Antimicrobial peptides, isolated from horseshoe crab hemocytes, tachyplesin II, and polyphemusins I and II: chemical structures and biological activity

Tachyplesin is an antimicrobial peptide recently found in the acid extract of hemocytes from the Japanese horseshoe crab (Tachypleus tridentatus) [Nakamura, T. et al. (1988) J. Biol. Chem. 263, 16709-16713]. In our continuing studies on the peptide, we have found an isopeptide, tachyplesin II, and also polyphemusins I and II in hemocytes of the American horseshoe crab (Limulus polyphemus). The complete primary structures of these peptides, which are very similar to that of the previously isolated peptide, now named tachyplesin I, were determined to be as follows: (Table: see text). The isopeptide, tachyplesin II, consists of 17 residues with a COOH-terminal arginine alpha-amide. On the other hand, both polyphemusins I and II were found to contain 18 residues due to an additional Arg residue at the NH2-terminal end as well as a COOH-terminal arginine alpha-amide. The disulfide linkages for polyphemusin I consisted of two bridges between Cys-4 and Cys-17 and between Cys-8 and Cys-13, which was identical to in the case of tachyplesin I. Moreover, all of these peptides inhibited the growth of not only Gram-negative and -positive bacteria but also fungi, such as Candida albicans M9. Furthermore, complex formation between these peptides and bacterial lipopolysaccharides was also observed in a double diffusion test. These results suggest that tachyplesins and polyphemusins are probably located in the hemocyte membrane, where they act on antimicrobial peptides as a self-defense mechanism in the horseshoe crab against invading microorganisms.     

Antimicrobial tachyplesin peptide precursor. cDNA cloning and cellular localization in the horseshoe crab (Tachypleus tridentatus)

The hemocytes of the horseshoe crab have been found to contain a new family of Arthropodous antibiotics, termed the "tachyplesin family." These peptides are composed of 17-18 amino acid residues with a carboxyl-terminal arginine alpha-amide. We report here the entire cDNA sequence coding for the tachyplesin precursors and their distribution in various tissues of the horseshoe crab. Sequence analysis of the cloned cDNAs revealed that the tachyplesin precursors consist of 77 amino acids with 23 residues in a presegment, and that there are two types of mRNAs corresponding to the isopeptides tachyplesins I and II. Both precursors contain a putative signal peptide, a processing peptide sequence and a carboxyl-terminal amidation signal "Gly-Lys-Arg" connected to the mature tachyplesin peptide. Moreover, an unusual acidic amino acid cluster, Asp-Glu-Asp-Glu-Asp-Asp-Asp-Glu-Glu-COOH, is present in the carboxyl-terminal portions of both precursors. These results suggest that the two types of tachyplesin precursors are first synthesized as preproproteins and are then incorporated into the intracellular organelle, accompanied by various processing events. Northern blot analysis on a total RNA from various tissues of the horseshoe crab revealed that the tachyplesin precursors are expressed mainly in hemocytes and cardiac and brain tissues. Tachyplesin was immunohistochemically localized in the smaller dense granules rather than the typical large granules present in abundance in the hemocytes.     

Functional conversion of hemocyanin to phenoloxidase by horseshoe crab antimicrobial peptides

Arthropod hemocyanins and phenoloxidases serve different physiological functions as oxygen transporters and enzymes involved in defense reactions, respectively. However, they are equipped with a structurally similar oxygen-binding center. We have shown that the clotting enzyme of the horseshoe crab, Tachypleus tridentatus, functionally converts hemocyanin to phenoloxidase by forming a complex without proteolytic cleavage (Nagai, T., and Kawabata, S. (2000) J. Biol. Chem. 275, 35297-35301). Here we show that chitin-binding antimicrobial peptides of the horseshoe crab induce the intrinsic phenoloxidase activity of hemocyanin. Tachyplesin, a major Tachypleus antimicrobial peptide with an amphiphilic structure, converted the hemocyanin to phenoloxidase. Surface plasmon resonance analysis revealed the specific interaction of tachyplesin with hemocyanin at K(d) = 3.4 x 10(-)6 m. The chemical modification of Trp or Tyr in tachyplesin, but not Lys or Arg, dramatically reduced the affinity to hemocyanin, suggesting that the binding site is located in the hydrophobic face of tachyplesin. Hemocyanin has no affinity with chitin, but it significantly binds to tachyplesin-coated chitin, leading to the expression of phenoloxidase activity. The chitin coated with antimicrobial peptides may serve as a scaffold for the binding of hemocyanin, and the resulting phenoloxidase activity appears to function as a trigger of exoskeleton wound healing.     

