Location of intrinsic and inducible phenoloxidase activity in molluscan hemocyanin

The phenoloxidase (PO) activity of the hemocyanins (Hcs) from two molluscan species, the gastropod Helix pomatia (Hp) and the cephalopod Sepia officinalis (So), was studied. With catechol as substrate the Hcs showed a weak o-diPO activity, which was moderately enhanced on limited proteolysis with subtilisin. The sites in the Hc molecules mainly responsible for this activity were identified. The highest intrinsic o-diPO activity and also by far the highest level of induction were found in the functional units (FUs) Hp f and So g, isolated from Hp beta-Hc and So Hc (subunit 2), respectively. The results thus support the earlier conclusion, made on the basis of sequence homology between molluscan Hcs, that Hp f and So g are functional and structural analogues. The subtilisin treatment of Hp f also induced monoPO activity, considered to be at the origin of browning of the sample.     

Characterization of two crustin antimicrobial peptides from the freshwater crayfish Pacifastacus leniusculus

The two bacteria-induced crustin genes, Plcrustin1 and Plcrustin2, previously found in the hemocyte cDNA library of Pacifastacus leniusculus, contain the open reading frames of 357 bp encoding a putative protein of 118 amino acid residues and 330 bp encoding a putative protein of 109 amino acid residues, respectively. The carboxyl-terminal part of the two crustins possesses, respectively, 7 and 8 conserved cysteine residues representation of a WAP domain that is found in carcinins and crustins in other several crustaceans. The amino acid sequences of Plcrustin1 and Plcrustin2 show that they belong to type I crustins. In order to characterize their properties and biological activities, the two recombinant crustin proteins were produced in the Escherichia coli expression system. Antimicrobial assays showed that the growth of only one Gram-positive bacterium, Micrococcus luteus M1 11, was inhibited by the recombinant Plcrustin1 and Plcrustin2 with MIC of about 0.07-0.27 μM and 3.5-8 μM, respectively. In addition, the study of inhibition mechanism revealed that the antimicrobial activity of the two recombinant crustin proteins was a result of bactericidal effect. However, the two crustins did not exhibit the inhibitory activities against trypsin, chymotrypsin, elastase and subtilisin A.     

Types and arrangements of neurons in the crayfish optic lamina

Cell and Tissue Research - The neural arrangements in the optic lamina of the crayfish Pacifastacus leniusculus Dana have been studied by light microscopy by means of silver impregnation...     

Latent phenoloxidase activity and N-terminal amino acid sequence of hemocyanin from Bathynomus giganteus,a primitive crustacean

N-terminal amino acid sequences for the two hemocyanin subunits from the deep-sea crustacean Bathynomus giganteus have been determined by Edman degradation, providing the first sequence information for a hemocyanin from an isopod. In addition, purified hemocyanin from B. giganteus exhibited phenoloxidase activity in the presence of sodium dodecyl sulfate. Although a natural activator has not yet been identified, a preliminary study of the enzyme indicated a K(m) of 5mM for dopamine and an initial rate of 0.1 micromol per min per mg protein, values consistent with a significant role for this enzyme in the innate immune system of B. giganteus. Moreover, after separation of hemolymph by alkaline polyacrylamide gel electrophoresis, the only detectable phenoloxidase activity coincided with the two hemocyanin subunits. The hemocyanin of this primitive crustacean may fulfill dual functions, both as oxygen carrier and as the phenoloxidase crucial for host defense.     

红螯螯虾酚氧化酶原及其特性研究

采用同源克隆策略和RACE技术, 从红螯螯虾Cherax quadricarinatus血细胞中克隆得到酚氧化酶原基因的全长cDNA序列, 共2951 bp, 开放读码框为1995 bp, 编码665个氨基酸. 预测的分子量和等电点分别为75.7 kD和6.23. 酚氧化酶原含有两个推测的tyrosinase copper-binding motifs (带有六个组氨酸残基)和一个thiol-ester-like motif, 这些特征和其他甲壳动物的酚氧化酶原特征相同. 红螯螯虾酚氧化酶原氨基酸序列与通讯螯虾Pacifastacus leniusculus、欧洲龙虾Homarus gammarus、美洲龙虾Homarus americanus 和克氏原螯虾Procambarus clarkii 酚氧化酶原的相似率分别为68%、63%、63%和59%. 酚氧化酶原基因双酶切后连接入pET-28a原核表达载体, 转化到大肠杆菌BL21后重组表达酚氧化酶原蛋白. 在重组蛋白纯化后, 免疫新西兰大耳兔制备得到的酚氧化酶原多克隆抗体, 其效价大于1:12800. 红螯螯虾血淋巴、肝和鳃组织中的酚氧化酶原mRNA表达和酚氧化酶活性较高, 而神经、心、肠和肌肉中较低. 中华绒螯蟹螺原体和嗜水气单胞菌免疫红螯螯虾后, 血淋巴细胞、肝和鳃组织中的酚氧化酶原和酚氧化酶活性在免疫后的不同时间均出现了显著性的增加, 此结果表明酚氧化酶原和酚氧化酶在红螯螯虾对抗细菌感染的过程中起到重要的免疫作用. 此结果为进一步深入研究酚氧化酶原基因和酚氧化酶的功能及其调控机理奠定基础.       

