Cloning of novel bombesin precursor cDNAs from skin of Bombina maxima

Amphibian skin is a rich resource of bioactive peptides like proline-rich bombesin from frog Bombina maxima. A novel cDNA clone encoding a precursor protein that comprises proline-rich bombesin and a novel peptide, designated as bombestatin, was isolated from a skin cDNA library of B. maxima. The predicted primary structure of the novel peptide is WEVLLNVALIRLELLSCRSSKDQDQKESCGMHSW, in which two cysteines form a disulfide bond. A BLAST search of databases did not detect sequences with significant similarity. Bombestatin possesses dose-dependent contractile activity on rat stomach strips. The differences between cDNAs encoding PR-bombesin plus bombestatin and PR-bombesin alone are due to fragment insertions located in 3'-coding region and 3'-untranslational region, respectively. (c) 2005 Elsevier B.V. All rights reserved.

An anionic antimicrobial peptide from toad Bombina maxima

Amphibian skin is a rich resource of antimicrobial peptides like maximins and maximins H from toad Bombina maxima. A novel cDNA clone encoding a precursor protein that comprises maximin 3 and a novel peptide. named maximin H5. was isolated from a skin cDNA library of B. maxima. The predicted primary structure of maximin H5 is ILGPVLGLVSDTLDDVLGIL-NH2,. Containing three aspartate residues and no basic amino acid residues. maximin H5 is characterized by an anionic property. Different from cationic maximin H peptides. only Gram-positive strain Staphylococcus aureus was sensitive to maximin H5. while the other bacteria] and fungal strains tested ere resistant to it. The presence of metal ions. like Zn2+ and Mg2+, did not increase its antimicrobial potency. Maximin H5 represents the first example of potential anionic antimicrobial peptides from amphibians, The results provide the first evidence that. together kith cationic antimicrobial peptides. anionic antimicrobial peptides may also exist naturally as part of the innate defense system. (C), 2002 Elsevier Science (USA). All rights reserved.

Maximin 9, a novel free thiol containing antimicrobial peptide with antimycoplasma activity from frog Bombina maxima

Amphibian skin is a rich resource of antimicrobial peptides, like maximins and maximin Hs from frog Bombina maxima. Novel cDNA clones encoding a precursor protein, which comprises a novel maximin peptide (maximin 9) and reported maximin H3, were isolated from two constructed skin cDNA libraries of B. maxima. The predicted primary structure of maximin 9 is GIGRKFLGGVKTTFRCGVKDFASKHLY-NH2. A surprising substitution is at position 16, with a free cysteine in maximin 9 rather than usual conserved glycine in other reported maximins. Maximin 9, the homodimer form and its Cys(16) to Gly(16) mutant were synthesized and their antimicrobial activities were evaluated. Unlike previously reported maximin 3, the tested bacterial and fungal strains were resistant to maximin 9, its homodimer and the Cys(16) to Gly(16) mutant (with MICs > 100 mu M). On the other hand, interestingly, while eight clinical Mollicutes strains were generally resistant to maximin 9 homodimer and its Cys(16) to Gly(16) mutant, most of them are sensitive to maximin 9 at a peptide concentration of 30 mu M, especially in the presence of dithiothreitol. These results indicate that the presence of a reactive Cys residue in maximin 9 is important for its antimycoplasma activity. The diversity of antimicrobial peptide cDNA structures encountered in B. maxima skin cDNA libraries and the antimicrobial specificity differences of the peptides may reflect well the species' adaptation to the unique microbial environments. (c) 2005 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Molecular characterization of a weak fibrinogen-clotting enzyme from Trimeresurus jerdonii venom

A fibrinogen-clotting enzyme designed as jerdonobin-II was isolated from the venom of Trimeresurus jerdonii. It differed in molecular weight and N-terminal sequence with the previously isolated jerdonobin, a thrombin-like enzyme from the same venom. The enzyme consists of a single polypeptide chain with molecular weights of 30,000 and 32,000 under non-reducing and reducing conditions, respectively. Jerdonobin-II showed weak fibrinogen clotting activity and its activity unit on fibrinogen was calculated to be less than one unit using human thrombin as standard. The precursor protein sequence of jerodonobin-II was deduced from cloned cDNA sequence. The sequence shows high similarity (identity = 89%) to TSV-PA, a specific plasminogen activator from venom of T stejnegeri. Despite of the sequence similarity, jerdonobin-II was found devoid of plasminogen activating effect. Sequence alignment analysis suggested that the replacement of Lys(239) in TSV-PA to Gln(239) in jerdonobin-II might play an important role on their plasminogen activating activity difference. (C) 2005 Elsevier Ltd. All rights reserved.

