Showing papers by "J. Michael Conlon published in 2010"
TL;DR: The cyclic peptide urotensin II (UII) was originally isolated from the urophysis of teleost fish on the basis of its ability to contract intestinal smooth muscle and, later on, the pre‐proUII cDNA has been characterized in mammals, including humans.
Abstract: The cyclic peptide urotensin II (UII) was originally isolated from the urophysis of teleost fish on the basis of its ability to contract intestinal smooth muscle. The UII peptide has subsequently been isolated from frog brain and, later on, the pre-proUII cDNA has been characterized in mammals, including humans. A UII paralog called urotensin II-related peptide (URP) has been identified in the rat brain. The UII and URP genes originate from the same ancestral gene as the somatostatin and cortistatin genes. In the central nervous system (CNS) of tetrapods, UII is expressed primarily in motoneurons of the brainstem and spinal cord. The biological actions of UII and URP are mediated through a G protein-coupled receptor, termed UT, that exhibits high sequence similarity with the somatostatin receptors. The UT gene is widely expressed in the CNS and in peripheral organs. Consistent with the broad distribution of UT, UII and URP exert a large array of behavioral effects and regulate endocrine, cardiovascular, renal, and immune functions.
95 citations
TL;DR: This chapter describes the use of norepinephrine (injection or immersion) to stimulate peptide release from granular glands in the skin in procedures that do not appear to cause distress to the animals.
Abstract: Skin secretions from many species of anurans (frogs and toads) are a rich source of peptides with broad-spectrum antimicrobial activities that may be developed into agents with therapeutic potential, particularly for topical applications. This chapter describes the use of norepinephrine (injection or immersion) to stimulate peptide release from granular glands in the skin in procedures that do not appear to cause distress to the animals. The peptide components in the secretions are separated using reversed-phase HPLC on octadecylsilyl-silica (C(18)) columns after partial purification on Sep-Pak C(18) cartridges. Peptides with antimicrobial activity are then identified by demonstration of their abilities to inhibit growth of Gram-negative (Escherichia coli) and Gram-positive (Staphylococcus aureus) bacteria in liquid phase microtiter plate assays. Individual peptides with activity are purified to near homogeneity by further chromatography on butylsilyl-(C(4)) and diphenylmethylsilyl-silica columns and characterized structurally by automated Edman degradation and mass spectrometry.
57 citations
TL;DR: The data indicate that nonfunctionalization has been the most common fate of duplicated antimicrobial peptide genes following the polyploidization events in the X. amieti lineage, and the very low antimicrobial activity of the magainin-AM1 and PGLa-AM2 paralogs suggests the possibility that certain peptides may have evolved toward a new, as yet undetermined, function (neofunctionalization).
54 citations
TL;DR: Circulating concentrations of chromogranin B-like immunoreactivity (CgB-LI) are not elevated in non-neoplastic diseases and measurements of CCB, the COOH-terminal fragment of CgB, may be useful as a biochemical marker for neuroendocrine differentiation in lung tumors.
47 citations
TL;DR: Nine peptides with differential growth inhibitory activity against Escherichia coli and Staphylococcus aureus were isolated from norepinephrine-stimulated skin secretions of the tetraploid frog Xenopus borealis Parker, 1936 (Pipidae).
Abstract: Nine peptides with differential growth inhibitory activity against Escherichia coli and Staphylococcus aureus were isolated from norepinephrine-stimulated skin secretions of the tetraploid frog Xenopus borealis Parker, 1936 (Pipidae). Structural characterization of the peptides demonstrated that they were orthologous to magainin-2 (1 peptide), peptide glycine-leucine-amide, PGLa (2 peptides), caerulein-precursor fragments, CPF (4 peptides), and xenopsin-precursor fragments, XPF (2 peptides), previously isolated from Xenopus laevis and X. amieti. In addition, a second magainin-related peptide (G**KFLHSAGKFGKAFLGEVMIG) containing a two amino acid residue deletion compared with magainin-2 was identified that had only weak antimicrobial activity. The peptide with the greatest potential for development into a therapeutically valuable anti-infective agent was CPF-B1 (GLGSLLGKAFKIGLKTVGKMMGGAPREQ) with MIC=5 microM against E. coli, MIC=5 microM against S. aureus, and MIC=25 microM against Candida albicans, and low hemolytic activity against human erythrocytes (LC(50)>200 microM). This peptide was also the most abundant antimicrobial peptide in the skin secretions. CPF-B1 was active against clinical isolates of the nosocomial pathogens, methicillin-resistant S. aureus (MRSA) and multidrug-resistant Acinetobacter baumannii (MDRAB) with MIC values in the range 4-8 microM.