Diurnal variations in physiology and behaviour of the noble crayfish Astacus astacus and the signal crayfish Pacifastacus leniusculus

Heart rate, locomotor activity, and oxygen consumption were recorded simultaneously and continuously in seven individuals of the noble crayfish Astacus astacus (Linneus 1758) and seven individuals of the signal crayfish Pacifastacus leniusculus (Dana 1852). The recordings were made in the laboratory over 7 days at 15°C under a 12?:?12?h dark?:?light regime. Circadian rhythms in heart rate, locomotor activity and oxygen consumption were found both in A. astacus and P. leniusculus. Increased heart rate, locomotor activity, and oxygen consumption levels during night time in both A. astacus and P. leniusculus illustrated expression of nocturnal behaviour. No differences in oxygen consumption levels were observed between A. astacus and P. leniusculus. Also, no significant difference between heart rate levels or heart rate variances was found in A. astacus and P. leniusculus at night. During day, however, heart rate levels, heart rate variances and locomotor activity were higher in P. leniusculus than in A. astacus. The higher activity level in P. leniusculus than in A. astacus during daytime indicates that P. leniusculus is less strictly nocturnal than is A. astacus.     

The 76 kD cell-adhesion factor from crayfish haemocytes promotes encapsulation in vitro

Summary Semigranular cells from the crayfish, Pacifastacus leniusculus, were separated by Percoll gradient centrifugation and were used to study the encapsulation of foreign particles. The semigranular cells were found strongly to encapsulate glass beads coated with haemocyte lysate in which the prophenoloxidase-activating system had been activated with laminarin or with a low concentration of calcium ions. The granular cells only weakly encapsulated these particles. The encapsulationpromoting factor was purified from haemocyte lysates and found to be a 76 kD protein which was recognized by an antiserum to the previously described 76 kD cell-adhesion factor. After the last step in purification (Con A-Sepharose chromatography), the flowthrough consisted of several proteins, which had some, but less, encapsulation-promoting activity and contained a 30 kD band that was also recognized by the antiserum to the 76 kD cell-adhesion factor. If the haemocyte lysate prepared in low [Ca²⁺] was incubated with a -1,3-glucan prior to purification, no 76 kD protein could be isolated but only a 30 kD protein. The 30 kD protein thus seems to be a degradation product of the 76 kD cell-adhesion factor. We conclude that the 76 kD protein which is released from degranulating haemocytes, and to a lesser extent its 30 kD fragment, can promote encapsulation. Phenoloxidase did not have any encapsulation-promoting activity.     

A challenging insight on the structural unit 1 of molluscan Rapana venosa hemocyanin

Hemocyanins of mollusks are high molecular mass glycoproteins with a complex quaternary structure which still remains to be defined in detail for most of its species as far as number, spatial distribution and interactions of their structural units is concerned. In the present study, we isolated the functional units of the structural subunit RvH1 of Rapana venosa hemocyanin, combining enzymatic and non-enzymatic methods. Our results suggest that Hc's carbohydrate moieties play a basic role in the organization of the structural units, resulting from post-translational polymerization of the 50 kDa functional units and involving sugar moieties that link between them.     

Exocytosis and uptake of bacteria by isolated haemocyte populations of two crustaceans: evidence for cellular co-operation in the defence reactions of arthropods