Antimicrobial peptide, tachyplesin I, isolated from hemocytes of the horseshoe crab (Tachypleus tridentatus). NMR determination of the beta-sheet structure

The conformation of tachyplesin I, an antimicrobial cationic peptide of 17 residues found in the hemocyte debris of horseshoe crab, was investigated using two-dimensional NMR spectroscopy. The 1H NMR spectrum of tachyplesin I in aqueous solution could be completely assigned, and the secondary structure was substantiated by interpretation of the nuclear Overhauser effect, coupling constant, amide exchange rate, and temperature dependence of the amide chemical shift. Tachyplesin I takes on a fairly rigid conformation constrained by two disulfide bridges and adopts a conformation consisting of an anti-parallel beta-sheet (residues 3-8 and 11-16) connected by a beta-turn (residues 8-11). In this planar conformation, five bulky hydrophobic side groups are localized in one side of the plane and six cationic side groups are distributed at the "tail" part of the molecule (residues 1-5 and 14-17). This amphipathic structure of the molecule is presumed to be closely associated with the bactericidal activity.     

Isolation and characterization of gomesin, an 18-residue cysteine-rich defense peptide from the spider Acanthoscurria gomesiana hemocytes with sequence similarities to horseshoe crab antimicrobial peptides of the tachyplesin family

We have purified a small size antimicrobial peptide, named gomesin, from the hemocytes of the unchallenged tarantula spider Acanthoscurria gomesiana. Gomesin has a molecular mass of 2270.4 Da, with 18 amino acids, including a pyroglutamic acid as the N terminus, a C-terminal arginine alpha-amide, and four cysteine residues forming two disulfide bridges. This peptide shows marked sequence similarities to antimicrobial peptides from other arthropods such as tachyplesin and polyphemusin from horseshoe crabs and androctonin from scorpions. Interestingly, it also shows sequence similarities to protegrins, antimicrobial peptides from porcine leukocytes. Gomesin strongly affects bacterial growth, as well as the development of filamentous fungi and yeast. In addition, we showed that gomesin affects the viability of the parasite Leishmania amazonensis.     

The amino acid sequence of coagulogen isolated from southeast Asian horseshoe crab, Tachypleus gigas

The amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab (Tachypleus gigas) has been determined. The NH2-terminal sequence of the first 51 residues was obtained by automated Edman degradation. The intact protein was then treated with a Tachypleus clotting enzyme, to form a gel and to remove an internal peptide C (28 residues) located near the NH2-terminal portion. The gel protein, which consisted of A chain (18 residues) and B chain (129 residues), was S-alkylated and the resulting two chains were separated by acetone precipitation. Among these segments, A chain and peptide C were assigned to the NH2-terminal portion of whole coagulogen, as judged from their amino acid compositions. On the other hand, the covalent structure of B chain was determined by sequencing the peptides obtained from its tryptic digest. The alignments of the tryptic peptides were deduced from the sequence homology in comparison with the previously established B chain sequence of Japanese horseshoe crab (T. tridentatus) coagulogen. T. gigas coagulogen had a total of 175 amino acids and a calculated molecular weight of 19,770. When the sequence was compared with those of Japanese and American horseshoe crab (Limulus polyphemus) coagulogens, extensive structural homology was found: T. tridentatus/T. gigas, 87% and L. polyphemus/T. gigas, 67%. This comparison suggests that Japanese and Southeast Asian horseshoe crabs have a crab, based on amino acid sequence data.     