46 citations
TL;DR: Phylogenetic analysis based upon the amino acid sequences of 47 brevinin-2 peptides from 17 Asian species belonging to the family Ranidae provides support for the placement of H. erythraea in the genus Hylarana.
32 citations
TL;DR: Synthetic alyteserin-1c displayed potent activity against clinical isolates of MDRAB while displaying low hemolytic activity against human erythrocytes and retained antimicrobial activity but showed dramatically increased hemolyic activities (>40- and >13-fold, respectively).
31 citations
TL;DR: It is proposed that the marked decrease in haemolytic activity produced by the substitution Gly(4)-->Lys in XT-7 arises from a decrease in both helicity and hydrophobicity.
21 citations
TL;DR: In tissue expression analyses, preprobrevinin-1, preprotemporin, and preproranatuerin-2 gene transcripts were detected at higher levels in brain compared with peripheral tissues (heart, small intestine, kidney, liver lung, skeletal muscle, stomach, and testis).
18 citations
TL;DR: This chapter describes the use of norepinephrine (injection or immersion) to stimulate peptide release in a procedure that does not appear to cause distress to the animals.
Abstract: Skin secretions from anurans (frogs and toads), particularly those species belonging to the Hylidae and Ranidae families, are a rich source of biologically active peptides. Cytolytic peptides with broad-spectrum antimicrobial activities and highly variable amino acid sequences are often released into these secretions in high concentrations. Identification and characterization of these components can prove to be valuable in species identification, elucidation of evolutionary histories and phylogenetic relationships between species, and may lead to development of agents with potential for therapeutic application. This chapter describes the use of norepinephrine (injection or immersion) to stimulate peptide release in a procedure that does not appear to cause distress to the animals. The peptide components in the secretions are separated by reversed-phase HPLC on octadecylsilyl silica (C(18)) columns under standard conditions after partial purification on Sep-Pak cartridges. Individual peptides are identified by determination of their molecular masses by MALDI-TOF mass spectrometry and from their retention times. The use of mixtures of synthetic peptides of appropriate molecular mass as calibration standards enables mass determination to a high degree of precision.
17 citations
TL;DR: The results indicate that the expression of amphibian AMP genes is correlated with metamorphosis but is subjected to differential regulation.
Abstract: Previous studies led to the isolation of antimicrobial peptides (AMPs) of the brevinin-2, palustrin-2, and ranatuerin-2 families from skin extracts and/or skin secretions of the Japanese mountain brown frog, Rana ornativentris. In the present study, we cloned cDNAs encoding the precursors of brevinin-2Oc, palustrin-2Oa, and ranatuerin-2Ob and -2Oe from skin total RNA preparations from adult R. ornativentris and established a semi-quantitative RT-PCR system to measure the concentrations of these mRNAs. The levels of preprobrevinin-2 and preproranatuerin-2 mRNAs in the skin specimens of developing R. ornativentris larva were detectable only at stages later than the onset of metamorphosis and reached peaks at the stage of metamorphic climax. In contrast, prepropalustrin-2 mRNA was detected prior to the onset of metamorphosis and levels peaked at stages earlier than those of the other two mRNAs. In adult animals, preprobrevinin-2 and preproranatuerin-2 gene transcripts were detected at low levels in the small intestine and skeletal muscle but not in the stomach, liver, or kidney, whereas prepropalustrin-2 gene transcripts were detected at relatively high concentrations in all tissues examined. These results indicate that the expression of amphibian AMP genes is correlated with metamorphosis but is subjected to differential regulation.
