A cDNA encoding vitellogenin (Vg) in the giant freshwater prawn, Macrobrachium rosenbergii, was cloned based on the cDNA sequence of vitellin (Vn) fragments A-N and B-42 determined previously, and its amino acid sequence deduced. The open reading frame (ORF) encoded 2,537 amino acid residues and its deduced amino acid sequence possessed three consensus cleavage sites, R-X-R-R, similar to those reported in Vgs of insects. The deduced primary structure of Vg in M. rosenbergii was seen to be similar to that of Penaeus japonicus, especially in the N-terminal region. It is therefore likely that Vgs in crustacean species including prawns and other related decapods exhibit a similar structural pattern. Based on the deduced primary structure of Vg and analysis of the various Vg and Vn subunits found in the hemolymph and ovary during ovarian maturation, we demonstrated the post-translational processing of Vg in M. rosenbergii. This is the first time that Vg processing has been clearly demonstrated in a crustacean species. Vg, after being synthesized in the hepatopancreas, is considered to be cleaved by a subtilisin-like endoprotease to form two subunits, A and proB, which are then released into the hemolymph. In the hemolymph, proB is possibly cleaved by a processing enzyme of unknown identity to give rise to subunits B and C/D. The three processed subunits A, B, and C/D are sequestered by the ovary to give rise to three yolk proteins, Macr-VnA, VnB, and VnC/D.

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Defensin from a beetle, Allomyrina dichotoma, is known to have anti-bacterial activity against Gram-positive bacteria. This peptide, which comprises 43 amino acid residues, was effective against methicillin-resistant Staphylococcus aureus. We identified the active site of beetle defensin by measuring anti-bacterial activity against S. aureus of 64 overlapping 12-mer peptides with either a free carboxylate or a free amide group at their C-termini. An LCAAHCLAIGRR-NH2 (19L–30R-NH2) fragment showed the greatest activity of the synthetic oligopeptides. The 19L–30R-NH2 fragment was effective against both Gram-positive and Gram-negative bacteria. CD spectra showed that the 19L–30R-NH2 fragment formed an α-helical structure in the lipidic environment. The anti-bacterial effect of the 19L–30R-NH2 fragment was due to its interaction with bacterial membranes, judging from the leakage of liposome-entrapped glucose. Its anti-bacterial activity was increased when certain amino acid residues were replaced. Truncated peptides having had some amino acids removed from the N-terminus of the 19L–30R-NH2 fragment (8–10-mer peptides) still had strong anti-bacterial activity. Deleting some amino acids from the C-terminal region of the fragment dramatically reduced activity, indicating that the C-terminal region of the 19L–30R-NH2 fragment, i.e. RR-NH2, is important for exerting anti-bacterial activity. The AHCLAIGRR-NH2 (22A–30R-NH2) fragment and its analogues exhibited about 3-fold and 9–12-fold higher activity against S. aureus than did the 19L–30R-NH2 fragment, and these analogues were effective against methicillin-resistant S. aureus and Pseudomonas aeruginosa isolated from patients. These oligopeptides showed no haemolytic activity and did not inhibit the growth of murine fibroblast cells.

A novel member of the insect defensins, a family of antibacterial peptides, was purified from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros, immunized with Escherichia coli. A full-size cDNA was cloned by combining reverse-transcription PCR (RT-PCR), and 5 H -and 3 H -rapid amplification of cDNA ends (RACE). Analysis of the O. rhinoceros defensin gene expression showed it to be expressed in the fat body and hemocyte, midgut and Malpighian tubules. O. rhinoceros defensin showed strong antibacterial activity against Staphylococcus aureus. A 9-mer peptide amidated at its C-terminus, AHCLAICRK-NH 2 (Ala22±Lys30-NH 2 ), was synthesized based on the deduced amino-acid sequence, assumed to be an active site sequence by analogy with the sequence of a defensin isolated from larvae of the beetle Allomyrina dichotoma. This peptide showed antibacterial activity against S. aureus, methicillin-resistant S. aureus, E. coli and Pseudomonas aeruginosa. We further modified this oligopeptide and synthesized five 9-mer peptides, ALRLAIRKR-NH 2 , ALLLAIRKR-NH 2 , AWLLAIRKR-NH 2 , ALYLAIRKR-NH 2 and ALWLAIRKR-NH 2 . These oligopeptides showed strong antibacterial activity against Gram-negative and Gram-positive bacteria. The antibacterial effect of Ala22±Lys30-NH 2 analogues was due to its interaction with bacterial membranes, judging from the leakage of liposome-entrapped glucose. These Ala22±Lys30-NH 2 analogues did not show haemolytic activity and did not inhibit the growth of murine fibroblast cells or macrophages, except for AWLLAIRKR-NH 2 .Keywords: Oryctes rhinoceros; antibacterial peptide; cDNA cloning; gene expression; synthetic oligopeptides.Insect antibacterial peptides have unique properties that disrupt bacterial membranes via peptide±lipid interactions [1], and some are known to be effective against antibiotic-resistant pathogenic bacteria such as methicillin-resistant S. aureus (MRSA) and P. aeruginosa [2±4], suggesting their potential use as therapeutic agents. To develop novel antibiotics, many trials have been conducted using insect antibacterial peptides. Shortened cecropin A±mellitin hybrid 15-mer peptides, for example, have shown strong antibacterial activity [5]. Truncated antibacterial peptides have also been synthesized, and some of them were effective against bacteria [6±8]. Amphipathic a-helical regions have been identified as active sites of antibacterial peptides [9±11]. Modifications of these peptides can lead to greater and broader antibacterial activity than the original peptide [12,13]. The C-terminal b-sheet domain of an antibacterial peptide was found to be an active site and showed activity against fungi and Gram-positive and Gram-negative bacteria [14].We have designed and synthesized oligopeptides based on the amino-acid sequence of Allomyrina dichotoma defensin [15]. These oligopeptides (8-to 12-mer peptides) were effective against both Gram-positive and Gram-negative bacteria including MRSA and P. aeruginosa isolated from patients. The antibacterial effect was due to their interact...