基于MAXENT模型评估北部湾潮间带中国鲎和圆尾鲎稚鲎的潜在地理分布及种群保育对策

鲎具有极高的经济价值和科研意义。近年来由于过度捕捞和栖息地受损等原因,亚洲鲎种群数量正急剧下降。鲎漫长的生命周期使得鲎资源的保护和增殖迫在眉睫。生态位模型已经广泛应用于物种的潜在地理分布预测。基于实地调研数据和公开发表的北部湾中国海域中国鲎和圆尾鲎地理分布数据,运用MAXENT模型得到中国鲎和圆尾鲎在广西北部湾(中国部分)的栖息地适宜度指数(Habitat suitability index, HSI),确定了这两种稚鲎在北部湾中国海域潜在适生区。模型分析结果表明,潮间带坡度和地形指数是影响中国鲎分布的主要环境因子,而潮间带底质的有机物含量和植被指数是影响圆尾鲎分布的主要环境因子,根据研究结果建议在两种稚鲎适生区建立保护区,进行人工放流稚鲎,加强对海草和红树林的生态建设,进而促进鲎资源种群恢复和发展。     

Modified Cysteine-Deleted Tachyplesin (CDT) Analogs as Linear Antimicrobial Peptides: Influence of Chain Length,Positive Charge,and Hydrophobicity on Antimicrobial and Hemolytic Activity

Due to increasing resistance of bacteria to traditional antibiotics, antimicrobial peptides are being investigated as a promising alternative. Tachyplesin, an antimicrobial peptide isolated from horseshoe crab, inhibits the growth of many different types of bacteria with its ability to permeabilize the cell membrane. Starting with a previously reported linear tachyplesin analog lacking cysteine (cysteine-deleted tachyplesin, CDT, KWFRVYRGIYRRR-CONH₂), this study examines the systematic deletion of the C-terminal arginines and the N-terminal lysine, addition of positively charged N-and C-terminal residues, replacement of arginine with similarly-charged lysine, and replacement of hydrophobic residues with aliphatic, aromatic, fluoro-substituted aromatic, and bicyclic amino acids to examine effects on activity. The 16 modified CDT analogs were tested for their ability to disrupt model liposomes, and minimum inhibitory concentrations were determined for gram-positive and gram-negative bacterial strains. Hemolytic activity also was assessed. Overall, results indicate that elimination of two C-terminal arginine residues results in a peptide ([des-Arg^12,13]CDT) with preserved antimicrobial activity but a reduction in hemolysis, a selectivity desirable for a therapeutic agent. Additional deletion was not tolerated, nor was addition of residues at the termini. Analysis of the 16 analogs also reveals the importance of hydrophobicity, not necessarily aromaticity, as an analog with hydrophobic isoleucine residues placed throughout the sequence ([Ile^2,3,6,10]CDT) displayed comparable antimicrobial activity to CDT with lower hemolysis, representing a promising antimicrobial peptide with lowered toxicity.     

Temporal and spatial patterns of organic carbon are linked to egg deposition by beach spawning horseshoe crabs (<Emphasis Type="Italic">Limulus polyphemus</Emphasis>)

The spring spawning by the American horseshoe crab (Limulus polyphemus L.) results in temporally and spatially discrete inputs of eggs onto sandy beaches in Delaware Bay, USA. We tested the hypothesis that seasonal patterns of sediment organic carbon on Delaware Bay beaches is linked to this pulsed input of horseshoe crab eggs. At a location with minimal horseshoe crab spawning activity (Higbee Beach), there was little seasonal variation in sediment organic carbon, no distinction between organic carbon levels as a function of shoreline position or sediment depth, and no significant correlation between the abundance of crab eggs and percent organic carbon. Conversely, at a prime horseshoe crab spawning habitat (North Beach), organic carbon levels were seasonally pulsed and were correlated with egg abundance. Moreover, the strongest evidence of seasonality was seen at the middle foreshore location at the 15–20 cm depth, consistent with the highest input of horseshoe crab eggs. Although some of the organic carbon contributed by horseshoe crab eggs in May–June leaves the beach in the form of hatched larvae later in the year, there is a net input of organic carbon to the system in the form of unfertilized and/or dead eggs, egg membranes, and embryonic molts. We suggest that the inputs of eggs from horseshoe crabs and other beach spawning animals, such as grunion and capelin, make significant contributions to the energy budget of sandy beaches.     

