, representing a sunflower gene whose expression is enhanced during early stages of the incompatible interaction, was isolated. Different timing of RNA accumulation is observed between compatible and incompatible combinations. Sequence analysis and database search revealed significant homology with auxin-induced genes from plants. The expression of this gene, is also induced after treatment with 2,4-dichlorophenoxyacetic acid (2,4-D), salicylic acid (SA) and wounding.

2,4-Dichlorophenoxyacetic Acid↗

PubMed1998

Solution conformations of deltorphin-I obtained from combined use of quantitative 2D-NMR and energy calculations: a comparison with dermenkephalin.

Deltorphin-I, Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2 and dermenkephalin, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, two highly related opioid peptides from frog skin, display very similar N-termini but strikingly different C-terminal tails. Nevertheless, both peptides are highly potent at, and exquisitely selective for the delta-opioid receptor. To identify common determinants concuring to the remarkably efficient targeting of deltorphin-I and dermenkephalin, combined use of quantitative two-dimensional nuclear magnetic resonance (53 dipolar interactions studied at four temperatures) and energy calculations using simulated annealing generated five groups of deltorphin-I conformers. These groups were pooled into two families whose overall conformation could be described either by a left-handed helix (Family I) or by a big loop (Family II), both stabilized by H-bonds. Proximity of D-Ala2-Phe3-Asp4 and Val5-Val6-Gly7 triads is an obvious structural similarity between almost all groups in both families of structures. Whereas differences between the two families originated mostly from a transition at psi Asp4 backbone dihedral angle, the backbone structures at segment 1-4 are similar and spatial arrangements of Tyr1 (t) and Phe3 (g-) are identical in one group of each family. Moreover, these two groups have a N-terminal tetrapeptide whose conformation most closely resembles that of a well-defined group of structures for dermenkephalin. Altogether, these results suggest that conformational attributes that are common to dermenkephalin and deltorphin-I, i.e., the backbone conformation of the N-terminal tetrapeptide and preferential orientations in the side-chain of Tyr1 (t) and Phe3 (g-) underlie their ability to bind with high selectivity to the delta-opioid receptor.

Analgesics, Opioid↗

PubMed1998

Assembly of connexins and MP26 in lens fiber plasma membranes studied by SDS-fracture immunolabeling.

The SDS-fracture immunolabeling technique, unlike conventional freeze-fracture, provides direct evidence for the biochemical nature of membrane constituents. SDS-fracture immunolabeling shows that during differentiation of lens fiber cells the onset of junctional assembly is characterized by the presence of small clusters and linear arrays comprising connexins alpha3 and alpha8. At this initial stage MP26, a major fiber membrane constituent, appears to be colocalized with these two connexins. The application of double-immunogold labeling reveals that when large junctional plaques are assembled MP26 becomes mainly associated with the periphery of the junctional domains. This type of distribution suggests that MP26 may play a role in the clustering and gathering of connexons. In aged nuclear fiber membranes connexins, MP26 and their proteolytic derivatives form an orthogonal lattice of repeating subunits.

Animals↗

PubMed1998

Clinical pharmacokinetics of diacerein.

Diacerein is a drug for the treatment of patients with osteoarthritis. This drug is administered orally as 50 mg twice daily. Diacerein is entirely converted into rhein before reaching the systemic circulation. Rhein itself is either eliminated by the renal route (20%) or conjugated in the liver to rhein glucuronide (60%) and rhein sulfate (20%); these metabolites are mainly eliminated by the kidney. The pharmacokinetics characteristics of diacerein are about the same in young healthy volunteers and elderly people with normal renal function, both after a single dose (50 mg) or repeated doses (25 to 75 mg twice daily). Rhein kinetics after single oral doses of diacerein are linear in the range 50 to 200 mg. However, rhein kinetics are time-dependent, since the nonrenal clearance decreases with repeated doses. This results in a moderate increase in maximum plasma concentration, area under the plasma concentration-time curve and elimination half-life. Nevertheless, the steady-state is reached by the third administration and the mean elimination half-life is then around 7 to 8 hours. Taking diacerein with a standard meal delays systemic absorption, but is associated with a 25% increase in the amount absorbed. Mild-to-severe (Child Pugh's grade B to C) liver cirrhosis does not change the kinetics of diacerein, whereas mild-to-severe renal insufficiency (creatinine clearance < 2.4 L/h) is followed by accumulation of rhein which justifies a 50% reduction of the standard daily dosage. Rhein is highly bound to plasma proteins (about 99%), but this binding is not saturable so that no drug interactions are likely to occur, in contrast to those widely reported with nonsteroidal anti-inflammatory drugs. Except for moderate and transient digestive disturbances (soft stools, diarrhoea), diacerein is well tolerated and seems neither responsible for gastrointestinal bleeding nor for renal, liver or haematological toxicity.

Aged↗

PubMed1998

[Standardization of the Neisseria meningitidis antibiogram. Detection of strains relatively resistant to penicillin].

Studying the susceptibility of 189 Neisseria meningitidis strains to penicillin, amoxicillin, cefotaxime, ceftriaxone, chloramphenicol and rifampicin by determination of minimum inhibitory concentrations (MICs) by agar dilution (reference method), E-test and disc diffusion method on Mueller-Hinton agar at 37 degrees C with 5% CO2 enabled us to standardize the antibiograms. While MIC determination by agar dilution is still the reference method, it is possible to obtain exact or approximate MIC values using the E-test. For laboratories that cannot determine penicillin MICs, it is impossible to detect strains that are relatively resistant to penicillin (RRP strains: 0.1 < or = MIC < or = 1 mg/l) using a 10-U penicillin disc. A 1 microgram-oxacillin disc allows MIC to be determined in most cases when the oxacillin inhibition zone is < or = 10 mm. Such strains must be sent to a reference laboratory for exact MIC determination. Based on our results and literature data on pharmacokinetics, we propose critical concentrations for these various antibiotics as well as critical diameters for chloramphenicol and rifampicin discs.

Amoxicillin↗