The dynamics of vitellogenin mRNA expression during ovarian maturation in Macrobrachium rosenbergii were examined by measuring hemolymph vitellogenin (Vg) levels and Vg mRNA expression in the hepatopancreas and ovary at differing reproductive stages in both intact and eyestalk ablated animals. Vg mRNA was quantified using real-time RT-PCR and hemolymph Vg was measured by enzyme immunoassay. In intact animals, Vg mRNA levels in the hepatopancreas and hemolymph Vg levels showed a gradual increase during the molt cycle concomitant with increasing gonadosomatic index (GSI), with Vg levels decreasing prior to ecdysis although GSI continued to increase. Eyestalk ablation was seen to accelerate Vg synthesis as well as ovarian maturation, although it did not alter the overall pattern of Vg expression. Vg mRNA expression was negligible in the ovary of both intact and eyestalk ablated animals, confirming that the hepatopancreas is the principal site of Vg synthesis in M. rosenbergii with the ovary being only a minor contributor. This study has shown that Vg synthesis is correlated to ovarian maturation and the molt cycle in M. rosenbergii.

In order to determine the primary structure of vitellogenin in a protandric species, the coonstriped shrimp Pandalus hypsinotus, we previously purified four vitellin components (designated as VnA, VnB, VnC, and VnD, respectively), and chemically analyzed their partial amino acid sequences. In this study, we subsequently cloned a cDNA encoding vitellogenin in this species based on the N-terminal and internal amino acid sequences of VnA, as well as the N-terminal sequence of VnC. The open reading frame of this cDNA encoded a pro-vitellogenin in which vitellins were arranged as follows: NH2-VnA-VnB-VnC/D-COOH. The deduced amino acid sequence possessed a single consensus cleavage sequence, R-X-K/R-R, along the lines of vitellogenins reported in other crustaceans and insects, and the N-terminal sequence of VnB was immediately preceded by this sequence. The comparison of primary structures revealed the existence of a basic and characteristic structure for the vitellogenin molecule in decapod crustacean species, and phylogenetic analysis reflected the current taxonomic classifications of Crustacea. An approximately 8 kb-long transcript of the vitellogenin gene was detected in the hepatopancreas of female shrimps having a gonadosomatic index higher than 1.0 by Northern blot analysis, but was not observed in the hepatopancreas and gonads of male shrimps and the hepatopancreas of female shrimps having a gonadosomatic index lower than 1.0. These results indicate that the hepatopancreas is responsible for vitellogenin synthesis.

Defensin from a beetle, Allomyrina dichotoma, is known to have anti-bacterial activity against Gram-positive bacteria. This peptide, which comprises 43 amino acid residues, was effective against methicillin-resistant Staphylococcus aureus. We identified the active site of beetle defensin by measuring anti-bacterial activity against S. aureus of 64 overlapping 12-mer peptides with either a free carboxylate or a free amide group at their C-termini. An LCAAHCLAIGRR-NH2 (19L-30R-NH2) fragment showed the greatest activity of the synthetic oligopeptides. The 19L-30R-NH2 fragment was effective against both Gram-positive and Gram-negative bacteria. CD spectra showed that the 19L-30R-NH2 fragment formed an alpha-helical structure in the lipidic environment. The anti-bacterial effect of the 19L-30R-NH2 fragment was due to its interaction with bacterial membranes, judging from the leakage of liposome-entrapped glucose. Its anti-bacterial activity was increased when certain amino acid residues were replaced. Truncated peptides having had some amino acids removed from the N-terminus of the 19L-30R-NH2 fragment (8-10-mer peptides) still had strong anti-bacterial activity. Deleting some amino acids from the C-terminal region of the fragment dramatically reduced activity, indicating that the C-terminal region of the 19L-30R-NH2 fragment, i.e. RR-NH2, is important for exerting anti-bacterial activity. The AHCLAIGRR-NH2 (22A-30R-NH2) fragment and its analogues exhibited about 3-fold and 9-12-fold higher activity against S. aureus than did the 19L-30R-NH2 fragment, and these analogues were effective against methicillin-resistant S. aureus and Pseudomonas aeruginosa isolated from patients. These oligopeptides showed no haemolytic activity and did not inhibit the growth of murine fibroblast cells.

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