Biomedical subjects

Ren Lai

Publications and source records attributed to Ren Lai.

28 records · Page 2Linked to original sources

PubMed2004

A thrombin inhibitor from the ixodid tick, Amblyomma hebraeum.

A novel thrombin inhibitor named Amblin was identified from the haemolymph of the ixodid (hard) tick, Amblyomma hebraeum, and the coding cDNA was isolated from a tick cDNA library. This cDNA codes for a preprotein of 166 amino acids, including a predicted signal peptide composed of 15 amino acids N-terminal to the mature Amblin. The 151-amino-acid mature Amblin contains 14 cysteines and two Kunitz-like domains. It displays high sequence similarity with a tissue factor pathway inhibitor (TFPI), Ixolaris, from the ixodid tick, Ixodes scapularis, which has 10 cysteines, and a thrombin inhibitor, Boophilin, from the ixodid tick, Boophilus microplus, which has 12 cysteines. Recombinant Amblin specifically inhibited thrombin as efficiently as native Amblin did. This is the first report of a thrombin inhibitor from tick haemolymph.

Amino Acid Sequence↗

PubMed2004

A new type of antimicrobial protein with multiple histidines from the hard tick, Amblyomma hebraeum.

A novel 11 kDa antimicrobial protein, named as hebraein, and having a unique amino acid sequence, was purified from the hemolymph of fed female Amblyomma hebraeum ticks. A full-length cDNA clone encoding hebraein was isolated from a cDNA library made from tick synganglia. Hebraein consists of 102 amino acids, including 6 cysteine residues; has 9 histidines in its C-terminal domain that are mainly present as HX repeats; and has no significant similarity to any known protein. The secondary structure prediction is very clearly all alpha-helical (4-6 helices) except for a very short extension at the C terminus. Such high alpha-helical content is quite different from known antimicrobial proteins. Recombinant hebraein and a mutant lacking the histidine residues in the C-terminal domain were constructed and expressed. Assayed at the slightly acidic pH equivalent of fed female tick hemolymph, the wild-type and the histidine-rich recombinant hebraein had stronger antimicrobial activities than the histidine-deficient mutant. The pH-dependent properties of histidine-rich antimicrobial proteins may allow the design of agents that would function selectively in specific pH environments. The results from protein profiling of hemolymph, analyzed by surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry combined with ProteinChip technology and RT-PCR analysis suggested that this antimicrobial protein was up-regulated by blood feeding. Our findings describe a new type of antimicrobial protein with multiple cysteine and histidine residues, and with unique secondary structure.

Amino Acid Sequence↗

PubMed2004

Two novel non-cationic defensin-like antimicrobial peptides from haemolymph of the female tick, Amblyomma hebraeum.

Two non-cationic defensin-like antimicrobial peptides, named Amblyomma defensin peptide 1 and Amblyomma defensin peptide 2, were identified from the hard tick, Amblyomma hebraeum, by a combination of suppression subtractive hybridization for differentially expressed genes and proteomics. cDNA clones encoding each of these two defensin-like antimicrobial peptides were isolated from the differentially expressed cDNA library of the tick synganglia (central nervous system). The preproproteins deduced from the cDNA sequences each have 92 amino acid residues. Amblyomma defensin peptide 2 was purified from the haemolymph of fed female ticks. The purified peptide displayed antibacterial activity against Gram-negative and Gram-positive bacteria. Amblyomma defensin peptide 1 was further identified by protein chip capture combined with SELDI-TOF (surface-enhanced laser desorption/ionization-time-of-flight) MS. By screening for differentially expressed proteins, it was found that the expression of Amblyomma defensin peptide 1 was upregulated during 4 days post-feeding. Our findings firstly provide two defensin-like antimicrobial peptides that are particularly novel in being anionic, together with corresponding cDNA sequences, in hard ticks, and prove that the combination of suppression subtractive hybridization and protein profiling is a powerful method to study differentially expressed proteins, especially for organisms without available genome sequence information.

Amino Acid Sequence↗

PubMed2003

Bombinakinin M gene associated peptide, a novel bioactive peptide from skin secretions of the toad Bombina maxima.

A novel 28-amino acid peptide, termed bombinakinin-GAP, was purified and characterized from skin secretions of the toad Bombina maxima. Its primary structure was established as DMYEIKQYKTAHGRPPICAPGEQCPIWV-NH(2), in which two cysteines form a disulfide bond. A FASTA search of SWISS-PROT databank detected a 32% sequence identity between the sequences of the peptide and a segment of rat cocaine- and amphetamine-regulated transcript (CART). Intracerebroventricular (i.c.v.) administration of the peptide induced a significant decrease in food intake in rats, suggesting that it played a role in the control of feeding by brain. Analysis of its cDNA structure revealed that this peptide is coexpressed with bombinakinin M, a bradykinin-related peptide from the same toad. Bombinakinin-GAP appears to be the first example of a novel class of bioactive peptides from amphibian skin, which may be implicated in feeding behavior.

Amino Acid Sequence↗

PubMed2003

Two novel Bv8-like peptides from skin secretions of the toad Bombina maxima.