Tachyplesin and CyLoP-1 as efficient anti-mycobacterial peptides: A novel finding

Mycobacterium tuberculosis is an etiological agent of tuberculosis (TB) known to be a highly contagious disease and is the major cause of mortality from a single infectious agent worldwide. Emergence of multi-drug resistant and extremely drug resistant strains of M. tuberculosis has made TB management extremely challenging eliciting the urgent need for alternative therapeutics. Peptide based therapeutic strategies are an emerging area that can be employed as a prospective alternative to the currently existing therapeutic regime for TB treatment. Here, we are reporting the anti-mycobacterial activity of two peptides, Tachyplesin and CyLoP-1, derived from marine horseshoe crab and snake toxin respectively, with potent anti-mycobacterial activity against various mycobacterium species. Both the peptides exhibit appreciable antimicrobial and anti-biofilm activities against mycobacterium species with minimum cytotoxicity towards macrophage cells. They are also effective in eliminating mycobacterium cells from infected macrophage cells. Tachyplesin acts on mycobacterium cells in a lytic manner with outer membrane disruption confirmed by propidium iodide uptake with slight membrane depolarization and reactive oxygen species (ROS) production. CyLoP-1, on the other hand, does not rupture the mycobacterium cells even at high concentrations. It seems to follow intracellular pathway of killing mycobacterium cells by production of more ROS and membrane depolarization. Both the peptides do not lead to apoptotic way of mycobacterium cell death. These results suggest an effective peptide-based antimicrobial strategy for development of future anti-TB therapeutics.     

Complete cDNA sequence of SAP-like pentraxin from Limulus polyphemus: implications for pentraxin evolution

The serum amyloid P component (SAP)-like pentraxin Limulus polyphemus SAP is a recently discovered, distinct pentraxin species, of known structure, which does not bind phosphocholine and whose N-terminal sequence has been shown to differ markedly from the highly conserved N terminus of all other known horseshoe crab pentraxins. The complete cDNA sequence of Limulus SAP, and the derived amino acid sequence, the first invertebrate SAP-like pentraxin sequence, have been determined. Two sequences were identified that differed only in the length of the 3' untranslated region. Limulus SAP is synthesised as a precursor protein of 234 amino acid residues, the first 17 residues encoding a signal peptide that is absent from the mature protein. Phylogenetic analysis clusters Limulus SAP pentraxin with the horseshoe crab C-reactive proteins (CRPs) rather than the mammalian SAPs, which are clustered with mammalian CRPs. The deduced amino acid sequence shares 22% identity with both human SAP and CRP, which are 51% identical, and 31-35% with horseshoe crab CRPs. These analyses indicate that gene duplication of CRP (or SAP), followed by sequence divergence and the evolution of CRP and/or SAP function, occurred independently along the chordate and arthropod evolutionary lines rather than in a common ancestor. They further indicate that the CRP/SAP gene duplication event in Limulus occurred before both the emergence of the Limulus CRP variants and the mammalian CRP/SAP gene duplication. Limulus SAP, which does not exhibit the CRP characteristic of calcium-dependent binding to phosphocholine, is established as a pentraxin species distinct from all other known horseshoe crab pentraxins that exist in many variant forms sharing a high level of sequence homology.     