TL;DR: Five peptides with antimicrobial activity were isolated from an extract of the skins of specimens of Rana tagoi okiensis collected on the Oki Islands, Japan and determined that they belong to the ranatuerin-2 family, two peptides to the temporin family, and one peptide to the brevinin-1 family.
TL;DR: Low-temperature pyrolysis of methionine-enkephalin-Arg-Gly-Leu has been carried out and the non-volatile residues have been analyzed and a complete or partial loss of an amino-acid side group was observed.
Abstract: Low-temperature pyrolysis of methionine-enkephalin-Arg-Gly-Leu has been carried out and the non-volatile residues have been analyzed. The fragments were separated and characterized by LC–UV/Vis–MS/MS. Two major types of pyrolysis products were identified by matching the experimental results with a theoretical list that contains the expected fragments. These products were mainly composed of cyclic oligopeptides and linear fragments produced from the peptide backbone. These fragments have preserved the sequence of amino acids in the peptide. In some cases, a complete or partial loss of an amino-acid side group was observed. Tandem mass spectrometry and cyanogen bromide cleavage experiments were used to confirm the nature of the cyclic and linear pyrolysates, in addition to chromatographic and mass spectrometric data of actual standard synthetic cyclic peptides. Copyright © 2010 John Wiley & Sons, Ltd.
TL;DR: Chromatographic analysis of norepinephrine-stimulated skin secretions from L. onca led to the identification of six peptides with antimicrobial activity, which indicate a close phylogenetic relationship between L.onca and L. yavapaiensis but suggest that they are not conspecific species.
Abstract: The phylogenetic relationship between the relict leopard frog Lithobates (Rana) onca (Cope, 1875) and the lowland leopard frog Lithobates (Rana) yavapaiensis (Platz and Frost, 1984) is unclear. Chromatographic analysis of norepinephrine-stimulated skin secretions from L. onca led to the identification of six peptides with antimicrobial activity. Determination of their primary structures indicated that four of the peptides were identical to brevinin-1Ya, brevinin-1Yb, brevinin-1Yc and ranatuerin-2Ya previously isolated from skin secretions of L. yavapaiensis. However, a peptide belonging to the temporin family (temporin-ONa: FLPTFGKILSGLF.NH2) and an atypical member of the ranatuerin-2 family containing a C-terminal cyclic heptapeptide domain (ranatuerin-2ONa: GLMDTVKNAAKNLAGQMLDKLKCKITGSC) were isolated from the L. onca secretions but were not present in the L. yavapaiensis secretions. Ranatuerin-2ONa inhibited the growth of Escherichia coli (MIC = 50 μM) and Candida albicans (MIC = 100 μM ) and showed hemolytic activity (LC50 = 90 μM) but was inactive against Staphylococcus aureus. The data indicate a close phylogenetic relationship between L. onca and L. yavapaiensis but suggest that they are not conspecific species.
TL;DR: By means of in situ hybridization using digoxigenin-labeled cRNA probes for preprotemporin-CBa and preprochensirin-2CBa, it is demonstrated for the first time in an amphibian the presence of mRNAs encoding these two precursors in the cytoplasm of the glandular cells in the bullfrog Harderian gland.
Abstract: The Harderian gland is an orbital gland found in many tetrapod species that possess a nictitating membrane. While the main role of the Harderian gland is lubrication of the eyeballs, numerous other functions are attributed to this gland. In amphibians, mast cells have been detected in the Harderian gland, suggesting that the gland is involved in the host's system of innate immunity defending against microbial invasions. Using reverse-transcription polymerase chain reaction, we cloned from the bullfrog Harderian gland total RNA preparations, cDNAs encoding biosynthetic precursors for the antimicrobial peptides temporin-CBa (FLPIASLLGKYL-NH 2 ), previously isolated from an extract of bullfrog skin, and chensirin-2CBa (IIPLPLGYFAKKP) that contained the amino acid substitution Thr 13 → Pro compared with chensirin-2 from the Chinese brown frog, Rana chensinensis. By means of in situ hybridization using digoxigenin-labeled cRNA probes for preprotemporin-CBa and preprochensirin-2CBa, we have demonstrated for the first time in an amphibian the presence of mRNAs encoding these two precursors in the cytoplasm of the glandular cells in the bullfrog Harderian gland.