Summary The role of exocytosis in the cellular defence reactions of arthropods was investigated using in vitro cultures of isolated haemocytes (blood cells) from the freshwater crayfish Pacifastacus leniusculus, and the shore crab Carcinus maenas. In both species, activated lysates of those cell types that contain the prophenoloxidase activating system (granular cells of crab and crayfish and semigranular cells of crayfish) were found to induce degranulation (exocytosis) of semigranular and granular cells. A cell lysate, in which the prophenoloxidase system was kept inactive, did not have this effect. Limited degranulation of granular cells of crab was also induced by lipopolysaccharides as has earlier been shown for crayfish semigranular cells. The phagocytic capability of semigranular cells from crayfish was lost after exocytosis induced by the Ca²⁺ ionophore A23187, and under no conditions were the granular cells of crabs or crayfish seen to ingest bacteria in vitro. An opsonic function for the attaching proteins of a 1,3-glucan-activated haemocyte lysate was demonstrated using the phagocytic hyaline cells from crabs. Phenoloxidase appeared to lack opsonic properties.We suggest that, in crustaceans, opsonization takes place through hierarchically stimulated exocytotic release, and biochemical activation of the prophenoloxidase activating system: first from lipopolysaccharide-sensitive cells (semigranular cells of crayfish or granular cells of crabs) and then from granular cells, triggered by the initially released and activated prophenoloxidase system. Finally, sticky proteins of the activated prophenoloxidase system coat the invader, rendering it susceptible to the phagocytes (hyaline cells in both crab and crayfish and, to a lesser extent, semigranular cells of crayfish). These processes would, together, constitute a cellular communication pathway not previously demonstrated for invertebrates.Abbreviations DMSO dimethyl sulfoxide - L-DOPA L-dihydroxy-phenylalanine - GLS granular cell lysate supernatant - HLS haemocyte lysate supernatant - HyLS hyaline cell lysate supernatant - LPS lipopolysaccharide - proPO prophenoloxidase - SGLS semigranular cell lysate supernatant - SITS 4-acetamido-4-isothiocyanatostilbene-2,2-disulfonic acid disodium salt     

Functional properties of hemocyanin from Oncomelania hupensis, the intermediate host of Schistosoma japonicum

The gastropod mollusc, Oncomelania hupensis is a unique intermediate host for the human parasite Schistosoma japonicum. It is a primary factor for the epidemic of schistosomiasis and its distribution is consistent with the epidemic area of schistosomiasis. Here we report the functional properties of hemocyanin of O. hupensis (OhH), a copper-containing respiratory protein which was isolated from its hemolymph and purified by ammonium sulfate fractionation and ultracentrifugation. We identified the protein characters including UV absorption at 340 nm, copper content and quaternary structure. Furthermore, by induction of phenoloxidase and enzyme-linked immunosorbent assay we show that OhH exhibited o-diphenoloxidase activity after limited proteolysis, and shared carbohydrate epitopes with glycoconjugates of S. japonicum.     

The potential use of PIT telemetry for identifying and tracking crayfish in their natural environment

A method for tracking crayfish and other benthic animals in rivers and streams, based on passive integrated transponder (PIT) technology, using a portable detector was investigated. The effect of implanting crayfish with PIT tags and the efficiency of the PIT tag detector system at locating tags is described. In a laboratory study 30 signal crayfish Pacifastacus leniusculus (>33.7 mm carapace length) were internally implanted with PIT tags (12-mm long×2.1-mm diameter) and 30 crayfish matched for size and sex were kept as controls and maintained for 6 months. Tagging had no significant effect on survival, moulting or growth of crayfish, and tag retention was 100%. The reader unit consists of an antenna coil mounted on a pole and designed to be moved over the streambed to search for tagged crayfish. Efficiency testing indicated that more than 80% of tags were identified and located when hidden within different stream microhabitats.     

Glycan structures and antiviral effect of the structural subunit RvH2 of Rapana hemocyanin

Molluscan hemocyanins are very large biological macromolecules and they act as oxygen-transporting glycoproteins. Most of them are glycoproteins with molecular mass around 9000 kDa. The oligosaccharide structures of the structural subunit RvH2 of Rapana venosa hemocyanin (RvH) were studied by sequence analysis of glycans using MALDI-TOF-MS and tandem mass spectrometry on a Q-Trap mass spectrometer after enzymatical liberation of the N-glycans from the polypeptides. Our study revealed a highly heterogeneous mixture of glycans of the compositions Hex_0-9 HexNAc_2-4 Hex_0-3 Pent_0-3 Fuc_0-3. A novel type of N-glycan, with an internal fucose residue connecting one GalNAc(β1-2) and one hexuronic acid, was detected, as also occurs in subunit RvH1. A glycan with the same structure but with two deoxyhexose residues was observed as a doubly charged ion. Antiviral effects of the native molecules of RvH and also of Helix lucorum hemocyanin (HlH), of their structural subunits, and of the glycosylated functional unit RvH2-e and the non-glycosylated unit RvH2-c on HSV virus type 1 were investigated. Only glycosylated FU RvH2-e exhibits this antiviral activity. The carbohydrate chains of the FU are likely to interact with specific regions of glycoproteins of HSV, through van der Waals interactions in general or with certain amino acid residues in particular. Several clusters of these residues can be identified on the surface of RvH2-e.     