Isolation and Characterization of a Sialic Acid-specific Lectin from Hemolymph of the Southeast Asian Horseshoe Crab Tachypleus gigas

A lectin was isolated from hemolymph of the Southeast Asian horseshoe crab Tachypleus gigas by using glycophorin HA affinity chromatography and Sephacryl S-300 gel filtration. The purified lectin had a molecular mass of approximately 396 kDa and was composed of 13 identical subunits with molecular masses of 31 kDa. The serological specificity of the purified lectin was specifically inhibited by sialic acids sialoglycoproteins, but not by neutral sugars, hexosamines, N-acetylhexosamines, or asialoglycoproteins. Although the N-terminal amino acid sequence of the lectin from T. gigas was identical to that from American horseshoe crab (liphemin) by the same purification method and cross reacted with the anti-liphemin serum, the calcium concentration of hemagglutinating activity of the purified lectin showed a smaller optimal concentration than that of liphemin.     

Induction of Secondary Embryos by Intra- and Interspecific Grafts of Center Cells under the blastopore in Horseshoe Crabs

When the cell mass (center cells) of the early gastrulae in both American and Asian horseshoe crabs was grafted into the embryo of the homologous species, secondary embryos were formed as a result of these grafts. Secondary embryos were also formed in a similar way when the center cells of heterologous embryos were grafted between the American and Asian horseshoe crab embryos. The characteristics of the secondary embryos were similar to the host embryos in both cases, indicating that the center cells played the roles same as those by the amphibian organizer. The homogenate of center cells also induced the formation of secondary embryos. This is the first published study in which secondary embryos of horseshoe crabs have been induced by grafting. The fact may mean that this type of embryonic induction is widespread in the animal kingdom.     

Characterization of a complementary deoxyribonucleic acid for the coagulogen of Limulus polyphemus

An 869-nucleotide-long cDNA clone for the coagulogen from Limulus amebocyte has been isolated and its nucleotide sequence has been determined. The deduced amino-acid sequence revealed a signal peptide, 20 amino acids long, and a mature protein of 175 amino acids. The amino-acid sequence of the coagulogen was compared to all known proteins by two computer programs. Using these programs, Limulus coagulogen showed 70% homology with the coagulogen of Tachypleus tridentatus (Japanese horseshoe crab). Further computer analysis showed no statistically significant homology to support an evolutionary origin of the horseshoe crab coagulogen common to other protein families. These results place horseshoe crab coagulogen in a new superfamily unrelated to any other proteins investigated. RNA blot analysis of Limulus RNA indicated that the coagulogen mRNA was about 900 bases long and represented an abundant species in the amebocyte while detected only in small quantities in the hepatopancreas. Besides mature RNA, high-molecular-weight forms of coagulogen RNA were also observed. Southern blot analysis of Limulus DNA digested with restriction endonucleases suggested that the Limulus coagulogen gene contains at least three introns, or belongs to a multigene family.     

Solution and micelle-bound structures of tachyplesin I and its active aromatic linear derivatives

Tachyplesin I is a 17-residue peptide isolated from the horseshoe crab, Tachypleus tridentatus.It has high antimicrobial activity and adopts a beta-hairpin conformation in solution stabilized by two cross-strand disulfide bonds. We report an NMR structural investigation of wild-type tachyplesin I and three linear derivatives (denoted TPY4, TPF4, and TPA4 in which the bridging cysteine residues are uniformly replaced with tyrosine, phenylalanine, and alanine, respectively). The three-dimensional aqueous solution structures of the wild type and the active variant TPY4 reveal very similar beta-hairpin conformations. In contrast, the inactive variant TPA4 is unstructured in solution. The arrangement of the tyrosine side chains in the TPY4 structure suggests that the beta-hairpin is stabilized by aromatic ring stacking interactions. This is supported by experiments in which the beta-hairpin structure of TPF4 is disrupted by the addition of phenol, but not by the addition of an equimolar amount of cyclohexanol. We have also determined the structures of wild-type tachyplesin I and TPY4 in the presence of dodecylphosphocholine micelles. Both peptides undergo significant conformational rearrangement upon micelle association. Analysis of the micelle-associated peptide structures shows an increased level of exposure of specific hydrophobic side chains and an increased hydrophobic integy moment. Comparison of the structures in micelle and aqueous solution for both wild-type tachyplesin I and TPY4 reveals two requirements for high antimicrobial activity: a beta-hairpin fold in solution and the ability to rearrange critical side chain residues upon membrane association.     