PLCß介导的钙调腺苷酸环化酶在突触可塑性相关基因转录中的作用和FE65对海马依赖学习及LTP的调节

Gene regulation is required for activity-dependent changes in synaptic plasticity and remodeling. The metabotropic glutamate receptors (mGluRs) contribute to different brain functions, including learning/memory, mental disorders, drug addiction, and persistent pain in the CNS. We found that Gp I mGluRs activate PLCß through Gq and then lead to activation of several calcium-dependent signaling pathways, including ERK, which play an important role in gene transcription. These findings support a calcium-dependent role for Gq in release of Calcium and activation of calcium-stimulated adenylyl cyclases I in activity-dependent transcription in response to application of group I metabotropic glutamate receptors agonist and may provide insights into group I mGluRs-dependent synaptic plasticity through MAP kinases signaling. Moreover, the present study investigated the transcription-dependent changes of Arc in response to the activation of group I mGluRs and suggested the central role of ERK1/2 in group I mGluR-mediated Arc transcription. Further, we selected APP-interaction protein FE65 to investigate the mechanism of transcription-related process in synaptic plasticity. FE65 is expressed predominantly in the brain, and interacts with the C-terminal domain of β-amyloid precursor protein (APP). We examined hippocampus-dependent memory and in vivo long-term potentiation (LTP) at the CA1 synapses with the isoform-specific FE65 knock-out (p97FE65-/-) mice. p97FE65 knock-out mice showed impaired short-term memory for both TDPA and CFC when tested 10min after training, which is transcription-independent. Consistently, at the Schaffer collateral-CA1 synapses, p97FE65 knock-out mice showed defective early phase LTP. These results demonstrate novel roles of FE65 in synaptic plasticity, acquisition, and retention for certain forms of memory formation.

Accelerated evolution of the pituitary adenylate cyclase-activating polypeptide precursor gene during human origin

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide abundantly expressed in the central nervous system and involved in regulating neurogenesis and neuronal signal transduction. The amino acid sequence of PACAP is extremely conserved across vertebrate species, indicating a strong functional constraint during the course of evolution. However, through comparative sequence analysis, we demonstrated that the PACAP precursor gene underwent an accelerated evolution in the human lineage since the divergence from chimpanzees, and the amino acid substitution rate in humans is at least seven times faster than that in other mammal species resulting from strong Darwinian positive selection. Eleven human-specific amino acid changes were identified in the PACAP precursors, which are conserved from murine to African apes. Protein structural analysis suggested that a putative novel Deuropeptide might have originated during human evolution and functioned in the human brain. Our data suggested that the PACAP precursor gene underwent adaptive changes during human origin and may have contributed to the formation of human cognition.

Molecular cloning and mRNA expression of peptidoglycan recognition protein (PGRP) gene in bay scallop (Argopecten irradians, Lamarck 1819)

Peptidoglycan recognition proteins (PGRPs) are a type of pattern recognition molecules (PRM) that recognize the unique cell wall component peptidoglycan (PGN) of bacteria and are involved in innate immunity. The first bivalve PGRP cDNA sequence was cloned from bay scallop Argopecten irradians by expressed sequence tag (EST) and PCR technique. The full-length cDNA of bay scallop PGRP (designated AiPGRP) gene contained 10 18 bp with a 615-bp open reading frame that encoded a polypeptide of 205 amino acids. The predicted amino acid sequence of AiPGRP shared high identity with PGRP in other organisms, such as PGRP precursor in Trichoplusia ni and PGRP SC2 in Drosophila melanogaster. A quantitative reverse transcriptase Real-Time PCR (qRT-PCR) assay was developed to assess the mRNA expression of AiPGRP in different tissues and the temporal expression of AiPGRP in the mixed primary cultured hemocytes challenged by microbial components lipopolyssacharide (LPS) from Escherichia coli and PGN from Micrococcus luteus. Higher-level mRNA expression of AiPGRP was detected in the tissues of hemocytes, gonad and kidney. The expression of AiPGRP in the mixed primary cultured hemocytes was up regulated after stimulated by PGN, while LPS from E. coli did not induce AiPGRP expression. The results indicated that AiPGRP was a constitutive and inducible expressed protein that was mainly induced by PGN and could be involved in scallop immune response against Gram-positive bacteria infection. (c) 2006 Elsevier Ltd. All rights reserved.