Winter movements and activity of signal crayfish Pacifastacus leniusculus in an upland river,determined by radio telemetry

Radio-telemetry was used to study the late autumn and winter movements of twenty adult signal crayfish Pacifastacus leniusculus (32.9–63.8 mm carapace length) an introduced exotic crayfish species, in the upland River Wharfe, northern England. The distances moved during the study varied greatly between individuals (0–328 m). Movements were generally sporadic; crayfish would remain in one position for several weeks and make occasional movements to new locations. Total distances travelled, linear range and ranging area did not differ significantly between males and females. The distance travelled in upstream and downstream directions did not differ significantly and there was no correlation between distance travelled and crayfish size. Several high flow events occurred during the study, but these did not cause any mortality or apparent displacement of crayfish downstream, suggesting that this is not a significant factor in downstream dispersal or mortality of adults of this invasive crayfish species in winter. A marked reduction in large-scale movements occurred in mid-December which coincided with a decline in water temperature. There was a less distinct pattern in local activity which was strongly correlated with water temperature and varied before and after mid-December.     

Involvement of glycan chains in the antigenicity of Rapana thomasiana hemocyanin

Functional unit (FU) RtH2-e from Rapana thomasiana hemocyanin (Hc) was degraded into small fragments with chymotrypsin. The glycopeptides were separated from the non-glycosylated peptides by chromatography on Concanavalin-A-Sepharose and characterized by mass spectrometry. The glycan part of the glycopeptides (all with common peptide stretch of 14 amino acids) consists of the classical trimannosyl-N,N-diacetylchitobiose core for N-glycosylation, predominantly extended with a unique tetrasaccharide that is branched on fucose. In inhibition ELISA experiments, the glycopeptides interfered in the complex formation between FU RtH2-e and rabbit antibodies against Rapana Hc (about 30% of inhibition). The inhibition also was retained after treatment of the glycopeptides with pronase in order to completely destroy the peptide part. The inhibitory effect of the non-glycosylated peptides, on the other hand, was very low. This study thus demonstrates that the glycans attached to FU RtH2-e contribute to the antigenicity of Rapana Hc.     

cDNA cloning and sequencing of tarantula hemocyanin subunits

Summary Tarantula heart cDNA libraries were screened with synthetic oligonucleotide probes deduced from the highly conserved amino acid sequences of the two copper-binding sites, copper A and copper B, found in chelicerate hemocyanins. Positive cDNA clones could be obtained and four different cDNA types were characterized.     

The artificial incubation of crayfish eggs: review and report from an experimental study concerning the effects of offspring origin (maternal or artificial incubation) on the survival and growth of juvenile signal crayfish (Pacifastacus leniusculus, Astacidae)

The development of artificial incubation techniques in astacid crayfish has attracted attention from scientists in many countries ever since the nineteenth century. It is only in the last few years that these techniques, along with studies on egg storage and transport, have provided reliable options for improving the reproductive phase in farming. The juveniles produced need to be reared until they reach a sufficient size both for restocking and for growing purposes. In view of the current level of knowledge of rearing juvenile astacids, two 80-day experiments were carried out under controlled conditions to compare the survival and growth of Stage 2 juvenile signal crayfish (Pacifastacus leniusculus) from two origins: maternal or artificial incubation. In the first experiment, three treatments were tested: juveniles from artificially incubated eggs with formaldehyde treatments, juveniles from maternal incubation and a mixture from both origins (50% each). Survival rates ranged from 87.8% to 93.3% with no significant differences among treatments. Crayfish from artificial incubation grew significantly faster (11.47 mm carapace length (CL), 373.80 mg weight) than crayfish from maternal incubation. In the second experiment, a bifactorial design included four treatments: the crayfish was derived from artificial or from maternal incubation and was fed once a day or twice a day. Final survival rates ranged from 68.89% to 77.78%, with no significant differences among treatments. Crayfish from artificial incubation grew significantly faster than crayfish from maternal incubation. The highest CL (14.54 mm) and weight (780.13 mg) were reached by the juveniles from artificial incubation that were fed once a day. No significant differences were found between the two feeding frequencies. Results showed that artificial incubation with formaldehyde treatments had no harmful effects and made it feasible to get a better performance from the juveniles obtained.     