Further studies on lipopolysaccharide-sensitive serine protease zymogen (factor C): its isolation from Limulus polyphemus hemocytes and identification as an intracellular zymogen activated by alpha-chymotrypsin, not by trypsin

An intracellular serine protease zymogen, factor C, is an initiator in the hemolymph coagulation system of horseshoe crab. We purified this zymogen from the hemocytes of the American horseshoe crab, Limulus (L.) polyphemus, the objective being to compare its properties with those of the Japanese horseshoe crab, Tachypleus (T.) tridentatus, factor C. The purified zymogen L.-factor C showed similar properties to those of T.-factor C, in terms of molecular mass (123,000), amino acid composition (1,011 residues), subunit structure (two chains), and antigenicity. Like the zymogen T.-factor C, this zymogen was also activated autocatalytically in the presence of bacterial lipopolysaccharide (LPS) and its synthetic lipid A analogue. A most interesting finding is that both protease zymogens are rapidly activated by alpha-chymotrypsin or rat mast cell chymase, but not by trypsin. The active enzyme factor C showed alpha-thrombin-like specificity toward synthetic tripeptide substrates. This factor C was also strongly inhibited by an alpha-thrombin inhibitor, D-Phe-Pro-Arg-chloromethyl ketone. Thus, the enzymatic properties of factor C are similar to those of mammalian alpha-thrombin. On the other hand, the coagulation cascade system present in the hemocyte lysate was activated when chymotrypsin, free from LPS, was added to the lysate used to detect the endotoxins. The implication of our findings is that the chymotrypsin-catalyzed initiation of the horseshoe crab coagulation system is unique, since all known mammalian coagulation, fibrinolysis and complement systems are initiated by trypsin-like enzymes.     

Design,synthesis and antibacterial activity of a class of novel molecular engineering analogues of Tachyplesin I

Tachyplesin I (TP-I) is a marine-derived antimicrobial peptide isolated from the hemocyte membrane of Japanese horseshoe crab (Tachypleus tridentatus) with broad antimicrobial spectrum. It consists of 17 amino acid residues and shows a characteristic structure with three tandem repeats of a tetrapeptide sequence. The three tandem repeats of a tetrapeptide sequence are the key components of the amphipathic β-hairpin structure of TP-I and we proposed that these fragments were critical for the antimicrobial activity, and designed a class of novel molecular engineering analogs of TP-I via re-combination of the three tetrapeptide fragments with different numbers of disulfide bonds in a search of therapeutically valuable lead compound. The synthesis of cyclic peptides with 1/2 disulfide bond(s) are challenging and we successfully prepared most of them using the orthogonal Cys protection strategy. The antimicrobial assay result revealed that the molecular engineering analogs of TP-I via fragment re-combination with 2 disulfide bonds maintained the antimicrobial potency across the range of microbes and confirmed the importance of tetrapeptide fragments and disulfide bonds. Of particular interest, analogue TP-I-R2 exhibited good antimicrobial activity against most of the tested microbes with MIC ranging from 4 to 128 μg/mL. Moreover, TP-I-R1, TP-I-R1b and TP-I-R3 particularly exhibited higher potency of more than 16/16/8-fold against Grampositive Bacillus pumilus strains than TP-I, TP-I-R2 and TP-I-R4 showed comparable potency against Gramnegtive Salmonella typhimurium strains with TP-I. These compounds may be considered as promising lead for further development toward new treatments for infections caused by multiple bacterial pathogens.     