Two novel bioactive peptides were purified from skin secretions of the toad Bombina maxima. The partial N-terminal sequences of these two peptides were determined by automated Edman degradation. This allowed the cloning of full-length cDNAs encoding these two peptides from a cDNA library prepared from the toad skin. The deduced complete amino acid sequences indicate that both peptides are composed of 77 amino acids. A FASTA search in the databanks revealed that they exhibit 86-91% sequence identity with Bv8, a peptide originally isolated from skin secretions of Bombina variegata. They were thus named as Bv8-like peptide 1 (Bv8-LP1) and Bv8-like peptide 2 (Bv8-LP2), respectively. Sequence differences between Bv8-LP1 and 2 were due to six amino acid substitutions at positions 6, 11, 23, 24, 62 and 63. Bv8-LP1 and 2 differed from Bv8 with eleven and seven amino acid substitutions, respectively. Like Bv8, Bv8-LP1 and 2 possessed contractile activity on isolated guinea pig ileum. Additionally, they stimulated contraction of rabbit aortic rings in a dose-dependent manner at nanomolar concentrations.

Amino Acid Sequence↗

PubMed2002

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 bacterial and fungal strains tested were 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 with cationic antimicrobial peptides, anionic antimicrobial peptides may also exist naturally as part of the innate defense system.

Amino Acid Sequence↗

PubMed2002

Antimicrobial peptides from skin secretions of Chinese red belly toad Bombina maxima.

Two groups of antimicrobial peptides have been isolated from skin secretions of Bombina maxima. Peptides in the first group, named maximins 1, 2, 3, 4 and 5, are structurally related to bombinin-like peptides (BLPs). Unlike BLPs, sequence variations in maximins occurred all through the molecules. In addition to the potent antimicrobial activity, cytotoxicity against tumor cells and spermicidal action of maximins, maximin 3 possessed a significant anti-HIV activity. Maximins 1 and 3 were toxic to mice with LD(50) values of 8.2 and 4.3 mg/kg, respectively. Peptides in the second group, termed maximins H1, H2, H3 and H4, are homologous with bombinin H peptides. cDNA sequences revealed that one maximin peptide plus one maximin H peptide derived from a common larger protein.

Amino Acid Sequence↗

PubMed2002

A novel proline rich bombesin-related peptide (PR-bombesin) from toad Bombina maxima.

A novel bombesin-related peptide was isolated from skin secretions of Chinese red belly toad Bombina maxima. Its primary structure was established as pGlu-Lys-Lys-Pro-Pro-Arg-Pro-Pro-Gln-Trp-Ala-Val-Gly-His-Phe-Met-NH(2.) The amino-terminal (N-terminal) 8-residue segment comprising four prolines and three basic residues is extensively different from bombesins from other Bombina species. The peptide was thus named proline rich bombesin (PR-bombesin). PR-bombesin was found to elicit concentration-dependent contractile effects in the rat stomach strip, with both increased potency and intrinsic activity as compared with those of [Leu(13)]bombesin. Analysis of different bombesin cDNA structures revealed that an 8 to 14- nucleotide fragment replacement in the peptide coding region (TGGGGAAT in the cDNAs of multiple bombesin forms from Bombina orientalis and CACCCCGGCCACCC in the cDNA of PR-bombesin) resulted in an unusual Pro-Pro-Arg-Pro-Pro motif in the N-terminal part of PR-bombesin.

Amino Acid Sequence↗

PubMed2002

Identification and cloning of a trypsin inhibitor from skin secretions of Chinese red-belly toad Bombina maxima.

A novel trypsin inhibitor was identified and purified from skin secretions of Chinese red-belly toad Bombina maxima. The partial N-terminal 29 amino acid residues of the peptide, named BMTI, were determined by automated Edman degradation. This allowed the cloning of a full-length cDNA encoding BMTI from a cDNA library prepared from the toad skin. The deduced complete amino acid sequence of BMTI indicates that mature BMTI is composed of 60 amino acids. A FASTA search in the databanks revealed that BMTI exhibits 81.7% sequence identity with BSTI, a trypsin/thrombin inhibitor from European toad Bombina bombina skin secretions. Sequence differences between BMTI and BSTI were due to 11 substitutions at positions 2, 9, 25, 27, 36-37, 39, 41-42, 50 and 56. BMTI potently inhibited trypsin with a K(i) value of 0.06 microM, similar to that of BSTI. However, unlike BSTI, which also inhibited thrombin with a K(i) value of 1 microM, no inhibitory effect of BMTI on thrombin was observed under the assay conditions.

Amino Acid Sequence↗

PubMed2002

Biological activities of skin secretions of the salamander Tylototriton verrucosus.

Water-soluble skin secretions of salamander Tylototriton verrucosus, first described by Anderson in 1871, were studied for their biological and enzymatic activities. They were found to be toxic to mice with an intraperitoneal LD50 of 11.5 mg/kg. Using Sephadex G-75 gel filtration, it was proven that the toxic components of the secretions are proteins with molecular weights ranging from 30,000 to 50,000 Da. The secretions of T. verrucosus display a wide spectrum of antimicrobial activities and also contain both proteolytic activity and trypsin inhibitory activity. In contrast, neither hemolytic nor hemorrhagic activities were found. The secretions were determined to have phospholipase A2 activity; however, no acetylcholine esterase activity was detectable under the assay conditions.

Animals↗