Role of the tertiary structure in the diphenol oxidase activity of Octopus vulgaris hemocyanin

The functional differences between the oxygen transport protein Hemocyanin and the enzymes Tyrosinase and Catechol oxidase are believed to be governed, at least in part, by the tertiary structure, which differs in these molecules and controls the accessibility of their copper containing active site for substrate(s). Accordingly, Octopus vulgaris Hemocyanin catalyses the o-diphenol oxidation to o-quinone at a very low rate. The crystallographic structure of one of the functional units (called Odg) of O. dofleini Hemocyanin shows two domains, a mainly α-helical domain that directly binds the copper ions of the reaction center and a β-strand domain that precludes access to the active site to ligands bigger than molecular oxygen. In this work, we have first cleaved the whole protein and then purified different oxygen binding functional units from O. vulgaris Hemocyanin. These functional units were used in activity assays with l-DOPA, the paradigmatic substrate for Catechol oxidase. All functional units show a negligible enzymatic activity. The procedure to generate the functional units induces in only one of them a proteolytic cleavage. Amino terminal sequencing and mass spectroscopy of the fragments allow to place the cleavage site between the alpha and beta domains of the functional unit homologous to Odd, in the O. dofleini sequence. An increase, up to three orders of magnitude, of Tyrosinase-like activity was observed when the cleaved Odd-like was incubated with the substrate in the presence of trifluoroethanol or hexafluoroisopropanol.     

New records and distributions of two North American branchiobdellidan species (Annelida: Clitellata) from introduced signal crayfish, <Emphasis Type="Italic">Pacifastacus leniusculus,</Emphasis> in Japan

This is the first report of two North American branchiobdellidans, Sathodrilus attenuatus Holt, 1981, and Xironogiton victoriensis Gelder and Hall, 1990, on the signal crayfish, Pacifastacus leniusculus (Dana, 1852) introduced into Japan from the Columbia River system, northwestern North America. Signal crayfish from 12 localities in eastern and northern Hokkaido, Japan, were examined and each supported S. attenuatus. In addition, an individual of this species was found on preserved material from Ishikawa Prefecture, central Honshu. All of these branchiobdellidans reported in Hokkaido most probably came from the original population of signal crayfish introduced into Lake Mashu, Hokkaido, Japan, in 1930. It is suggested that the use of non-pathogenic branchiobdellidans, when present, provides an easy method for tracing the spread of crayfishes around Japan and could also be applied in other countries and continents. Specimens of X. victoriensis were only found on crayfish in a stream at Akashina in Nagano Prefecture, central Honshu, Japan. Although the signal crayfish appears to be displacing the endemic Japanese crayfish, C. japonicus, no native branchiobdellidans were found on any of the introduced signal crayfish examined.     

Hemocyanin-derived phenoloxidase activity with broad temperature stability extending into the cold environment in hemocytes of the hair crab Erimacrus isenbeckii

Phenoloxidase (PO) activity is a major component of the innate immune response in arthropods. In this study, we characterized PO activity from the hair crab Erimacrus isenbeckii, which inhabits very cold regions (2.4-3.4°C) of the Bering Sea. Hemocyte lysate supernatant (HLS) prepared from E. isenbeckii was inactive HLS until activated by nonspecific agents such as sodium dodecyl sulfate and trypsin, and elicitors such as lipopolysaccharide and lipoteichoic acid from the cell wall constituent of bacteria. The PO activity was maximal at 4°C, decreased slightly at temperatures up to 60°C, and fell rapidly at 80°C. Both L-DOPA and catechol were efficient substrates for the PO (EC 1.10.3.1), with K(m) values of 0.96 and 1.15mM, respectively, whereas tyrosine and hydroquinone were not. We isolated a protein fraction from HLS as a hexamer of 75kDa units with 216.7-fold higher PO activity than that of the HLS. The N-terminal amino acid analysis of an isolated protein revealed 80% sequence identity to hemocyanins from other crabs. These results suggest that cold-adapted hemocyanin-derived PO activity is important to the survival of these crabs. This is the first report of a crab PO activity with broad temperature stability extending into the cold environment.     

Battle and balance at mucosal surfaces - The story of Shigella and antimicrobial peptides

Shigella is a major cause of morbidity and mortality for children in many developing countries. Emergence of antibiotic-resistance among Shigella demands the development of effective medicines. Antimicrobial peptides (AMPs) are expressed in phagocytes and at epithelial surfaces and are important effector molecules of innate immunity. We have found that pathogens are able to turn off the endogenous expression of AMPs, resulting in serious infections such as shigellosis. A therapeutic rationale to prevent microbial invasion would be to strengthen the epithelial line of defence through enhancing AMP expression. We have identified several inducers of AMP-production, including butyrate, phenylbutyrate and vitamin D, which have been investigated in animal models of shigellosis as well as in clinical trials. We believe that the conceptual framework presented here can be applied to additional clinical entities and that this novel approach can be an alternative or complement to traditional antibiotics in the future.