Biomolecular characterization of allergenic proteins in snow crab (Chionoecetes opilio) and de novo sequencing of the second allergen arginine kinase using tandem mass spectrometry

Snow crab (Chionoecetes opilio) proteins have been recognized as an important source of both food and occupational allergens. While snow crab causes a significant occupational allergy, only one novel allergen has recently been fully characterized. The muscle proteins from snow crab legs were profiled by SDS-PAGE. Several of these proteins were characterized using tandem mass spectrometry. Five proteins were identified; sarcoplasmic Ca-binding (20kDa), arginine kinase (40), troponin (23kDa) and α-actine (42kDa) and smooth endoplasmic reticulum Ca(2+)ATPase (113kDa). Immunoblotting using serum of sixteen allergic patients resulted in strong reactivity with the 40-kDa protein in seven patients (43%). This protein was purified by chromatography and subsequently de novo sequenced using matrix assisted laser desorption ionization and electrospray tandem mass spectrometry. We identified a second important allergen, arginine kinase, in snow crab, designated Chi o 3. Based on identity and homology analysis, using bioinformatics tools, a signature peptide was identified as a chemical surrogate for arginine kinase. The suitability of this signature peptide was tested for analytically representing the arginine kinase, by performing a multi-reaction monitoring tandem mass spectrometry approach on actual air filter samples collected from a simulated crab processing plant.     

Identification of the C-terminally alpha-amidated amino acid in peptides by high-performance liquid chromatography

A sensitive method for the rapid identification of the C-terminally amidated amino acid in peptides is described. Peptides containing the alpha-amide group at the C-terminus were cleaved with endopeptidases. The fragments released (oligopeptides, amino acids and the C-terminally amidated residue) are coupled to phenylisothiocyanate. The phenylthiocarbamoyl derivative of the amino acid alpha-amide is selectively extracted from the mixture by alkaline butyl acetate and identified by a high-performance liquid chromatography system that enables rapid and complete separation of the derivatives of 17 amino acid amides at a detection limit of 20-50 pmol. The C-terminal alpha-amides of neurokinin-A (Met-NH2), mammalian secretin (Val-NH2), pancreatic polypeptide (Tyr-NH2) and peptide HI (Ile-NH2) are unequivocally determined at a level of 0.5-2 nmol per peptide. This method was used to characterize a crude peptide fraction prepared from porcine brain. Cholecystokinin-58 was identified in this fraction by detection of phenylthiocarbamoyl-phenylalaninamide. The method is suitable for the identification of the C-terminal alpha-amidated residue of purified peptides, but can also be used as a screening strategy to isolate from complex biological extracts novel peptides containing an alpha-amidated amino acid at the C-terminus.     

Sufficiency of horseshoe crab eggs for red knots during spring migration stopover in Delaware Bay USA

Horseshoe crab (Limulus polyphemus) eggs are a dietary staple of the red knot (Calidris canutus) during its spring stopover on the Delaware Bay. Numbers of knots stopping in Delaware Bay declined in the 1990s concurrent with a decline in horseshoe crabs, leading to the hypothesis that reduced horseshoe crab egg abundance limited the red knot population. Management efforts, including a seasonal harvest moratorium in the Delaware Bay, have been instituted to restore crab populations to levels of sustainable use by multiple users, including migratory birds. Our objective was to evaluate the sufficiency of horseshoe crab eggs in Delaware Bay in May–June 2004 and 2005 for knots to refuel for their migratory flight to the Arctic breeding grounds. We examined egg counts to determine if there were fewer high egg-density sites later than earlier in the day and season, as migrating birds might deplete this resource. We studied foraging rates at red knot locations to determine if foraging probes increased with time of day and season as birds depleted surface eggs by pecking, then began probing for subsurface eggs. Finally, we experimentally tested whether red knots and their competitors depleted horseshoe crab eggs. Crab egg numbers at knot foraging sites did not decline throughout the day or season in 2004. In both years, we found no evidence that knots switched from pecking to probing with increases in time since sunrise or start of the stopover. Egg numbers were similar in exclosed and accessible plots on crab nesting depressions and in areas of open intertidal zone, but were significantly lower in accessible than in exclosed plots in the wrack line. Our results indicate that horseshoe crab eggs in Delaware Bay were sufficient to support the refueling of the present-day stopover population of red knots. If an increase in the availability of crab eggs to foraging birds does not result in an increase in knot numbers, managers must prioritize mitigation of limiting factors at other historically important spring stopovers and on the poorly understood breeding and wintering grounds in addition to the Delaware Bay. © 2011 The Wildlife Society.